ovalbumin and Skin-Neoplasms

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8

Topics: Animals; Antigens, Neoplasm; Antimetabolites, Antineoplastic; Bromodeoxyuridine; CD8-Positive T-Lymphocytes; Interferon-gamma; Luciferases; Luminescent Measurements; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; NK Cell Lectin-Like Receptor Subfamily K; Ovalbumin; Skin Neoplasms; Tumor Microenvironment; Vaccination

The presence and activity of CD8

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Granzymes; Humans; Immunotherapy, Adoptive; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Ovalbumin; Programmed Cell Death 1 Receptor; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Skin Neoplasms; Tumor Microenvironment

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune-related adverse events have been reported. Therefore, more effective and antigen-specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS-ML). In this study, we generated OX40L-overexpressing iPS-ML (iPS-ML-Zsgreen-OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS-ML-Zsgreen-OX40L suppressed the progression of B16-BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)-expressing B16 melanoma (MO4). The number of antigen-specific CD8

Topics: Animals; Antigens, Neoplasm; Cell Proliferation; Cross-Priming; Cytokines; Induced Pluripotent Stem Cells; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Mice, Inbred C57BL; Models, Biological; Myeloid Cells; Neoplasm Staging; Ovalbumin; OX40 Ligand; Peptides; Peritoneum; Skin Neoplasms; Spleen; T-Lymphocytes; Up-Regulation

Topics: Administration, Cutaneous; Allografts; Animals; CD27 Ligand; CD40 Ligand; Cytotoxicity, Immunologic; Gene Expression; Graft Rejection; Imiquimod; Immunization; Immunologic Memory; Immunotherapy; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Ovalbumin; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Toll-Like Receptor 7

Immune-checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti-Toll-like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell-surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti-TLR4 mAb in T-cell-mediated antitumour immunity. The anti-TLR4 mAb induced the activation of antigen-specific T-cells in adoptive transfer studies. The growth of ovalbumin (OVA)-expressing tumours was significantly suppressed by administration of OVA and the anti-TLR4 mAb in combination, but not individually. The antitumour effect of anti-PD-1 mAb was enhanced in mice administered with OVA plus the anti-TLR4 mAb. The OVA-specific IFN-γ-producing CD8 T-cells were induced by administration of OVA and the anti-TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T-cells. The inflammatory response to the anti-TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF-κB activation and production of TNF-α, IL-6 and IL-1β. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti-TLR4 mAb) plus OVA induced no or less-marked activation of OVA-specific T-cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti-TLR4 mAb induces potent CD8 T-cell-dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti-TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antineoplastic Agents, Immunological; CD8-Positive T-Lymphocytes; Immunization; Immunotherapy; Interferon-gamma; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Lymphocyte Activation; Macrophages; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B; Ovalbumin; Primary Cell Culture; Programmed Cell Death 1 Receptor; Skin Neoplasms; Th1 Cells; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

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

The epidermal application of the Toll Like Receptor 7 agonist imiquimod and a T-cell peptide epitope (transcutaneous immunization, TCI) mediates systemic peptide-specific cytotoxic T-cell (CTL) responses and leads to tumor protection in a prophylactic tumor setting. However, it does not accomplish memory formation or permanent defiance of tumors in a therapeutic set-up. As a distinct immunologic approach, CTLA-4 blockade augments systemic immune responses and has shown long-lasting effects in preclinical experiments as well as in clinical trials.. The study investigates the vaccination capacity of TCI in combination with the checkpoint inhibitor CTLA-4 in matters of primary response, memory formation and tumor protection and characterizes the role of regulatory T cells (Tregs).. After performing TCI with IMI-Sol (containing 5% Imiquimod) and the model epitope SIINFEKL, 6-8 week old C57BL/6 mice received anti-CTLA-4 antibody either s.c or i.p. The CTL responses and frequency of peptide specific CD8. The combination of s.c. anti-CTLA-4 antibody and TCI leads to an enhanced systemic cytotoxic response, to memory formation and allows significantly improved survival in a tumor setting with B16 OVA melanoma. Towards the mechanism, we show that in this vaccination protocol the CTLA-4 antibody acts mainly Treg-independent.. We demonstrate that the combination of TCI with IMI-Sol and anti-CTLA-4 can confer potent immune responses and tumor-protection. These results might contribute to the development of advanced vaccination approaches targeting tumors or persistent infectious diseases.

Topics: Adjuvants, Immunologic; Aminoquinolines; Animals; Antineoplastic Agents, Immunological; CTLA-4 Antigen; Drug Synergism; Flow Cytometry; Humans; Imiquimod; Immunologic Memory; Immunotherapy; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Skin Neoplasms; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Toll-Like Receptor 7; Xenograft Model Antitumor Assays

Development of vaccines to prevent and treat emerging new pathogens and re-emerging infections and cancer remains a major challenge. An attractive approach is to build the vaccine upon a biocompatible NP that simultaneously acts as accurate delivery vehicle and radiotracer for PET/SPECT imaging for ultrasensitive and quantitative in vivo imaging of NP delivery to target tissues/organs. Success in developing these nanovaccines will depend in part on having a "correct" NP size and accommodating and suitably displaying antigen and/or adjuvants (e.g., TLR agonists). Here we develop and evaluate a NP vaccine based on iron oxide-selective radio-gallium labeling suitable for SPECT((67)Ga)/PET((68)Ga) imaging and efficient delivery of antigen (OVA) and TLR 9 agonists (CpGs) using lipid-coated magnetite micelles. OVA, CpGs and rhodamine are easily accommodated in the hybrid micelles, and the average size of the construct can be controlled to be ca. 40 nm in diameter to target direct lymphatic delivery of the vaccine cargo to antigen presenting cells (APCs) in the lymph nodes (LNs). While the OVA/CpG-loaded construct showed effective delivery to endosomal TLR 9 in APCs, SPECT imaging demonstrated migration from the injection site to regional and nonregional LNs. In correlation with the imaging results, a range of in vitro and in vivo studies demonstrate that by using this microdosed nanosystem the cellular and humoral immune responses are greatly enhanced and provide protection against tumor challenge. These results suggest that these nanosystems have considerable potential for image-guided development of targeted vaccines that are more effective and limit toxicity.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Antigens; Cancer Vaccines; Dendritic Cells; Gallium Radioisotopes; Gene Expression; Immunity, Cellular; Lymph Nodes; Magnetite Nanoparticles; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Micelles; Oligodeoxyribonucleotides; Ovalbumin; Positron-Emission Tomography; Rhodamines; Skin Neoplasms; T-Lymphocytes; Theranostic Nanomedicine; Toll-Like Receptor 9

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

The generation of CTLs is crucial in the immunological fight against cancer and many infectious diseases. To achieve this, vaccine Ags need to be targeted to the cytosol of dendritic cells, which can activate CD8 T cells via MHC class I (MHCI). Therefore, such targeting has become one of the major objectives of vaccine research. In this study, we aimed to bypass the unwanted and default MHC class II Ag presentation and trigger MHCI presentation by using a photosensitizer that, upon light activation, would facilitate cytosolic targeting of codelivered Ag. Poly(lactide-co-glycolide) microparticles ∼1 μm size were loaded with OVA and the photosensitizer tetraphenyl chlorine disulphonate (TPCS2a) and administered intradermally in mice, which were illuminated 1 d later for activation of the photosensitizer. Immunization in the presence of TPCS2a significantly increased activation of CD8 T cells compared with immunization without TPCS2a and as measured by CD8 T cell proliferation, production of proinflammatory IFN-γ, TNF-α, and IL-2, and prevention of tumor growth. Cytotoxicity was demonstrated by granzyme B production in vitro and by in vivo killing of CFSE-labeled targets. CD4-dependent Ab responses were abrogated in mice immunized with TPCS2a-containing particles, suggesting that photosensitization facilitated a shift from default MHC class II toward MHCI Ag presentation. Hence, vaccine particles with Ag and photosensitizers proved an effective vehicle or adjuvant for stimulation of CTLs, and they may find potential application in therapeutic cancer vaccination and in prophylactic and therapeutic vaccination against intracellular infections.

Topics: Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Cancer Vaccines; Cytosol; Dendritic Cells; Drug Carriers; Female; Granzymes; Immunization; Injections, Intradermal; Interleukin-2; Lactic Acid; Light; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Neoplasm Transplantation; Ovalbumin; Photosensitizing Agents; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Porphyrins; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8(+) OT-I or CD4(+) OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, CD; Biological Transport; Bone Marrow Cells; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cytokines; Dendritic Cells; Dextrans; Fluorescein-5-isothiocyanate; Gene Expression; Lectins, C-Type; Lipopolysaccharides; Lymphocyte Culture Test, Mixed; Melanoma, Experimental; Mice; Mice, Knockout; Minor Histocompatibility Antigens; Ovalbumin; Peptides; Poly I-C; Proto-Oncogene Proteins c-cbl; Receptors, Cell Surface; Skin Neoplasms; Ubiquitin-Protein Ligases

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

Background We previously have shown that nona-arginine protein transduction domain (R9-PTD) induced efficient protein-antigen (Ag) transduction of dendritic cells (DCs) in vitro, resulting in the efficient induction of strong Ag-specific immune responses mediated by CD8+ and CD4+ T cells and in superior antitumour effects in vivo in cancer-bearing mice. Objectives The Ag-specific immune responses caused by intradermal (i.d.) injections of R9-PTD-containing protein Ags without DC preparation were investigated. We also investigated the antitumour effects by intratumoral (i.t.) injections of rR9-containing protein Ags. Methods Synthesized SIINFEKL peptide, or recombinant ovalbumin fusion proteins (rOVA, rR9-OVA), were directly injected into abdominal skin in naïve C57BL/6 mice. OVA-specific cytotoxic T lymphocyte (CTL) activity, serum IgG titre and cytokine profiles were investigated. Histopathological analyses were also performed. In a cancer vaccination model, EG.7 (OVA-cDNA transfectants thymoma) cells were inoculated intradermally in C57BL/6 mice, and the antitumour effects were evaluated by i.t. injections of rR9-OVA in a treatment setting. Results i.d. injections of rR9-OVA into naïve C57BL/6 mice elicited OVA-specific CTLs and produced IgG2-dominant immunoglobulin. The i.d. injections of rR9-OVA also induced inflammatory cell infiltrates containing neutrophils, monocytes and lymphocytes, as well as production of inflammatory cytokines such as interferon (IFN)-gamma, interleukin-2 and IFN-inducible protein 10, with presenting SIINFEKL epitopes on major histocompatibility complex (MHC) class I molecules at the injection area. i.t. injections of rR9-OVA into EG.7 tumour mass significantly suppressed tumour growth, and these effects were completely abrogated by the depletion of CD8+ T cells. These antitumour effects were superior to those elicited by i.t. injections of rR9-OVA-treated DCs. Conclusions i.d. injections of rR9-containing immunogenic Ag without adjuvants simultaneously induce dual immunological effects: the induction of Tc1- and Th1-dominant immune responses, and the induction of inflammatory and CTL-mediated immune responses at the injection area by expressing Ag epitopes on MHC class I molecules as targets. This simple vaccination approach with R9-PTD-containing fusion proteins might be useful as prophylactic immunotherapy for cancer or infectious diseases.

Topics: Animals; Antigen Presentation; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytokines; Female; Histocompatibility Antigens; Immunoglobulin G; Injections, Intradermal; Mice; Mice, Inbred BALB C; Oligopeptides; Ovalbumin; Recombinant Fusion Proteins; Skin; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Th1 Cells

Immunostimulatory mAb as vaccine adjuvants for the treatment of cancer hold considerable potential for boosting weak responses when used against immunogenic tumours, or in combination with various other vaccines. We now show that when administered with OVA, the combination of anti-4-1BB mAb with anti-CD40, anti-OX40 or anti-CD25 resulted in a fourfold enhancement in the antigen-specific T-cell response compared with anti-4-1BB mAb alone, with a similar enhancement in memory responses following rechallenge with OVA. Although the number of antigen-specific T-cells generated after treatment with each of the combinations was similar, marked functional differences were detected. In particular, anti-4-1BB/anti-CD25 resulted in excellent expansion of specific CD8+ T cells but produced fewer IFN-gamma-secreting effector cells than the other combinations. Anti-4-1BB/anti-OX40 proved to be the most potent, inducing the most effective T-cell responses in the RIPmOVA diabetes model with adoptively transferred OVA-specific T cells, and, when given with a peptide vaccine, protecting mice against the poorly immunogenic B16-F10 tumour. Overall the results suggest that although these combinations of mAb look promising in terms of their therapeutic potential, further functional assays are needed to compare their effects.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Cancer Vaccines; CD40 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Interleukin-2 Receptor alpha Subunit; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, OX40; Skin Neoplasms; Tumor Necrosis Factor Receptor Superfamily, Member 9

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

Priming of CTLs at mucosal sites, where various tumors are originated, seems critical for controlling tumors. In the present study, the effect of the oral administration of OVA plus adjuvant cholera toxin (CT) on the induction of Ag-specific mucosal CTLs as well as their effect on tumor regression was investigated. Although OVA-specific TCRs expressing lymphocytes requiring in vitro restimulation to gain specific cytotoxicity could be detected by OVA peptide-bearing tetramers in both freshly isolated intraepithelial lymphocytes and spleen cells when OVA was orally administered CT, those showing direct cytotoxic activity without requiring in vitro restimulation were dominantly observed in intraepithelial lymphocytes. The magnitude of such direct cytotoxicity at mucosal sites was drastically enhanced after the second oral administration of OVA with intact whole CT but not with its subcomponent, an A subunit (CTA) or a B subunit (CTB). When OVA plus CT were orally administrated to C57BL/6 mice bearing OVA-expressing syngeneic tumor cells, E.G7-OVA, in either gastric tissue or the dermis, tumor growth was significantly suppressed after the second oral treatment; however, s.c. or i.p. injection of OVA plus CT did not show any remarkable suppression. Those mucosal OVA-specific CTLs having direct cytotoxicity expressed CD8alphabeta but not CD8alphaalpha, suggesting that they originated from thymus-educated cells. Moreover, the infiltration of such OVA-specific CD8(+) CTLs was observed in suppressed tumor tissues. These results indicate that the growth of ongoing tumor cells can be suppressed by activated CD8alphabeta CTLs with tumor-specific cytotoxicity via an orally administered tumor Ag with a suitable mucosal adjuvant.

Topics: Animals; Antigens, Neoplasm; CD8 Antigens; Cell Line, Tumor; Cholera Toxin; Cytotoxicity, Immunologic; Female; Intestinal Mucosa; Lymphocyte Activation; Lymphocyte Subsets; Mice; Mice, Inbred C57BL; Mouth Mucosa; Ovalbumin; Skin Neoplasms; Stomach Neoplasms; T-Lymphocytes, Cytotoxic

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Cancer Vaccines; Carbohydrate Sequence; Dendritic Cells; Erythrocyte Membrane; Galactosyltransferases; Glycolipids; Immunotherapy; Injections; Lymph Nodes; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Mice; Mice, Knockout; Molecular Sequence Data; Ovalbumin; Rabbits; Skin Neoplasms; Transplantation, Heterologous; Trisaccharides; Xenograft Model Antitumor Assays

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

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.

Topics: Animals; Anterior Chamber; CD11b Antigen; Cell Line, Tumor; Cell Movement; Cell Proliferation; Eye Neoplasms; Female; Immunosuppression Therapy; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myeloid Cells; Neoplasm Transplantation; Ovalbumin; Skin Neoplasms; 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