CD8 T cell help for innate antitumor immunity.

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.

Monoclonal antibodies for the diagnosis of canine mastocytoma.

Mastocytomas are the most common malignant neoplasm in the dog; they are more aggressive than the mast cell tumors of other species. Therefore, it is imperative to develop a highly sensitive and specific immunoassay for clinical diagnosis of canine mastocytoma. The production and characterization of new mouse monoclonal antibodies (MAb 9-3 and MAb 80) directed against canine mastocytoma are reported here. By immunohistochemistry using fresh frozen tissue of tissue impression smears, we observed that the antigen recognized by MAb 9-3 is expressed exclusively on the surface and cytoplasmic granules of canine mastocytoma but not on the mast cells in normal canine skin. MAb 80 did not compete for binding to mast cells in normal canine skin. Western blot assays performed with canine mastocytoma indicated that MAb 9-3 recognized the 74 kDa band, and MAb 80 recognized the 167 and 248 kDa bands. We studied the immunostaining pattern of impression smears with MAb 9-3 from 36 benign and malignant canine masses, including eight samples of mastocytoma that were positive and other tumor samples that were negative by MAb 9-3. This report for the first time precisely characterizes a monoclonal antibody specific for canine mastocytoma, facilitating clinical and molecular investigation of canine mastocytoma.

Tumor necrosis factor receptor 2-mediated tumor suppression is nitric oxide dependent and involves angiostasis.

Tumor necrosis factor (TNF) binds to two different receptors. Although most of its functions are attributed to TNF receptor 1 (TNFR1), the independent role of TNFR2 is still largely unknown. Using TNFR single or double knock-out mice, we show here that the expression of TNFR2 alone on host cells was sufficient to suppress the growth of TNF-secreting tumors in both immune competent and T/B lymphocyte-deficient severe combined immunodeficiency (SCID) mice. Histologic studies showed that TNF recruited, via TNFR2, large numbers of macrophages and efficiently inhibited angiogenesis in the tumor. In vitro, TNF activated TNFR1-deficient macrophages to produce nitric oxide (NO). Treatment of TNFR1 knock-out mice with L-NAME, a specific NO synthase inhibitor, almost completely eliminated TNF-induced angiostasis and tumor suppression. Moreover, L-NAME acted only during the first few days of tumor growth. Our results show for the first time that TNFR2 expressed on host innate immune cells is sufficient to mediate the antitumor effect of TNF, and NO is necessary for this process, possibly by inhibition of angiogenesis in the tumor.

[F-18]-Fluoro-2-deoxy-D: -glucose positron emission tomography as a tool for early detection of immunotherapy response in a murine B cell lymphoma model.

[F-18]-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) is a non-invasive imaging technique which has recently been validated for the assessment of therapy response in patients with aggressive non-Hodgkin's lymphoma. Our objective was to determine its value for the evaluation of immunotherapy efficacy in immunocompetent Balb/c mice injected with the A20 syngeneic B lymphoma cell line. The high level of in vitro FDG uptake by A20 cells validated the model for further imaging studies. When injected intravenously, the tumour developed as nodular lesions mostly in liver and spleen, thus mimicking the natural course of an aggressive human lymphoma. FDG-PET provided three-dimensional images of tumour extension including non-palpable lesions, in good correlation with ex vivo macroscopic examination. When mice were pre-immunized with an A20 cell lysate in adjuvant before tumour challenge, their significantly longer survival, compared to control mice, were associated with a lower incidence of lymphoma visualized by PET at different time points. Estimation of tumour growth and metabolism using the calculated tumour volumes and maximum standardized uptake values, respectively, also demonstrated delayed lymphoma development and lower activity in the vaccinated mice. Thus, FDG-PET is a sensitive tool relevant for early detection and follow-up of internal tumours, allowing discrimination between treated and non-treated small animal cohorts without invasive intervention.

B7-H3 enhances tumor immunity in vivo by costimulating rapid clonal expansion of antigen-specific CD8+ cytolytic T cells.

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

Promiscuity of MHC class Ib-restricted T cell responses.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.

Individual analysis of mice vaccinated against a weakly immunogenic self tumor-specific antigen reveals a correlation between CD8 T cell response and antitumor efficacy.

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.

Functional alterations in CD11b(+)Gr-1(+) cells in mice injected with allogeneic tumor cells and treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure results in an increased percentage of CD11b(+) (Mac-1(+)) cells in the spleens of mice challenged with P815 tumor cells, coincident with a failure of the mice to generate allospecific CD8(+) CTL activity. Since CD11b(+)Gr-1(+) myeloid suppressor cells (MSC) have been described as that which prevent cytotoxic T lymphocyte (CTL) development in a variety of disease states, we hypothesized that TCDD promoted MSC development, leading to suppression of CTL activity. The purpose of the present studies was to compare the phenotypic and functional characteristics of CD11b(+) cells in vehicle- and TCDD-treated mice during the P815 tumor allograft response to determine their potential to function as MSC. Initial studies showed that virtually all splenic CD11b(+) cells in both vehicle- and TCDD-treated mice co-expressed Gr-1. Consistent with MSC activity, CD11b(+)Gr-1(+) cells isolated from TCDD- but not vehicle-treated mice suppressed the development of CTL activity when added in vitro to mixed lymphocyte-P815 tumor cell cultures. Also consistent with MSC activity, this suppressive effect in vitro required cell-to-cell contact. Surprisingly, however, in vivo depletion of CD11b(+)Gr-1(+) cells failed to affect TCDD-induced suppression of the CTL response, arguing against an immunoregulatory role for the cells in vivo. Immunohistochemical analysis of the spleen showed that CD11b(+)Gr-1(+) cells were localized in the red pulp, and physically separated from the T cells in the white pulp. The localization of CD11b(+)Gr-1(+) cells in the red pulp was indicative of extramedullary myelopoiesis and suggested that TCDD enhanced myelopoiesis. A significantly enhanced neutrophilia in the blood of TCDD-treated mice supported this conclusion. CD11b(+)Gr-1(+) cells isolated from the blood or spleen of TCDD-treated mice produced up to fivefold higher levels of superoxide following PMA stimulation when compared with cells from vehicle-treated mice. However, unlike vehicle-treated mice, CD11b(+)Gr-1(+) cells from TCDD-treated mice were unable to kill YAC-1 target cells. These results indicate that TCDD exposure alters the host response to allogeneic tumor growth, resulting in enhanced myelopoiesis perhaps as a compensatory response to the suppressed T cell-mediated immunity in the face of an increasing P815 tumor burden. Furthermore, within the context of the P815 response, TCDD appears to alter the functional capabilities of mature neutrophils, by enhancing their oxidative burst capacity but reducing their tumoricidal response.

TRAIL: a mechanism of tumor surveillance in an immune privileged site.

TRAIL is a recently described member of the TNF superfamily. The ability of TRAIL to induce apoptosis in a large number of tumors has stimulated interest in TRAIL as a tumor therapeutic agent. Although TRAIL mRNA is expressed in a number of tissues, its functional significance to various organs is unknown. Because tumors rarely develop in the eye, we have examined this organ for functional TRAIL expression. Our analysis revealed that TRAIL mRNA and protein are constitutively expressed on numerous ocular structures, including the cornea and retina. More importantly, ocular tissue displays functional TRAIL as determined by in vitro killing of TRAIL-sensitive tumor cell lines. Previous studies have shown that ocular tissue also expresses functional Fas ligand (FasL). To assess the contribution of TRAIL and FasL for tumor cell killing in the eye, cell lines susceptible to both TRAIL and FasL were examined. The results show that ocular tissue kills via either ligand, suggesting a compensatory mechanism between TRAIL and FasL. Collectively, these results provide physiological evidence for ocular TRAIL expression, and suggest a role for this molecule in tumor surveillance in an immune privileged site.

TNF-mediated toxicity after massive induction of specific CD8+ T cells following immunization of mice with a tumor-specific peptide.

We immunized mice with antigenic peptide P815E, which is presented by H-2K(d) and recognized by tumor-specific CTL raised against P815 tumor cells. This peptide is encoded by the ubiquitously expressed gene MsrA and carries a mutated residue conferring tumor specificity. Unexpectedly, we observed a severe toxicity occurring in the early hours after the third injection, resulting in the death of most mice within 24 h. The toxic syndrome was reminiscent of TNF-induced shock, and the sera of ill mice contained high levels of TNF. Toxicity was prevented by injection of neutralizing anti-TNF Abs, confirming the involvement of TNF. Depletion of CD8+ T cells could also prevent toxicity, and ex vivo experiments confirmed that CD8+ lymphocytes were the major cellular source of TNF in immunized mice. Tetramer analysis of the lymphocytes of immunized mice indicated a massive expansion of P815E-specific T cells, up to >60% of circulating CD8+ lymphocytes. A similar toxicity was observed after massive expansion of specific CD8+ T cells following immunization with another P815 peptide, which is encoded by gene P1A and was injected in a form covalently linked to an immunostimulatory peptide derived from IL-1. We conclude that the toxicity is caused by specific CD8+ lymphocytes, which are extensively amplified by peptide immunization in a QS21-based adjuvant and produce toxic levels of TNF upon further stimulation with the peptide. Our results suggest that immunotherapy trials involving new peptides should be pursued with caution and should include a careful monitoring of the T cell response.

Inflammation and cancer, the mastocytoma P815 tumor model revisited: triggering of macrophage activation in vivo with pro-tumorigenic consequences.

Subcutaneous in vivo injections of cells of the mastocytoma line P815 in syngenic DBA/2 mice induce locally fast growing solid tumors. These have been used extensively as a cancer model to analyze and manipulate the relationship between tumor cells and host's immune defenses. We report that progression of P815 tumors in vivo was accompanied by a burst (Days 5-7) of local inflammatory cells recruitment and angiogenesis observed histologically, corroborated in vivo by MRI with gadolinium, overtranscription of macrophage activation marker genes, secretion of TNF-alpha by regional lymph node cells and concomitant systemic inflammation. No substantial overtranscriptions of either VEGF or IL-10 or TGF-beta genes were observed. Induction of COX-2 gene was a late event. To establish a possible relationship between the tumor-induced local, regional and systemic increase of pro-inflammatory mediators and progression of tumors in vivo, we carried out experiments deliberately modulating the inflammatory status of the recipient animals. Pretreatment of recipient animals by i.p. injection of thioglycolate accelerated P815 tumor growth. At the opposite, treatment of mice with either a COX-1 + COX-2 inhibitor (aspirin, 1 mg/day/mouse) or a specific COX-2 inhibitor (celecoxib, 0.13 mg/day/mouse) for 2 weeks after injection of tumor cells, significantly reduced the size and growth rate of tumors compared to control mice. Experiments carried out in vitro indicated that peritoneal macrophages from untreated animals were strongly activated by live P815 cells and by P815 membrane preparations. The tumor-induced inflammatory reaction could establish a local micro environment favoring tumor progression. The P815 tumor model might be helpful to recognize important factors controlling host/tumor relationship.

Evaluation of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture.

OBJECTIVE: To assess expression and function of cell-surface IgE receptors on the canine mastocytoma cell line C2 maintained in continuous culture. SAMPLE POPULATION: C2 cells maintained in medium lacking IgE for up to 10 passages before being stored at -80 C. PROCEDURE: Cells were thawed, cultured in medium without IgE for 1 to 3 passages, sensitized for 7 days with IgE-rich serum from dogs naturally sensitized to Ascaris suum, and stimulated with antigen Asc S1 from A suum, goat polyclonal anti-canine IgE, or calcium ionophore and phorbol myristate acetate (PMA). Percentage of intracellular beta-hexosaminidase released and concentration of tumor necrosis factor-alpha (TNF-alpha) synthesized after stimulation were determined. Expression of cell-surface IgE receptors was assessed by use of a flow cytometry. RESULTS: Immunologic stimulation (antigen or anti-IgE) failed to induce release or synthesis of detectable amounts of beta-hexosaminidase or TNF-alpha. In contrast, nonimmunologic stimulation (calcium ionophore and PMA) led to release of beta-hexosaminidase (mean +/- SEM maximum release, 23.95+/-1.96%) and synthesis of TNF-alpha (maximum concentration, 34.34+/-2.34 pg/10(6) cells). As revealed by use of flow cytometry, C2 cells expressed surface IgE receptors that bound canine IgE in vitro. CONCLUSIONS: Continuous culture of the canine mastocytoma cell line C2 in medium without exogenous IgE or cytokines and other growth factors resulted in cell-surface expression of nonfunctional IgE receptors. However, C2 cells maintained in continuous culture may still be a useful tool for the evaluation of mast cell responses to nonimmunologic stimulation and IgE receptor differentiation and maturity.

Glycolipid-anchored IL-12 expressed on tumor cell surface induces antitumor immune response.

Systemic or local administration of cytokine has been used as a mode to enhance the antitumor immune response induced by many cancer vaccines. We have investigated whether the expression of cytokines on the tumor cell surface as a glycolipid (GPI)-anchored form will be effective in inducing antitumor immune response using a GPI-anchored interleukin (IL)-12 (GPI-IL-12) as a model. GPI-IL-12-induced the proliferation of concanavalin A-activated T cells and induced IFN-gamma secretion by activated and allogeneic T cells, indicating that the membrane-expressed IL-12 can stimulate T cells. GPI-IL-12 expressed on the tumor cell surface prevented tumor growth in mice in a highly tumorigenic murine mastocytoma model. These results suggest that the cell surface-expressed GPI-IL-12 can be effective in inducing antitumor immune response, and GPI-anchored cytokines expressed on the tumor cell surface may be a novel approach to deliver cytokines at the immunization site during vaccination against cancer. Furthermore, purified GPI-anchored cytokines can be used to quickly modify tumor membranes by the protein transfer method to express the desired cytokines for vaccine development.

Down-regulation of antihost alloreactivity after bone marrow transplant permits relapse of hematological malignancy.

Relapse of leukemia remains a common event after allogeneic bone marrow transplantation, despite potential donor antihost alloreactivity present in most transplants. This work examined posttransplant relapse of the DBA/2 P815 mastocytoma in a murine model of MHC-matched, minor histocompatibility antigen (mHAg)-mismatched bone marrow transplantation (BALB/c donors into DBA/2 recipients). Antihost alloreactivity was associated with reduction of posttransplant tumor burden and prolongation of survival, but posttransplant relapse commonly occurred. No evidence of acquired resistance to immune control was found in 12 relapse reisolates. Relapse tumors remained sensitive to donor antihost CTLs in vitro, suggesting continued expression of mHAgs. Reisolates also continued to express Fas. However, loss of posttransplant alloreactivity was observed at 3 weeks. This was temporally associated with the time of relapse. Antihost alloreactivity could be reactivated in stable graft-versus-host disease-free recipients by immunization with host cells. The results of this study suggest that one mechanism for relapse after bone marrow transplant is acquired tolerance of allogeneic minor histocompatibility antigens and that posttransplant immunotherapy directed against mHAgs may induce antitumor activity.

Characterisation of tryptase and a granzyme H-like chymase isolated from equine mastocytoma tissue.

Mast cell proteinases are important inflammatory mediators in man and other species, but until now there has been no investigation of the nature of equine mast cell proteinases. These studies describe the purification and characterisation of two proteolytic components from equine mastocytoma tissue, detected using chromogenic substrates for trypsin and chymotrypsin. Following chromatographic purification, the trypsin-like component was found to be equine mast cell tryptase by N-terminal amino acid sequencing, showing a close similarity with human tryptase-beta (85% identity over 20 residues). It also had similar subunit molecular size (34-36kDa by SDS-PAGE) and substantially similar cleavage specificity to human tryptase-beta with the substrates tested. A 32kDa chymotrypsin-like component was also purified from mastocytoma extract, and termed equine mast cell proteinase-1 (eqMCP-1). The N-terminal amino acid sequence of eqMCP-1 was very similar to human granzyme H (95% over 19 residues). Rabbit antisera directed against tryptase and eqMCP-1 both detected equine mast cells by immunohistochemistry, and will be of use in future clinical studies of the relevance of mast cell proteinases in equine allergic disease.

A stable single-chain variable fragment expressing transfectoma demonstrates induction of idiotype-specific cytotoxic T-cells during early growth stages of a murine B-lymphoma.

The idiotypic determinants associated with the variable regions of antibody molecules are known to function as tumor-associated antigens (TAAs). However, there is no clear-cut evidence documenting their efficacy in inducing TAA-specific cytotoxic T-lymphocytes (CTLs). In most previous studies, idiopeptides were implicated in elicitation of TAA-specific CD4+ T-cells. Using a murine B-cell lymphoma, 2C3, we earlier demonstrated induction of splenic CD4+ and CD8+ T-lymphocytes directed to idiotypic Ig of the tumor. In the present study, we provide more direct evidence of the existence of Id-specific CTLs in the spleens of 2C3 bearing BALB/c mice using an scFv-transfectoma, P815A4, as a target. While both P815A4 and 2C3 cells were equally susceptible to cytolysis by the effector cells, lysis was evident only during early tumor progression. Moribund animals at the late stage of tumor growth failed to demonstrate any significant cytotoxic immune response against either tumor. Antibodies to MHC class I alleles Kd, Dd, Ld, beta2m and CD8 molecules all inhibited cytotoxicity. The CTL population from early tumor-bearers recognized 2C3 tumor in the context of all major H-2d alleles; however, in case of P815A4 cells, it was restricted to Kd and Dd alleles only. Based on these antibody inhibition studies, it appears that the idiopeptides generated in both tumors are in some way different, yet they were recognized equally by CTLs not only from the tumor-bearers but also by CTLs from 2C3-hyperimmune mice. It appears that scFv-containing transfectomas expressing antibody variable region epitopes would be useful for both elucidating CTL-defined idiopeptides and monitoring TAA-specific CTL response in tumor-bearing animals.

Induction of cytotoxic T lymphocyte responses against hepatitis delta virus antigens which protect against tumor formation in mice.

The cellular immune response is a crucial defense mechanism against hepatotropic viruses and in chronic viral hepatitis prevention. Moreover, hepatitis delta virus (HDV) immunogenicity may be an important component in the development of prophylactic and therapeutic vaccines. Therefore, we evaluated the immunogenicity of the small (HDAg) or large delta antigen (LHDAg) to be used as a DNA-based vaccine. We immunized different mouse haplotypes, determined cellular immune responses, and tested protection of animals against tumor formation using syngeneic tumor cells stably expressing the delta antigens. Both LHDAg and HDAg primed CD4+ and CD8+ T cell immunity against both forms of delta antigens. CD8+ T cell frequencies were about 1% and antigen-specific CD8+ T cells remained detectable directly ex vivo for at least 35 days post-injection. No anti-delta antibody responses could be detected despite multiple detection systems and varied immunization approaches. We observed protection against syngeneic tumor formation and growth in mice immunized with DNA plasmids encoding secreted or intracellular forms of HDAg and LHDAg but not with recombinant HDAg establishing the generation of significant cellular immunity in vivo. Both CD4+ and CD8+ T cells were required for antitumoral activity as determined by in vivo T cell depletion experiments. The results indicate that DNA-based immunization with genes encoding LHDAg and HDAg induces strong T cell responses and, therefore, is an attractive approach for the construction of therapeutic and prophylactic T cell vaccines against HDV.

Mechanism of melphalan-induced B7-1 gene expression in P815 tumor cells.

We have previously shown that exposure of P815 tumor cells to melphalan (L-phenylalanine mustard; L-PAM) leads to up-regulation of B7-1 surface expression, and this L-PAM-induced up-regulation requires de novo RNA synthesis and is associated with accumulation of B7-1 mRNA. Here we show that the effect of L-PAM on B7-1 surface expression can be mimicked by exposing P815 tumor cells to oxidative stress but not to heat shock. Moreover, the antioxidant N-acetyl-L-cysteine prevented the L-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells, suggesting that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced B7-1 gene expression. Although AP-1 and NF-kappaB are regarded as redox-sensitive transcription factors and the promoter/enhancer region of the B7-1 gene contains an AP-1 and an NF-kappaB binding site, exposure of P815 tumor cells to L-PAM led to rapid and transient activation only of NF-kappaB, but not AP-1, that bound specifically to a probe containing the respective binding site in the murine or human B7-1 gene. Moreover, exposure of P815 tumor cells to a cell-permeable peptide that selectively inhibits NF-kappaB activation by blocking the activation of the IkappaB-kinase complex was found to inhibit the L-PAM-induced B7-1 mRNA accumulation, indicating that NF-kappaB activation is essential for the L-PAM-induced B7-1 gene expression. Taken together, these results indicate that L-PAM leads to activation of B7-1 gene expression by activating NF-kappaB via a pathway that involves reactive oxygen species.

Epitope spreading upon P815 tumor rejection triggered by vaccination with the single class I MHC-restricted peptide P1A.

Epitope spreading has been best characterized as an exacerbating factor in CD4(+) T cell-dependent autoimmune disease models and is believed to occur via presentation of antigens liberated by tissue destruction initiated by CD4(+) T cells specific for a primary epitope. The growing evidence that exogenous antigens can also be processed and presented by class I MHC molecules has suggested that epitope spreading could occur for CD8(+) cytotoxic T lymphocyte (CTL) responses as well. In the context of anti-tumor immunity, expansion of a CTL response to include secondary epitopes could improve the efficacy of therapeutic vaccines. To determine directly whether epitope spreading can occur during an anti-tumor immune response, two defined class I MHC-binding peptides in the P815 tumor model were utilized. We observed that immunization against the single tumor peptide, P1A, followed by rejection of a P1A(+) tumor, subsequently yielded CTL activity and tumor protection against a P1A(-) tumor variant. P1A immunized mice that subsequently rejected tumor challenge developed CTL against a second defined epitope, P1E. These results indicate that, as for class II-restricted peptides in autoimmune disease, epitope spreading can occur for class I-restricted peptides during tumor rejection. A broadened CTL response may help eliminate outgrowth of antigen-negative tumor variants.