ovalbumin and Melanoma

We developed an orally delivered nanoemulsion that induces cancer immunization. It consists of tumor antigen-loaded nano-vesicles carrying the potent invariant natural killer T-cell (iNKT) activator α-galactosylceramide (α-GalCer), to trigger cancer immunity by effectively activating both innate and adaptive immunity. It was validated that adding bile salts to the system boosted intestinal lymphatic transport as well as the oral bioavailability of ovalbumin (OVA) via the chylomicron pathway. To increase intestinal permeability further and amplify the antitumor responses, an ionic complex of cationic lipid 1,2-dioleyl-3-trimethylammonium propane (DTP) with sodium deoxycholate (DA) (DDP) and α-GalCer were anchored onto the outer oil layer to form OVA-NE#3. As expected, OVA-NE#3 exhibited tremendously improved intestinal cell permeability as well as enhanced delivery to mesenteric lymph nodes (MLNs). Subsequent activation of dendritic cells and iNKTs, in MLNs were also observed. Tumor growth in OVA-expressing mice with melanoma was more strongly suppressed (by 71%) after oral administration of OVA-NE#3 than in untreated controls, confirming the strong immune response induced by the system. The serum levels of OVA-specific IgG1 and IgG2a were 3.52- and 6.14-fold higher than in controls. Treating OVA-NE#3 increased the numbers of tumor-infiltrating lymphocytes, including cytotoxic T-cell and M1-like macrophage. Antigen- and α-GalCer-associated enrichment of dendritic cells and iNKTs in tumor tissues also increased after OVA-NE#3 treatment. These observations indicate that our system induces both cellular and humoral immunity by targeting the oral lymphatic system. It may offer a promising oral anti-cancer vaccination strategy that involves the induction of systemic anti-cancer immunization.

Topics: Animals; Antigens, Neoplasm; Immunization; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin

Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8. CD4

Topics: Adenocarcinoma; Animals; B7-H1 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Epitopes, T-Lymphocyte; Glycoproteins; Humans; Immune Checkpoint Inhibitors; Immunotherapy; Melanoma; Mice; Ovalbumin; Programmed Cell Death 1 Receptor; Receptors, Antigen, T-Cell; Tumor Microenvironment; Vaccination

Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva). Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova. T cells from Ova-specific OT-II but not from C57BL/6 or SKH-1 wild type mice presents enhanced activation after Ova addition. OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon-gamma production, a known anti-melanoma agent. OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova-expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen-presenting cells when compared to their Ova-vaccinated counterparts. Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi-ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; Disease Models, Animal; Inflammation; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin; Oxidative Stress; Plasma Gases; Reactive Oxygen Species; T-Lymphocytes

Substantial progress has been made with cancer immunotherapeutic strategies in recent years, most of which mainly rely on enhancing the T cell response. However, sufficient tumor antigen information often cannot be presented to T cells, resulting in a failed effector T cell response. The innate immune system can effectively recognize tumor antigens and then initiate an adaptive immune response. Here, we developed a spontaneous multifunctional hydrogel (NOCC-CpG/OX-M, Ncom Gel) vaccine to amplify the innate immune response and harness innate immunity to launch and maintain a powerful adaptive immune response.

Topics: Adaptive Immunity; Animals; Cancer Vaccines; Cell Line, Tumor; Chitosan; Dendritic Cells; Female; Hydrogels; Immunity, Innate; Immunotherapy; Inflammation; Macrophages; Mannans; Melanoma; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Ovalbumin; Rheology; Schiff Bases; T-Lymphocytes; Tumor Microenvironment

Despite recent advances in cancer therapy using vaccines, the efficacy of vaccine regimens remains to be improved. Cutaneous transportation of biomolecules, particularly DNA vaccines, has potentially improved the therapeutic efficacy and has been found to be an appealing approach in cancer immunotherapy. Nevertheless, the effectiveness of transdermal vaccination is limited by the lack of efficacious immune stimulation. Here, to elicit strong immunogenicity in target cells, we propose an array of dissolving microneedle cocktails for pain-free implantation and triggered release of vaccines and adjuvants at cutaneous tissues. The microneedle cocktails comprising a bioresorbable polypeptide matrix with a nanopolyplex, which include cationic amphiphilic conjugates with ovalbumin-expressing plasmid OVA (pOVA) and immunostimulant-polyinosinic:polycytidylic acid (poly(I:C)), were prepared using a one-pot synthesis. The cationic nanopolyplex effectively transported pOVA and poly(I:C) into the intracellular compartments of dendritic cells and macrophages. Cutaneous implantation of microneedle cocktails on mice elicits a stronger antigen-specific antibody response than subcutaneous administration of the microneedle-free nanopolyplex. Compared with traditional vaccination, the dissolving microneedle cocktails enhanced the antibody recall memory after challenge; remarkably, the cocktail-based therapeutic vaccination also resulted in enhanced lung clearance of cancer cells. The dissolving microneedle cocktail therapy based on the triggered release of immunomodulators and adjuvants synergistically augmented the therapeutic effect in B16/OVA melanoma tumors.

Topics: Adjuvants, Immunologic; Animals; Cell Survival; Cells, Cultured; Female; HEK293 Cells; Humans; Immunotherapy; Injections, Intradermal; Injections, Intravenous; Melanoma; Mice; Mice, Inbred BALB C; Needles; Ovalbumin; Particle Size; Peptides; RAW 264.7 Cells; Surface Properties; Vaccination

Tumor vaccines to induce robust immunity for cancer treatment have attracted tremendous interests in cancer immunotherapy. In this work, a type of cancer vaccine is prepared by using nanoscale coordination polymer (NCP) formed between Mn

Topics: Animals; Cancer Vaccines; Dendritic Cells; Diaminopimelic Acid; Immunotherapy; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin; Polymers; Tumor Cells, Cultured

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the Braf

Topics: Animals; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Costimulatory and Inhibitory T-Cell Receptors; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Drug Screening Assays, Antitumor; Humans; Male; MAP Kinase Signaling System; Melanoma; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Ovalbumin; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Skin Neoplasms

Messenger RNA encoding tumor antigens has the potential to evoke effective antitumor immunity. This study reports on a nanoparticle platform, named mRNA Galsomes, that successfully co-delivers nucleoside-modified antigen-encoding mRNA and the glycolipid antigen and immunopotentiator α-galactosylceramide (α-GC) to antigen-presenting cells after intravenous administration. By co-formulating low doses of α-GC, mRNA Galsomes induce a pluripotent innate and adaptive tumor-specific immune response in mice, with invariant natural killer T cells (iNKT) as a driving force. In comparison, mRNA Galsomes exhibit advantages over the state-of-the-art cancer vaccines using unmodified ovalbumin (OVA)-encoding mRNA, as we observed up to seven times more tumor-infiltrating antigen-specific cytotoxic T cells, combined with a strong iNKT cell and NK cell activation. In addition, the presence of suppressive myeloid cells (myeloid-derived suppressor cells and tumor-associated macrophages) in the tumor microenvironment was significantly lowered. Owing to these antitumor effects, OVA mRNA Galsomes significantly reduced tumor growth in established E.G7-OVA lymphoma, with a complete tumor rejection in 40% of the animals. Moreover, therapeutic vaccination with mRNA Galsomes enhanced the responsiveness to treatment with a PD-L1 checkpoint inhibitor in B16-OVA melanoma, as evidenced by a synergistic reduction of tumor outgrowth and a significantly prolonged median survival. Taken together, these data show that intravenously administered mRNA Galsomes can provide controllable, multifaceted, and effective antitumor immunity, especially when combined with checkpoint inhibition.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Female; Galactosylceramides; Immunity, Cellular; Kaplan-Meier Estimate; Killer Cells, Natural; Liposomes; Lymphocyte Activation; Lymphoma; Melanoma; Melanoma, Experimental; Mice; Natural Killer T-Cells; Ovalbumin; RNA, Messenger; T-Lymphocytes

The goal of present study to assess the antigen specific immunopotentiation effect of mannose functionalized endosomolytic and conventional nanocomposite(s) based combination approach using C57BL/6 mice melanoma model. Endosomolytic and conventional nanocomposite(s) were prepared by double emulsification method. The optimized formulation was extensively characterized for average particle size, zeta potential and PDI of nanocomposite(s) which were measured in range of ≈200 nm, 0.111 ± 0.024, -23.4 ± 2.0 mV, respectively. pH-dependent morphological changes in the surface of MRPRPNs and PRPNs were analyzed by using surface electron microscopy at different time intervals. The cellular uptake assessment of developed formulations were followed by using RAW 264.7 macrophage cell lines. Results revealed that after immunizing B16F10 melanoma cells implanted C57BL/6 mice with combination [endosomolytic and conventional nanocomposite(s)] of nanocomposite(s), a significant increase in the interleukins level i.e. IL-2, IFN-ϒ, IL-12 and IL-6 and OVA Ag(s) specific antibody responses were recorded. Consequently, a strong immunological response was elicited with specific polarization contributing to humoral and activation of CD8

Topics: Animals; Antibodies; Antineoplastic Agents; Cell Line; Cell Line, Tumor; Disease Models, Animal; Endosomes; Female; Mannose; Melanoma; Mice; Mice, Inbred C57BL; Nanocomposites; Nanoparticles; Ovalbumin; Particle Size; RAW 264.7 Cells

Although powerful adjuvants hold promise of vaccines for cancer immunotherapy, cumbersome preparation processes, elusive mechanisms and failure to induce T cell responses have largely limited their clinical translation. Due to their ease of synthesis, good biocompatibility and designable bioactivity, peptide derivatives-based supramolecular nanomaterials have attracted increasing interest in improving the immunogenicity of cancer vaccines.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cross-Priming; Cytokines; Female; Humans; Immunotherapy; Melanoma; Mice; Mice, Inbred C57BL; NF-kappa B; Ovalbumin

Archaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

Topics: Adjuvants, Immunologic; Animals; Archaea; Disease Models, Animal; Drug Carriers; Female; Glycolipids; Immunity, Cellular; Intramolecular Oxidoreductases; Liposomes; Melanoma; Mice, Inbred C57BL; Ovalbumin; Treatment Outcome; Vaccines

Topics: Adenylate Cyclase Toxin; Adjuvants, Immunologic; Animals; Antigens, Neoplasm; Cancer Vaccines; Disease Models, Animal; DNA-Binding Proteins; Drug Carriers; Female; Immunologic Memory; Injections, Intradermal; Melanoma; Mice, Inbred C57BL; Neoplasm Proteins; Oncogene Proteins, Viral; Ovalbumin; Ovarian Neoplasms; Papillomavirus E7 Proteins; Papillomavirus Infections; Papillomavirus Vaccines; T-Lymphocytes; Treatment Outcome; Uterine Cervical Neoplasms; Vaccines, Synthetic

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This "photochemical internalisation" resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important.

Topics: Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Proliferation; Cross-Priming; Dendritic Cells; Histocompatibility Antigens Class I; Interferon-gamma; Light; Melanoma; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Photosensitivity Disorders; Photosensitizing Agents; Porphyrins; Skin Neoplasms; Spleen

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; Cytokines; Disease Models, Animal; Female; gp100 Melanoma Antigen; Granzymes; Immunoglobulin G; Ligands; Male; Mannose; MART-1 Antigen; Melanoma; Mice, Inbred C57BL; Mice, Transgenic; Nanoparticles; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Poly I-C; Polymers; Toll-Like Receptors; Tumor Burden

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

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b(+)Ly-6G/C(+) myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chemokines; Dendritic Cells; Gold; Hyperthermia, Induced; Immunotherapy, Adoptive; Inflammation Mediators; Lung Neoplasms; Lymph Nodes; Melanoma; Melanoma, Experimental; Mice; Myeloid Cells; Nanoshells; Ovalbumin; Phototherapy; Recurrence; T-Lymphocytes

Axitinib, a tyrosine kinase inhibitor of vascular endothelial growth factor receptors, has demonstrated modest efficacy when applied as a single agent in the setting of advanced-stage melanoma. On the basis of the reported ability of axitinib to 'normalize' the tumor vasculature, we hypothesize that combination therapy using axitinib plus specific peptide-based vaccination would promote superior activation and recruitment of protective T cells into the melanoma microenvironment, leading to enhanced treatment benefit. Using a subcutaneous M05 (B16.OVA) melanoma model, we observed that a treatment regimen consisting of a 7-day course of axitinib (0.5 mg/day provided orally) combined with a subcutaneous vaccine [ovalbumin (OVA) peptide-pulsed syngenic dendritic cells adenovirally engineered to produce IL-12p70] yielded superior protection against melanoma growth and extended overall survival when compared with animals receiving either single modality therapy. Treatment benefits were associated with: (a) a reduction in suppressor cell (myeloid-derived suppressor cells and Treg) populations in the tumor, (b) activation of tumor vascular endothelial cells, and (c) activation and recruitment of type-1, vaccine-induced CD8 T cells into tumors. These results support the therapeutic superiority of combined vaccine+axitinib immunotherapy and the translation of such approaches into the clinic for the treatment of patients with advanced-stage melanoma.

Topics: Angiogenesis Inhibitors; Animals; Axitinib; Cancer Vaccines; Cell Line, Tumor; Combined Modality Therapy; Dendritic Cells; Endothelial Cells; Female; Imidazoles; Indazoles; Interleukin-12; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Protein Kinase Inhibitors; Skin Neoplasms; T-Lymphocytes; Time Factors; Tumor Burden; Tumor Microenvironment

In experimental tumor immunotherapy, incomplete Freund's adjuvant (IFA) has been considered as the "gold standard" for T-cell vaccination in mice and humans in spite of its considerable adverse effects. Recently, we succeeded in eliciting strong CTL responses in mice after vaccination with biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS). In our study, we compared the immune response to IFA and PLGA-MS containing ovalbumin (OVA) and CpG-oligodeoxynucleotide (MS-OVA/CpG) or we used a mixture of MS-OVA/CpG and MS-polyI:C. A single vaccination with MS-OVA/CpG elicited long-lasting titers of IgG1 and IgG2a, but only low IgE titers, and also the T-cell response was biased toward Th(1) differentiation. Antigen presentation to CD4(+) and CD8(+) cells and activation of a cytotoxic T-cell response in mice vaccinated with PLGA-MS and IFA lasted for over 3 weeks. Preconditioning of the injection site with TNF-α and heterologous prime-boost regimen further enhanced the cytotoxic response. PLGA-MS were as efficient or superior to IFA in eradication of preexisting tumors and suppression of lung metastases. Taken together, PLGA-MS are well-defined, biodegradable and clinically compatible antigen carrier systems that compare favorably with IFA in their efficacy of tumor immunotherapy in mouse models and hence deserve to be tested for their effectiveness against human malignant diseases.

Topics: Animals; CD8-Positive T-Lymphocytes; Disease Models, Animal; Freund's Adjuvant; Immunotherapy; Lactic Acid; Lipids; Melanoma; Mice; Mice, Inbred C57BL; Microspheres; Oligodeoxyribonucleotides; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Treatment Outcome

Active vaccination against melanoma requires tolerance break as melanoma-associated antigens (MAA) used in vaccine formula are mostly self-antigens. While tolerance to MAA in the CD8(+) T cell compartment is well characterized, it is still not the case for the CD4(+) T cell compartment. Here, we analysed CD4(+) T cell tolerance to such antigens in mice genetically engineered to express ovalbumin (OVA) in melanocytes (Tyr-OVA mice). When we crossed Tyr-OVA mice with DO11.10 and OT-II mice transgenic for an OVA-specific TCR restricted by MHC class II, we observed different tolerization levels. Central tolerance was complete for high avidity DO11.10 CD4(+) T cells, but absent for low avidity OT-II CD4(+) T cells. OT-II CD4(+) T cells also ignored OVA in the periphery of Tyr-OVA mice, albeit being potently reactive to vaccination. OVA challenge in single transgenic Tyr-OVA mice confirmed the existence of OVA-reactive CD4(+) T cells with the induction of efficient T helper cells for antibody production and anti-tumour T cell response. In total, our study demonstrates the existence of low avidity MAA-specific CD4(+) T cells escaping by ignorance central and peripheral tolerance, but valuable in the context of vaccination against melanoma.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; CD4-Positive T-Lymphocytes; Immune Tolerance; Melanocytes; Melanoma; Mice; Mice, Transgenic; Ovalbumin

Although CD8+ T cells are usually considered antitumoral, several recent studies report that the cells can also promote tumor progression. Using the melanoma cell line B16 as a murine model of pulmonary metastasis, we examined whether the pro- versus antitumoral effects of CD8+ T cells relate to their Ag specificity. Results of the study indicate that although CD8+ T cells specific for tumor Ags promote tumor rejection, CD8+ T cells specific for unrelated Ags promote tumor progression. We found the effect to be partly attributable to CD8+ T cells dampening effective antitumor NK cell responses. Notably, activation of CD8+ T cell responses by an unrelated stimulus, in this case infection with influenza virus, increased the number of pulmonary tumor nodules. These data provide a rationale for previously unexplained data identifying contrasting roles for CD8+ T cells in tumor progression.

Topics: Animals; Antigens; Antigens, Neoplasm; Antigens, Viral; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytotoxicity, Immunologic; Disease Progression; Immunologic Memory; Killer Cells, Natural; Lung Neoplasms; Melanoma; Mice; Ovalbumin; T-Cell Antigen Receptor Specificity

Tumor-bearing individuals have been reported to harbor increased numbers of Foxp3(+) regulatory T cells (Treg), which prevent the development of efficient antitumor immune responses. Thus, Treg depletion has already been tested as a promising therapeutic approach in various animal models and entered clinical trials. However, the use of nonspecific Treg targeting agents such as CD25 depleting antibodies, which in addition to CD25(+) Tregs also deplete recently activated CD25(+) effector T cells, potentially masked the tremendous potential of this therapeutic strategy. To avoid such nonspecific effects, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3(+) Tregs even during ongoing immune responses. We showed that Foxp3(+) Treg depletion induced partial regression of established ovalbumin (OVA)-expressing B16 melanoma, which was associated with an increased intratumoral accumulation of activated CD8(+) cytotoxic T cells. The antitumor effect could be significantly enhanced when Treg depletion was combined with vaccination against OVA. To further assess whether this therapeutic approach would break self-tolerance, we crossed DEREG mice with RipOVA(low) mice, expressing OVA as neo-self-antigen under control of the rat insulin promoter. In these mice, combined Treg depletion and vaccination also induced tumor regression without the onset of diabetes. Together, our data suggest that selective Treg targeting strategies combined with vaccinations against tumor-associated (self) antigens have the potential to evoke efficient antitumor responses without inducing overt autoimmunity. These findings might have implications for future therapeutic interventions in cancer patients.

Topics: Animals; Autoantigens; Cancer Vaccines; Crosses, Genetic; Disease Models, Animal; Forkhead Transcription Factors; Humans; Immunohistochemistry; Interleukin-2 Receptor alpha Subunit; Lymphocyte Depletion; Lymphocytes, Tumor-Infiltrating; Melanoma; Melanoma, Experimental; Mice; Mice, Mutant Strains; Ovalbumin; Prognosis; T-Lymphocytes; T-Lymphocytes, Regulatory

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

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

Topics: Animals; Antigen Presentation; Autoantigens; Immunotherapy; Lymph Nodes; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Ovalbumin; 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

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Immunization; Langerhans Cells; Melanoma; Mice; Mice, Transgenic; Ovalbumin; Skin; Skin Neoplasms

Transfection of dendritic cells (DCs) with messenger RNAs (mRNAs) of tumor-associated antigens (TAAs) is a promising strategy for cancer vaccines. TAA mRNA can be generated by in vitro transcription using DNA encoding the TAA gene as a template. A cap analog is usually added upon in vitro transcription to stabilize mRNA and enhance the efficiency of mRNA translation. However, the inclusion of the cap analog correlates with significantly lower-yield mRNA transcription, potentially leading to an expensive vaccine manufacturing process. To solve this problem, we present a novel approach in which DNA templates are modified with an internal ribosomal entry site (IRES) sequence inserted upstream of the gene of interest to replace the use of the cap analog. The presence of IRES greatly enhanced transcription for the mRNA in vitro compared with the cap analog. Also, higher transgene expression was achieved using luciferase (Luc) mRNA with IRES than using capped Luc mRNA to transfect DCs. Immunization of mice with DCs transfected with IRES-containing mRNA encoding chicken ovalbumin (OVA) induced significant levels of antigen-specific interferon gamma-producing CD8(+) T cells and in vivo killing of antigen-bearing cells. Consistently, mice immunized with IRES-containing OVA mRNA-transfected DCs were protected from pulmonary metastasis of melanoma cells injected intravenously. We suggest that IRES can be used for the production of larger quantities of mRNA and that such IRES-containing mRNAs may be useful for DC-based antitumor immunotherapy.

Topics: 5' Untranslated Regions; Animals; Antigens, Neoplasm; Cancer Vaccines; Dendritic Cells; Female; Luciferases; Lung Neoplasms; Melanoma; Mice; Ovalbumin; Plasmids; Protein Biosynthesis; RNA Cap Analogs; Transfection

In situ tumor cell killing by the herpes simplex virus thymidine kinase (HSVtk) gene can effectively prime antitumor T-cell responses, at least in part through local induction of a pro-inflammatory environment. Therefore, we reasoned that tumor-associated HSVtk expression would significantly enhance the efficacy of adoptive T-cell transfer (ACT) of (tumor) antigen-specific T cells into tumor-bearing hosts. When B16ovaHSVtk tumors were treated with ganciclovir (GCV), along with suboptimal numbers of activated OT-1T cells, complete tumor regressions were observed where GCV, or ACT, alone was completely ineffective. To our surprise, analysis of regressing tumors showed no increases in intratumoral OT-1T cell trafficking. However, the intratumoral percentages of both OT-1 and endogenous natural killer (NK) cells were substantially increased over controls. Depletion of endogenous NK cells abrogated the efficacy of the combination therapy and reduced the percentages of interferon-gamma(IFNgamma)-secreting OT-1T cells in mice that received combined therapy to levels similar to those of control mice. These data suggest that even relatively low levels of gene transfer of suicide genes into tumors may have therapeutic value as an adjuvant for other T-cell therapies, by providing immunological signals that support T-cell activation and expansion in vivo.

Topics: Adoptive Transfer; Animals; Antiviral Agents; Combined Modality Therapy; Female; Ganciclovir; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Interferon-gamma; Killer Cells, Natural; Melanoma; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Simplexvirus; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Thymidine Kinase; Transduction, Genetic

We have previously shown preferential tumor-homing and therapeutic efficacy of adoptively transferred type 1 CTL (Tc1) when compared with type 2 CTL (Tc2) in mice bearing intracranial ovalbumin-transfected melanoma (M05). Further characterizing the expression of a panel of homing receptors on Tc1 and Tc2 cells, we found that very late antigen (VLA)-4 (a heterodimer of CD49d and CD29), but none of other receptors evaluated, was expressed at significantly higher levels on Tc1 cells than on Tc2 cells. Although CD49d (alpha(4) integrin) can form heterodimers with both beta(1) (CD29) and beta(7) integrins, alpha(4)beta(7) complexes were not expressed by either Tc1 or Tc2 cells, suggesting that CD49d is solely expressed in VLA-4 complexes. VLA-4 expression on Tc2 cells was down-regulated in an interleukin (IL)-4 dose-dependent manner but not by other type 2 cytokines, such as IL-10 and IL-13, suggesting that IL-4 uniquely down-regulates VLA-4 expression on these cells. In accordance with the differential expression of VLA-4 on Tc1 versus Tc2 cells, Tc1 cells alone were competent to adhere to plate-bound VCAM-1-Ig fusion protein. Finally, the efficient trafficking of Tc1 cells into intracranial M05 lesions in vivo was efficiently blocked by administration of monoclonal antibodies against CD49d or VCAM-1 or small interfering RNA-mediated silencing of CD49d on Tc1 cells. Collectively, these data support the critical role of VLA-4 in the effective intracranial tumor homing of adoptive-transferred, antigen-specific Tc1 cells and suggest that more effective vaccine and/or ex vivo T-cell activation regimens may be developed by promoting the generation of VLA-4(+) antitumor Tc1 cells.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Central Nervous System Neoplasms; Gene Expression Regulation, Neoplastic; Integrin alpha4; Integrin alpha4beta1; Integrin beta1; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin; RNA, Small Interfering; Signal Transduction; T-Lymphocytes, Cytotoxic

Dendritic cell (DC) and DC-derived exosomes (EXO) have been used extensively for tumor vaccination. However, its therapeutic efficiency is limited to only production of prophylactic immunity against tumors. T cells can uptake DC-released EXO. However, the functional effect of transferred exosomal molecules on T cells is unclear. In this study, we demonstrated that OVA protein-pulsed DC-derived EXO (EXO(OVA)) can be taken up by Con A-stimulated, nonspecific CD4(+) T cells derived from wild-type C57BL/6 mice. The active EXO-uptaken CD4(+) T cells (aT(EXO)), expressing acquired exosomal MHC I/OVA I peptide (pMHC I) complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of stimulating OVA-specific CD8(+) T cell proliferation in vitro and in vivo and inducing efficient CD4(+) Th cell-independent CD8(+) CTL responses in vivo. The EXO(OVA)-uptaken CD4(+) aT(EXO) cell vaccine induces much more efficient CD8(+) T cell responses and immunity against challenge of OVA-transfected BL6-10 melanoma cells expressing OVA in wild-type C57BL/6 mice than EXO(OVA). The in vivo stimulatory effect of the CD4(+) aT(EXO) cell to CD8(+) T cell responses is mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and pMHC I complexes, respectively. In addition, CD4(+) aT(EXO) vaccine stimulates a long-term, OVA-specific CD8(+) T cell memory. Therefore, the EXO(OVA)-uptaken CD4(+) T cells may represent a new, effective, EXO-based vaccine strategy in induction of immune responses against tumors and other infectious diseases.

Topics: Animals; Antigens, Neoplasm; B7-1 Antigen; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD40 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Concanavalin A; Dendritic Cells; Histocompatibility Antigens Class I; Humans; Immunologic Memory; Melanoma; Mice; Mice, Knockout; Mitogens; Ovalbumin; Peptides; Signal Transduction; Time Factors

A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune escape mechanisms. Here we report a conceptually new approach of cancer immunotherapy based on in vivo infection of tumors and killing of infected tumor cells. Attenuated but still invasive, Salmonella typhimurium can be successfully exploited to invade melanoma cells that can present antigenic determinants of bacterial origin and become targets for anti-Salmonella-specific T cells. However, to fully appreciate the anticancer therapeutic properties of S. typhimurium, tumor-bearing mice need to be vaccinated against S. typhimurium before intratumoral Salmonella injection. Tumor infection when coupled to anti-Salmonella vaccination leads to 50% to 100% tumor-free mice with a better outcome on larger tumors. Invasive Salmonella also exert an indirect toxic effect on tumor cells through the recruitment of inflammatory cells and the cross-presentation of tumor antigens, which allow induction of tumor-specific immune response. This is effective in retarding the growth of untreated established distant tumors and in protecting the mice from subsequent tumor challenges.

Topics: Animals; Antigens, Bacterial; Epitopes, T-Lymphocyte; Female; Humans; Immunotherapy; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Ovalbumin; Salmonella Infections; Salmonella typhimurium; Salmonella Vaccines; T-Lymphocytes, Cytotoxic

Pseudomonas exotoxin (PE) is a 66 kDa bacterial toxin that is able to bind to mammalian cells, undergo receptor mediated endocytosis, and translocate its C-terminal catalytic domain into the cytosol. We investigated whether PE could be used in vivo to deliver CD8+ T-cell epitopes to the MHC-class I antigen presentation pathway to trigger a specific cytotoxic T-lymphocyte (CTL) response.. Amino acid 553 of PE was deleted to eliminate toxin catalytic activity, and amino acids 204-386 of ovalbumin were fused near the nontoxic PE C-terminus to produce PE(D)-OVA200. Mice were vaccinated with 100 microg of PE(D)-OVA200 3 times at 21 day intervals. Splenocytes were harvested 1 week later, and stimulated in vitro with ovalbumin expressing EG7 murine thymoma cells. In vivo tumor protection experiments were performed by vaccinating groups of mice as before, followed by a lethal dose of ovalbumin expressing tumor cells (MO5) injected subcutaneously.. Splenocytes from PE(D)-OVA200 vaccinated mice lysed (51)Cr labeled EG7 cells but not the untransfected EL4 parent cell line, whereas splenocytes from mice immunized with PBS, PE(D), or ovalbumin were unable to lyse EG7 cells. Cytotoxicity in vitro was mediated by CD8+ T-cells. PE(D)-OVA200 vaccinated mice survived (88%) a lethal subcutaneous challenge of ovalbumin expressing MO5 cells. Depletion of CD8+ cells from PE(D)-OVA200 vaccinated mice abolished this protection, indicating that this cell population is required for tumor rejection in vivo.. Our results indicate that PE(D) may be used as a vehicle to stimulate a protective CTL response to heterologous antigen in vivo.

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cloning, Molecular; Exotoxins; Female; Genetic Vectors; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; Plasmids; Pseudomonas aeruginosa Exotoxin A; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Viral Fusion Proteins; Virulence Factors

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.

Topics: Antigens, CD; Antigens, Neoplasm; Cancer Vaccines; Cell Movement; Cryopreservation; Dendritic Cells; Flow Cytometry; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Melanoma; Ovalbumin; Receptors, Chemokine; Tumor Cells, Cultured

Tumor cells transfected with strong antigenic epitopes were able to stimulate humoral response against relevant wild-type tumors. Crossreacting immune sera have been obtained by immunizing C57BL/6 mice with OVA-transfected leukemia EL-4 (subclone E.G7) and OVA-transfected melanoma B16 (subclone MO.5) stimulated with interferon-gamma. Tumors were significantly inhibited when antisera were injected subcutaneously close to the site of solid wild-type tumors EL-4 and B16 grafted onto C57BL/6 mice.

Topics: Animals; Antibody Formation; Cross Reactions; Fluorescent Antibody Technique, Indirect; Leukemia, Experimental; Melanoma; Mice; Mice, Inbred C57BL; Ovalbumin; Tumor Cells, Cultured

Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze thaw lysis for DC priming. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-gamma release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-gamma production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in two of three cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in two analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge. These data demonstrate that the method of tumor cell preparation clearly influences the ability of DCs to present antigen to T cells. Correlation of in vitro data with the generation of protective immunity in vivo suggests the utility of irradiated tumor-primed DCs as a means to generate protective immunity in patients with solid malignancies.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Apoptosis; Cancer Vaccines; Carcinoma, Squamous Cell; Dendritic Cells; Freezing; Heating; Humans; Immunotherapy, Adoptive; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Necrosis; Neoplasms, Experimental; Ovalbumin; T-Lymphocytes, Cytotoxic; Thymoma; Tumor Cells, Cultured

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.

Topics: Animals; Antibodies, Viral; Genetic Vectors; Immunization, Secondary; Melanoma; Mice; Ovalbumin; Poliovirus; Poliovirus Vaccine, Oral; T-Lymphocytes, Cytotoxic; Vaccination; Vaccines, Synthetic

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Female; Immunoglobulin G; Interleukin-4; Lung Neoplasms; Lymph Nodes; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; Ovalbumin; Tumor Cells, Cultured

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.

Topics: Adoptive Transfer; Animals; Antigens; Antigens, Neoplasm; Epitopes, T-Lymphocyte; Female; Immunophenotyping; Interferon-gamma; Lung Neoplasms; Lymphocyte Activation; Melanoma; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

The 175-kDa mannose receptor is one of the receptors that mediates the clearance of tissue-type plasminogen activator (t-PA). The affinity of t-PA for the mannose receptor is much higher than the affinity of other high-mannose-type oligosaccharide-containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t-PA with the isolated human mannose receptor in several in vitro binding assays. t-PA showed a high affinity (Ki = 0.2 nM) for the mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the mannose receptor. The interaction was not affected by non-glycosylated t-PA. The high affinity differed slightly between t-PAs synthesized by various cell types (range Ki 0.2-0.7 nM) and between various glycoforms of t-PA. No statistically significant difference in affinity between t-PA and t-PA complexed to inhibitors was observed. In contrast to intact t-PA, a trypsin digest of t-PA had a low affinity (Ki = 0.5 microM) for the mannose receptor. Both intact and trypsin digests of the high-mannose-type oligosaccharide-containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5-1.5 microM) for the mannose receptor. We conclude that neither protein-protein interactions, nor the complex-type oligosaccharides and the fucose residue on t-PA contribute significantly to the high-affinity binding of t-PA. We suggest that the conformation of the high-mannose-type oligosaccharide on t-PA is influenced by the protein moiety of t-PA in such a way that the oligosaccharide has a high affinity for the mannose receptor.

Topics: Binding, Competitive; Biotin; Female; Fucose; Glycoproteins; Humans; Lectins, C-Type; Mannans; Mannose Receptor; Mannose-Binding Lectins; Melanoma; Oligosaccharides; Ovalbumin; Placenta; Pregnancy; Protease Inhibitors; Receptors, Cell Surface; Ribonucleases; Tissue Plasminogen Activator; Trypsin; Tumor Cells, Cultured

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These "empty" MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8+ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when "sub-optimal" antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; B7-1 Antigen; CD8 Antigens; Epitopes; Female; Histocompatibility Antigens Class I; Melanoma; Mice; Molecular Sequence Data; Ovalbumin; Peptide Fragments; T-Lymphocytes, Cytotoxic; Transfection; Tumor Cells, Cultured

Surface receptor sites for the Fc portion of antigen-antibody complexes were demonstrated on cells derived from three methylcholanthrene-induced fibrosarcomas, one of strain C3H and two of strain BALB/c origin, two spontaneously occurring malignant melanomas (B16 in strain C57BL/6 and Harding-Passey in strain BALB/c mice), a Moloney sarcoma virus-induced tumor of strain BALB/c origin and the Walker 256 carcinosarcoma of Holtzman rats. Primary cell cultures derived from these tumors adsorbed technetium-99m labeled, antibody-sensitized sheep erythrocytes (99mTc EA) as determined either by visual scoring of adherence or radioisotopic quantitation. Depending upon the tumor tested, from 20% to greater than 95% of the target cells absorbed 99mTc EA. All cells lost their reactivity after 1 or 2 passages in vitro, but this was regained after a single passage in vivo. Indicator erythrocytes coated with F(ab')2 fragments of the sensitizing sheep erythrocytes (SRBC) antiserum did not adhere thereby demonstrating that the hemadsorption required an intact Fc portion of the antibody molecule. Adherence of 99mTc EA was blocked by soluble immune complexes prepared with ovalbumin and rabbit antibody directed against it and Escherichia coli 055:B5 lipopolysaccharide and mouse antibody directed against it. Normal rabbit or mouse serum, immune serum, or antigen alone did not block adherence of 99mTc EA thereby demonstrating that the receptors had greater affinity for immune complexes than for either antigen or antibody alone. The existence of membrane receptors on tumor-derived cells which react with the Fc portion of antigen-antibody complexes may provide an explanation for the mechanism by which immune complexes are capable of blocking cell-mediated tumor cell destruction irrespective of whether the receptors are on the tumor cells themselves or on admixed lymphocytes and macrophages.

Topics: Animals; Antigen-Antibody Complex; Binding Sites, Antibody; Binding, Competitive; Culture Techniques; Erythrocytes; Escherichia coli; Fibrosarcoma; Hemadsorption; Immune Sera; Immunization; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Melanoma; Mice; Neoplasms, Experimental; Ovalbumin; Rabbits; Sarcoma, Experimental; Sheep; Technetium

Topics: Animals; Chemical Phenomena; Chemistry; DNA; Iodine; Iodine Isotopes; Melanins; Melanoma; Mice; Neoplasms, Experimental; Ovalbumin; Quinones; Radionuclide Imaging

Topics: Animals; Antibodies; Fluorescent Antibody Technique; gamma-Globulins; Humans; In Vitro Techniques; Kidney; Melanoma; Ovalbumin; Rabbits; Rats; Skin; Sodium Chloride; Tissue Extracts

Topics: Animals; Antigen-Antibody Reactions; Antigens; Humans; Immunoglobulin G; In Vitro Techniques; Kidney; Melanoma; Ovalbumin; Passive Cutaneous Anaphylaxis; Rabbits; Serum Albumin; Skin; Sodium Chloride; Tissue Extracts

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