Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.     

Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells

Vaccination with dendritic cells (DCs) pulsed with antigenic peptides derived from various tumor antigens has great, but as yet significantly unrealized, potential in cancer treatment. Here, we describe a strategy for prolonged presentation of an MHC class I-restricted self-peptide on DCs through linkage of it to a cell penetrating peptide (CPP). DCs loaded with a peptide derived from tyrosinase-related protein 2 (TRP2) covalently linked to a CPP1 sequence retained full capacity to stimulate T cells for at least 24 h, completely protected immunized mice from subsequent tumor challenge, and significantly inhibited lung metastases in a 3-day tumor model. DCs pulsed with TRP2 alone failed to provide any of these protections. In addition, we demonstrate that both CD4+ and CD8+ T cells were required for potent antitumor immunity. This CPP-based approach may be generally applicable to enhance the efficacy of DC-based peptide vaccines against cancer and other diseases.     

Provision of granulocyte-macrophage colony-stimulating factor converts an autoimmune response to a self-antigen into an antitumor response

Many tumor Ags recognized by T cells are self-Ags. Because high avidity, self-reactive T cells are deleted in the thymus, any residual self-reactive T cells existing in the periphery are likely to be low avidity and nonresponsive due to peripheral tolerance mechanisms. Activation of these residual T cells is critical for targeting tumors for immunotherapy. In this study, we studied immune responses against the murine B16 melanoma using a tyrosinase-related protein 2 (TRP-2) peptide as a model tumor/self-Ag. Our results showed that TRP-2 peptide vaccination alone elicited a weak T cell response and modestly decreased B16 lung tumor nodules. The combination of peptide vaccination and treatment with an Ab directed against the inhibitory receptor CTLA-4 enhanced the immune response against TRP-2 peptide, inducing autoimmune depigmentation and further decreasing lung tumor nodules. However, both vaccination methods failed to protect against orthotopic (s.c.) B16 tumor challenge. The addition of an irradiated GM-CSF-expressing, amelanotic tumor cell vaccine significantly delayed s.c. B16 tumor growth. Subsequent studies revealed that provision of GM-CSF increased dendritic cell numbers in lymph nodes and spleen. Furthermore, addition of CTLA-4 blockade increased the frequency of TRP-2-specific, IFN-secreting T cells in spleen and lymph nodes. Overall, our results indicate that combining enhancement of Ag presentation with removal of CTLA-4-mediated inhibition can convert a "weaker" autoimmune response into a more potent antitumor immune response.     

TNF-alpha-treated DC exacerbates disease in a murine tumor metastasis model

Due to the pivotal role that dendritic cells (DC) play in eliciting functional anti-tumor T cell responses, immunotherapeutic approaches utilizing DC-based vaccines have readily been exploited. It has been argued that, in the setting of immunotherapy, mature DC will be more efficient at T cell priming and, therefore, required for effective vaccination. As TNF-alpha is commonly used as a DC maturation factor, we have examined the efficacy of treatment with DC matured with TNF-alpha (DC-TNF) in a murine model of melanoma. We have now shown that treatment with DC-TNF leads to an increase in the number of lung metastases as compared to mice treated with immature DC. No differences in the number of CD4⁺CD25⁺ T-regulatory cells were measured in the lungs of DC-TNF-treated mice. On examination of the infiltrating lymphocytes, an enhanced secretion of IL-10 and a higher percentage of CD4⁺IL-10⁺ T cells were measured in the lungs of DC-TNF-treated mice. However, treatment with DC-TNF did not enhance the number of melanoma lesions in the lungs of IL-10 knockout mice or in mice depleted of CD4⁺ T cells. Together, these studies indicate that treatment of melanoma-bearing mice with DC treated with TNF-alpha can induce IL-10 production by resident cells at the tumor site, leading to immune tolerance and exacerbation of disease.     

CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

High-affinity HLA-A(*)02.01 peptides from parathyroid hormone-related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.     

Enhanced Responses to Tumor Immunization Following Total Body Irradiation Are Time-Dependent

The development of successful cancer vaccines is contingent on the ability to induce effective and persistent anti-tumor immunity against self-antigens that do not typically elicit immune responses. In this study, we examine the effects of a non-myeloablative dose of total body irradiation on the ability of tumor-naïve mice to respond to DNA vaccines against melanoma. We demonstrate that irradiation followed by lymphocyte infusion results in a dramatic increase in responsiveness to tumor vaccination, with augmentation of T cell responses to tumor antigens and tumor eradication. In irradiated mice, infused CD8⁺ T cells expand in an environment that is relatively depleted in regulatory T cells, and this correlates with improved CD8⁺ T cell functionality. We also observe an increase in the frequency of dendritic cells displaying an activated phenotype within lymphoid organs in the first 24 hours after irradiation. Intriguingly, both the relative decrease in regulatory T cells and increase in activated dendritic cells correspond with a brief window of augmented responsiveness to immunization. After this 24 hour window, the numbers of dendritic cells decline, as does the ability of mice to respond to immunizations. When immunizations are initiated within the period of augmented dendritic cell activation, mice develop anti-tumor responses that show increased durability as well as magnitude, and this approach leads to improved survival in experiments with mice bearing established tumors as well as in a spontaneous melanoma model. We conclude that irradiation can produce potent immune adjuvant effects independent of its ability to induce tumor ablation, and that the timing of immunization and lymphocyte infusion in the irradiated host are crucial for generating optimal anti-tumor immunity. Clinical strategies using these approaches must therefore optimize such parameters, as the correct timing of infusion and vaccination may mean the difference between an ineffective treatment and successful tumor eradication.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

Induction of anti-tumor immunity by vaccination with dendritic cells pulsed with anti-CD44 IgG opsonized tumor cells

Due to the pivotal role that dendritic cells (DC) play in eliciting and maintaining functional anti-tumor T cell responses, these APC have been exploited against tumors. DC express several receptors for the Fc portion of IgG (Fcγ receptors) that mediate the internalization of antigen-IgG complexes and promote efficient MHC class I and II restricted antigen presentation. In this study, the efficacy of vaccination with DC pulsed with apoptotic B16 melanoma cells opsonized with an anti-CD44 IgG (B16-CD44) was explored. Immature bone marrow derived DC grown in vitro with IL-4 and GM-CSF were pulsed with B16-CD44. After 48 h of pulsing, maturation of DC was demonstrated by production of IL-12 and upregulation of CD80 and CD40 expression. To test the efficacy of vaccination with DC+B16-CD44, mice were vaccinated subcutaneously Lymphocytes from mice vaccinated with DC+B16-CD44 produced IFN-γ in response to B16 melanoma lysates as well as an MHC class I restricted B16 melanoma-associated peptide, indicating B16 specific CD8 T cell activation. Upon challenge with viable B16 cells, all mice vaccinated with DC alone developed tumor compared to 40% of mice vaccinated with DC+B16-CD44; 60% of the latter mice remained tumor free for at least 8 months. In addition, established lung tumors and distant metastases were significantly reduced in mice treated with DC+B16-CD44. Lastly, delayed growth of established subcutaneous tumors was induced by combination therapy with anti-CD44 antibodies followed by DC injection. This study demonstrates the efficacy of targeting tumor antigens to DC via Fcγ receptors.     

Vaccination of a melanoma patient with mature dendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumor cells

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of approximately 10(-5) among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2x10(-4) among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression.     

Synergism between CpG-containing oligodeoxynucleotides and IL-2 causes dramatic enhancement of vaccine-elicited CD8+ T cell responses

Novel anticancer vaccination regimens that can elicit large numbers of Ag-specific T cells are needed. When we administered therapeutic vaccines containing the MHC class I-presented self-peptide tyrosinase-related protein (TRP)-2(180-188) and CpG-containing oligodeoxynucleotides (CpG ODN) to mice, growth of the TRP-2-expressing B16F1 melanoma was not inhibited compared with growth in mice that received control vaccinations. When we added systemic IL-2 to the TRP-2(180-188) plus CpG ODN vaccines, growth of B16F1 was inhibited in a CD8-dependent, epitope-specific manner. Vaccines containing TRP-2(180-188) without CpG ODN did not cause epitope-specific tumor growth inhibition when administered with IL-2. The antitumor efficacy of the different regimens correlated with their ability to elicit TRP-2(180-188)-specific CD8+ T cell responses. When we administered TRP-2(180-188) plus CpG ODN-containing vaccines with systemic IL-2, 18.2% of CD8+ T cells were specific for TRP-2(180-188). Identical TRP-2(180-188) plus CpG ODN vaccines given without IL-2 elicited a TRP-2(180-188)-specific CD8+ T cell response of only 1.1% of CD8+ T cells. Vaccines containing TRP-2(180-188) without CpG ODN elicited TRP-2(180-188)-specific responses of 2.8% of CD8+ T cells when administered with IL-2. There was up to a 221-fold increase in the absolute number of TRP-2(180-188)-specific CD8+ T cells when IL-2 was added to TRP-2(180-188) plus CpG ODN-containing vaccines. Peptide plus CpG ODN vaccines administered with IL-2 generated epitope-specific CD8+ T cells by a mechanism that depended on endogenous IL-6. This is the first report of synergism between CpG ODN and IL-2. This synergism caused a striking increase in vaccine-elicited CD8+ T cells and led to epitope-specific antitumor immunity.     

Simultaneous and Dose Dependent Melanoma Cytotoxic and Immune Stimulatory Activity of Betulin

Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.     

IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response

IL-21, a newly described cytokine belonging to the IL-2 gamma-chain receptor cytokine family (that includes IL-2, IL-7, and IL-15), has been described as an important regulator of the cellular immune response. In this study, the role of IL-21 in the generation of a human Ag-specific CD8+ T cell response is characterized by tracking a rare, but measurable population of self-Ag-specific T cells in vitro. Autologous dendritic cells pulsed with the melanoma antigen recognized T cells 1 self-peptide were used to stimulate CD8+ T cells from HLA-A2+ healthy donors and melanoma patients. We demonstrate that exposure to IL-21 increased the total number of MART-1-specific CD8+ T cells that could be elicited by >20-fold and, at the clonal level, enriched for a population of high-affinity CD8+ T cells with a peptide dose requirement more than 1 log(10)-fold less than their untreated counterparts. Phenotypic analysis of T cells from IL-21-treated cultures revealed a unique population of CD45RO+ CD28(high) CD8+ T cells, a phenotype that was stable for at least 4 wk after IL-21 exposure. These CD28(high) CD8+ T cells produced IL-2 upon Ag stimulation and represent potential helper-independent CTLs. Our studies demonstrate a significant role for IL-21 in the primary Ag-specific human CTL response and support the use of IL-21 in the ex vivo generation of potent Ag-specific CTLs for adoptive therapy or as an adjuvant cytokine during in vivo immunization against tumor Ags.     

Tumors hamper the immunogenic competence of CD4+ T cell-directed dendritic cell vaccination

Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44(high)CD62L(low), IL-2(+), IFN-gamma(+)) and central memory-like lymphocytes (CD44(high)CD62L(high), only IL-2(+)) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

Mechanisms of murine dendritic cell antitumor dysfunction in aging

Effective cancer immunotherapy depends on the body’s ability to generate tumor antigen-presenting cells and tumor-reactive effector lymphocytes. As the most potent antigen presenting cells (APCs), dendritic cells (DCs) are capable of sensitizing T cells to new and recall antigens. Clinical trials of antigen-pulsed autologous DCs have been conducted in patients with a number of hematological and solid cancers, including malignant melanoma, lymphoma, myeloma, and non-small cell lung cancer. These studies suggest that antigen-loaded DC vaccination is a potentially safe and effective cancer therapy. However, the clinical results have been variable. Since the elderly are preferentially affected by diseases targeted by DC-directed immunotherapy, it is quite striking that few studies to date have focused on the effect of aging on DC function, a key aspect of optimal immunotherapy design in an aging population. In the present paper, we will discuss the consequences of aging on murine bone marrow-derived DC function and their use in cancer immunotherapy.     

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

A B16 melanoma-specific CD8⁺ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor V_β11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2K^b gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2K^b monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2_181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type V_β11 ⁺ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2_181–188 peptide in an H-2K^b-restricted manner. Received: 4 June 1998 / Accepted: 21 July 1998