ovalbumin and Neoplasms

Langerhans cells (LCs) are antigen-presenting dendritic cells (DCs) that reside in epithelia. The best studied example is the LC of the epidermis. By electron microscopy, their identifying feature is the unique rod- or tennis racket-shaped Birbeck granule. The phenotypic hallmark is their expression of the C-type lectin receptor langerin/CD207. Langerin, however, is also expressed on a recently discovered population of DC in the dermis and other tissues of the body. These 'dermal langerin(+) dendritic cells' are unrelated to LCs. The complex field of langerin-negative dermal DCs is not dealt with here. In this article, we briefly review the history, ontogeny, and homeostasis of LCs. More emphasis is laid on the discussion of functional properties in vivo. Novel models using genetically engineered mice are contributing tremendously to our understanding of the role of LCs in eliciting adaptive immune responses against pathogens or tumors and in inducing and maintaining tolerance against self antigens and innocuous substances in vivo. Also, innate effector functions are increasingly being recognized. Current activities in this area are reviewed, and possibilities for future exploitation of LC in medicine, e.g. for the improvement of vaccines, are contemplated.

Topics: Adaptive Immunity; Animals; Antigen Presentation; Antigens, CD; Antigens, Surface; Cell Lineage; Communicable Diseases; Dermatitis, Contact; Disease Models, Animal; Homeostasis; Humans; Immune Tolerance; Immunity, Innate; Langerhans Cells; Lectins, C-Type; Mannose-Binding Lectins; Mice; Mice, Transgenic; Neoplasms; Ovalbumin; Phenotype; Skin; Vaccines

A successful vaccine for immunotherapy, particularly for solid tumors or viral infections, requires a suitable target antigen and the production of a cytotoxic T-cell response. In addition, CD4 T cells play an important role in cellular immunity. Here, we briefly discuss methods by which T cells are measured in vitro after vaccination.

Topics: Animals; Cancer Vaccines; CD4-Positive T-Lymphocytes; Hematopoietic Stem Cells; Humans; Neoplasms; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Vaccination

Serine protease inhibitors (serpins) are a family of proteins that are important in the regulation of several biological processes. This mainly involves the inhibition of serine proteases, although some serpins inhibit a different class of proteases or even function without inhibitory activity. In contrast to other protease inhibitor families, serpins inhibit their target proteases by a specific mechanism, which depends on a change in conformation. This review primarily focuses on one subgroup of serpins--ovalbumin (ov)-serpins. Different than most members of the family, this group of serpins lacks secretion signal sequences and therefore, mainly functions intracellularly. In addition to expression in most normal tissues, ov-serpins can be found in multiple different cells of the immune system. Interestingly, expression of ov-serpins in these cells is tightly regulated, indicating a role for these serpins in the regulation of immune responses. The role of serpins in the immune response will be the topic of this review.

Topics: CD8-Positive T-Lymphocytes; Humans; Immunologic Memory; Neoplasms; Ovalbumin; Serpins; T-Lymphocytes

Topics: Adenine Phosphoribosyltransferase; Animals; Cell Differentiation; DNA; DNA (Cytosine-5-)-Methyltransferases; Eukaryotic Cells; Genes; Growth Hormone; Humans; Insulin; Methylation; Methyltransferases; Neoplasms; Ovalbumin; Species Specificity; Vitellogenins

Topics: Amyloidosis; Animals; Antigens; Basement Membrane; Blood Group Antigens; Brain Chemistry; Carbohydrates; Cell Membrane; Collagen; Connective Tissue; Cystic Fibrosis; Diabetic Angiopathies; Enzymes; gamma-Globulins; Glycoproteins; Hormones; Humans; Mucins; Mucous Membrane; Neoplasms; Ovalbumin

Recent studies have shown the potent efficacy of peptide-based vaccines for cancer immunotherapy. Immunological performance is optimized through the co-delivery of adjuvant and antigenic peptide molecules to antigen-presenting cells simultaneously. In our previous study, we showed that a conjugate consisting of 40-mer CpG-DNA and an antigenic ovalbumin peptide through disulfide bonding could efficiently induce ovalbumin-specific cytotoxic T lymphocyte (CTL) responses in vivo. In this study, based on the conjugation design, we prepared a conjugate consisting of 30-mer CpG-DNA (CpG30) and a cancer antigenic peptide of Tyrosinase-related protein 2 (TRP2

Topics: Animals; Antigens; CpG Islands; Cysteine; DNA; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Neoplasms; Ovalbumin; Peptide Fragments; Peptides; T-Lymphocytes, Cytotoxic

As metal ions play important roles in the process of immunomodulation, immunotherapy based on metal ions has attracted tremendous interests in recent years. Here, we screened common metal ions and found that Mn

Topics: Animals; Antigen Presentation; Antigens; Cancer Vaccines; Dendritic Cells; Hydrogen-Ion Concentration; Immunotherapy; Manganese; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Signal Transduction

Adjuvants play an important role in enhancing vaccine-induced immune protection. Adequate cellular uptake, robust lysosomal escape, and subsequent antigen cross-presentation are critical steps for vaccine adjuvants to effectively elicit cellular immunity. Here, a fluorinated supramolecular strategy to generate a series of peptide adjuvants by using arginine (R) and fluorinated diphenylalanine peptide (DP) is adopted. It is found that the self-assembly ability and antigen-binding affinity of these adjuvants increase with the number of fluorine (F) and can be regulated by R. By comparison, 4RDP(F5) shows the strongest binding affinity with model antigen ovalbumin (OVA) and the best performance in dendritic cells maturation and antigen's lysosomal escape, which contributes to the subsequent antigen cross-presentation. As a consequence, 4RDP(F5)-OVA nanovaccine generates a strong cellular immunity in a prophylactic OVA-expressing EG7-OVA lymphoma model, leading to long-term immune memory for resisting tumor challenge. What's more, 4RDP(F5)-OVA nanovaccine in combination with anti-programmed cell death ligand-1 (anti-PD-L1) checkpoint blockade could effectively elicit anti-tumor immune responses and inhibit tumor growth in a therapeutic EG7-OVA lymphoma model. Overall, this study demonstrates the simplicity and effectiveness of fluorinated supramolecular strategies for constructing adjuvants and might provide an attractive vaccine adjuvant candidate for cancer immunotherapy.

Topics: Adjuvants, Immunologic; Antigen Presentation; Antigens; Cancer Vaccines; Humans; Neoplasms; Ovalbumin

Nanovaccines have attracted intense interests for efficient antigen delivery and tumor-specific immunity. It is challenging to develop a more efficient and personalized nanovaccine to maximize all steps of the vaccination cascade by exploiting the intrinsic properties of nanoparticles. Here, biodegradable nanohybrids (MP) composed of manganese oxide nanoparticles and cationic polymers are synthesized to load a model antigen ovalbumin to form MPO nanovaccines. More interestingly, MPO could serve as autologous nanovaccines for personalized tumor treatment taking advantage of in situ released tumor-associated antigens induced by immunogenic cell death (ICD). The intrinsic properties of MP nanohybrids including morphology, size, surface charge, chemical, and immunoregulatory functions are fully exploited to enhance of all steps of the cascade and induce ICD. MP nanohybrids are designed to efficiently encapsulate antigens by cationic polymers, drain to lymph nodes by appropriate size, be internalized by dendritic cells (DCs) by rough morphology, induce DC maturation through cGAS-STING pathway, and enhance lysosomal escape and antigen cross-presentation through the "proton sponge effect". The MPO nanovaccines are found to efficiently accumulate in lymph nodes and elicit robust specific T-cell immune responses to inhibit the occurrence of ovalbumin-expressing B16-OVA melanoma. Furthermore, MPO demonstrate great potential to serve as personalized cancer vaccines through the generation of autologous antigen depot through ICD induction, activation of potent antitumor immunity, and reversal of immunosuppression. This work provides a facile strategy for the construction of personalized nanovaccines by exploiting the intrinsic properties of nanohybrids.

Topics: Antigens, Neoplasm; Humans; Immunogenic Cell Death; Neoplasms; Ovalbumin; Polymers; Vaccination

Antigen cross-presentation by dendritic cells is an important pathway to prime CD8

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Dendritic Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin

Mild-temperature photothermal therapy (mild PTT) is a safe and efficient antitumor therapy. However, mild PTT alone usually fails to activate the immune response and prevent tumor metastasis. Herein, a photothermal agent, copper sulfide@ovalbumin (CuS@OVA), with an effective PTT effect in the second near-infrared (NIR-II) window, is developed. CuS@OVA can optimize the tumor microenvironment (TME) and evoke an adaptive immune response. Copper ions are released in the acidic TME to promote the M1 polarization of tumor-associated macrophages. The model antigen OVA not only acts as a scaffold for nanoparticle growth but also promotes the maturation of dendritic cells, which primes naive T cells to stimulate adaptive immunity. CuS@OVA augments the antitumor efficiency of the immune checkpoint blockade (ICB) in vivo, which suppresses tumor growth and metastasis in a mouse melanoma model. The proposed therapeutic platform, CuS@OVA nanoparticles, may be a potential adjuvant for optimizing the TME and improving the efficiency of ICB as well as other antitumor immunotherapies. STATEMENT OF SIGNIFICANCE: Mild-temperature photothermal therapy (mild PTT) is a safe and efficient antitumor therapy, but usually fails to activate the immune response and prevent tumor metastasis. Herein, we develop a photothermal agent, copper sulfide@ovalbumin (CuS@OVA), with an excellent PTT effect in the second near-infrared (NIR-II) window. CuS@OVA can optimize the tumor microenvironment (TME) and evoke an adaptive immune response by promoting the M1 polarization of tumor-associated macrophages and the maturation of dendritic cells. CuS@OVA augments the antitumor efficiency of the immune checkpoint blockade (ICB) in vivo, suppressing tumor growth and metastasis. The platform may be a potential adjuvant for optimizing the TME and improving the efficiency of ICB as well as other antitumor immunotherapies.

Topics: Animals; Cell Line, Tumor; Copper; Hyperthermia, Induced; Immune Checkpoint Inhibitors; Immunotherapy; Mice; Multifunctional Nanoparticles; Nanoparticles; Neoplasms; Ovalbumin; Phototherapy; Photothermal Therapy; Sulfides; Tumor Microenvironment

Tumor-specific cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens presented on major histocompatibility complex (MHC) class I molecules. However, it is difficult to induce potent CTLs by vaccination because the antigenicity is not so high, compared with that of foreign antigens derived from viruses and microbes. The affinity of binding to MHC class I molecules is proportional to the antigenicity of the antigen that they are presenting. Here, we prepared several conjugates consisting of hyaluronic acid (HA) as a carrier to cancer cells and ovalbumin (OVA) as a foreign protein and changed the antigens on cancer cells from intrinsic antigens to OVA fragments. The conjugate containing multiple HA and OVA molecules (100k4HA-3OVA) adopted a highly condensed structure and was well recognized by recombinant CD44 molecules in quartz crystal microbalance analysis and incorporated into cancer cells (CT26 cells). A mixture of CT26 cells treated with 100k4HA-3OVA and splenocytes including OVA-specific CTLs induced abundant secretion of IFN-γ into the supernatant. At 48 h after mixing with the CTLs, almost all CT26 cells had died. These results indicate that 100k4HA-3OVA is actively internalized into the cells through interaction between HA and CD44. Subsequently, CT26 cells present not only self-antigens, but also OVA fragments on MHC class I molecules and are recognized by OVA-specific CTLs. We thus succeeded in modifying the antigenicity from self- to non-self-antigens on cancer cells. Therefore, this foreign-antigen delivery using HA to cancer cells, followed by antigen replacement, could be used as a novel strategy for treating cancers.

Topics: Animals; Antigens; Antigens, Neoplasm; Histocompatibility Antigens Class I; Humans; Hyaluronic Acid; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Developing low-cost, easy-to-prepare, biocompatible and highly efficient vaccine carriers is a promising approach to realize practical cancer immunotherapy. In this study, through facile modification of mPEG5k-4-toluenesulfonate (mPEG5k-OTs) on PEI25k under mild conditions, a series of "stealth" mPEG5k-PEI25k polymers (PP1, PP2 and PP3) were prepared, their structures and physicochemical properties were characterized and theoretically analyzed. The polymers could bind/load ovalbumin (OVA) to form mPEG5k-PEI25k/OVA complexes as negatively charged nanoparticles with small hydrodynamic particle size (80-210 nm) and narrow size distribution. Compared to PEI25k/OVA, lower cytotoxicity could be achieved on mPEG5k-PEI25k/OVA complexes in dendritic cells (DCs). In DCs-RF 33.70 T-cells co-culture system, the mPEG5k-PEI25k/OVA complexes could bring about higher IL-2 production /secretion than that of PEI25k/OVA, notably, the optimum IL-2 secretion could reach 9.3-folds of the PEI25k/OVA under serum condition (10% FBS). Moreover, the cell biological features could be optimized by selecting suitable mPEG5k-grafting ratios and/or mPEG5k-PEI25k/OVA weight ratios. Intracellular imaging results showed that the mPEG5k-PEI25k(PP3)/Rhodamine-OVA complexes mainly localized inside lysosomes. Taken together, this work provided a facile method to prepare "stealth" PEGylated-PEI25k polymers with reduced cytotoxicity, promoted OVA cross-presentation efficiency and improved serum compatibility towards cancer immunotherapy.

Topics: Humans; Interleukin-2; Neoplasms; Ovalbumin; Polyethylene Glycols; Polyethyleneimine; Polymers; Vaccines

Immunoadjuvants, as an indispensable component of tumor vaccines, can observably enhance the magnitude, breadth, and durability of antitumor immunity. However, current immunoadjuvants suffer from different issues such as weak immunogenicity, inadequate cellular internalization, poor circulation time, and mono-functional bioactivity.

Topics: Adjuvants, Immunologic; Antigens; Cancer Vaccines; Cell Line, Tumor; Ferric Compounds; Humans; Immunogenic Cell Death; Immunotherapy; Nanomedicine; Neoplasms; Ovalbumin; Tissue Distribution; Tumor Microenvironment; Vaccination

The delivery location of traditional vaccines can impact immune responses and resulting efficacy. Cryogel-based cancer vaccines, which are typically injected near the inguinal lymph nodes (iLNs), recruit and activate dendritic cells (DC) in situ, induce DC homing to the iLNs, and have generated potent anti-tumor immunity against several murine cancer models. However, whether cryogel vaccination distance to a draining LN affects the kinetics of DC homing and downstream antigen-specific immunity is unknown, given the heightened importance of the scaffold vaccine site. We hypothesized that vaccination near the iLNs would lead to more rapid DC trafficking to the iLNs, thereby inducing faster and stronger immune responses. Here, mice were injected with cryogel vaccines against ovalbumin either adjacent or distal to the iLNs, and the resultant DC trafficking kinetics, T cell phenotypes, antigen-specific T cell and humoral responses, and prophylactic efficacy in an ovalbumin-expressing tumor model were assessed. Cryogel vaccines induced potent, long-lasting antigen-specific immune responses independent of distance to the iLNs, with no significant differences in DC trafficking kinetics, ovalbumin-specific T cell and antibody responses, or prophylactic efficacy. Moreover, DC trafficking and activation state were not impacted when cryogels were injected near a tumor. These results demonstrate a flexibility in vaccination location for scaffold-based vaccines, independent of draining LN distance.

Topics: Animals; Antigens; Cancer Vaccines; Cryogels; Dendritic Cells; Immunity; Lymph Nodes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin

Although therapeutic cancer vaccines have been gaining substantial ground, the development of cancer vaccines is impeded because of the undegradability of delivery systems, ineffective delivery of tumor antigens and weak immunogenicity of adjuvants. Here, we made use of a whole glucan particle (WGP) to encapsulate ovalbumin (OVA), thereby formulating a novel cancer vaccine. Results from in vitro experiments showed that WGP-OVA not only induced the activation of bone marrow-derived macrophages (BMDMs) including driving M0 BMDM polarization to the M1 phenotype, upregulating the costimulatory molecules and inducing the generation of cytokines, but also facilitated antigen presentation. After oral administration of the WGP-OVA formulation to mice with OVA-expressing tumors, these particles can increase tumor-infiltrating OVA-specific CD8

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Cancer Vaccines; Glucans; Macrophages; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin

Induction of tumor-specific CD8 + T cell responses is known as a major challenge for cancer vaccine development; here we presented a strategy to improve peptide nanofibers-mounted antitumor immune responses. To this end, peptide nanofibers bearing class I (Kb)-restricted epitope (Epi-Nano) were formulated with polyethylene imine backbone (Epi-Nano-PEI), and characterized using morphological and physicochemicalcharacterizationtechniques. Nanofibers were studied in terms of their uptake by antigen-presenting cells (APCs), antigen cross-presentation capacity, and cytotoxic activity. Furthermore, nanofibers were assessed by their potency to induce NLRP3 inflammasome-related cytokines and factors. Finally, the ability of nanofibers to induce tumor-specific CD8 T cells and tumor protection were investigated in tumor-bearing mice. The formulation of Epi-Nano with PEI led to the formation of short strand nanofibers with a positive surface charge, a low critical aggregation concentration (CAC), and an increased resistancetoproteolytic degradation. Epi-Nano-PEI was significantly taken up more efficiently by antigen-presenting cells (APCs), and was more potent in cross-presentation when compared to Epi-Nano. Moreover, Epi-Nano-PEI, in comparison to Epi-Nano, efficiently up-regulated the expression of NLRP3, caspase-1, IL-1b, IL18 and IL-6. Cell viability analysis showed that formulation of PEI with Epi-Nano not only abolished its cytotoxic activity, but surprisingly induced macrophage proliferation. Furthermore, it demonstrated that Epi-Nano-PEI triggered robust antigen-specific CD8

Topics: Animals; Antigen-Presenting Cells; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Epitopes; Female; Mice, Inbred C57BL; Nanofibers; Neoplasms; Ovalbumin; Peptides; Polyethyleneimine

Adjuvant loaded nanoparticles are a potent strategy for developing effective combined cancer immunotherapies. A polyinosinic-polycytidylic acid (poly I:C) is a ligand for toll-like receptor 3 and a promising cancer adjuvant. However, regarding intravenous administration, the potential for and the mechanism of poly I:C loaded nanoparticles as a cancer vaccine are largely unknown. We investigated the effects of using a combination of poly I:C and an antigen loaded liposome for cancer immunotherapy and a key process for achieving effective antitumor immunity of the liposome system under conditions of intravenous vaccination. A poly I:C and ovalbumin (OVA) loaded octaarginine (R8) modified liposome (PoIC/OVA-R8L) drastically inhibited the systemic cytokine production derived from the poly I:C intravenous injection. Treatment with PoIC/OVA-R8L improved the immune status in B16-OVA tumors to an inflamed immune status and induced a significant combined antitumor effect with the anti-programmed cell death 1 ligand (PD-L1). In a mechanistic analysis compared with a high dose of the free form of poly I:C, interestingly, local cytokine production, maturation of antigen presenting cells and antigen presentation were comparable. Conversely, significant differences were identified in the processes after OVA-specific CTL generation. Collectively, our findings have implications for the development of intravenous liposomal vaccines.

Topics: Adjuvants, Immunologic; Administration, Intravenous; Animals; Antigen Presentation; Cancer Vaccines; Cytokines; Dendritic Cells; Ligands; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Poly I-C

Topics: Adjuvants, Immunologic; Animals; Antigens; Cancer Vaccines; Immunity, Cellular; Mice; Mice, Inbred C57BL; Nanogels; Neoplasms; Ovalbumin; Toll-Like Receptor 7; Toll-Like Receptor 8

Enhancement of drug efficacy is essential in cancer treatment. The immune stimulator ovalbumin (Ova)-coated citric acid (AC-)-stabilized iron oxide nanoparticles (AC-IO-Ova NPs) and enhanced permeability and retention (EPR)-based tumor targeted 4.5 generation poly(amidoamine) dendrimer(4.5GDP)-cisplatin (Cis-pt) nanocomplex (NC) (4.5GDP-Cis-pt NC) were used for enhanced anticancer efficiency. The formations of 4.5GDP-Cis-pt NC, AC-IO, and AC-IO-Ova NPs were examined via FTIR spectroscopy, X-ray diffraction, Raman spectroscopy, and X-ray photoelectron spectroscopy. The conjugation of Cis-pt with 4.5GDP was confirmed using carbon NMR spectroscopy. The tumor-specific 4.5GDP-Cis-pt NC provided 45%and 28% cumulative cisplatin release in 72 h at pH 6.5 and 7.4, respectively. A significant immune response with high TNF-α and IL-6 cytokine secretion was confirmed for the co-incubation of AC-IO-Ova with RAW 264.7 or HaCaT cells. AC-IO-Ova NPs were biocompatible with different cell lines, even at a high concentration (200 µg mL

Topics: Cell Survival; Cisplatin; Dendrimers; Humans; Magnetic Iron Oxide Nanoparticles; Nanoparticles; Neoplasms; Ovalbumin; Polyamines

"Foreignization" of tumor cells via delivery of a non-self foreign antigen (Ag) into tumors is an appealing strategy to initiate anti-tumor immunity that can facilitate tumor rejection by pre-existing foreign-Ag-reactive T cells. However, the immune-suppressive factors in the tumor microenvironment (TME) limit the durable and potent immune response of these cells against tumor antigens, stressing the need for improved tumor-foreignization strategies. Here, we demonstrate that blockade of programmed cell death ligand 1 (PD-L1) on both tumor cells and dendritic cells (DCs) can markedly potentiate the induction of tumor-reactive T cells, thereby strengthening the anti-tumor immunity ignited by tumor-foreignization. Specifically, we developed a polymeric nanoconjugate (PEG-HA-OVA/PPLs), consisting of siPD-L1-based polyplexes, PEGylated hyaluronic acid as the CD44-targeting moiety, and ovalbumin (OVA) as a model foreign antigen. Notably, PEG-HA-OVA/PPLs were simultaneously delivered into CD44

Topics: Animals; Antigens, Neoplasm; B7-H1 Antigen; Hyaluronic Acid; Immunotherapy; Mice; Mice, Inbred C57BL; Nanoconjugates; Neoplasms; Ovalbumin; RNA, Small Interfering; Tumor Microenvironment

Phototherapy-triggered immunogenic cell death (ICD) rarely elicits a robust antitumour immune response, partially due to low antigen exposure and inefficient antigen presentation. To address these issues, we developed novel methylene blue-loaded ovalbumin/polypyrrole nanoparticles (MB@OVA/PPY NPs) via oxidative polymerization and π-π stacking interactions.. The as-prepared MB@OVA/PPY NPs with outstanding photothermal conversion efficiency (38%) and photodynamic properties were readily internalized into the cytoplasm and accumulated in the lysosomes and mitochondria. Upon 808 nm and 660 nm laser irradiation, the MB@OVA/PPY NPs not only ablated tumour cells by inducing local hyperthermia but also damaged residual tumour cells by generating a large amount of reactive oxygen species (ROS), finally triggering the release of many damage-associated molecular patterns (DAMPs). Moreover, the MB@OVA/PPY NPs synergized with DAMPs to promote the maturation and improve the antigen presentation ability of DCs in vitro and in vivo.. This work reported a PPY NPs-based nanoplatform to encapsulate the therepeutic proteins and absorb the functional molecules for combination therapy of tumours. The results demonstrated that the prepared MB@OVA/PPY NPs could be used as effective nanotherapeutic agents to eliminate solid tumours and trigger a powerful antitumour immune response.

Topics: Humans; Methylene Blue; Nanoparticles; Neoplasms; Ovalbumin; Phototherapy; Polymers; Pyrroles

As two of the most widely used adjuvants, aluminum hydroxide and the oil-in-water emulsion MF59 have their intrinsic limitations: classical aluminum gel induces only weak cellular immune responses while MF59 cannot be used as an antigen delivery system due to its poor physical interaction with antigen molecules. Herein, we combined these two adjuvants and constructed a novel nano-vaccine delivery system by inserting aluminum hydroxide into the surface of a modified MF59 nano-emulsion (AlNEs). A model antigen ovalbumin (OVA) and an immune potentiator CpG were adsorbed on the surface of AlNEs (hereinafter AlNEs-OVA-CpG) through a facile mixing step. After subcutaneous injection, AlNEs-OVA-CpG effectively drained to lymph nodes, delivered both cargos into lymph node-resident antigen presenting cells (APCs), and escaped from lysosomes into the cytoplasm, resulting in enhanced antigen cross-presentation. Finally, AlNEs-OVA-CpG induced potent antigen-specific humoral and cellular immune responses, which significantly inhibited tumor growth and prolonged mice survival in a EG7-OVA tumor model. In sum, our results suggested that AlNEs have a great prospect to induce CD8

Topics: Adjuvants, Immunologic; Aluminum; Aluminum Hydroxide; Animals; Antigens; Immunity, Cellular; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Polysorbates; Squalene; Water

The targeted delivery of messenger RNA (mRNA) to desired organs remains a great challenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted delivery to the lymph node (LN) is predicted to reduce side effects and increase the immune response. In this study, we explored an endogenously LN-targeting lipid nanoparticle (LNP) without the modification of any active targeting ligands for developing an mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expression in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased the CD8

Topics: Amino Alcohols; Animals; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Decanoates; Immunologic Memory; Liposomes; Lymph Nodes; Mice; mRNA Vaccines; Nanoparticles; Neoplasm Metastasis; Neoplasms; Ovalbumin

Activation of the stimulator of interferon genes (STING) is essential for blocking viral infections and eliciting antitumor immune responses. Local injection of synthetic STING agonists, such as 2'3'-cGAMP [cGAMP = cyclic 5'-guanosine monophosphate (cGMP)-adenosine monophosphate (AMP)], is a promising approach to enhance antiviral functions and cancer immunotherapy. However, the application of such agonists has been hindered by complicated synthetic procedures, high doses, and unsatisfactory systemic immune responses. Herein, we report the design and synthesis of a series of 2'3'-cGAMP surrogates in nanoparticle formulations formed by reactions of AMP, GMP, and coordinating lanthanides. These nanoparticles can stimulate the type-I interferon (IFN) response in both mouse macrophages and human monocytes. We further demonstrate that the use of europium-based nanoparticles as STING-targeted adjuvants significantly promotes the maturation of mouse bone-marrow-derived dendritic cells and major histocompatibility complex class I antigen presentation. Dynamic molecular docking analysis revealed that these nanoparticles bind with high affinity to mouse STING and human STING. Compared with soluble ovalbumin (OVA), subcutaneously immunized europium-based nanovaccines exhibit significantly increased production of primary and secondary anti-OVA antibodies (∼180-fold) in serum, as well as IL-5 (∼28-fold), IFN-γ (∼27-fold), and IFN-α/β (∼4-fold) in splenocytes ex vivo. Compared with the 2'3'-cGAMP/OVA formulation, subcutaneous administration of nanovaccines significantly inhibits B16F10-OVA tumor growth and prolongs the survival of tumor-bearing mice in both therapeutic and protective models. Given the rich supramolecular chemistry with lanthanides, this work will enable a readily accessible platform for potent humoral and cellular immunity while opening new avenues for cost-effective, highly efficient therapeutic delivery of STING agonists.

Topics: Adenosine Monophosphate; Animals; Europium; Humans; Interferon Type I; Interferon-beta; Lanthanoid Series Elements; Membrane Proteins; Mice; Molecular Docking Simulation; Nanoparticles; Neoplasms; Nucleotides; Nucleotides, Cyclic; Ovalbumin

As a representative of tumor immunotherapy, tumor vaccine can inhibit tumor growth by activating tumor-specific immune response, which has the advantages of relatively low toxicity and high efficiency, and has attracted much attention in recent years. However, there are still difficulties in how to effectively deliver tumor vaccines in vivo and make them work efficiently. It is a relatively mature method to load tumor specific antigens with suitable carriers to produce tumor vaccines. Here, a generally minimalist construction method of tumor nanovaccine was developed. A high-efficiency tumor nanovaccine (NV) was prepared in one step by a biomineralization-like method, which contained ovalbumin (OVA, model antigen), unmethylated cytosine-phosphate-guanine (CpG, adjuvant) and Mn-NP (carrier and adjuvant). NV not only showed good tumor preventive effect, but also could successfully inhibited tumor development and metastasis when combined with anti-PD-L1, and induced long-term immune memory effect. However, the method of screening tumor specific antigen to construct nanovaccine is cumbersome and tumors are heterogeneous. Therefore, surgically resected tumor tissue is the best source of antigens for preparing tumor vaccines. Next, based on the strong loading ability of the carrier, we designed a personalized tumor nanovaccine (PNV) using the supernatant of tumor abrasive fluid (STAF) as antigen based on the generally minimalist tumor nanovaccine construction strategy. PNV combined with anti-PD-L1 could successfully inhibit post-surgical tumor recurrence and induce strong and durable immune memory effects. This study presents a novel, general, and minimalist strategy to construct high-efficiency personalized nanovaccine, which has a wide range of potential applications in the field of tumor treatment.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cytosine; Guanine; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Phosphates

Cancer vaccines have recently garnered tremendous interest. However, the targeted delivery of antigens and adjuvants to dendritic cells (DCs) still remains challenging. In this study, we developed glycosylated poly(lactic-co-glycolic acid) nanoparticles (NPs) loaded with the SIINFEKL peptide (OVA) as a tumor-specific antigen and CpG oligodeoxynucleotide (CpG) as an adjuvant for an effective DC-targeted cancer vaccine. Surface modification of NPs with galactose (Gal) or mannose (Man) was carried out by a double-emulsion solvent evaporation method, and the products were respectively named OVA-CpG Gal-NPs and OVA-CpG Man-NPs. They exhibited a uniform particle size, high loading capacity, robust stability, and extended release. The OVA-CpG Gal-NPs were found to rapidly enhance antigen uptake and DC maturation. In the in vivo study, OVA-CpG Gal-NPs via intravenous (i.v.), intranasal (i.n.) and subcutaneous (s.c.) routes had rapidly accumulated in the spleen. Moreover, the non-glycosylated OVA-CpG NPs after s.c. immunization could rapidly be trafficked to distal lymph nodes and sustained higher levels. All of these formulations increased the level of cluster of differentiation 4-positive (CD4

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Cancer Vaccines; Dendritic Cells; Glycosylation; Humans; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Polylactic Acid-Polyglycolic Acid Copolymer; Vaccination

Topics: Animals; Autoantigens; Disease Models, Animal; Inflammation; Mice; Myeloid-Derived Suppressor Cells; Neoplasms; Ovalbumin

Osteosarcoma is a malignant osteogenic tumor with a high metastatic rate commonly occurring in adolescents. Although radiotherapy is applied to treat unresectable osteosarcoma with radiation resistance, a high dose of radiotherapy is required, which may weaken the immune microenvironment. Therefore, there is an urgent need to develop novel agents to maximize the radiotherapeutic effects by eliciting immune activation effects. In this study, we synthesized therapeutic gadolinium-based metal-bisphosphonate nanoparticles (NPs) for osteosarcoma treatment that can be combined with radiotherapy. The gadolinium ion (Gd) was chelated with zoledronic acid (Zol), a commonly used drug to prevent/treat osteoporosis or bone metastases from advanced cancers, and stabilized by ovalbumin (OVA) to produce OVA-GdZol NPs. OVA-GdZol NPs were internalized into K7M2 osteosarcoma cells, showing a high sensitization effect under X-ray irradiation. Cell pretreatment of OVA-GdZol NPs significantly enhanced the radiation therapeutic effect in vitro by reducing the cell colonies and increased the signal of γH2AX-positive cells. More importantly, OVA-GdZol NPs promoted the maturation of bone marrow-derived dendritic cells (BMDCs) and M1 polarization of macrophages. The inhibitory effect on K7M2 osteosarcoma of OVA-GdZol NPs and X-ray radiation was evident, indicated by a significantly reduced tumor volume, high survival rate, and decreased lung metastasis. Meanwhile, both innate and adaptive immune systems were activated to exert a strong antitumor effect. The above results highly suggest that OVA-GdZol NPs serve as both radiosensitizers and immune adjuvants, suitable for the sequential combination of vaccination and radiotherapy.

Topics: Adolescent; Diphosphonates; Gadolinium; Humans; Nanoparticles; Neoplasms; Ovalbumin; Tumor Microenvironment

Induction of cellular immunity is important for effective cancer immunotherapy. Although various antigen carriers for cancer immunotherapy have been developed to date, balancing efficient antigen delivery to antigen presenting cells (APCs) and their activation

Topics: Animals; Antigen-Presenting Cells; beta-Glucans; Biocompatible Materials; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytokines; Female; Hydrogen-Ion Concentration; Immunity, Cellular; Immunotherapy; Liposomes; Macrophage Activation; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Tumor Microenvironment

Peptides and proteins represent an emerging class of powerful therapeutics. Peptide and protein nanogels are attractive carriers for the transport and delivery of biologically active peptides and proteins because they allow essentially quantitative encapsulation of these biologics. One interesting field of use of peptide and protein nanogels is the transport of antigens and adjuvants in cancer immunotherapy. This study demonstrates the use of reduction-sensitive protein nanogels for the delivery of ovalbumin and oxidized tumor lysate-based antigens to mouse and human-donor-derived dendritic cells. Challenging mouse-derived and human dendritic cells with reduction-sensitive ovalbumin nanogels was found to significantly enhance antigen uptake as compared to the use of the corresponding free protein antigen. The experiments with mouse-derived dendritic cells further showed that the administration of ovalbumin in the form of reduction-sensitive nanogels enhanced dendritic cell maturation as well as the presentation of the SIINFEKL epitope as compared to experiments that use free ovalbumin. In addition to ovalbumin as a model antigen, the feasibility of reduction-sensitive nanogels was also demonstrated for the delivery of oxidized, whole tumor lysate-based cancer antigens. In experiments with dendritic cells harvested from human donors, dendritic cell uptake of the oxidized tumor lysate antigen was significantly enhanced in experiments that used oxidized tumor lysate nanogels as compared to the free antigen.

Topics: Animals; Antigens, Neoplasm; Dendritic Cells; Humans; Mice; Nanogels; Neoplasms; Ovalbumin; Peptides

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; Tumor Microenvironment

Amino acid-tuned self-assembly has become an attractive strategy for constructing various functional materials. Here, a series of dibenzocyclooctyne (DIBO) functionalized amphiphilic amino acid derivatives are designed and screened as building blocks of functional supramolecular self-assembly nanoparticles for cancer immunotherapy. One top-performing supramolecular self-assembly material (named DA6C1) is identified through combinatorial screening, and spherical nanoparticles can be easily prepared by this material tuned multicomponent synergistic self-assembly of ovalbumin (OVA) and CpG oligonucleotide. DA6C1 based nanovaccine can significantly enhance the cellular uptake of OVA and CpG into the same bone marrow derived dendritic cells (BMDCs) and greatly improve the activation of DCs. Moreover, after subcutaneous injection, this nanovaccine flows rapidly to the lymph nodes and elicits strong immune responses to achieve effective prophylactic and therapeutic effect. Therefore, our work highlights the great potential of clickable amino acid derivatives as a convenient and powerful tool to construct nanovaccine for effective immunotherapy.

Topics: Amino Acids; Animals; Cancer Vaccines; Dendritic Cells; Immunotherapy; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin

For mRNA mediated cancer immunotherapy, Pam3 was incorporated as an adjuvant within lipid nanoparticles (LNPs) with OVA mRNA. The developed Pam3 incorporated LNPs showed successful expression of tumor antigens with enhanced immune stimulation. We demonstrated that the synergies of Pam3 LNPs could greatly improve the efficacy of tumor prevention by mRNA vaccines.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line; Humans; Immunotherapy; Injections, Intramuscular; Lipids; Lipopeptides; Mice; Nanoparticles; Neoplasms; Ovalbumin; Particle Size; RNA, Messenger; Xenograft Model Antitumor Assays

Flow cytometry has revolutionized the field of molecular immunology, enabling the monitoring and characterization of immune events at the single-cell level. Here, we describe a flow cytometry-based workflow to quantify the activation of specific immune cell subsets in mice in response to a molecular intervention. Compared to laborious long-term disease models, this technique allows for relatively rapid evaluation of candidate therapeutics designed to elicit a targeted immune response. This approach has the range to address both disease applications in which an immunostimulatory effect would be desired (e.g., cancer, infectious disease) or those in which an immunosuppressive effect would be desired (e.g., autoimmune disorders, transplantation medicine). Overall, our technique presents a powerful and accessible strategy for preliminary in vivo assessment of potential immunotherapeutics.

Topics: Adoptive Transfer; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Flow Cytometry; Immunophenotyping; Mice; Mice, Transgenic; Neoplasms; Ovalbumin; Single-Cell Analysis; Workflow

Aimed at evoking an adequate anti-tumor immune response via oral administration route, this study constructed functionally and structurally mimicking-bacteria-membrane (MBM) nano-vesicle (RGD-PEOP) to orally deliver ovalbumin (OVA) antigen. In terms of simulating bacterial membrane structure, we creatively designed this nano-vesicle to have phospholipid-like octadecylphosphoethanolamine groups in vesicle membrane to improve OVA loading by means of specific interactions including salt bridge and hydrogen bond interaction. For simulating bacterial membrane function, the RGD peptide was modified onto the nano-vesicle surface, and the resulting vector displayed a good transport ability with a 3.4-fold higher than free OVA. In vitro and in vivo assay showed that the expression of co-stimulatory molecules and MHC class II complexes was significantly enhanced by MBM nano-vesicle. IFN-γ and IL-4 levels also increased several folds in the MBM nano-vesicle group. Consequently, MBM nano-vesicle achieved the highest in vivo inhibition rate of 69% against E.G7-OVA tumors among all the oral groups. These results suggest that this MBM nano-vesicle may be a promising vector to orally deliver OVA antigen for cancer immunotherapy. STATEMENT OF SIGNIFICANCE: Developing an effective non-bacterial carrier for oral cancer immunotherapy remains challenging. This work constructed a mimicking-bacteria-membrane nano-vesicle based on phospholipid-like amphiphilic polyphosphazenes for oral delivery of ovalbumin antigen. With the considerable capability to load ovalbumin antigen and target M cells, the nano-vesicle produced remarkable tumor suppression in vivo by evoking anti-tumor immune response.

Topics: Administration, Oral; Animals; Antineoplastic Agents; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Drug Carriers; Female; Immunotherapy; Liposomes; Mice, Inbred BALB C; Neoplasms; Organophosphorus Compounds; Ovalbumin; Polymers; Surface-Active Agents

DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of

Topics: Animals; Cancer Vaccines; Dendritic Cells; DNA; Female; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Immunity, Cellular; Immunization; Lipids; Liposomes; Lymph Nodes; Macrophages; Mice, Inbred BALB C; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Plasmids; Protozoan Infections; Vaccines, DNA; Vitamin E

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.

Topics: Acids; Animals; Antigen Presentation; Antigens; Antigens, CD; Bone Marrow Cells; Cell Proliferation; Dendritic Cells; Endocytosis; Endosomes; Histocompatibility Antigens Class I; Kinesins; Mice, Knockout; Mice, Transgenic; Microtubules; Neoplasms; Ovalbumin; Solubility

The immunotherapy played a vital role in the treatment of metastatic tumor. To further enhance the effect of the immunotherapy, the combination of photothermal effect can not only eradicate the tumor cells by hyperthermia, but also improved the antigen release in vivo to achieve enhanced immune responses. In this study, a core-shell structured nanocomplex was developed by loading of ovalbumin (OVA) and copper sulfide nanoparticles (CuS-NPs) into the poly(lactide-co-glycolide acid) nanoparticles (PLGA-NPs). The CuS-NPs exhibited favorable photothermal effect, which significantly kill the 4T1 tumor cells in vitro. The photothermal effect of the CuS-NPs accelerated the OVA release, which led to higher levels of IL-6, IL-12 and TNF-α, and activation of CD8

Topics: Animals; Cell Line, Tumor; Copper; Dose-Response Relationship, Drug; Female; Immunotherapy; Light; Metal Nanoparticles; Mice; Mice, Inbred BALB C; Neoplasms; Ovalbumin; Photothermal Therapy; Polylactic Acid-Polyglycolic Acid Copolymer; RAW 264.7 Cells; Xenograft Model Antitumor Assays

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Disease Models, Animal; Female; Freund's Adjuvant; Humans; Immunogenicity, Vaccine; Lipids; Melanins; Mice; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 9; Vaccines, Subunit

Hollow Gd2O3 nanospheres significantly promote the cellular uptake of a tumor antigen by antigen presenting cells, exhibit pH-dependent alteration of the MR signal intensity and markedly enhance the antitumor immunity. Hollow Gd2O3 nanospheres are promising as magnetic resonance imaging (MRI)-visible cancer immunoadjuvants for cancer immunotherapy.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cell Membrane Permeability; Contrast Media; Drug Discovery; Gadolinium; Humans; Immunotherapy; Interleukin-1beta; Lymph Nodes; Magnetic Resonance Imaging; Mice; Nanospheres; Neoplasms; Ovalbumin; T-Lymphocytes

Exogenous antigens processed in the cytosol and subsequently cross-presented on major histocompatibility complex class I (MHC-I) molecules activate cytotoxic CD8

Topics: Animals; Antigen Presentation; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell-Penetrating Peptides; Cross-Priming; Mice; Nanoparticles; Neoplasms; Ovalbumin; Polylactic Acid-Polyglycolic Acid Copolymer; Vaccines; Xenograft Model Antitumor Assays

The effective induction of an antigen-specific T cell immune response through dendritic cell activation is one of the key goals of tumor immunotherapy.. In this study, efficient antigen-delivery carriers using silica-coated magnetic nanoparticles were designed and, their antigen-specific T cell immune response through dendritic cell activation investigated.. The results showed that the silica-coated magnetic nanoparticles with conjugated ovalbumin enhanced the production of cytokines and antigen uptake in bone marrow-derived dendritic cells. Also, this induced an antigen-specific cytotoxic T lymphocyte (CTL) immune response and activated antigen-specific Th1 cell responses, including IL-2 and IFN-γ production and proliferation. We proved that the immune-stimulatory effects of silica-coated magnetic nanoparticles with conjugated ovalbumin were efficient in inhibiting of tumor growth in EG7-OVA (mouse lymphoma-expressing ovalbumin tumor-bearing mice model).. Therefore, the silica-coated magnetic nanoparticles with conjugated ovalbumin are expected to be useful as efficient anti-cancer immunotherapy agents.

Topics: Animals; Antigens; Chickens; Cytokines; Dendritic Cells; Female; Immunity; Immunotherapy; Magnetite Nanoparticles; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Silicon Dioxide

Antigen presenting cells (APCs) initiate the immune response against cancer by engulfing and presenting tumor antigens to T cells. Our lab has recently developed a liposomal nanoparticle that binds complement C3 proteins, allowing it to bind to the complement C3 receptors of APCs and directly deliver antigenic peptides. APCs were shown to internalize and process complement C3-bound liposomes containing ovalbumin (OVA), resulting in a significant increase in activated T cells that recognize OVA. Mice bearing A20-OVA lymphoma tumors were treated with OVA-loaded C3-liposomes, which led to reduced tumor growth in both treated and distal tumors in all mice. Peripheral blood from treated mice had a lower percentage of immunosuppressive myeloid derived suppressor cells (MDSCs), a higher percentage of B cells, and increased anti-OVA IgG

Topics: Animals; Antigen-Presenting Cells; Cell Line, Tumor; Cell Proliferation; Complement C3; Dendritic Cells; Endocytosis; Humans; Immunoglobulin G; Liposomes; Liver; Lymphocyte Activation; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Neoplasms; Ovalbumin; T-Lymphocytes; Tumor Burden

Metal-organic frameworks (MOFs) have high surface area, tunable pore size, and high loading capacity, making them promising for drug delivery. However, their synthesis requires organic solvents, high temperature and high pressure that are incompatible with biomacromolecules. Zeolitic imidazole frameworks (ZIF-8) which forms through coordination between zinc ions and 2-methylimidazole (MeIM) have emerged as an advanced functional material for drug delivery due to its unique features such as high loading and pH-sensitive degradation. In this study, we took advantage of a natural biomineralization process to create aluminum-containing nanoZIF-8 particles for antigen delivery. Without organic solvents or stabilizing agent, nanoparticles (ZANPs) were synthesized by a mild and facile method with aluminum, model antigen ovalbumin (OVA) and ZIF-8 integrated. A high antigen loading capacity (%) of 30.6% and a pH dependent antigen release were achieved. A Toll-like receptor 9 agonist cytosine-phosphate-guanine oligodeoxynucleotides (CpG) was adsorbed on the surface of ZANPs (hereafter CpG/ZANPs) to boost the immune response. After subcutaneous injection in vivo, CpG/ZANPs targeted lymph nodes (LNs), where their cargo was efficiently internalized by LN-resident antigen-presenting cells (APCs). ZANPs decomposition in lysosomes released antigen into the cytoplasm and enhanced cross-presentation. Moreover, CpG/ZANPs induced strong antigen-specific humoral and cytotoxic T lymphocyte responses that significantly inhibited the growth of EG7-OVA tumors while showing minimal cytotoxicity. We demonstrate that ZANPs may be a safe and effective vehicle for the development of cancer vaccines.

Topics: Adjuvants, Immunologic; Aluminum Chloride; Animals; Antigens; Cell Line; Female; Imidazoles; Immunity, Cellular; Immunity, Humoral; Lymph Nodes; Metal-Organic Frameworks; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes, Cytotoxic; Zeolites

MHC I-restricted epitopes of chicken ovalbumin (OVA) were originally identified using CD8 T cells as probes. Here, using bioinformatics tools, we identify four additional epitopes in OVA in addition to a cryptic epitope. Each new epitope is presented in vivo, as deduced from the lack of CD8 response to it in OVA-transgenic mice. In addition, CD8 responses to the known and novel epitopes are examined in C57BL/6 mice exposed to the OVA-expressing tumor E.G7 in several ways. No responses to any epitope including SIINFEKL are detected in mice with growing E.G7 or mice immunized with the tumor. Only in E.G7-bearing mice treated with an anti-CTLA4 antibody which depletes tumor-infiltrating regulatory T cells, CD8 responses to SIINFEKL and the novel epitope EKYNLTSVL are detected. Finally, all epitopes fails to treat mice with pre-existing tumors. These observations force an important re-consideration of the common assumptions about the therapeutic value of neoepitopes detected by CD8 responses in tumor-bearing hosts.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Computational Biology; Disease Models, Animal; Epitope Mapping; Epitopes, T-Lymphocyte; Female; Histocompatibility Antigens Class I; Humans; Mice; Mice, Transgenic; Neoplasms; Ovalbumin

Strengthening the antitumor immune response to surpass the activation energy barrier associated with the immunosuppressive tumor microenvironment is an active area of cancer immunotherapy. Emerging evidence suggests that delivery of immunostimulatory molecules with the aid of a carrier system is essential for cancer immunotherapy. However, the size-dependent effect of the delivery system on immune-targeted sites and anticancer immune responses is yet to be comprehensively understood. Herein, to clarify the size-dependent effect of the delivery system on the underlying anticancer immune mechanism, rod-shaped hydroxyapatite (HA) particles with lengths from 100 nm to 10 μm are designed. HA rods stimulate anticancer immunity in a size-dependent manner. Shorter HA rods with lengths ranging from 100 to 500 nm promote antigen cellular uptake, dendritic cell (DC) maturation, and lymph node targeting antigen. In contrast, longer HA rods with lengths ranging from 500 nm to 10 μm prolong antigen retention and increase DC accumulation. Medium-sized HA rods with a length of 500 nm, taking advantage of both short and long rods, show optimized antigen release and uptake, increased DCs accumulation and maturation, highest CD4

Topics: Adsorption; Animals; Antigens, Neoplasm; Antineoplastic Agents; Biocompatible Materials; Cells, Cultured; Drug Delivery Systems; Durapatite; Female; Immunotherapy; Injections, Subcutaneous; Lymph Nodes; Mice; Mice, Inbred C57BL; Neoplasms; Optical Imaging; Ovalbumin; Particle Size; Surface Properties

Prolonged vaccine release enables gradual immunostimulation, providing long-term immunity. Herein, Vitamin E-PEG-Vitamin E triblock 'ABA' hydrogel, which is formed through physical cross-linking of flower-shaped micelles and can reside in vivo for >17 weeks, was employed for delivery of cancer preventive vaccines to provide sustained anticancer immunity. Mice vaccinated with hydrogel formulations produced a significantly higher quantity of antibodies compared to solution formulations. OVA was used to study EG.7-OVA tumor rejection in vaccinated mice. Among all formulations, OVA-loaded hydrogel containing aluminum-based adjuvant had the best therapeutic outcome, and only 2/10 mice developed solid tumors with significantly smaller tumor size. Moreover, no adverse effect on liver and kidney was detected with the hydrogel formulation. In a lymphoma metastasis mouse model, vaccination with the OVA-loaded hydrogel and adjuvant resulted in increased survival (66.7%) compared to other formulations (12.5-50%) over 100 days. This hydrogel is a promising formulation for sustained delivery of vaccines.

Topics: Adjuvants, Immunologic; Animals; Biodegradable Plastics; Cancer Vaccines; Drug Carriers; Humans; Hydrogels; Immunity, Cellular; Kidney; Liver; Mice; Neoplasms; Ovalbumin; Vitamin E

The skin is a very suitable organ for the induction of immune responses to vaccine antigens. Antigen delivery systems to the skin by needle and syringe directly deposit the antigen into the epidermal-dermal compartment, one of the most immunocompetent sites due to the presence of professional antigen-presenting cells aimed at the induction of antigen-specific T cells. In this study, we analyzed the amount of ovalbumin as an antigen delivered to the skin by a microneedle. When ovalbumin protein as an antigen was delivered to the skin of mice using a dissolving microneedle, it induced an immune response through the enhanced proliferation and cytokines production by the splenocytes and lymph nodes. Also, it effectively increased the ovalbumin-specific CD8+ T cell and CD4+ T cell population and induced an ovalbumin-specific CTL response against the graft of ovalbumin-expressing EG7 tumor cells in the immunized mice. Also, we identified the inhibition of tumor growth and prevention of tumor formation in the context of the therapeutic and prophylactic vaccine, respectively through EG-7 tumor mouse model. Finally, these data show the potential of patches as attractive antigen delivery vehicles.

Topics: Administration, Cutaneous; Animals; Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Drug Delivery Systems; Immunity; Immunotherapy; Mice; Needles; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; Transdermal Patch; Treatment Outcome

Tumor-specific CD8

Topics: Animals; Antigen Presentation; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Epitopes; Female; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Vaccines, Subunit

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs were found to be able to target cells that express Toll-like receptor 9 to modulate innate and adaptive immune reactions. But their in vivo application in immunotherapy against cancer has not been successful. We attempted in this study to examine polyethylene-glycol (PEG) conjugated CpG ODNs and investigated their mechanism of immune modulation in anti-cancer therapy.. CpG-PEG conjugates with different PEG lengths were synthesized. In vitro activity as well as in vivo pharmacokinetics and pharmacodynamics properties were evaluated.. CpG-PEG20Ks were found to be able to persist longer in circulation and activate various downstream effector cells. After intravenous injection, they resulted in higher levels of IL-12p70 in the circulation and lower M-MDSC infiltrates in the tumor microenvironment. Such activities were different from those of CpG ODNs without PEGylation, suggesting different PK-PD profiles systemically and locally.. Our data support the development of CpG-PEGs as a new therapeutic agent that can be systemically administered to modulate immune responses and the microenvironment in tumor tissues.

Topics: Adaptive Immunity; Animals; Cell Line, Tumor; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Drug Compounding; Drug Evaluation, Preclinical; Female; Humans; Injections, Intravenous; Interleukin-12; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Polyethylene Glycols; Primary Cell Culture; Tumor Microenvironment

Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunologic outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here, we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells, subsequent increased antitumor T-cell priming in draining lymph nodes, and enhanced effector T-cell infiltration in the tumor microenvironment. These results reinforce the concept that i.t. delivery of TLR9 agonists alters the tumor microenvironment by improving the antitumor activity of both innate and adaptive immune cells.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Communication; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Histocompatibility Antigens Class II; Humans; Immune Tolerance; Immunotherapy; Injections, Intralesional; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasms; Neutrophils; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; T-Lymphocytes; Toll-Like Receptor 9; Tumor Microenvironment

Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Their proper delivery to dendritic cells (DCs) requires their protection against RNase degradation and more specificity for dose reduction. Lipid-Polymer-RNA lipopolyplexes (LPR) are attractive mRNA delivery systems and their equipment with mannose containing glycolipid, specific of endocytic receptors present on the membrane of DCs is a valuable strategy. In this present work, we evaluated the capacity of LPR functionalized with a tri-antenna of α-d-mannopyranoside (triMN-LPR) concerning (i) their binding to CD209/DC-SIGN and CD207/Langerin expressing cell lines, human and mouse DCs and other hematopoietic cell populations, (ii) the nature of induced immune response after in vivo immunization and (iii) their therapeutic anti-cancer vaccine efficiency. We demonstrated that triMN-LPR provided high induction of a local inflammatory response two days after intradermal injection to C57BL/6 mice, followed by the recruitment and activation of DCs in the corresponding draining lymph nodes. This was associated with skin production of CCR7 and CXCR4 at vaccination sites driving DC migration. High number of E7-specific T cells was detected after E7-encoded mRNA triMN-LPR vaccination. When evaluated in three therapeutic pre-clinical murine tumor models such as E7-expressing TC1 cells, OVA-expressing EG7 cells and MART-1-expressing B16F0 cells, triMN-LPR carrying mRNA encoding the respective antigens significantly exert curative responses in mice vaccinated seven days after initial tumor inoculation. These results provide evidence that triMN-LPR give rise to an efficient stimulatory immune response allowing for therapeutic anti-cancer vaccination in mice. This mRNA formulation should be considered for anti-cancer vaccination in Humans.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cell Movement; Dendritic Cells; Female; Humans; Injections, Intradermal; Lipids; Lymph Nodes; Mannose; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; RNA, Messenger; Vaccination

Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam

Topics: Animals; Antibodies; Antigen-Presenting Cells; Antigens, Tumor-Associated, Carbohydrate; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Female; Humans; Immunogenicity, Vaccine; Mice; Mice, Inbred C57BL; Mucin-1; Neoplasms; Ovalbumin; Rhamnose

Adoptive immunotherapy is based on

Topics: Animals; Biomarkers; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CCL21; Cytotoxicity, Immunologic; Immunotherapy, Adoptive; Intercellular Adhesion Molecule-1; Lymphocyte Activation; Mice; Neoplasms; Ovalbumin; T-Cell Antigen Receptor Specificity

Our previous study demonstrated that the incorporation of a tumor antigen into a self-assembling DNA hydrogel, comprised of a DNA containing un-methylated cytosine-phosphate-guanine (CpG) dinucleotides (CpG DNA), efficiently induced antigen-specific tumor immunity after intra-tumoral injection into tumor-bearing mice. We hypothesized that the additional incorporation of immune cells, the target for the antigen and immunostimulatory CpG DNA, would increase the antitumor response. To prove this, immune cells were also encapsulated into the CpG DNA hydrogel and delivered along with the antigen. Mouse dendritic DC2.4 cells maintained their form even after incorporation into the DNA hydrogel. The incorporation of mouse macrophage-like J774.1 cells and RAW264.7 cells into CpG DNA hydrogel did not significantly affect their viability. J774.1, RAW264.7, DC2.4, and mouse bone marrow-derived dendritic cells (BMDCs) were efficiently activated when incorporated into the CpG DNA hydrogel. The CpG DNA hydrogel incorporated with both the tumor antigen and BMDCs effectively induced antigen-specific immune responses, and retarded tumor growth following intradermal administration before and after tumor inoculation without severe local and systemic adverse events. These data indicate that the combined delivery of a tumor antigen and immune cells using an immunostimulatory CpG DNA hydrogel is effective in inducing antigen-specific antitumor immunity.

Topics: Animals; Antigens, Neoplasm; Cell Line; Cell Survival; CpG Islands; Dendritic Cells; DNA; Hydrogels; Interferon-gamma; Macrophages; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Toll-Like Receptor 9

Enhancing cytosol delivery of exogenous antigens in antigen presenting cells can improve cross-presentation and CD8+ T cell-mediated immune response. The antigen cytosol delivery speed, which has great importance on the rate of MHC class I molecules (MHC I) antigen presentation pathway and cytotoxic T lymphocytes (CTLs) induction, has not been well studied. We hypothesized that micelle-tailored vaccine with multiple cascaded lysosomal responsive capabilities could accelerate lysosomal escape and enhance cancer immunotherapy. To test our hypothesis, we created a novel micellar cancer vaccine (ovalbumin-loaded pH/redox dual-sensitive micellar vaccine, OLM-D) by cleavable conjugation of an antigen with house-made amphiphilic poly(l-histidine)-poly(ethylene glycol) (PLH-PEG) in current study. OLM-D was supposed to achieve cascade cytosol delivery of ovalbumin through three steps in terms of (i) initial redox triggered ovalbumin release, (ii) promoted proton inflow and micelle disassembly, and (iii) speeded proton sponge effect and lysosome bulging/broke. Redox-sensitive antigen release and consequently accelerative OLM-D disassembly were confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), transmission electronic microscopy (TEM), particle sizes, zeta potentials, and in vitro Ova release evaluation. The speeded cytosol delivery of ovalbumin was visualized under a confocal laser scanning microscope (CLSM). The ability of OLM-D to increase the MHC I molecule combination rate and antigen cross-presentation efficiency was identified by antigen presentation assay and maturation assay in bone marrow-derived dendritic cells (BMDCs). In vivo, the capability of OLM-D to accumulate in draining lymph nodes (LNs) after injection was visualized by real-time near infrared fluorescence imaging (NIRF) and the distribution order in different LNs was first observed (a, d, c, b). Enhanced cancer immunity of OLM-D was confirmed by increased CD3+CD8+ T cell quantity, CD3+CD8+25D11.6+ T cells quantity, and IFN-γ, IL-2 secretion post subcutaneous or intraperitoneal injection ( p < 0.05). Taken together, our results indicated that OLM-D provided a promising cascade cytosol delivery strategy, which held great potential to guide further design of nano-particulate cancer vaccines for efficient cancer immunotherapy.

Topics: Animals; Antigen-Presenting Cells; Cancer Vaccines; Cytosol; Dendritic Cells; Drug Delivery Systems; Electrophoresis, Polyacrylamide Gel; Histidine; Histocompatibility Antigens Class I; Humans; Immunotherapy; Lysosomes; Mice; Micelles; Nanoparticles; Neoplasms; Ovalbumin; Particle Size; Polyethylene Glycols; T-Lymphocytes, Cytotoxic

Synthetic imidazoquinoline-based toll-like receptor (TLR) 7/8 bi-specific agonists are promising vaccine adjuvants that can induce maturation of dendritic cells (DCs) and activate them to secrete pro-inflammatory cytokines. However, in vivo efficacy of these small molecule agonists is often hampered by their fast clearance from the injection site, limiting their use to topical treatments. In this study, we investigated the use of acidic pH-responsive poly(lactide-co-glycolide) (PLGA) nanoparticles for endo-lysosome specific release of 522, a novel TLR7/8 agonist. Bicarbonate salt was incorporated into the new formulation to generate carbon dioxide (CO2) gas at acidic pH, which can disrupt the polymer shell to rapidly release the payload. Compared to conventional PLGA nanoparticles, the pH responsive formulation resulted in 33-fold higher loading of 522. The new formulation demonstrated acid-responsive CO2 gas generation and drug release. The acid-responsive formulation increased the in vitro expression of co-stimulatory molecules on DCs and improved antigen-presentation via MHC I, both of which are essential for CD8 T cell priming. In vivo studies showed that the pH-responsive formulation elicited stronger antigen-specific CD8 T cell and natural killer (NK) cell responses than conventional PLGA nanoparticles, resulting in enhanced anticancer efficacy in a murine melanoma tumor model. Our results suggest that acidic-pH responsive, gas-generating nanoparticles are an efficient TLR7/8 agonist delivery platform for cancer immunotherapy.

Topics: Animals; Antigen Presentation; Bone Marrow Cells; Carbon Dioxide; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cryoelectron Microscopy; Dendritic Cells; Drug Carriers; Hydrogen-Ion Concentration; Immunotherapy; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Polylactic Acid-Polyglycolic Acid Copolymer; Toll-Like Receptor 7; Toll-Like Receptor 8

The success of immunotherapeutic vaccines is often limited by their inability to activate the cytotoxic T lymphocyte (CTL)-inducing Th1 pathway. We investigated the ability of self-assembled nanogels (CHP or CH-CDex) to activate this pathway, and characterised them chemically and biologically. Once loaded with antigen (ovalbumin, OVA) their OVA encapsulation and dissociation rates suggested the possibility of effective antigen delivery. The DC2.4 dendritic cell line took up either vaccine time-dependently, but both vaccines required CpG DNA for class I MHC presentation. The nanogel vaccines interacted with RAW264.7, a Balb/c mouse-derived macrophage cell line, and co-localised with lysosomes, suggesting their endocytotic internalization in RAW264.7. Both vaccines activated CTLs better than OVA alone. Unlike OVA alone, the nanogel vaccines induced IgG2a antibody production in mice, whereas the former induced IgG1 antibodies. OVA-nanogel delivery to the draining lymph nodes (DLNs) was higher than that for OVA alone, reaching a deeper medullary area. Furthermore, Langerin

Topics: Animals; Antigen Presentation; Antigens; Disease Models, Animal; Gels; Humans; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Nanostructures; Neoplasms; Ovalbumin; Polysaccharides; T-Lymphocytes, Cytotoxic; Vaccines; Xenograft Model Antitumor Assays

In this study, synthetic vaccine nanoparticles (SVNPs) that efficiently targeted lymph nodes, where immune responses against foreign antigens are primed, were developed to enhance antitumor immunity. The size (20-70 nm) and surface character (amination) of poly(γ-glutamic acid)-based SVNPs were selected for effective loading and delivery (i.e., migration and retention) of model tumor antigen (OVA) and toll-like receptor 3 agonist (poly (I:C)) to immune cells in lymph nodes. Antigen-presenting cells treated with SVNP-OVA and SVNP-IC showed higher uptake of OVA and poly (I:C) and higher secretion of inflammatory cytokines (TNF-α, IL-6) and type I interferon (IFN-α, IFN-β) than those treated with OVA and poly (I:C) alone. In vivo analysis revealed higher levels of activation markers, inflammatory cytokines, and type I IFNs in the lymph nodes of mice immunized with SVNP-IC compared to those of mice in other groups. SVNP-IC-treated mice showed significantly greater in vivo natural killer cell expansion/activation (NK1.1

Topics: Adaptive Immunity; Animals; Cancer Vaccines; Chickens; Immunity, Innate; Immunotherapy, Adoptive; Lymph Nodes; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Poly I-C; Vaccines, Synthetic

During human pregnancy, paternally inherited antigens expressed by the fetal-placental unit can elicit expansion of antigen-specific CD8+ T cells. These cells can persist for years as memory T cells, but their effects on long-term maternal health are unknown. Shared placenta/tumor-associated antigens are expressed by placenta and tumors, but are minimally expressed or absent in normal adult tissues. We hypothesized that maternal T cells elicited against these antigens can alter risk of cancers expressing the same antigen after pregnancy, and tested this in mice using chicken ovalbumin (OVA) as a surrogate shared placenta/tumor antigen. Hemizygous OVA transgenic males were bred to wild-type C57BL/6 females (H2b haplotype) such that the fetuses inherited and expressed OVA. Maternal OVA/H2Kb-specific CD8+ T cells became detectable during gestation, and persisted in some animals for up to 24 weeks. To determine whether these cells might influence growth of OVA-expressing tumors in OVA-bred females, E.G7-OVA thymoma cells were inoculated subcutaneously in OVA-bred, wild-type bred, and virgin females, and monitored for growth. OVA-bred mice had prolonged survival as compared to virgin mice and the progression of tumors was delayed in comparison to wild-type bred and virgin females. Thus, paternally inherited OVA antigen elicited a CD8+ T cell response during pregnancy that was associated with delayed growth of OVA-expressing tumors following pregnancy. These data suggest a possible role of antigen-specific T cells in protecting parous females against tumors bearing shared placenta/tumor antigens.

Topics: Animals; CD8-Positive T-Lymphocytes; Exosomes; Female; Lymphoma; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Placenta; Pregnancy; Pregnancy, Animal

Topics: Alginates; Animals; Antigens; Cell Line, Tumor; Cytokines; Dendritic Cells; Female; Glucuronic Acid; Hexuronic Acids; Immunotherapy; Mannose; Mice, Inbred BALB C; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Although it has been shown that the size of nanoparticle-based vaccines is a key determining factor for the induction of immune responses, few studies have provided detailed analyses of thresholds or critical sizes of nanoparticle vaccines. Here we report effects of the size of gold nanoparticle (GNP)-based vaccines on their efficiency of delivery to lymph nodes (LNs) and induction of CD8

Topics: Animals; Antigens; Cell Line; Cell Survival; Dendritic Cells; Escherichia coli; Female; Gold; Lymph Nodes; Metal Nanoparticles; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Particle Size; Recombinant Proteins; T-Lymphocytes, Cytotoxic; Tumor Burden; Vaccines

Developing potent adjuvants for the stimulation of robust immune response is central for effective cancer immunotherapy. Double-shelled dendritic mesoporous organosilica hollow spheres are an excellent adjuvant and provide superior immunity in cancer immunotherapy, and better than their counterparts either with a pure silica composition or a single-walled architecture. This study provides new insights in the rational design of effective nanostructured adjuvants for vaccine developments.

Topics: Adjuvants, Immunologic; Adsorption; Animals; Dendrimers; Humans; Immunotherapy; Mice; Microscopy, Confocal; Neoplasms; Organosilicon Compounds; Ovalbumin; Particle Size; Porosity; RAW 264.7 Cells; Surface Properties

Efficient vaccine carriers for cancer immunotherapy require two functions: antigen delivery to dendritic cells (DCs) and the activation of DCs, a so-called adjuvant effect. We previously reported antigen delivery system using liposomes modified with pH-sensitive polymers, such as 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG), for the induction of antigen-specific immune responses. We reported that inclusion of cationic lipids to MGlu-HPG-modified liposomes activates DCs and enhances antitumor effects. In this study, CpG-DNA, a ligand to Toll-like receptor 9 (TLR9) expressing in endosomes of DCs, was introduced to MGlu-HPG-modified liposomes containing cationic lipids using two complexation methods (Pre-mix and Post-mix) for additional activation of antigen-specific immunity. For Pre-mix, thin membrane of lipids and polymers were dispersed by a mixture of antigen/CpG-DNA. For Post-mix, CpG-DNA was added to pre-formed liposomes. Both Pre-mix and Post-mix delivered CpG-DNA to DC endosomes, where TLR9 is expressing, more efficiently than free CpG-DNA solution did. These liposomes promoted cytokine production from DCs and the expression of co-stimulatory molecules in vitro and induced antigen-specific immune responses in vivo. Both Pre-mix and Post-mix exhibited strong antitumor effects compared with conventional pH-sensitive polymer-modified liposomes. Results show that inclusion of multiple adjuvant molecules into pH-sensitive polymer-modified liposomes and suitable CpG-DNA complexation methods are important to design potent vaccine carriers.

Topics: Animals; Antigens; Cations; Cell Line; CpG Islands; Delayed-Action Preparations; Dendritic Cells; DNA; Drug Delivery Systems; Female; Hydrogen-Ion Concentration; Immunity; Immunotherapy; Lipids; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Propylene Glycols; Toll-Like Receptor 9

The purpose of present approach is to target C-Type lectin (CTL) receptors for preferential uptake by the macrophages/dendritic cells and improving the cross-presentation of ovalbumin.. Conventional and engineered nanoliposomes (MPNLs) were fabricated and extensively characterized. The nanoliposome(s) was spherical in shape; and their ζ potential, size and ovalbumin loading efficiency were recorded to be 268 ± 4.15 nm, 23.4 ± 0.35 mV, 46.65 ± 1.84%, respectively.. The findings demonstrate that MPNLs significantly improved the antigen uptake and its cross-presentation to evoke Th CD8. In a nutshell, this engineered approach mannose surface modification for active targeting to dendritic cells/macrophages and pH-dependent quick endosomal antigen release is a promising system for efficient cancer immunotherapy.

Topics: Animals; Dendritic Cells; Drug Liberation; Humans; Immunity, Cellular; Immunotherapy; Lectins, C-Type; Liposomes; Macrophages; Mannose; Mice, Inbred C57BL; Molecular Targeted Therapy; Nanoparticles; Neoplasms; Ovalbumin; Particle Size; Surface Properties

The cell-free approach to foreignizing tumor cells with non-self antigens has received increasing attention as a method to induce cytotoxic T lymphocyte (CTL)-mediated immunological rejection of tumors, because the clinical translation of the conventional CTL-based cancer immunotherapies has been limited by a complicated manufacturing process and autotransplantation. In this study, we prepared matrix metalloproteinase 9 (MMP9)-responsive polymeric conjugates consisting of PEGylated hyaluronic acid (HA) as the targeting moiety and ovalbumin (OVA) as the model foreign antigen. The MMP9-cleavable linker was introduced between PEG and the HA backbone to facilitate the detachment of the PEG corona from the conjugate at the tumor site. From the in vitro cellular uptake study, it was revealed that the conjugate was effectively taken up by the CD44-expressing TC-1 cancer cells in the presence of MMP9 via receptor-mediated endocytosis. When the conjugate was systemically administered into the tumor-bearing mice with endogenous OVA-specific CTLs, the tumor growth was markedly inhibited, which was attributed to the significant antigen presentation on the tumor cells. Overall, the MMP9-responsive conjugates bearing foreign antigens might have the potential as an alternative to CTL-based cancer immunotherapeutics.

Topics: Animals; Antigens; Apoptosis; Cell Line, Tumor; Female; Hyaluronic Acid; Immunotherapy; Matrix Metalloproteinase 9; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Neoplasms; Ovalbumin; Polyethylene Glycols; T-Lymphocytes, Cytotoxic

Cancer vaccine has the ability to directly eradicate tumor cells by creating and activating cytotoxic T lymphocytes (CTLs). To achieve efficient CTL activity and to induce Th1 responses, it is essential to administer an appropriate adjuvant as well as an antigen. CpG-ODN is known as a ligand of Toll-like receptor 9 (TLR9) and strongly induces Th1 responses. In our previous study, we developed a CpG-ODN delivery system by use of the formation of complexes between ODN and a β-glucan SPG, denoted as CpG/SPG, and demonstrated that CpG/SPG induces high Th1 responses. In this study, we created a nanogel made from CpG/SPG complexes through DNA-DNA hybridization (cross-linked (CL)-CpG). Immunization with CL-CpG induced much stronger antigen-specific Th1 responses in combination with the antigenic protein ovalbumin (OVA) than that with CpG/SPG. Mice preimmunized with CL-CpG and OVA exhibited a long delay in tumor growth and an improved survival rate after tumor inoculation. These immune inductions can be attributed to the improvement of cellular uptake by the combination of increased size and the cluster effect of the β-glucan recognition site in the nanogel structure. In other words, the particle nature of CL-CpG, instead of the semiflexible rod conformation of CpG/SPG, enhanced the efficacy of a cancer vaccine. The present results indicate that CL-CpG can be used as a potent vaccine adjuvant for the treatment of cancers and infectious diseases.

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Cancer Vaccines; Cross-Linking Reagents; Cytokines; Drug Delivery Systems; Gels; Immunization; Mice; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; RAW 264.7 Cells; Toll-Like Receptor 9

The goal of this research was to study the effect of polyanhydride chemistry on the immune response induced by a prophylactic cancer vaccine based on biodegradable polyanhydride particles. To achieve this goal, different compositions of polyanhydride copolymers based on 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), 1,6-bis-(p-carboxyphenoxy)-hexane (CPH), and sebacic anhydride (SA) were synthesized by melt polycondensation, and polyanhydride copolymer particles encapsulating a model antigen, ovalbumin (OVA), were then synthesized using a double emulsion solvent evaporation technique. The ability of three different compositions of polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. In addition, the impact of unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunological adjuvant, on the immune response was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8. Compared to soluble cancer vaccine formulations, tumor antigens encapsulated in biodegradable polymeric particles have been shown to sustain antigen release and provide long-term protection against tumor challenge by improving the immune response towards the antigen. Treatment of mice with cancer vaccines based on different polyanhydride copolymers encapsulating OVA resulted in stimulation of tumor-specific immune responses with different magnitudes. This clearly indicates that polyanhydride chemistry plays a substantial role in stimulating the immune response. Vaccination with 20:80 CPTEG:CPH/OVA, the most hydrophobic formulation, stimulated the strongest cellular and humoral immune responses and provided the longest survival outcome without adding any other adjuvant. The most important finding in this study is that the copolymer composition of polyanhydride particle-based vaccines can have a direct effect on the magnitude of the antitumor immune response and should be selected carefully in order to achieve optimal cancer vaccine efficacy.

Topics: Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Female; Immunoglobulin G; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Particle Size; Polyanhydrides; Proton Magnetic Resonance Spectroscopy; Static Electricity; Survival Analysis; Vaccination

Much effort has been put in the development of specific anti-tumour immunotherapies over the last few years, and several studies report on the use of liposomal carriers for tumour-associated antigens. In this work, the use of lipid implants, prepared using two different extruders, was investigated for sustained delivery in tumour therapy. The implants consisted of cholesterol, soybean lecithin, Dynasan 114, trehalose, ovalbumin (OVA) or a TRP2 peptide, and Quil-A. Implants were first produced on a Haake Minilab extruder, and then a scale-down to minimal quantities of material on a small scale ZE mini extruder was performed. All formulations were characterised in terms of extrudability, implant properties and in vitro release behaviour of the model antigen ovalbumin. The type of extruder used to produce the implants had a major influence on implant properties and the release behaviour, demonstrating that extrusion parameters and lipid formulations have to be individually adapted to each extrusion device. Subsequently, lipid implants containing TRP-2 peptide were extruded on the ZE mini extruder and investigated in vitro and in vivo. The in vivo study showed that mice having received TRP2 loaded implants had delayed tumour growth for 3days compared to groups having received no TRP2.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Delayed-Action Preparations; Drug Compounding; Drug Implants; Excipients; Immunotherapy; Lipids; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Ovalbumin; Peptide Fragments; Quillaja Saponins

Conjugation of a vaccine adjuvant to an antigen enhances anti-tumor immune responses. Direct chemical conjugation, however, may limit their processing by the antigen-presenting cell for immune stimulation. To test this hypothesis, antigen-adjuvant conjugates were designed to be cleaved by an intracellular trigger to release antigen and adjuvant from each other. The different reductive environment inside and outside antigen-presenting cells was used as a trigger for targeted intracellular release. Two redox-responsive disulphide linkers were used to conjugate the model antigen ovalbumin to CpG. In vitro stability assays with the reductant glutathione showed that one conjugate (SS) was cleaved by glutathione concentrations of the extra- and intracellular compartments. A second conjugate (HYN-SS) was only cleaved at the higher intracellular glutathione concentration. In vitro cell culture studies showed that high T cell responses were generated by the HYN-SS and the stable conjugate HYN. The SS conjugate induced a lower T cell response similar to a mixture of CpG and ovalbumin. An in vivo therapeutic tumor trial demonstrated a superior survival rate of 9/10 for mice vaccinated with HYN-SS conjugate compared to HYN (6/10), SS (2/10), and the mixture (2/10). This intracellular cleavable conjugation strategy represents a promising approach to improve cancer immunotherapy of soluble vaccines.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Antigens; Cancer Vaccines; Disease Models, Animal; Melanoma, Experimental; Mice; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocyte Subsets

Nanotechnology offers invaluable tools to tailor cancer vaccines in order to generate robust antitumor immune response. Among the types of vehicles for cancer vaccines, nanoparticles (NPs) are easier to produce with better scalability. Several nanostructures have been discussed in literature as potential delivery systems for cancer antigens. Here, we focus on polymeric NPs fabricated from poly(D,L-lactic-co-glycolic) acid (PLGA). We describe how to prepare and characterize such NPs loaded with ovalbumin (OVA) antigen and immune adjuvant monophosphoryl lipid A (MPLA). We further describe methods to test the immune efficacy of such NPs in vitro and in vivo.

Topics: Animals; Cancer Vaccines; Dendritic Cells; Disease Models, Animal; Humans; Immunotherapy; Lactic Acid; Lymphocyte Activation; Melanoma, Experimental; Nanoparticles; Nanotechnology; Neoplasms; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; T-Lymphocyte Subsets; Vaccination

Topics: Animals; Antigen Presentation; Antigens; Cancer Vaccines; Cell Line, Tumor; Cytokines; Dendritic Cells; Disease Models, Animal; DNA-Binding Proteins; Female; Histocompatibility Antigens Class I; Immunization; Liposomes; Mice; Neoplasms; Ovalbumin; Peptides; T-Lymphocytes, Cytotoxic

An adenovirus vector carrying the human Reduced Expression in Immortalized Cell (REIC)/Dkk-3 gene (Ad-REIC) mediates simultaneous induction of cancer-selective apoptosis and augmentation of anticancer immunity. In our preclinical and clinical studies, in situ Ad-REIC gene therapy showed remarkable direct and indirect antitumor effects to realize therapeutic cancer vaccines. We herein aimed to confirm the induction of tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) by Ad-REIC. Using an ovalbumin (OVA), a tumor-associated antigen, expressing E.G7 tumor-bearing mouse model, we investigated the induction and expansion of OVA-specific CTLs responsible for indirect, systemic effects of Ad-REIC. The intratumoral administration of Ad-REIC mediated clear antitumor effects with the accumulation of OVA-specific CTLs in the tumor tissues and spleen. The CD86-positive dendritic cells (DCs) were upregulated in the tumor draining lymph nodes of Ad-REIC-treated mice. In a dual tumor-bearing mouse model in the left and right back, Ad-REIC injection in one side significantly suppressed the tumor growth on both sides and significant infiltration of OVA-specific CTLs into non-injected tumor was also detected. Consequently, in situ Ad-REIC gene therapy is expected to realize a new-generation cancer vaccine via anticancer immune activation with DC and tumor antigen-specific CTL expansion.

Topics: Adaptor Proteins, Signal Transducing; Adenoviridae; Animals; Antigens, Neoplasm; Apoptosis; Cancer Vaccines; Chemokines; Dendritic Cells; Gene Expression Regulation, Neoplastic; Genetic Therapy; Genetic Vectors; Humans; Intercellular Signaling Peptides and Proteins; Mice; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Topics: Amino Acid Sequence; Animals; Cancer Vaccines; Cations; CD8-Positive T-Lymphocytes; Female; Immunity, Cellular; Immunization; Lactic Acid; Liposomes; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Ovalbumin; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Vaccines, Synthetic

Immune adjuvants are important components of current and prospective cancer vaccines. In this study, we aimed at evaluating the use of a synthetic lipid A derivative, pentaerythritol lipid A (PET lipid A), loaded into poly(lactic-co-glycolic acid) particles, as a potential cancer vaccine adjuvant. Poly(lactic-co-glycolic acid) particles (size range: 250-600 nm) were successfully formulated to include PET lipid A and/or the model tumor antigen, chicken ovalbumin (OVA). It was shown that particulated PET lipid A had a distinct advantage at promoting secretion of the immune potentiating cytokine, IL-12p70, and upregulating key costimulatory surface proteins, CD86 and CD40, in murine dendritic cells in vitro. In a murine tumor model, involving prophylactic vaccination with various permutations of soluble versus particulated formulations of OVA with or without PET lipid A, modest benefit was observed in terms of OVA-specific cell-mediated immune responses when PET lipid A was delivered in particles. These findings translated into a corresponding trend toward increased survival of mice challenged with OVA-expressing tumor cells (E.G7). In terms of translation of safe adjuvants into the clinic, these results promote the concept of delivering toll-like receptor-4 agonists in particles because doing so improves their adjuvant properties, while decreasing the chances of adverse effects due to off-target uptake by nonphagocytic cells.

Topics: Adjuvants, Immunologic; Animals; Antigens; Biocompatible Materials; Cancer Vaccines; Chemistry, Pharmaceutical; Cytokines; Dendritic Cells; Female; Lactic Acid; Lipid A; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Propylene Glycols; Toll-Like Receptors

Synthetic liposomes provide a biocompatible and biodegradable approach for delivering drugs and antigens. In addition, self-adjuvanting recombinant lipoproteins (rlipoproteins) can enhance Th1 anti-tumor immune responses via the TLR2 signaling pathway. To generate a liposomal rlipoprotein for a cancer immunotherapeutic vaccine, we assessed 3 types of synthetic liposomes for use with the rlipoproteins rlipoE7m and rlipoOVA. We determined that the cationic liposome DOTAP could stabilize anionic rlipoproteins and delay rlipoprotein release. Surprisingly, rlipoproteins and DOTAP could synergistically up-regulate CD83 expression in bone marrow-derived dendritic cells (BMDCs). Compared with other liposome formulations, the rlipoprotein/DOTAP formulation elicited higher cytotoxic T-lymphocyte (CTL) responses. To explore the mechanism of BMDC activation by rlipoprotein/DOTAP, we assessed the production of reactive oxygen species (ROS) and the TNF-α secretion of BMDCs. We observed that rlipoprotein/DOTAP induced ROS to the same extent as DOTAP did. In addition, TLR2 signaling was also required for the TNF-α secretion of rlipoprotein/DOTAP-treated BMDCs. Moreover, compared with rlipoOVA-treated BMDCs, rlipoOVA/DOTAP-treated BMDCs increased the levels of IFN-γ produced by OVA-specific T cells. We also observed that rlipoE7m/DOTAP treatment but not rlipoE7m treatment delayed tumor growth. These results indicate that the rlipoprotein/DOTAP formulation can synergistically activate BMDCs via ROS and the TLR2 signaling pathway. In summary, rlipoprotein/DOTAP is a novel and stable formulation for cancer immunotherapy.

Topics: Allergens; Animals; Cancer Vaccines; Cell Line, Tumor; Cell Survival; Dendritic Cells; Fatty Acids, Monounsaturated; Lipoproteins; Liposomes; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Papillomavirus E7 Proteins; Quaternary Ammonium Compounds; Reactive Oxygen Species; Recombinant Proteins; Tumor Burden

Micro- and nanometer-size particles have become popular candidates for cancer vaccine adjuvants. However, the mechanism by which such particles enhance immune responses remains unclear. Here, we report a porous silicon microparticle (PSM)-based cancer vaccine that greatly enhances cross-presentation and activates type I interferon (IFN-I) response in dendritic cells (DCs). PSM-loaded antigen exhibited prolonged early endosome localization and enhanced cross-presentation through both proteasome- and lysosome-dependent pathways. Phagocytosis of PSM by DCs induced IFN-I responses through a TRIF- and MAVS-dependent pathway. DCs primed with PSM-loaded HER2 antigen produced robust CD8 T cell-dependent anti-tumor immunity in mice bearing HER2+ mammary gland tumors. Importantly, this vaccination activated the tumor immune microenvironment with elevated levels of intra-tumor IFN-I and MHCII expression, abundant CD11c+ DC infiltration, and tumor-specific cytotoxic T cell responses. These findings highlight the potential of PSM as an immune adjuvant to potentiate DC-based cancer immunotherapy.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Antigens, Neoplasm; Cancer Vaccines; Cross-Priming; Dendritic Cells; Female; Immunity, Innate; Interferon Type I; Mammary Neoplasms, Animal; Mice, Inbred C57BL; Microspheres; Neoplasms; Ovalbumin; Phagocytosis; Porosity; Receptor, ErbB-2; Signal Transduction; Silicon; T-Lymphocytes, Cytotoxic; Tumor Microenvironment

The development of vaccines inducing efficient CD8(+) T cell responses is the focus of intense research. Dendritic cells (DCs) expressing the XCR1 chemokine receptor, also known as CD103(+) or CD8α(+) DCs, excel in the presentation of extracellular Ags to CD8(+) T cells. Because of its high numbers of DCs, including XCR1(+) DCs, the skin dermis is an attractive site for vaccine administration. By creating laser-generated micropores through the epidermis, we targeted a model protein Ag fused to XCL1, the ligand of XCR1, to dermal XCR1(+) DCs and induced Ag-specific CD8(+) and CD4(+) T cell responses. Efficient immunization required the emigration of XCR1(+) dermal DCs to draining lymph nodes and occurred irrespective of TLR signaling. Moreover, a single intradermal immunization protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. The mild inflammatory milieu created in the dermis by skin laser microporation itself most likely favored the development of potent T cell responses in the absence of exogenous adjuvants. The existence of functionally equivalent XCR1(+) dermal DCs in humans should permit the translation of laser-assisted intradermal delivery of a tumor-specific vaccine targeting XCR1(+) DCs to human cancer immunotherapy. Moreover, considering that the use of adjuvants in vaccines is often associated with safety issues, the possibility of inducing protective responses against melanoma tumor growth independently of the administration of exogenous adjuvants should facilitate the development of safer vaccines.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Cancer Vaccines; Chemokines, C; Dendritic Cells; Disease Models, Animal; Injections, Intradermal; Melanoma, Experimental; Mice; Mice, Knockout; Myeloid Differentiation Factor 88; Neoplasms; Ovalbumin; Protein Binding; Receptors, Chemokine; T-Lymphocyte Subsets; Tumor Burden

λ-Carrageenan is a seaweed polysaccharide which has been generally used as proinflammatory agent in the basic research, however, how the immunomodulating activity of λ-carrageenan affects tumor microenvironment remains unknown. In this study, we found that intratumoral injection of λ-carrageenan could inhibit tumor growth in B16-F10 and 4T1 bearing mice and enhance tumor immune response by increasing the number of tumor-infiltrating M1 macrophages, DCs and more activated CD4(+)CD8(+) T lymphocytes in spleen. In addition, λ-carrageenan could enhance the secretion of IL17A in spleen and significantly increase the level of TNF-α in tumor, most of which was secreted by infiltrating macrophages. Moreover, λ-carrageenan exhibited an efficient adjuvant effect in OVA-based preventative and therapeutic vaccine for cancer treatment, which significantly enhanced the production of anti-OVA antibody. The toxicity analysis suggested that λ-carrageenan was with a good safety profile. Thus, λ-carrageenan might be used both as a potent antitumor agent and an efficient adjuvant in cancer immunotherapy.

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Cancer Vaccines; Carrageenan; Cell Death; Cell Proliferation; Cell Survival; Immunity; Immunotherapy; Injections, Intralesional; Interleukin-17; Melanoma, Experimental; Mice; Neoplasms; Organ Specificity; Ovalbumin; Tumor Necrosis Factor-alpha

T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8(+) T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for Gαi-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8(+) effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8(+) effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.

Topics: Adoptive Transfer; Animals; CD8-Positive T-Lymphocytes; Cell Movement; Female; Gene Expression Regulation; Melanoma; Melanoma, Experimental; Mice; Mice, Knockout; Mice, Transgenic; Neoplasms; Ovalbumin; Receptors, CCR2; Receptors, CCR5; Receptors, CXCR3; Signal Transduction

Potentiation of pH-sensitive liposome-based antigen carriers with IFN-γ gene lipoplexes was attempted to achieve efficient induction of tumor-specific immunity. 3-Methylglutarylated poly(glycidol) (MGluPG)-modified liposomes and cationic liposomes were used, respectively, for the delivery of antigenic protein ovalbumin (OVA) and IFN-γ-encoding plasmid DNA (pDNA). The MGluPG-modified liposomes and the cationic liposome-pDNA complexes (lipoplexes) formed hybrid complexes via electrostatic interactions after their mixing in aqueous solutions. The hybrid complexes co-delivered OVA and IFN-γ-encoding pDNA into DC2.4 cells, a murine dendritic cell line, as was the case of MGluPG-modified liposomes for OVA or the lipoplexes for pDNA. Both the lipoplexes and the hybrid complexes transfected DC2.4 cells and induced IFN-γ protein production, but transfection activities of the hybrid complexes were lower than those of the parent lipoplexes. Subcutaneous administration of hybrid complexes to mice bearing E.G7-OVA tumor reduced tumor volumes, which might result from the induction of OVA-specific cytotoxic T lymphocytes (CTLs). However, the hybrid complex-induced antitumor effect was the same level of the MGluPG-modified liposome-mediated antitumor immunity. In contrast, an extremely strong antitumor immune response was derived when these liposomes and lipoplexes without complexation were injected subcutaneously at the same site of tumor-bearing mice. Immunohistochemical analysis of tumor sections revealed that immunization through the liposome-lipoplex combination promoted the infiltration of CTLs to tumors at an early stage of treatment compared with liposomes, resulting in strong therapeutic effects.

Topics: Animals; Antigens; Cell Line, Tumor; Dendrites; Drug Delivery Systems; Electrophoresis; Female; Fluorescent Antibody Technique; Hydrogen-Ion Concentration; Immunotherapy; Interferon-gamma; Liposomes; Mice, Inbred C57BL; Microscopy, Confocal; Neoplasms; Ovalbumin; Particle Size; Polymers; Propylene Glycols; Spleen; Static Electricity; Subcutaneous Tissue; T-Lymphocytes, Cytotoxic

Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.

Topics: Amino Acid Sequence; Animals; Bone Marrow Cells; Carrier Proteins; Cell Proliferation; Cell-Penetrating Peptides; Cytotoxicity, Immunologic; Dendritic Cells; Endocytosis; Epitopes, T-Lymphocyte; Immunity, Cellular; Immunization; Interferon-gamma; Lymphocyte Activation; Mice, Inbred C57BL; Molecular Sequence Data; Mucin-1; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; tat Gene Products, Human Immunodeficiency Virus

In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow-derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors.

Topics: Animals; Capsid; Capsid Proteins; Cell Line, Tumor; Dependovirus; Genetic Therapy; Genetic Vectors; Immunotherapy; Male; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Prostatic Neoplasms; T-Lymphocytes; Transduction, Genetic

Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-β. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

Topics: Animals; Antineoplastic Agents; CD40 Ligand; Cell Proliferation; CTLA-4 Antigen; Female; Forkhead Transcription Factors; Humans; Immunotherapy; Interferon-gamma; Interleukin-17; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Interleukin-6; Jurkat Cells; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; NFATC Transcription Factors; Niacinamide; Ovalbumin; Peptide Fragments; Phenylurea Compounds; Promoter Regions, Genetic; Receptors, Antigen, T-Cell; Sorafenib; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Cancer vaccines are considered to be a promising tool for cancer immunotherapy. However, a well-designed cancer vaccine should combine a tumor-associated antigen (TAA) with the most effective immunomodulatory agents and/or delivery system to provoke intense immune responses against the TAA. In the present study, we introduced a new approach by conjugating the immunomodulatory molecule LD-indolicidin to the hydrophilic chain end of the polymeric emulsifier poly(ethylene glycol)-polylactide (PEG-PLA), allowing the molecule to be located close to the surface of the resulting emulsion. A peptide/polymer conjugate, named LD-indolicidin-PEG-PLA, was synthesized by conjugation of the amine end-group of LD-indolicidin to the N-hydroxysuccinimide-activated carboxyl end-group of PEG. As an adjuvant for cancer immunotherapeutic use, TAA vaccine candidate formulated with the LD-indolicidin-PEG-PLA-stabilized squalene-in-water emulsion could effectively help to elicit a T helper (Th)1-dominant antigen-specific immune response as well as antitumor ability, using ovalbumin (OVA) protein/EG7 cells as a TAA/tumor cell model. Taken together, these results open up a new approach to the development of immunomodulatory antigen delivery systems for vaccine adjuvants and cancer immunotherapy technologies.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Antimicrobial Cationic Peptides; Antineoplastic Agents; Biocompatible Materials; Cancer Vaccines; Drug Delivery Systems; Emulsions; Female; Immunity; Immunomodulation; Immunotherapy; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptides; Polyethylene Glycols; Squalene; Succinimides

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that β-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases.

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Cancer Vaccines; Cells, Cultured; Immunization; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Mice, Inbred C57BL; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Peptide Fragments; Sizofiran; Spleen; T-Lymphocytes, Cytotoxic; Tissue Distribution; Tumor Burden

pH-Sensitive dextran derivatives having 3-methylglutarylated residues (MGlu-Dex) were prepared by reacting dextran with 3-methyl-glutaric anhydride. MGlu-Dex changed the protonation state and their characteristics from hydrophilic to hydrophobic in neutral and acidic pH regions. Surface modification of egg yolk phosphatidylcholine liposomes with MGlu-Dex produced highly pH-sensitive liposomes that were stable at neutral pH but which were destabilized strongly in the weakly acidic pH region. MGlu-Dex-modified liposomes were taken up efficiently by dendritic cells and delivered entrapped ovalbumin (OVA) molecules into the cytosol. When MGlu-Dex-modified liposomes loaded with OVA were administered subcutaneously to mice, the antigen-specific humoral and cellular immunity was induced more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of MGlu-Dex-modified liposomes loaded with OVA to mice bearing E.G7-OVA tumor significantly suppressed tumor growth and extended the mice survival. Results suggest that MGlu-Dex-modified liposomes are promising for the production of safe and potent antigen delivery systems that contribute to the establishment of efficient cancer immunotherapy.

Topics: Anhydrides; Animals; Antigens, Neoplasm; Cell Line; Chemical Precipitation; Dextrans; Female; Fluorescence; Fluorescent Dyes; Glutarates; Hydrogen-Ion Concentration; Immunity; Immunization; Immunotherapy; Interferon-gamma; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Particle Size; Pyrenes; Static Electricity; Subcutaneous Tissue; T-Lymphocytes, Cytotoxic; Titrimetry

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.

Topics: Acetylcysteine; Animals; Antigen Presentation; Cell Line, Tumor; Cells, Cultured; Cross-Priming; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Dendritic Cells; Flow Cytometry; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Humans; Interferon-gamma; Lipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Neoplasms; Ovalbumin; Oxidation-Reduction; Peptide Fragments

The expression of Adenovirus serotype 2 or serotype 5 (Ad2/5) E1A in tumor cells reduces their tumorigenicity in vivo by enhancing the NK cell mediated and T cell mediated anti-tumor immune response, an activity that correlates with the ability of E1A to bind p300. We determined if E1A could be used as a molecular adjuvant to enhance antigen-specific T cell responses to a model tumor antigen, ovalbumin (OVA). To achieve this goal, we stably expressed a fusion protein of E1A and OVA (MCA-205-E1A-OVA), OVA (MCA-205-OVA) or a mutant version of E1A unable to bind p300 and OVA (E1A-Δp300-OVA) in the B6-derived, highly tumorigenic MCA-205 tumor cell line. MCA-205-E1A-OVA tumor cells were over 10,000 fold less tumorigenic than MCA-205-OVA, MCA-205-E1A-Δp300-OVA, or MCA-205 in B6 mice. However, immunization of B6 mice with live MCA-205-OVA, MCA-205-E1A-Δp300-OVA and MCA-E1A-OVA tumor cells induced nearly equivalent OVA-specific CD4 T cells and CD8 CTL responses. Further studies revealed that mice with primary, enlarging MCA-205-OVA or MCA-205-E1A-Δp300-OVA tumors on one flank exhibited OVA-specific anti-tumor T cell responses that rejected a tumorigenic dose of MCA-205-OVA cells on the contralateral flank (concomitant tumor immunity). Next we found that tumor associated macrophages (TAMs) in progressive MCA-205-OVA tumors, but not MCA-205-E1A-OVA tumors that expressed high levels of arginase-1, which is known to have local immunosuppressive activities. In summary, immunization of mice with MCA-205 cells expressing OVA, E1A-Δp300-OVA or E1A-OVA induced equivalent OVA-specific CD4 and CD8 anti-tumor responses. TAMs found in MCA-205-OVA, but not MCA-205-E1A-OVA, tumors expressed high levels of arginase-1. We hypothesize that the production of arginase-1 by TAMs in MCA-205-OVA or MCA-205-E1A-Δp300-OVA tumor cells leads to an ineffective anti-tumor immune response in the tumor microenvironment, but does not result in inhibition of a systemic anti-tumor immunity.

Topics: Adenovirus E1A Proteins; Animals; Arginase; Carcinogenesis; CD3 Complex; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Immunity; Immunization; Macrophages; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Recombinant Fusion Proteins; Transfection

Golden vaccine for cancers. Gold nanoparticles enable efficient antigen delivery to dendritic cells and then activate the cells to facilitate cross-presentation, inducing antigen-specific cytotoxic T-lymphocyte responses for effective cancer therapy.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line; Dendritic Cells; Fibronectins; Gold; Immunotherapy; Interferon-gamma; Interleukin-2; Metal Nanoparticles; Mice; Mice, Inbred BALB C; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol=40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line; Cell Line, Tumor; Cyclic GMP; Female; Immunotherapy; Interferon-beta; Lipids; Liposomes; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Piperidines; T-Lymphocytes, Cytotoxic; Tumor Burden

The sentinel or tumor-draining lymph node (tdLN) serves as a metastatic niche for many solid tumors and is altered via tumor-derived factors that support tumor progression and metastasis. tdLNs are often removed surgically, and therapeutic vaccines against tumor antigens are typically administered systemically or in non-tumor-associated sites. Although the tdLN is immune-suppressed, it is also antigen experienced through drainage of tumor-associated antigens (TAA), so we asked whether therapeutic vaccines targeting the tdLN would be more or less effective than those targeting the non-tdLN. Using LN-targeting nanoparticle (NP)-conjugate vaccines consisting of TAA-NP and CpG-NP, we compared delivery to the tdLN versus non-tdLN in two different cancer models, E.G7-OVA lymphoma (expressing the nonendogenous TAA ovalbumin) and B16-F10 melanoma. Surprisingly, despite the immune-suppressed state of the tdLN, tdLN-targeting vaccination induced substantially stronger cytotoxic CD8+ T-cell responses, both locally and systemically, than non-tdLN-targeting vaccination, leading to enhanced tumor regression and host survival. This improved tumor regression correlated with a shift in the tumor-infiltrating leukocyte repertoire toward a less suppressive and more immunogenic balance. Nanoparticle coupling of adjuvant and antigen was required for effective tdLN targeting, as nanoparticle coupling dramatically increased the delivery of antigen and adjuvant to LN-resident antigen-presenting cells, thereby increasing therapeutic efficacy. This work highlights the tdLN as a target for cancer immunotherapy and shows how its antigen-experienced but immune-suppressed state can be reprogrammed with a targeted vaccine yielding antitumor immunity.

Topics: Animals; Antigen-Presenting Cells; Antigens, Neoplasm; Cancer Vaccines; Female; Humans; Immune Tolerance; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Mice; Myeloid Cells; Nanoparticles; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocyte Subsets; Tumor Burden

An impaired antitumor immunity is found in patients with cancer and represents a major obstacle in the successful development of different forms of immunotherapy. Signaling through Notch receptors regulates the differentiation and function of many cell types, including immune cells. However, the effect of Notch in CD8(+) T-cell responses in tumors remains unclear. Thus, we aimed to determine the role of Notch signaling in CD8(+) T cells in the induction of tumor-induced suppression. Our results using conditional knockout mice show that Notch-1 and Notch-2 were critical for the proliferation and IFNγ production of activated CD8(+) T cells and were significantly decreased in tumor-infiltrating T cells. Conditional transgenic expression of Notch-1 intracellular domain (N1IC) in antigen-specific CD8(+) T cells did not affect activation or proliferation of CD8(+) T cells, but induced a central memory phenotype and increased cytotoxicity effects and granzyme B levels. Consequently, a higher antitumor response and resistance to tumor-induced tolerance were found after adoptive transfer of N1IC-transgenic CD8(+) T cells into tumor-bearing mice. Additional results showed that myeloid-derived suppressor cells (MDSC) blocked the expression of Notch-1 and Notch-2 in T cells through nitric oxide-dependent mechanisms. Interestingly, N1IC overexpression rendered CD8(+) T cells resistant to the tolerogenic effect induced by MDSC in vivo. Together, the results suggest the key role of Notch in the suppression of CD8(+) T-cell responses in tumors and the therapeutic potential of N1IC in antigen-specific CD8(+) T cells to reverse T-cell suppression and increase the efficacy of T cell-based immunotherapies in cancer.

Topics: Animals; Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Immune Tolerance; Immunotherapy, Adoptive; Mice, Transgenic; Neoplasms; Ovalbumin; Receptor, Notch1; Receptor, Notch2; Signal Transduction; Tumor Burden

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies.

Topics: Animals; Antibodies; Antigens; Brentuximab Vedotin; Cancer Vaccines; Cell Line; Cells, Cultured; CTLA-4 Antigen; Cytokines; Dendritic Cells; Depsipeptides; Humans; Immunoconjugates; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Programmed Cell Death 1 Receptor; T-Lymphocytes; Tubulin Modulators; Tumor Burden

This study describes the synthesis of glycopeptides NHAc[βGal]-(Thr)2 -[αGalNAc]-(Thr)2 -[αGlcNAc]-(Thr)2 Gly-OVA (1-OVA) and NHAc[βGal-αGalNAc]-(Thr)3 -[αLacNAc]-(Thr)3 -Gly-OVA (2-OVA) as mimetics of both T. cruzi and tumor mucin glycoproteins. These glycopeptides were obtained by solid-phase synthesis, which involved the prior preparation of the protected glycosyl amino acids αGlcNAc-ThrOH (3), αGalNAc-ThrOH (4), βGal-ThrOH (5), αLacNAc-ThrOH (6), and βGal-αGalNAc-ThrOH (7) through glycosylation reactions. Immunizations of mice with glycopeptides 1-OVA and 2-OVA induced high antibody titers (1:16 000), as verified by ELISA tests, whereas flow cytometry assays showed the capacity of the obtained anti-glycopeptides 1-OVA and 2-OVA antibodies to recognize both T. cruzi and MCF-7 tumor cells. In addition, antisera induced by glycopeptides 1-OVA and 2-OVA were also able to inhibit T. cruzi fibroblast cell invasion (70 %) and to induce antibody-mediated cellular cytotoxicity (ADCC) against MCF-7 cells, with 50 % reduction of cell viability.

Topics: Amino Acid Sequence; Animals; Antibodies; Cell Line, Tumor; Cell Survival; Chagas Disease; Glycopeptides; Humans; Immunization; Mice; Mucins; Neoplasms; Ovalbumin; Trypanosoma cruzi

Europium-doped GdPO4 hollow spheres/polymer core-shell nanoparticles are functionalized with ovalbumin (OVA) as a model antigen and an oligonucleotide (CpG) that stimulates the immune response. These functionalized core-shell nanoparticles are used as vaccines, where they enable efficient delivery of an antigen to target sites, tracking of the vaccines using non-invasive clinical imaging technology.

Topics: Animals; Antibodies; Antigens; Cell Line, Tumor; Coordination Complexes; CpG Islands; Cytokines; Enzyme-Linked Immunosorbent Assay; Gadolinium; Lanthanoid Series Elements; Magnetic Resonance Imaging; Metal Nanoparticles; Mice; Mice, Inbred C57BL; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Radiography

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.

Topics: Animals; Antibodies, Bispecific; Antigens, CD20; B-Lymphocytes; Cell Line, Tumor; Cytotoxicity, Immunologic; Disease Models, Animal; Gene Order; Histocompatibility Antigens Class I; Humans; Immunoconjugates; Lymphocyte Activation; Lymphocyte Depletion; Mice; Neoplasms; Ovalbumin; Peptides; Protein Binding; Recombinant Fusion Proteins; Single-Chain Antibodies; T-Lymphocytes; T-Lymphocytes, Cytotoxic

For production of pH-sensitive liposomes, we developed pH-sensitive polymer-lipids that consists of pH-sensitive fusogenic polymer moieties such as 3-methyl glutarylated poly(glycidol) and 2-carboxycyclohexane-1-carboxylated poly(glycidol), connected to a phosphatidylethanolamine head group. Incorporation of these pH-sensitive polymer-lipids into egg yolk phosphatidylcholine liposomes produced highly pH-sensitive liposomes that were stable at neutral pH but which destabilized markedly in response to very small pH change in weakly acidic pH region. These liposomes delivered their contents (pyranine) into cytosol of dendritic cell-derived DC2.4 cells. When these polymer-lipid-incorporated liposomes loaded with antigenic protein ovalbumin (OVA) were administered subcutaneously to mice, the antigen-specific cellular immunity was induced efficiently in the mice. Furthermore, immunization of mice with these OVA-loaded pH-sensitive polymer-lipid-incorporated liposomes induced strong OVA-specific immunity, which achieved complete rejection of OVA-expressing E.G7-OVA cells and marked regression of E.G7-OVA tumors.

Topics: Animals; Antigens; Arylsulfonates; Cell Line; Female; Hydrogen-Ion Concentration; Immunity; Immunization; Immunotherapy; Lipids; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Phosphatidylethanolamines; Phycoerythrin; Polymers; Rhodamines; Temperature

This protocol describes a procedure to evaluate the ability of Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) isolated from the spleen or tumor of a tumor-bearing mouse to suppress antigen-specific T-cell proliferation. MDSCs are cultured with responder splenocytes from an OT-1 T-cell receptor transgenic mouse stimulated with cognate peptide (OVA-257 peptide) or with an irrelevant, control peptide. This protocol spans a 4-d period. The readout is T-cell proliferation by (3)[H]-thymidine incorporation during an 18-h pulse between Days 3 and 4.

Topics: Animals; Cell Proliferation; Immunosuppression Therapy; Mice; Mice, Transgenic; Neoplasms; Ovalbumin; Spleen; T-Lymphocytes

To develop a liposome formulation incorporating antigen-presenting cells (APCs) membrane microdomains with enriched epitope/MHC complexes to evaluate the activities of these liposomes (RAFTsomes) to activate T cells and prime immune responses.. We isolated membrane microdomain structures that contained the epitope/MHC complexes from ovalbumin (OVA) primed dendritic cells (DCs), and reconstituted them on liposomes surface by detergent dialysis. The resulted RAFTsomes were purified by density gradient centrifugation. Their T cell activation functions were evaluated by IL-2 secreting and proliferation assays in vitro. In vivo immune responses and the protective effect against OVA expressing EG.7 tumor challenge were also examined.. Membrane microdomains containing enriched epitope/MHC complexes can be reconstituted into liposomes with defined size and composition. The integrity and activities of these complexes after reconstitution were confirmed by in vitro T cell assays. OVA epitope loaded RAFTsomes injected in vivo resulted in high anti-OVA IgG production (predominantly IgG1). The immunized mice were protected from EG.7 tumor cell inoculation challenge.. Based on these findings, we propose that RAFTsomes can be prepared with unique properties that may be used as an antigen delivery system for immunotherapeutic applications.

Topics: Animals; Cancer Vaccines; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cells, Cultured; Dendritic Cells; Epitopes; Genes, MHC Class II; Interleukin-2; Liposomes; Lymphocyte Activation; Membrane Microdomains; Mice; Mice, Inbred BALB C; Neoplasms; Ovalbumin

Recent preclinical evidence substantially supports the successful combination of chemotherapies and active immunotherapy for cancer treatment. These data sustain the effect of sequential combination schemes (vaccine plus chemotherapy or vice versa), which could be difficult to implement in clinical practice. Since chemotherapy is the standard treatment for most cancers, ethical issues forbid its delay and make difficult the evaluation of other treatments such as using an immunotherapeutic agent. Besides, vaccines must be applied as soon as possible to advanced cancer patients, in order to give them time to develop an effective immune response. Thus, a clinically attractive scenario is the concomitant application of treatments. However, little is known about the specific effect of different chemotherapeutic agents when combined with a cancer vaccine in such concomitant treatment. In this work, we analyze the influence of high-dose carboplatin or paclitaxel in the generation of a specific immune response when administered concomitantly with an OVA vaccine. Interestingly, neither carboplatin nor paclitaxel affects the humoral and CTL in vivo response generated by the vaccine. Moreover, an enhancement of the overall anti-tumor effect was observed in animals treated with OVA/CF vaccine combined with cytotoxic drugs. Moreover, the effect of the concomitant treatment was tested using a tumor-related antigen, the epidermal growth factor (EGF). Animals administered with EGF-P64k/Montanide and cytotoxic agents showed an antibody response similar to that from control animals. Therefore, our study suggests that carboplatin and paclitaxel can be concomitantly combined with active immunotherapies in the clinical practice of advanced cancer patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cancer Vaccines; Carboplatin; Combined Modality Therapy; Epidermal Growth Factor; Female; Humans; Immunotherapy, Active; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Paclitaxel

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responses against cancer leading to reduced tumor growth and improved survival. In this study, various formulations of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant properties were evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vaccinated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), in combination with the Toll-like receptor-9 agonist, CpG oligonucleotide 1826 (CpG ODN). Mice treated with OVA-encapsulated CPTEG:CPH particles elicited the highest CD8(+) T cell responses on days 14 and 20 when compared to other treatment groups. This treatment group also displayed the most delayed tumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN generated the highest anti-OVA IgG(1) antibody responses in mice but these mice did not show significant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles can stimulate antigen-specific cellular responses and could therefore potentially be used to promote antitumor responses in cancer patients.

Topics: Animals; Antibody Specificity; Antigens; Antineoplastic Agents; Immunoglobulin G; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Particle Size; Polyanhydrides; Polyethylene Glycols; Static Electricity; Survival Analysis; T-Lymphocytes; Vaccination

Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Deoxycytidine; Epidermal Growth Factor; ErbB Receptors; Gemcitabine; Humans; Mice; Mice, Nude; Molecular Targeted Therapy; Nanoparticles; Neoplasms; Ovalbumin; Tumor Burden

To develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy.. The novel delivery system was constructed with antigen protein, benzalkonium chloride (BK), and γ-polyglutamic acid (γ-PGA), using ovalbumin (OVA) as a model antigen protein and evaluating its immune induction effects and utilities for cancer vaccine.. BK and γ-PGA enabled encapsulation of OVA and formed stable anionic particles at nanoscale, OVA/BK/γ-PGA complex. Complex was taken up by dendritic cell line DC2.4 cells efficiently. We subcutaneously administered the complex to mice and examined induction of IgGs. The complex induced not only Th2-type immunoglobulins but also Th1-type immunoglobulins. OVA/BK/γ-PGA complex inhibited tumor growth of E.G7 cells expressing OVA regularly; administered OVA/BK/γ-PGA complex completely rejected tumor cells.. The novel vaccine could be platform technology for a cancer vaccine.

Topics: Animals; Antigen-Presenting Cells; Benzalkonium Compounds; Cancer Vaccines; Cell Line; Dendritic Cells; Drug Delivery Systems; Immunoglobulin G; Male; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Polyglutamic Acid

Priming of CD8(+) T cells requires two signals, one produced by T-cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non-covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen-presenting cells (APC) to be presented by MHC I molecules to CD8(+) T cells, thus, signal 1 has been well characterized in this pathway of cross-presentation. Signal 2 is not fully understood, although there are reports that Toll-like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross-priming by a mycobacterial HSP70-antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP-mediated cross-priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross-priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4-dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden.

Topics: Animals; Antigen-Presenting Cells; CD8-Positive T-Lymphocytes; Cross-Priming; HSP70 Heat-Shock Proteins; Interleukin-1 Receptor-Associated Kinases; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Neoplasms; Ovalbumin; Recombinant Proteins; Toll-Like Receptors

Central memory CD8(+) T cells expressing the adhesion molecule CD62L (L-selectin) are potent mediators of anti-cancer immunity due to their ability to proliferate extensively upon antigen re-stimulation. The interaction of selectin with its ligands mediates leukocyte rolling along high endothelial venules. Mice deficient in α(1,3) Fucosyltransferase IV and VII (FtDKO) lack functional L, P and E selectin ligands. Thus, we addressed whether the lack of selectin ligand interactions alters tumor protection by CD8(+) T cells in FtDKO mice. Listeria monocytogenes-OVA (LM-OVA) infection evoked potent OVA-specific CD8(+) T cells that proliferated and contracted at similar kinetics and phenotype in FtDKO and wild-type mice. Additionally, OVA-specific CD8(+) T cells in both mouse strains exhibited similar phenotypic differentiation, in vivo cytolytic activity and IFN-γ expression. However, FtDKO mice succumbed to B16-OVA tumors significantly earlier than wild-type mice. In contrast, FtDKO mice evoked strong recall memory CD8(+) T cell responses and protection to systemic LM-OVA re-challenge. The diminished tumor protection in FtDKO mice was not related to defective antigen presentation by dendritic cells or reduced proliferation of antigen-specific CD8(+) T cells. However, WT or FtDKO OVA-specific CD8(+) T cells showed significantly reduced ability to traffic to lymph nodes upon adoptive transfer into naïve FtDKO recipients. Furthermore, FtDKO OVA-specific CD8(+) T cells displayed poor ability to infiltrate tumors growing in WT mice. These results reveal that selectin ligand expression on host endothelium as well CD8(+) T cells may be important for their efficient and continued extravasation into peripheral tumors.

Topics: Animals; Antigen Presentation; CD8-Positive T-Lymphocytes; Fucosyltransferases; Ligands; Mice; Mice, Knockout; Neoplasms; Ovalbumin; Selectins

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Antibodies; Antigens; Antigens, CD; Cross-Priming; Cytotoxicity, Immunologic; Dendritic Cells; Drug Delivery Systems; Immunoconjugates; Lectins, C-Type; Mice; Mice, Inbred C57BL; Minor Histocompatibility Antigens; Molecular Sequence Data; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Receptors, Cell Surface; Solubility; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 9

Vaccination with autologous cancer cells aims to enhance adaptive immune responses to tumour-associated antigens. The incorporation of Fms-like tyrosine kinase 3-ligand (FLT3L) treatment to the vaccination scheme has been shown previously to increase the immunogenicity of cancer vaccines, thereby enhancing their therapeutic potential. While evidence has been provided that FLT3L confers its effect through the increase of absolute dendritic cell (DC) numbers, it is currently unknown which DC populations are responsive to FLT3L and which effect FLT3L treatment has on DC functions. Here we show that the beneficial effects of FLT3L treatment resulted predominantly from a marked increase of two specific DC populations, the CD8 DCs and the recently identified merocytic DC (mcDC). These two DC populations (cross)-present cell-associated antigens to T cells in a natural killer (NK)-independent fashion. FLT3L treatment augmented the absolute numbers of these DCs, but did not change their activation status nor their capacity to prime antigen-specific T cells. While both DC populations effectively primed CD8(+) T cell responses to cell-associated antigens, only mcDC were capable to prime CD4(+) T cells to cell-associated antigens. Consequentially, the transfer of tumour vaccine-pulsed mcDC, but not of CD8 DCs, protected mice from subsequent tumour challenge in a vaccination model and resulted in eradication of established tumours in a therapeutic approach. These results show that the beneficial effect of FLT3L is associated with the induction of mcDC and suggests that selective targeting to mcDC or instilling mcDC 'characteristics' into conventional DC populations could significantly enhance the efficacy of tumour vaccines.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Antigens, Surface; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cytokines; Dendritic Cells; Immunodominant Epitopes; Interferon-gamma; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Transgenic; Natural Killer T-Cells; Neoplasms; Ovalbumin; Peptide Fragments; Spleen; Survival Analysis; Vaccination

Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

Topics: Animals; Antigens; CD8-Positive T-Lymphocytes; Cells, Cultured; Chickens; Communicable Diseases; Genetic Therapy; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Immunologic Memory; Immunotherapy, Adoptive; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; Time Factors

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.

Topics: Animals; Antennapedia Homeodomain Protein; Antigen Presentation; Antigens, Neoplasm; Carrier Proteins; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cell-Penetrating Peptides; Drosophila; Drosophila Proteins; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Interferon-gamma; Mice; Mice, Inbred C57BL; Mucin-1; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer

We previously reported strong induction of ovalbumin (OVA)-specific tumor immunity in mice injected subcutaneously with OVA-entrapping nanoparticles comprising amphiphilic poly(γ-glutamic acid) (OVA/γ-PGA NPs). In the present study we investigated antitumor efficacy and associated immune responses in mice vaccinated with OVA/γ-PGA NPs via the nasal cavity. Mice vaccinated intranasally with OVA/γ-PGA NPs resisted challenge by E.G7-OVA tumor cells, and lung metastasis of B16-OVA cells were significantly suppressed by three intranasal doses of OVA/γ-PGA NPs. Although the total serum anti-OVA IgG titer was equivalent between the OVA/γ-PGA NP- and OVA solution-immunized groups, intranasal vaccination with OVA/γ-PGA NPs efficiently induced cytotoxic T lymphocytes (CTLs) and interferon-γ-secreting cells specific for OVA in the spleen and lymph nodes. The antitumor activity induced by intranasal vaccination of OVA/γ-PGA NPs mainly required CD8(+) CTLs, and the development of long-term specific immunity was confirmed in rechallenge experiments. OVA/γ-PGA NPs administered via the nasal cavity were rapidly taken up by nasopharyngeal-associated lymphoid tissue and delivered to the cervical lymph nodes. Thus, nasal vaccination with antigen-entrapping γ-PGA NPs evokes tumor immunity by eliciting antigen-specific CTLs. γ-PGA NPs are a promising antigen delivery carrier for the development of non-invasive cancer vaccines.

Topics: Administration, Intranasal; Animals; Cancer Vaccines; Cell Line, Tumor; Drug Carriers; Female; Humans; Immunization; Immunoglobulin G; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Nanoparticles; Neoplasms; Ovalbumin; Polyglutamic Acid

The co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.. We find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.. This study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

Topics: Animals; Antigens, Differentiation; Antineoplastic Agents; Autoimmunity; Cell Proliferation; CTLA-4 Antigen; Cytokines; Interferon-gamma; Lectins, C-Type; Lymph Nodes; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Ovalbumin; Programmed Cell Death 1 Receptor; Receptors, Immunologic; T-Lymphocytes; Tumor Necrosis Factor Receptor Superfamily, Member 9

Estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is a tumor-promoting factor of largely unknown function. To assess a causative role of EBAG9 in advanced malignancies, we generated the EG7-OVA and MethA murine tumor cell lines that stably express full-length or truncated EBAG9 protein, using retroviral-mediated gene transduction. Upon subcutaneous inoculation into immunocompetent mice, both cell lines showed marked acceleration of in vivo tumor growth when full-length EBAG9 was overexpressed. Interestingly, deletion of the coiled-coil region, thereby producing truncated EBAG9 protein, abolished the tumor-acceleration effect, establishing the importance of this domain in EBAG9-mediated tumor promotion. However, there was no alteration in in vitro cell proliferation or expression levels of MHC class I and co-stimulatory molecules believed to play a role in immune evasion of tumor cells in these tumor cell lines expressing full-length or truncated EBAG9 protein. Furthermore, both full-length and truncated EBAG9 proteins showed a predominantly cytoplasmic localization in the tumor cells. Collectively, these results suggest that EBAG9 overexpression can be causative in enhancing the malignant properties of tumor cells, and that tumor promotion likely requires EBAG9 intracellular association with as yet unidentified binding partners via the coiled-coil region.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; B7-1 Antigen; B7-H1 Antigen; Carcinogens; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Female; Gene Expression; Gene Order; Genetic Vectors; H-2 Antigens; HEK293 Cells; Humans; Intracellular Space; Jurkat Cells; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptides; Protein Transport; Retroviridae; T-Lymphocytes; V-Set Domain-Containing T-Cell Activation Inhibitor 1

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antigen Presentation; Antigen-Presenting Cells; CD40 Antigens; Dendritic Cells; Immunoglobulin Fc Fragments; Lymphocyte Activation; Mice; Neoplasms; Ovalbumin; Receptors, IgG; Signal Transduction; T-Lymphocytes

B7-H3 is a B7-family co-stimulatory molecule and is broadly expressed on various tissues and immune cells. Transduction of B7-H3 into some tumours enhances anti-tumour responses. We have recently found that a triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a receptor for B7-H3. Here, we examined the roles of tumour-associated B7-H3 and the involvement of TLT-2 in anti-tumour immunity. Ovalbumin (OVA)(257-264)-specific OT-I CD8(+) T cells exhibited higher cytotoxicity against B7-H3-transduced OVA-expressing tumour cells (B7-H3/E.G7) in vitro and selectively eliminated B7-H3/E.G7 cells in vivo. The presence of B7-H3 on target cells efficiently augmented CD8(+) T-cell-mediated cytotoxicity against alloantigen or OVA, whereas the presence of B7-H3 in the priming phase did not affect the induced cytotoxicity. B7-H3 transduction into five tumour cell lines efficiently reduced their tumorigenicity and regressed growth. Treatment with either anti-B7-H3 or anti-TLT-2 monoclonal antibody accelerated growth of a tumour that expressed endogenous B7-H3, suggesting a co-stimulatory role of the B7-H3-TLT-2 pathway. The TLT-2 was preferentially expressed on CD8(+) T cells in regional lymph nodes, but was down-regulated in tumour-infiltrating CD8(+) T cells. Transduction of TLT-2 into OT-I CD8(+) T cells enhanced antigen-specific cytotoxicity against both parental and B7-H3-transduced tumour cells. Our results suggest that tumour-associated B7-H3 directly augments CD8(+) T-cell effector function, possibly by ligation of TLT-2 on tumour-infiltrating CD8(+) T cells at the local tumour site.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; B7 Antigens; B7-1 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cytokines; Cytotoxicity, Immunologic; Female; Interferon-gamma; Lectins, C-Type; Lymph Nodes; Lymphocyte Activation; Lymphocyte Depletion; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Nude; Mice, Transgenic; Neoplasms; Ovalbumin; Receptors, Immunologic; T-Lymphocytes, Cytotoxic; Transfection

Red blood cells (RBCs) were shown to be efficient antigen carriers to target dendritic cells (DCs) and induce cytotoxic T-cell responses. Mouse RBCs were loaded with ovalbumin (RBC-OVA) and injected with Poly (I:C) into mice. Phagocytosis of RBC-OVA by macrophages and DCs was demonstrated to induce OVA-specific CD4(+) and CD8(+) T cell activation. Moreover, these CD8(+) T cells produced IFN-gamma and were able to induce OVA-specific cell lysis. Finally, T-cell response was demonstrated to be dependent on the dose-amount of antigen entrapped and this response could be maintained for up to 30 days.

Topics: Animals; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dendritic Cells; Erythrocytes; Immunotherapy; Interferon-gamma; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Phagocytosis; T-Lymphocytes, Cytotoxic

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4-5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

Topics: Adoptive Transfer; Animals; Antigen Presentation; Antigen-Presenting Cells; beta 2-Microglobulin; Carcinoma, Lewis Lung; CD11a Antigen; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Movement; Cell Proliferation; DNA-Binding Proteins; Fingolimod Hydrochloride; Granzymes; Hyaluronan Receptors; Immunosuppressive Agents; Integrin alpha4; Interferon-gamma; Lymph Nodes; Lymphocyte Activation; Lysosomal Membrane Proteins; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Monophenol Monooxygenase; Neoplasms; Ovalbumin; Peptide Fragments; Propylene Glycols; Receptors, Antigen, T-Cell; Sphingosine; T-Lymphocyte Subsets

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4+ T cells, but also on MHC-class I molecules to CD8+ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4+ and CD8+ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin+ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin+ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4+ and CD8+ T cell responses emphasizes their potential for epicutaneous immunization strategies.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Epidermis; Humans; Immunotherapy, Active; Langerhans Cells; Lectins, C-Type; Mannose-Binding Lectins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Ovalbumin

In dendritic cell (DC)-based cancer immunotherapy, it is important that DCs present peptides derived from tumor-associated antigens on MHC class I, and activate tumor-specific cytotoxic T lymphocytes (CTLs). However, MHC class I generally present endogenous antigens expressed in the cytosol. We therefore developed an innovative approach capable of directly delivering exogenous antigens into the cytosol of DCs; i.e., a MHC class I-presenting pathway. In this study, we investigated the effect of antigen delivery using perfluoropropane gas-entrapping liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure on MHC class I presentation levels in DCs, as well as the feasibility of using this antigen delivery system in DC-based cancer immunotherapy. DCs were treated with ovalbumin (OVA) as a model antigen, BLs and US exposure. OVA was directly delivered into the cytosol but not via the endocytosis pathway, and OVA-derived peptides were presented on MHC class I. This result indicates that exogenous antigens can be recognized as endogenous antigens when delivered into the cytosol. Immunization with DCs treated with OVA, BLs and US exposure efficiently induced OVA-specific CTLs and resulted in the complete rejection of E.G7-OVA tumors. These data indicate that the combination of BLs and US exposure is a promising antigen delivery system in DC-based cancer immunotherapy.

Topics: Animals; Antigen Presentation; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Survival; Cytotoxicity Tests, Immunologic; Dendritic Cells; Endocytosis; Fluorocarbons; Immunotherapy, Active; Interleukin-2; Liposomes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Sodium Azide; Survival Analysis; T-Lymphocytes, Cytotoxic; Ultrasonics

Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-kappaB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-kappaB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8(+) T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8(+) T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-kappaB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-kappaB activator can improve the efficacy of a vaccine vector.

Topics: Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Leishmania donovani; Leishmaniasis; Lentivirus; Mice; Neoplasms; NF-kappa B; Ovalbumin; Receptors, Immunologic; Spleen; Survival Analysis; Viral Proteins; Viral Vaccines

Interleukin (IL-) 27 is a member of IL-12 cytokine family with Th1-promoting and anti-inflammatory effects. IL-27 has been shown to facilitate tumor-specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL-27 suppresses cytokine production of lymphocytes and antigen-presenting function of dendritic cells (DCs). To examine the in vivo role of IL-27 in generation of anti-tumor immunity, we examined IL-27-mediated antitumor-effects using WSX-1 (IL-27 receptor alpha chain)-deficient (WSX-1(-/-)) mice. In WSX-1(-/-) mice inoculated with B16 melanoma cells, tumor growth was higher than in wild-type (WT) mice. Accordingly, tumor-specific CTL generation was lower in WSX-1(-/-) mice than in WT mice. CTL induction in WSX-1(-/-) mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL-27 is directly required for generation of tumor-specific CTLs. However, when transferred into tumor-bearing mice, WSX-1(-/-) DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL-27 on DC function. Finally, the combination of WT CD8(+) T cells and KO DCs is more potent in generation of antigen-specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor-specific CD8(+) T cells were also the highest in the combination of WT CD8+ T cells and WSX-1(-/-) DCs. It was thus revealed that IL-27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL-27 signal-defective DCs may have therapeutic potential against tumors.

Topics: Angiogenesis Inhibitors; Animals; Dendritic Cells; Interleukin-17; Interleukin-2; Melanoma; Melanoma, Experimental; Mice; Mice, Knockout; Neoplasms; Neuroblastoma; Ovalbumin; Receptors, Cytokine; Receptors, Interleukin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; T-Lymphocytes, Cytotoxic; Th1 Cells

Heat shock protein 70 (Hsp70)-associated antigens in a soluble form have been shown to elicit strong antigen-specific cytotoxic T lymphocyte (CTL) responses following immunization without any adjuvants. In order to improve the potential of Hsp70, we genetically designed a novel Hsp70-based antigen delivery system, in which the model MHC class I epitope of ovalbumin (OVA) (SIINFEKL; OVA257-264) was fused to mouse Hsp70. To facilitate the cytosolic delivery of the peptide following Hsp receptor-mediated endocytosis, polyhistidine of 25 or 50 residues was further fused to the fusion protein. Each fusion protein was then expressed in E. coli and purified. When added to DC2.4 cells, a mouse dendritic cell line, the fusion protein containing polyhistidine of 25 residues was efficiently taken up by the cells and efficiently distributed to the cytosol. The fusion protein also exhibited a significantly improved efficacy of MHC class I-restricted presentation of antigen. Vaccination of mice with the polyhistidine fusion protein generated strong antigen-specific CTL responses and antitumor activity. These findings suggest that polyhistidine fusion is a useful strategy to increase the potential of Hsp-based vaccination.

Topics: Animals; Antigen Presentation; Cell Line, Tumor; Cell Survival; Cytosol; Dendritic Cells; Drug Delivery Systems; Epitopes; Escherichia coli; Female; Histidine; Histocompatibility Antigens Class I; HSC70 Heat-Shock Proteins; Interleukin-2; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Ovalbumin; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such "DC exhaustion" can be prevented by the presence of IFNgamma during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines.

Topics: Animals; Cancer Vaccines; Cell Polarity; Dendritic Cells; Female; Humans; Immunotherapy; Interferon-gamma; Interleukin-12; Interleukin-4; Lipopolysaccharides; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases.

Topics: Adenoviridae; Animals; Antineoplastic Agents; Antiviral Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Female; Hemagglutinins; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasms; Ovalbumin; Telomerase

Class A scavenger receptor (SRA), also known as CD204, has been shown to participate in the pathogenesis of atherosclerosis and the pattern recognition of pathogen infection. However, its role in adaptive immune responses has not been well defined. In this study, we report that the lack of SRA/CD204 promotes Toll-like receptor (TLR)4 agonist-augmented tumor-protective immunity, which is associated with enhanced activation of CD8(+) effector T cell and improved inhibition of tumor growth. Dendritic cells (DCs) deficient in SRA/CD204 display more effective immunostimulatory activities upon TLR4 engagement than those from wild-type counterparts. Silencing of SRA/CD204 by RNA interference improves the ability of DCs to prime antigen-specific CD8(+) T cells, suggesting that antigen-presenting cells, for example, DCs, play a major role in SRA/CD204-mediated immune modulation. Our findings reveal a previously unrecognized role for SRA/CD204, a non-TLR pattern recognition receptor, as a physiologic negative regulator of TLR4-mediated immune consequences, which has important clinical implications for development of TLR-targeted immunotherapeutic intervention.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line; Cell Proliferation; Dendritic Cells; Down-Regulation; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Ovalbumin; RNA Interference; Scavenger Receptors, Class A; Signal Transduction; Toll-Like Receptor 4

Induction of a functional CD8(+) T-cell response is the important criterion for cancer vaccines, and it is unclear whether acute or chronic live vectors are better suited for cancer antigen delivery. We have evaluated the tumor protective ability of two recombinant vectors, Listeria monocytogenes (LM) and Salmonella typhimurium (ST), both expressing ovalbumin (OVA). Although both vectors induced a similar OVA-specific CD8(+) T-cell response in the long term, LM-OVA induced mainly central-phenotype (T(CM), CD44(high)CD62L(high)), whereas ST-OVA induced mainly effector-phenotype (T(EM), CD44(high)CD62L(low)) cells. Both vectors induced functional OVA-specific CD8(+) T cells that expressed IFN-gamma and killed targets specifically in vivo. However, only LM-OVA-vaccinated mice were protected against B16-OVA tumors. This correlated to the ability of CD8(+) T cells generated against LM-OVA, but not against ST-OVA, to produce interleukin 2 and exhibit profound homeostatic and antigen-induced proliferation in vivo. Furthermore, adoptive transfer of memory CD8(+) T cells generated against LM-OVA (but not against ST-OVA) into recipient mice resulted in their trafficking to tumor-draining lymph nodes conferring protection. Although cytotoxicity and IFN-gamma production are considered to be the principal functions of memory CD8(+) T cells, the vaccine delivery strategy may also influence memory CD8(+) T-cell quality, and ability to proliferate and traffic to tumors. Thus, for efficacy, cancer vaccines should be selected for their ability to induce self-renewing memory CD8(+) T cells (CD44(high)IL-7Ralpha(high)CD62L(high)) besides their effector functions.

Topics: Adoptive Transfer; Animals; Autoantigens; CD8-Positive T-Lymphocytes; Crosses, Genetic; Female; Genetic Vectors; Humans; Immunologic Memory; Immunophenotyping; Immunotherapy; Listeria monocytogenes; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Neoplasms; Ovalbumin; Receptors, Antigen, T-Cell; Salmonella typhimurium; T-Cell Antigen Receptor Specificity

Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, Neoplasm; Cell Line, Tumor; Cross-Priming; Dendritic Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Peptide Fragments; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor Receptor Superfamily, Member 9

It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8(+) T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy.

Topics: Animals; Antigens; Cancer Vaccines; Dendritic Cells; Genetic Therapy; Genetic Vectors; HIV Integrase; HIV-1; Immunotherapy, Active; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Transduction, Genetic; Transgenes; Viral Envelope Proteins; Virus Integration

TNF receptor-2 (TNFR2) plays a critical role in promoting the activation and survival of naive T cells during the primary response. Interestingly, anti-CD3 plus IL-2 activated TNFR2(-/-) CD8 T cells are highly resistant to activation-induced cell death (AICD), which correlates with high expression levels of prosurvival molecules such as Bcl-2, survivin, and CD127 (IL-7Ralpha). We determined whether the resistance of activated TNFR2(-/-) CD8 T cells to AICD contributes to more effective protection against tumor cell growth. We found that during a primary tumor challenge, despite initial inferiority in controlling tumor cell growth, TNFR2(-/-) mice were able to more effectively control tumor burden over time compared with wild-type (WT) mice. Furthermore, vaccination of TNFR2(-/-) mice with recombinant Listeria monocytogenes that express OVA confers better protection against the growth of OVA-expressing E.G7 tumor cells relative to similarly vaccinated WT mice. The enhanced protection against tumor cell growth was not due to more effective activation of OVA-specific memory CD8 T cells in vaccinated TNFR2(-/-) mice. In vitro studies indicate that optimally activated OVA-specific TNFR2(-/-) CD8 T cells proliferated to the same extent and possess similar cytotoxicity against E.G7 tumor cells as WT CD8 T cells. However, relative to WT cells, activated OVA-specific TNFR2(-/-) CD8 T cells were highly resistant to AICD. Thus, the enhanced protection against E.G7 in TNFR2(-/-) mice is likely due to the recruitment and activation of OVA-specific memory TNFR2(-/-) CD8 T cells and their prolonged survival at the tumor site.

Topics: Animals; Apoptosis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Immunologic Memory; Interleukin-2; Lymphocyte Activation; Mice; Mice, Knockout; Mice, Mutant Strains; Mice, Transgenic; Neoplasms; Ovalbumin; Peptide Fragments; Receptors, Tumor Necrosis Factor, Type II

Exosomes have been proposed as candidates for therapeutic immunization. The present study demonstrates that incorporation of the G protein of vesicular stomatitis virus (VSV-G) into exosome-like vesicles (ELVs) enhances their uptake and induces the maturation of dendritic cells. Targeting of VSV-G and ovalbumin as a model antigen to the same ELVs increased the cross-presentation of ovalbumin via an endosomal acidification mechanism. Immunization of mice with VSV-G and ovalbumin containing ELVs led to an increased IgG2a antibody response, expansion of antigen-specific CD8 T cells, strong in vivo CTL responses, and protection from challenge with ovalbumin expressing tumor cells. Thus, incorporation of VSV-G and targeting of antigens to ELVs are attractive strategies to improve exosomal vaccines.

Topics: Animals; Antibodies; Antigen Presentation; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Dendritic Cells; Endosomes; Humans; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Protein Transport; Secretory Vesicles; Vaccines; Viral Envelope Proteins

Cancer immunosurveillance failure is largely attributed to insufficient activation signals and dominant inhibitory stimuli for tumor Ag (TAg)-specific CD8 T cells. CD4 T cells have been shown to license dendritic cells (DC), thereby having the potential for converting CD8 T cell responses from tolerance to activation. To understand the potential cooperation of TAg-specific CD4 and CD8 T cells, we have characterized the responses of naive TCR transgenic CD8 and CD4 T cells to poorly immunogenic murine tumors. We found that whereas CD8 T cells sensed TAg and were tolerized, the CD4 T cells remained ignorant throughout tumor growth and did not provide help. This disparity in responses was due to normal TAg MHC class I cross-presentation by immature CD8alpha+ DC in the draining lymph node, but poor MHC class II presentation on all DC subsets due to selective inhibition by the tumor microenvironment. Thus, these results reveal a novel mechanism of cancer immunosubversion, in which inhibition of MHC-II TAg presentation on DC prevents CD4 T cell priming, thereby blocking any potential for licensing CD8alpha+ DC and helping tolerized CD8 T cells.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytoplasm; Dendritic Cells; Histocompatibility Antigens Class II; Lymph Nodes; Mice; Mice, Transgenic; Neoplasm Transplantation; Neoplasms; Ovalbumin

B cells are antibody (Ab)-secreting cells as well as potent antigen (Ag)-presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti-CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-gamma-producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1(+) lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses.

Topics: Animals; Antibodies; Antigens, CD19; B-Lymphocytes; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cell Proliferation; Cell Survival; Clonal Anergy; Drug Screening Assays, Antitumor; Epitopes; Humans; Immune Tolerance; Immunoglobulin Isotypes; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred C57BL; Mucin-1; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes; Vaccination

T helper type 1 (Th1) cell-mediated immune responses play various roles in cellular immunity, including inducing cytotoxic T lymphocytes (CTLs) and they have been shown to be crucial in cancer immunotherapy. Previously, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) stimulated antigen-presenting cells to secrete IL-12, leading to enhanced Th1 cell responses. In this study, as a way of enhancing antigen-specific Th1 responses, mouse fibroblasts (H-2(b)) were genetically modified to express an AIMP1 and a costimulatory B7.1 (Fb/AIMP1/B7.1). Fb/AIMP1/B7.1 cells were then loaded with an ovalbumin epitope as a model antigen (Fb/AIMP1/B7.1/OVA), and tested to determine if they induced OVA-specific CTLs in C57BL/6 mice (H-2(b)). Immunization with Fb/AIMP1/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EG7 tumor cells, but not against other H-2(b) tumor cells. The levels of the cytotoxic response in the immunized mice with Fb/AIMP1/B7.1/OVA cells were significantly higher than the responses in mice immunized with other cell constructs. CD8(+) T cells were a major cell-type of OVA-specific antitumor immunity induced by Fb/AIMP1/B7.1/OVA cells. Furthermore, treatment with Fb/AIMP1/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. These results indicate that AIMP1-secreting, epitope-loaded fibroblasts efficiently induce antigen-specific CTL responses in mice.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; B7-1 Antigen; Cytokines; Epitopes; Female; Fibroblasts; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasms; Ovalbumin; RNA-Binding Proteins; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 x 10(4) adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Genetic Vectors; Immunocompetence; Luciferases, Firefly; Luminescent Measurements; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Protein Engineering; Retroviridae; T-Lymphocytes; Transduction, Genetic

Heat shock proteins (HSPs) induce cross-presentation of antigens by dendritic cells (DC) as well as DC maturation. These properties make HSP antigen complexes good candidates to prime CD8 T cell responses against tumor-associated antigens. In this study, we analyzed four different members of the HSP70 family fused to a fragment of ovalbumin (OVA) as a model tumor antigen. E. coli-derived recombinant HSP70-OVA fusion proteins efficiently primed antigen-specific cytotoxic T cells in short-term in vivo immunization assays. Because of concerns that the adjuvant effect of HSPs may be due to endotoxin contamination, we studied this issue in detail. Induction of OVA-specific cytotoxicity was significantly decreased in mice deficient for the LPS receptor, TLR4. After careful removal of endotoxins, immunization with HSP70-OVA failed to prime cytotoxic T cell responses. However, we obtained strong in vivo kill responses when endotoxin-depleted HSP70-OVA was used in combination with the TLR9 ligand CpG oligodeoxynucleotide 1668. Importantly, prophylactic and therapeutic treatment with endotoxin-depleted HSP70-OVA together with CpG significantly delayed the outgrowth of OVA-expressing B16 melanoma cells. However, we were unable to detect significant differences in the magnitudes of immune responses against endotoxin-depleted recombinant OVA vs. endotoxin-depleted HSP70-OVA fusion protein. Thus, immunization with recombinant HSP70-antigen fusion protein does not provide an advantage over recombinant antigen alone when combined with a suitable adjuvant. Altogether, our data suggest that the adjuvant effect of the HSP70 part of the fusion protein is completely lost after endotoxin removal.

Topics: Adjuvants, Immunologic; Animals; Antigens; CD8-Positive T-Lymphocytes; Cross-Priming; Cytotoxicity, Immunologic; Dendritic Cells; Endotoxins; HSP70 Heat-Shock Proteins; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Oligodeoxyribonucleotides; Ovalbumin; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; T-Lymphocytes, Cytotoxic

Steady-state dendritic cells (DCs) maintain peripheral T cell tolerance, whereas mature DCs generate immunity. CD70 is a costimulatory ligand acquired upon DC maturation. To determine its impact on T cell fate, we have generated mice that constitutively express CD70 in conventional DCs (cDCs). In these mice, naive CD4+ and CD8+ T cells spontaneously convert into effector cells. Administration of peptide without adjuvant, which is ordinarily tolerogenic, elicited tumor-eradicating CD8+ T cell responses and robust CD4+ T cell-independent memory. CD70 was also constitutively expressed in cDCs that inducibly present viral epitopes. In this case, tolerance induction was prevented as well. The antigen-presenting DCs generated protective immunity to virus infection and broke a pre-existing state of CD8+ T cell tolerance. Thus, the sole expression of CD70 by otherwise immature cDCs sufficed to convert CD8+ T cell tolerance into immunity, defining the importance of CD27-CD70 interactions at the interface between T cell and DC.

Topics: Animals; CD27 Ligand; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Dendritic Cells; Immune Tolerance; Immunologic Memory; Melanoma; Mice; Mice, Knockout; Mice, Transgenic; Neoplasms; Ovalbumin; Peptide Fragments; Tumor Necrosis Factor Receptor Superfamily, Member 7

We compared murine T-cell responses to synthetic lipopeptide vaccines in which the TLR2 ligand Pam(2)Cys was attached to co-linear CD4+ and CD8+ T-cell epitopes of ovalbumin (OVA) in a linear or branched configuration. Mice received OVA-specific transgenic CD8+ and CD4+ T-cells followed by one injection of vaccine. Although the branched lipopeptide was more potent in activating OVA-specific CD4+ and CD8+ T-cells in the primary response, both vaccines induced cytolytic T lymphocytes (CTL) that expressed perforin, granzyme A-C, and IFN-gamma mRNAs and conferred long-term protection of most mice against challenge with OVA-expressing tumor cells. OVA epitope display was reduced in tumors that developed in some mice, suggesting CD8+ T-cell dependent selection.

Topics: Animals; Cancer Vaccines; Carrier Proteins; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; Gene Expression; Granzymes; Immunologic Memory; Interferon-gamma; Lipoproteins; Lymphocyte Activation; Mice; Neoplasms; Ovalbumin; Perforin; Survival Analysis; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Vaccines, Subunit

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4(+) and CD8(+) T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Cell Proliferation; Columbidae; Cytochromes c; Dendritic Cells; Disease Models, Animal; DNA, Neoplasm; Epitopes; Female; Flow Cytometry; Immunohistochemistry; Interferon-gamma; Lymphocytes; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptide Fragments; Spleen

DNA formulations provide the basis for safe and cost efficient vaccines. However, naked plasmid DNA is only poorly immunogenic and new effective delivery strategies are needed to enhance the potency of DNA vaccines. In this study, we present a novel approach for the delivery of DNA vaccines using inert poly-L-lysine (PLL) coated polystyrene particles, which greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. Our data show that PLL-coated polystyrene nanoparticles of 0.05 microm but not 0.02 microm or 1.0 microm in diameter are highly effective for the delivery of DNA vaccines.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemistry, Pharmaceutical; Dendritic Cells; Drug Carriers; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Female; Immunity, Cellular; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Nanoparticles; Neoplasm Transplantation; Neoplasms; Ovalbumin; Particle Size; Plasmids; Polylysine; Polystyrenes; Vaccines, DNA

Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.

Topics: Amino Acid Motifs; Animals; Bone Marrow; Cell Differentiation; Cells, Cultured; Dendritic Cells; Female; Fibronectins; Humans; Interleukin-12; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Ovalbumin; Peptide Fragments; Protein Binding; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

One of the factors influencing the efficacy of tumour cell vaccines is the site of immunization. We have shown previously that gene-modified vaccines delivered directly inside the spleen induced antigen cross-presentation by splenic antigen-presenting cells (not B cells). Here, we examined the interaction between splenic CD11c(+) cells and antigen-specific CD4(+) T cells. We used tumour cells expressing ovalbumin (OVA), a situation where CD4(+) T-cell help is required for the generation of a cytotoxic T lymphocyte response. Using in vivo bioluminescence imaging of luciferase-expressing EL4-OVA cells, we could demonstrate that tumour cells were located exclusively inside the spleen following intrasplenic injection. We showed that after intrasplenic immunization with T/SA-OVA cells, splenic class I(+) class II(+) CD11c(+) cells engulfed and presented in vivo the OVA class I-restricted peptide SIINFEKL. However, in vivo previously adoptively transferred 5,6-carboxy-succinimidyl-fluorescein-ester-labelled transgenic CD4(+)KJI-26(+) cells specific for the class II OVA(323-339) peptide underwent abortive proliferation in the spleen. These CD4(+)KJI-26(+) cells were only transiently activated and produced IL-10 and IL-4 and not IFN-gamma. It appears that splenic CD11c(+) cells can downregulate splenic specific CD4(+) T-cell response thereby leading to a decrease in antitumour systemic immunity.

Topics: Adoptive Transfer; Animals; Cancer Vaccines; CD11c Antigen; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cross-Priming; Dendritic Cells; Flow Cytometry; Genetic Therapy; Immunization; Interleukin-10; Interleukin-4; Luminescent Measurements; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Spleen

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.

Topics: Animals; B7-1 Antigen; Cancer Vaccines; CD4 Antigens; CD8 Antigens; Cell Line, Tumor; Cell Proliferation; Dendritic Cells; Histocompatibility Antigens Class I; Immunologic Memory; Immunosuppression Therapy; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Mice; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

CD8+ cytotoxic T (Tc) cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8+ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8+ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin (OVA)-pulsed dendritic cell (DCOVA)-activated CD8+ type 1 Tc (Tc1) cells secreting IFN-gamma, and CD8+ type 2 Tc (Tc2) cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor (TCR) transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tc1 and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8+ Tc2, Tc1 cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8+ Tc1 and Tc2 cells skewed the phenotype of CD4+ T cells toward Th1 and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8+ Tc1 and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8+ T cells for adoptive T cell therapy of cancer.

Topics: Adoptive Transfer; Animals; CD8-Positive T-Lymphocytes; Cell Survival; Cytotoxicity, Immunologic; Immunotherapy; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Ovalbumin; Phenotype; T-Lymphocyte Subsets; Th1 Cells; Th2 Cells

Lentiviral vectors encoding antigens are promising vaccine candidates because they transduce dendritic cells (DC) in vivo and prime CTL responses. Here we examine their stimulation of antigen-specific CD4(+) T cells, critical for protective immunity against tumors or infectious disease. We constructed lentiviral vectors (lentivectors) expressing ovalbumin, which was secreted (OVA), cytoplasmic (OVAcyt), or fused to either invariant chain (Ii-OVA) or transferrin receptor (TfR-OVA) sequences, targeting the MHC class II presentation pathway. Murine DC infected with the various lentivectors could stimulate OT-I (CD8(+), OVA TCR transgenic) T cells and all except OVAcyt could also stimulate OT-II (CD4(+), OVA TCR transgenic) T cells in vitro. Direct injection of the OVA-, Ii-OVA-, or TfR-OVA-expressing vectors into mice resulted in a CD4(+) T cell response, as shown by expansion of adoptively transferred OT-II T cells and upregulation of CD44 on these cells. The Ii-OVA vector was the most potent inducer of IFN-gamma-secreting CD4(+) and CD8(+) T cells and was the only vector to protect mice completely from challenge with OVA-expressing tumor cells. Therefore directly injected lentivectors can stimulate CD4(+) T cells; both CD4(+) and CD8(+) responses can be enhanced by targeting the antigen to the MHC class II pathway.

Topics: Animals; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cell Line, Tumor; Cytokines; Epitopes, T-Lymphocyte; Genetic Vectors; Histocompatibility Antigens Class I; HIV-1; Humans; Immunotherapy, Adoptive; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; T-Lymphocytes, Helper-Inducer; Transgenes

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Dendritic Cells; Female; Genetic Therapy; Genetic Vectors; Immunologic Memory; Immunotherapy; Interferon-gamma; Lentivirus; Lymph Nodes; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Ovalbumin; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Cytotoxic; Transduction, Genetic

Protein transduction domains (PTDs) have been used increasingly to deliver reagents to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DCs) stimulated Ag-specific CD8+ and CD4+ T cells. Although the cytotoxic T lymphocytes (CTL) activity generated was sufficient to prevent engraftment of mice with Ag-expressing tumors, treatment of tumor-bearing mice with TAT-PTD Ag-transduced DCs resulted in tumor regression in some animals. Recently, several other PTDs were reported to promote higher transduction efficiencies than TAT-PTD. To evaluate the role of individual PTDs in induction of immune responses in tumor vaccination studies, we engineered recombinant fusion Ovalbumin (OVA) that contained three differrent PTDs, including the most efficacious known PTD (polyarginine (R9)-PTD). Our results demonstrated that R9-PTD-containing OVA transduced DCs most efficiently, and that transduction efficacy was closely correlated with the extent of Ag-specific CD4+ and CD8+ T-cell activation in vitro and in vivo. Repeated vaccination with R9-PTD-OVA-transduced DC in (OVA-expressing) tumor-bearing mice induced enhanced antitumor immunity, and elicited complete rejection of tumors when DC was co-injected with adjuvants. This vaccination strategy may be clinically applicable, and offers theoretical and practical advantages to those that are in current use.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Antineoplastic Agents; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Female; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms; Ovalbumin; Peptides; Picibanil; T-Lymphocytes, Cytotoxic

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DC(OVA)), and act as Th-APCs in stimulation of CD8+ CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DC(OVA) stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DC(OVA) and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).

Topics: Animals; Antigen-Presenting Cells; B7-1 Antigen; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line; Cell Proliferation; Dendritic Cells; Female; Histocompatibility Antigens Class I; Immunologic Memory; Intercellular Adhesion Molecule-1; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptides; Protein Binding

Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen-presenting cells (APCs) are critical in the induction of this unresponsive state. Here we show that in vitro treatment of APCs with the tyrosine kinase inhibitor, imatinib mesylate (STI-571), enhances the activation of naive antigen-specific T cells and restores the responsiveness of tolerant T cells from tumor-bearing hosts. Furthermore, in vivo treatment with STI-571 not only prevented the induction of tolerance in tumor-specific CD4(+) T cells, preserving their responsiveness to a subsequent immunization, but also resulted in enhanced vaccine efficacy. These findings demonstrate that tolerance to tumor antigens is not an insurmountable obstacle and points to modulation of APC function as a promising strategy in the immunotherapy of cancer.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Antineoplastic Agents; Benzamides; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Dendritic Cells; Imatinib Mesylate; Immune Tolerance; Lymphoma; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Ovalbumin; Peptide Fragments; Piperazines; Pyrimidines

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antigens; Antigens, Surface; Blastocyst; Carcinogens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Movement; Cell Physiological Phenomena; Cytoplasmic Granules; Dendritic Cells; Dose-Response Relationship, Drug; Electroporation; Embryo, Mammalian; Flow Cytometry; Genetic Vectors; Immunohistochemistry; Islets of Langerhans; Kinetics; Langerhans Cells; Lectins; Lectins, C-Type; Lymph Nodes; Mannose-Binding Lectins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron; Models, Genetic; Mutagenesis; Mutation; Neoplasms; Ovalbumin; Phenotype; Stem Cells

It is established that aging leads to declines in immune function. However, the mechanisms underlying remain poorly understood.. In this study, we compared the tumoriginecity of MO4 (ovalbumin-transfected) tumor cells, the efficacy of dendritic cell (DC) vaccination and cytotoxic T lymphocytes (CTLs), and the number of NK1.1+CD3+ NKT cells between aged mice and young mice using T cell proliferation and cytotoxicity assays and flow cytometry.. We showed that, in comparison to young mice, aged mice are 10-fold more susceptible to tumorigenicity of MO4 tumor cells. Aged mice immunized with bone marrow-derived DCs pulsed with ovalbumin (DCOVA) survived significantly shorter after challenge with MO4 tumor cells as compared to equally treated young mice. Furthermore, CTLs from aged mice immunized with DCOVA displayed 4-fold weaker cytotoxicity as compared to CTLs from immunized young mice. Interestingly, the number of NK1.1+CD3+ NKT cell significantly increase with aging (p < 0.05). Of particular importance, NK1.1+CD3+ NKT cells isolated from aged mice suppress the proliferation of T cells.. Based on these data, we conclude that NK1.1+CD3+ NKT cells from aged mice mediate immunosuppression, and further suggest that increased number of NK1.1+CD3+ NKT cells in aged mice might, among others, diminish their immune function by mediation of immunosuppression.

Topics: Aging; Animals; CD3 Complex; Cell Proliferation; Dendritic Cells; Disease Susceptibility; Flow Cytometry; Immunosuppression Therapy; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transfection; Vaccination

Dendritic cells (DC) genetically modified to present tumor-associated antigen are a promising means for anti-cancer immunotherapy. By introducing expression vectors into ES cells and subsequently inducing differentiation to DC (ES-DC), we can generate transfectant DC expressing the transgenes. In the future clinical application of this technology, the unavailability of human ES cells genetically identical to the patients will be a problem. However, in most cases, semi-allogeneic ES cells sharing some of HLA alleles with recipients are expected to be available. In the present study, we observed that model tumor antigen (OVA)-expressing mouse ES-DC transferred into semi-allogeneic mice potently primed OVA-reactive CTL and elicited a significant protection against challenge with OVA-expressing tumor. Genetic modification of ES-DC to overexpress SPI-6, the specific inhibitor of granzyme B, further enhanced their capacity to prime antigen-specific CTL in semi-allogeneic recipient mice. These results suggest the potential of ES-DC as a novel means for anti-cancer immunotherapy.

Topics: Animals; Antigens; Cell Differentiation; Cell Line; Dendritic Cells; Immunotherapy; Membrane Proteins; Mice; Neoplasms; Ovalbumin; Serine Endopeptidases; Serpins; Stem Cell Transplantation; Stem Cells; Survival Rate; T-Lymphocytes, Cytotoxic; Xenograft Model Antitumor Assays

NKG2D is an important activating/co-stimulatory receptor harnessed by NK and T cells in immune surveillance. In contrast to NK cells, T cells fail to express the activation-signaling molecule DAP12 even when activated and, therefore, ligation of NKG2D alone is insufficient to induce T cell cytolytic function. To test whether we could endow T cells with NK cell-like effector function, we have engineered DAP12 into T cells by retroviral transduction (T-DAP12). T-DAP12 cells were demonstrated to specifically secrete interferon-gamma following receptor ligation and to mediate potent and specific lysis of the NKG2D ligand (NKG2D-L) (Rae-1beta) expressing MHC class I-deficient and class I-sufficient tumors. To circumvent the inability of T-DAP12 cells to proliferate following NKG2D ligation by Rae-1beta expressing tumors, DAP12 was engineered into OT-1 cells with an endogenous T cell receptor specific for chicken ovalbumin peptide (amino acids 257-264). Importantly, following a period of proliferation through endogenous T cell receptor ligation, OT-1-DAP12 cells retained specificity against NKG2D-L expressing major histocompatibility complex class I-deficient tumor. In adoptive transfer experiments, T-DAP12 cells enhanced the survival of NK cell-depleted RAG-1-deficient mice inoculated with RMA-S-Rae-1beta but not parental RMA-S tumors. Overall, this study demonstrated the significant potential of suppressing tumors and other cellular targets expressing NKG2D-L by endowing T cells with innate NK cell-like function.

Topics: Adaptor Proteins, Signal Transducing; Animals; CD8-Positive T-Lymphocytes; Cell Line; Cell Line, Tumor; Cell Proliferation; Chickens; Cloning, Molecular; Cytokines; Epitopes; Flow Cytometry; Genetic Engineering; Immunotherapy; Interferon-gamma; Killer Cells, Natural; Ligands; Major Histocompatibility Complex; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; NK Cell Lectin-Like Receptor Subfamily K; Ovalbumin; Peptides; Polymerase Chain Reaction; Receptors, Immunologic; Receptors, Natural Killer Cell; Retroviridae; T-Lymphocytes; Time Factors

The close cooperation of both innate and acquired immunity is essential for the induction of truly effective antitumor immunity. We tested a strategy to enhance the cross-talk between NKT cells and conventional antigen-specific T cells with the use of alpha GalCer-loaded dendritic cells genetically engineered to express antigen plus chemokine, attracting both conventional T cells and NKT cells. DC genetically engineered to express a model antigen, OVA, along with SLC/CCL21 or monokine induced by IFN-gamma/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES-DC were loaded with alpha-GalCer and transferred to mice bearing MO4, an OVA-expressing melanoma, and their capacity to evoke antitumor immunity was evaluated. In vivo transfer of either OVA-expressing ES-DC, stimulating OVA-reactive T cells, or alpha-GalCer-loaded non-transfectant ES-DC, stimulating NKT cells, elicited a significant but limited degree of protection against the i.p. disseminated MO4. A more potent antitumor effect was observed when alpha-GalCer was loaded to ES-DC expressing OVA before in vivo transfer, and the effect was abrogated by the administration of anti-CD8, anti-NK1.1 or anti-asialo GM1 antibody. alpha-GalCer-loaded double transfectant ES-DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, alpha-GalCer-loaded ES-DC expressing tumor-associated antigen along with SLC can stimulate multiple subsets of effector cells to induce a potent therapeutic effect against peritoneally disseminated tumor cells. The present study suggests a novel way to use alpha-GalCer in immunotherapy for peritoneally

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Cell Communication; Cell Line, Tumor; Cell Survival; Chemokine CCL21; Chemokines, CC; Dendritic Cells; Galactosylceramides; Genetic Engineering; Granulocyte-Macrophage Colony-Stimulating Factor; Killer Cells, Natural; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Neoplasms; Ovalbumin; Recombinant Proteins; T-Lymphocytes

The lack of efficient T-cell infiltration of tumors is a major obstacle to successful adoptive T-cell therapy. We have previously demonstrated that adenovirus (AdV)-mediated transgene lymphotactin (Lptn) or IP-10 expression in tumors can significantly enhance T-cell tumor infiltration. In this study, active OVA-specific CD8+ T cells were prepared by coculturing naive OVA-specific CD8+ T cells from transgenic OT I mice with OVA-I peptide-pulsed dendritic cells in vitro. These XCR-1- and CXCR3-expressing T cells predominantly secreted IFN-gamma and displayed significant killing activity (84% at effector:target cell ratio of 1.5) against OVA-expressing EG7 tumor cells through perforin-mediated pathway. Our data also showed that chemokine Lptn and IP-10 not only can chemoattract, but also stimulate proliferation of CD8+ T cells in vitro, and that a mixture of Lptn and IP-10 can more efficiently chemoattract CD8+ T cells than either one of them. Furthermore, we demonstrated that the transferred CD8+ T cells detected in group of tumors treated with both AdVLptn and AdVIP-10 (group a) are around 4 and 2 times more than that in groups of tumors treated with control AdVpLpA (group b) and either AdVIP-10 (group c) or AdVLptn (group d), respectively. Around 87.5% of mice in group a were tumor-free compared to the aggressive tumor growth in all 8 mice of group b and 25% or 37.5% cured mice seen in groups c and d (p<0.05). Thus, our results indicate that enhancement of adoptive T-cell therapy can be obtained by double tranmsgene Lptn and IP-10 expression, which facilitates CD8+ T-cell tumor localization through proliferation and chemoattraction of the transferred CD8+ T cells by in situ chemokine transgene expressions in the tumors. Collectively, our data provide solid evidence of a potent synergy between adoptive T-cell therapy and adenovirus-mediated Lptn and IP-10 gene transfer into tumor tissues, which culminated in the T-cell tumor localization and eradication of well-established tumor masses.

Topics: Adenoviridae; Animals; CD8-Positive T-Lymphocytes; Cell Division; Chemokine CXCL10; Chemokines, C; Chemokines, CXC; Chemotaxis, Leukocyte; Cytotoxicity, Immunologic; Dendritic Cells; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Immunotherapy, Adoptive; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; Sialoglycoproteins; Transgenes; Tumor Cells, Cultured

The identification of tumor antigens has spurred the development of efficient adjuvants and novel delivery systems for cancer immunotherapy. To this end, a peptide-based vaccine consisting of the Antennapedia transduction sequence (ANTP) attached to an antigenic peptide was designed to enhance per-cutaneous delivery into cells of the epidermis and dermis. Here we show that the topical application of OVA(257-264) linked to ANTP in mice onto tape-stripped skin resulted in enhanced delivery of the antigen through the skin whereas OVA(257-264) alone remained distributed uniformly on the skin surface. This delivery correlated with an increase in the CTL response against OVA. When mixed with CpG oligodinucleotides (ODN), the recombinant antigen protected mice from tumor challenge. These data provide the first indication that in vivo use of a translocation sequence can enhance delivery of therapeutic peptides and increase anti-tumor immunity through a simple and safe mechanism involving enhanced penetration of the skin barrier.

Topics: Adjuvants, Immunologic; Administration, Cutaneous; Amino Acid Sequence; Animals; Antigens, Neoplasm; Cancer Vaccines; Epitopes; Flow Cytometry; Immunotherapy; Interferon-gamma; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasms; Oligonucleotides; Ovalbumin; Skin Absorption; T-Lymphocytes, Cytotoxic; Th1 Cells; Transduction, Genetic

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. In this study mouse fibroblasts (H-2(b)) were genetically modified to express a costimulatory B7.1 and a mature interleukin (IL)-18, and then loaded with an ovalbumin (OVA) epitope (SIINFEKL, H-2K(b) restricted) as a model antigen, and tested for the induction of OVA-specific CTLs in C57BL/6 mice (H-2(b)). The genetically modified fibroblasts lacking either IL-18 or B7.1 were also constructed. Immunization with the IL-18/B7.1-transfected fibroblasts induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells, but not against other H-2(b) tumor cells such as EL4, C1498, and B16F1 cells. The magnitude of the cytotoxic response in mice with the IL-18/B7.1-transfected fibroblasts was significantly higher than the response in mice immunized with any other cell constructs. CD8(+) T cells with OVA-specific cytotoxic activities were predominant in mice immunized with the IL-18/B7.1-transfected fibroblasts. Furthermore, treatment with the IL-18/B7.1-transfected fibroblasts significantly prolonged the survival period of EG7 tumor-bearing mice. Anti-tumor CTL immunity by the IL-18/B7.1-transfected fibroblasts could be induced without the help of host antigen-presenting cells (APCs) and NK1.1(+) cells, whereas partially decreased by the depletion of CD4(+) T cells at the inductive stage. These results support the ability of IL-18/B7.1 gene transfer to enhance the antigen-presenting capacity of fibroblasts for inducing antigen-specific CTL response.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Epitopes; Fibroblasts; Interleukin-18; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Peptide Fragments; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

Archaeal ether glycerolipid vesicles (archaeosomes) efficiently deliver exogenous antigen for induction of humoral and cell-mediated immunity. Because induction of CD8 cytotoxic T cells is critical for protective vaccination against tumors, we compared the ability of various archaeosome lipid compositions to evoke a strong CD8 CTL response to entrapped antigen. Subcutaneous immunization of mice with ovalbumin (OVA) entrapped in all archaeosome lipid compositions evoked a primary (day 10) splenic CTL response indicating processing for MHC class I presentation. Interestingly, several polar lipid compositions from halophilic archaea were very potent to adjuvant this early CTL response. Despite this, the lytic units reduced substantially by weeks 6-7. More importantly, at >50 weeks, only Methanobrevibacter smithii and Thermoplasma acidophilum both rich in bipolar membrane-spanning caldarchaeols, demonstrated recall memory CTLs. Immunization of mice with OVA entrapped in M. smithii, Halobacterium salinarum or T. acidophilum vesicles provided prophylactic protection against challenge with OVA-expressing solid tumors at 6 weeks. Even a dose of 3 microg OVA in archaeosomes significantly delayed tumor growth. Tumor protection was also noted in a therapeutic design wherein OVA-archaeosomes were injected concurrent with the tumor challenge. Interestingly, antigen-free T. acidophilum but not antigen-free H. salinarum archaeosomes provided innate therapeutic protection. Vaccination with a CTL peptide epitope from the melanoma differentiation antigen, tyrosinase-related protein 2, in archaeosomes induced a protective CD8 response against B16OVA metastasis, indicating potential for targeting self, tumor antigens. Thus, lipid structural properties of archaea may differentially modulate primary, long-term and/or innate immunity, impacting adjuvant choice for vaccine design.

Topics: Animals; Archaea; Cancer Vaccines; Cell Line, Tumor; Drug Carriers; Female; Immunity, Innate; Lipids; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Immunization with cytotoxic T cell epitope SPSYVYHQF (AH1), derived from MuLV gp70 envelope protein expressed by CT26 tumor cells, does not protect BALB/c mice against challenge with CT26 tumor cells. By contrast, immunization with AH1 plus T helper peptides OVA(323-337) or SWM(106-118) eliciting Th1 and Th0 profiles, protected 83% and 33% of mice, respectively. Interestingly, immunization with AH1 plus both helper peptides reverted the efficacy to 33%. We identified the endogenous T helper peptide p(320-333) from gp70 which elicits a Th1 profile and is naturally processed. As for OVA(323-337), immunization with p(320-333) alone did not protect against tumor challenge. However, p(320-333) plus AH1 protected 89% of mice at day 10 after vaccination. Only 20% of mice vaccinated with AH1 + OVA(323-337) or AH1 + p(320-333) were protected when challenged 80 days after immunization. Treatment with OVA(323-337) or with p(320-333) around established tumors delayed tumor growth. Our results show that tumor-related as well as tumor-unrelated but strong Th1 peptides may be useful for inducing CTL responses in tumor immunotherapy.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Epitopes, T-Lymphocyte; Female; Interferon-gamma; Interleukin-2; Interleukin-4; Lung Neoplasms; Mice; Mice, Inbred BALB C; Myoglobin; Neoplasms; Ovalbumin; Peptides; Retroviridae Proteins, Oncogenic; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Th1 Cells; Time Factors; Vaccination; Vaccines, Synthetic; Viral Envelope Proteins

BLK mouse fibroblasts (H-2b) were genetically engineered to express costimulatory B7.1 and interleukin-2 (BLK/IL2/B7.1). The BLK/IL2/B7.1 cells were then pulsed with an ovalbumin (OVA) epitope as a model antigen (Ag) (BLK/IL2/B7.1/OVA), and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2b). The genetically engineered fibroblasts lacking one or two of three factors (interleukin-2, B7.1, and OVA) were constructed and used as controls. Immunization with the BLK/IL2/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells, but not against other H-2b tumor cells, such as EL4, C1498 and B16F1 cells. The magnitude of the cytotoxic response in mice with the BLK/IL2/B7.1/OVA cells was significantly higher than the response in mice immunized with any other cell constructs. CD8+ T cells with OVA-specific cytotoxic activities were predominant in mice immunized with the BLK/IL2/B7.1/OVA cells. Furthermore, immunization with the BLK/IL2/B7.1/OVA cells significantly prolonged the survival of mice, compared with any other cell constructs, when the mice were challenged with EG7 tumor cells at 2 weeks postimmunization. Induction of antitumoral CTL immunity by the BLK/IL2/B7.1/OVA cells was independent of host Ag-presenting cells and of CD4+ T-cell and natural killer 1.1+ cell help. These results suggest that fibroblasts can be genetically modified to efficient Ag-presenting cells for the induction of an Ag-specific CTL response.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; B7-1 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Epitopes; Female; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Genetic Engineering; Genetic Therapy; Immunization; Immunotherapy; Interleukin-2; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

Molecular size is one of the key determinants of transvascular transport of therapeutic agents in tumors. However, there are no data in the literature on the molecular size dependence of microvascular permeability in tumors. Therefore, we measured microvascular permeability to various macromolecules in the human colon adenocarcinoma LS174T transplanted in dorsal skin chambers in severe combined immunodeficient mice. These molecules were fluorescently labeled and injected i.v. into mice. The microvascular permeability was calculated from the fluorescence intensity measured by the intravital fluorescence microscopy technique. The value of permeability varied approximately 2-fold in the range of molecular weight from 25,000 to 160,000. These data indicate that tumor vessels are less permselective than normal vessels, presumably due to large pores in the vessel wall. The transport of macromolecules appears to be limited by diffusion through these pores. The cutoff size of the pores was estimated by observations of transvascular transport of sterically stabilized liposomes of 100-600 nm in diameter. We found that tumor vessels in our model were permeable to liposomes of up to 400 nm in diameter, suggesting that the cutoff size of the pores is between 400 and 600 nm in diameter.

Topics: Animals; Capillary Permeability; Concanavalin A; Diffusion; Humans; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Liposomes; Mice; Mice, SCID; Molecular Weight; Neoplasms; Ovalbumin; Transplantation, Heterologous

The aim of this study was to investigate the compound 4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline (NOPYE) and some related compounds for skin sensitization in guinea pigs, as the first step in a search for more effective skin sensitizers for immunotherapy of cutaneous tumors. In guinea pigs, NOPYE and NOPYE-L-alanine produce far milder delayed hypersensitivity reactions than DNCB. Both NOPYE and DNCB fail to act as adjuvants for skin sensitization to tuberculin purified protein derivative (PPD) and ovalbumin (OV). This suggests an explanation for the lack of effectiveness of DNCB in immunotherapy of metastases: DNCB may be relatively ineffective as an adjuvant for production of specific antitumor immunity. Such adjuvant activity may be essential if the action of the immunotherapeutic reagent is not to be confined to its site of application but is to be effective at the site of distant metastases.

Topics: Adjuvants, Immunologic; Animals; Cross Reactions; Cyclohexanes; Dinitrochlorobenzene; Female; Guinea Pigs; Hypersensitivity, Delayed; Immunotherapy; Neoplasms; Ovalbumin; Phenylalanine; Pyrroles; Skin; Tuberculin

Topics: Alleles; Base Sequence; Cell Differentiation; DNA; Drosophila; Genes; Globins; Immunoglobulins; Neoplasms; Nucleic Acid Precursors; Ovalbumin; Phorbol Esters; RNA, Messenger; RNA, Transfer

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Autoantibodies; Epitopes; Fluorescent Antibody Technique; Humans; Neoplasms; Ovalbumin; Rats; Sepharose

The defined antigen substrate spheres (DASS) system was employed for analysing reactions between solid state antibody or antigen and soluble immune complexes. Sepharose beads covalently coupled with ovalbumin were used to represent tumour cells and beads coupled with antibody against ovalbumin were used to represent anti-tumour lymphocytes; the ovalbumin and corresponding antibody simulated tumour-derived antigen and antibody to tumour respectively. Binding of soluble complexes to the beads was measured by fluorimetry and/or radiometry of fluorescein or 125-I-labelled ovalbumin or antibody. Antigen-antibody complexes in antibody excess bound less effectively to the antibody beads than antigen alone, but complexes in slight or moderate antigen excess bound more effectively. Complexes in antibody excess were most effective in the complex before levelling off and then decreased in extreme antibody excess. The model demonstration of augmentation by antibody of antigen binding to solid state antibody might by analogy reflect a mechanism of inhibition of lymphocyte cytotoxicity. Complexes in a wide range of antibody excess should also be effective in blocking lymphocytotoxicity at the target cell level.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Antigen-Antibody Reactions; Antigens; Antigens, Neoplasm; Fluorescent Antibody Technique; Guinea Pigs; Lymphocytes; Models, Biological; Neoplasms; Ovalbumin; Sepharose

Topics: Animals; Biotin; Breast Neoplasms; Drug Synergism; Female; Humans; Laryngeal Neoplasms; Male; Neoplasm Metastasis; Neoplasms; Ovalbumin; Pharyngeal Neoplasms; Swine

Topics: ABO Blood-Group System; Antibodies; Chitin; Hemagglutination Inhibition Tests; Lipase; Neoplasms; Oligosaccharides; Ovalbumin; Periodic Acid; Polysaccharides, Bacterial; Triticum

Topics: Animals; Antineoplastic Agents; Chick Embryo; Cricetinae; Mammary Neoplasms, Animal; Mammary Neoplasms, Experimental; Melanoma; Mice; Neoplasms; Neoplasms, Experimental; Ovalbumin; Ovum; Research; Sarcoma; Sarcoma, Experimental

Topics: Animals; Anti-Bacterial Agents; Antibody Formation; Cattle; Chemotherapy, Cancer, Regional Perfusion; Cyclophosphamide; Diphtheria Toxoid; Fibrosarcoma; Geriatrics; Immunity; Immunosuppressive Agents; Mechlorethamine; Melanoma; Neoplasm Metastasis; Neoplasms; Ovalbumin; Pharmacology; Rabbits; Rats; Research; Sarcoma, Yoshida; Serum Albumin; Serum Albumin, Bovine; Toxicology