Dendritic cells, the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In three different well-characterized tumour models, naive mice injected with bone marrow-derived dendritic cells prepulsed with tumour-associated peptides previously characterized as being recognized by class I major histocompatibility complex-restricted cytotoxic T lymphocytes, developed a specific T-lymphocyte response and were protected against a subsequent lethal tumour challenge. Moreover, in the C3 sarcoma and the 3LL lung carcinoma murine models, treatment of animals bearing established macroscopic tumours (up to 1 cm2 in size) with tumour peptide-pulsed dendritic cells resulted in sustained tumour regression and tumour-free status in more than 80% of cases. These results support the clinical use of tumour peptide-pulsed dendritic cells as components in developing effective cancer vaccines and therapies.

Sera of patients with cancer contain membraneous microvesicles (MV) able to induce apoptosis of activated T cells by activating the Fas/Fas ligand pathway. However, the cellular origin of MV found in cancer patients’ sera varies as do their molecular and cellular profiles. To distinguish tumor-derived MV in cancer patients’ sera, we used MAGE 3/6+ present in tumors and MV. Molecular profiles of MAGE 3/6+ MV were compared in Western blots or by flow cytometry with those of MV secreted by dendritic cells or activated T cells. These profiles were found to be distinct for each cell type. Only tumor-derived MV were MAGE 3/6+ and were variably enriched in 42-kDa Fas ligand and MHC class I but not class II molecules. Effects of MV on signaling via the TCR and IL-2R and proliferation or apoptosis of activated primary T cells and T cell subsets were also assessed. Functions of activated CD8+ and CD4+ T lymphocytes were differentially modulated by tumor-derived MV. These MV inhibited signaling and proliferation of activated CD8+ but not CD4+ T cells and induced apoptosis of CD8+ T cells, including tumor-reactive, tetramer+CD8+ T cells as detected by flow cytometry for caspase activation and annexin V binding or by DNA fragmentation. Tumor-derived but not dendritic cell-derived MV induced the in vitro expansion of CD4+CD25+FOXP3+ T regulatory cells and enhanced their suppressor activity. The data suggest that tumor-derived MV induce immune suppression by promoting T regulatory cell expansion and the demise of antitumor CD8+ effector T cells, thus contributing to tumor escape.

SummaryAntigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic routine tumor models that bone marrow (BM)-derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J. I., T. Zorina, W.J. Storkus, C. Celluzzi, L. D. Falo, C.J. Melief, T. lldstad, W. M. Kast, A. B. DeLeo, and M. T. . Nature Med. 1:1297-1302. However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony-stimulating factorstimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W. J., H.J. Zeh III, R. D. Salter, and M. T. Lotze. 1993.J. lmmunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 3( 105 tumor peptide-pulsed DC dramatically suppressed the growth ofweaHy immunogenic tumors in day 4 to day 8 established MCA205 (H-2 b) and TS/A (H-2 a) tumor models, when applied in three biweeHy intravenous injections. Using the immunogenic C3 (H-2 b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4 + or CD8 + T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor-derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) ~/ production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DCinduced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1-associated cytokines (such as IL-12, tumor necrosis factor or, IFN-~/), and eventually, but not initially, blocked by anti-raiL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weaHy imnmnogenic tumors, and serves as a basis for designing clinical trials in cancer patients.R ecent advances in the understanding of antigen presentation, antigen reco...

SummaryCytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential imrnunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8 + T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other’s functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized “effector/memory” DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-γ, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, “helper” function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.

T helper type 1 (Th1)-type CD4+ antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4+ T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4+ T cells from human histocompatibility leukocyte antigens (HLA)-DRβ1*0401+ patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-γ and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6–derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus– or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4+ T cell secretion of IL-10 and transforming growth factor (TGF)-β1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4+ subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4+ T cell responses to provide optimal clinical benefit.

SUMMARY:Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. High frequencies of melanocyte-reactive cytotoxic T cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional T-cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. We isolated T cells from both perilesional and nonlesional skin biopsies obtained from five vitiligo patients, then cloned and analyzed their profile of cytokine production after short-term, nonspecific expansion in vitro. Perilesional T-cell clones (TCC) derived from patients with vitiligo exhibited a predominant Type-1-like cytokine secretion profile, whereas the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Detailed analysis of broad spectrum of cytokines produced by perilesional-and nonlesional-derived CD4 ϩ and CD8 ϩ TCC confirmed polarization toward Type-1-like in both CD4 and CD8 compartments, which paralleled depigmentation process observed locally in the skin. Furthermore, CD8 ϩ TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8 ϩ TCC isolated from perilesional biopsies of two patients with vitiligo. Finally, in two of five patients, tetramer analysis revealed presence of high frequencies of Mart-1-specific CD8 T cells in T-cell lines derived from perilesional skin. Altogether our data support the role of cellular mechanisms playing a significant part in the destruction of melanocytes in human autoimmune vitiligo. (Lab Invest 2003, 83:683-695).

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