In the past decade, a wealth of tumor-associated antigens have been identified that provide targets for CTL and CD4+
T cells in cancer patients (for reviews, see references 1
). Human melanoma has provided a paradigm in this respect, demonstrating that such tumor antigens can be derived from tissue-specific self-antigens, from tumor-specific mutated proteins, or from aberrantly expressed proteins that normally function only during embryonic development. Depigmentation or vitiligo (spontaneous or treatment related) appears to correlate with favorable prognosis and successful rejection of metastatic melanoma, indicating that self-antigens might play a role in antitumor responses 34
. Melanocyte proteins involved in pigment synthesis have frequently been shown to be targets for CTLs, CD4+
T cells, and antibodies in melanoma patients or in healthy donors. In vitiligo and melanoma patients, tumor-associated and potentially autoimmune T cell–mediated reactivity can be linked directly to destruction of pigmented cells in the skin 56
. As these pigmentation antigens are expressed by most melanomas even in advanced stages, they are considered to be good candidate targets for specific immunotherapy, provided that damage to healthy tissue is limited. Importantly, immunological tolerance to these melanocyte antigens is clearly incomplete or absent providing the rationale for clinical testing of vaccines consisting of pigmentation proteins or their T cell epitopes. A satisfactory animal model for melanoma treatment is essential for assessment of the risks and requirements for such immunotherapy to be successful.
T cell responses are regulated not only by antigen receptor signals, but also by positive and inhibitory costimulatory signals mediated by the interactions of CD28 and CTLA-4, respectively, with their cognate B7 ligands on antigen-presenting cells 7
. We and others have shown that administration of antibodies to block the inhibitory effects of CTLA-4 can enhance antitumor responses in several murine tumor models 891011121314
. Of particular relevance to melanoma is our demonstration that mice carrying a small load of B16 melanoma cells can be successfully treated with a combination vaccine consisting of GM-CSF–producing, irradiated B16 cells and CTLA-4 blockade 13
. Approximately 60% of the surviving mice developed lesions of depigmentation reminiscent of vitiligo. B16 therapy in this model absolutely requires the presence of CD8+
cells. Our finding that the combination therapy was CD8 dependent and resulted in depigmentation was especially interesting in light of previous studies using the GM-CSF–producing B16 vaccine as a single agent 15
. In these studies it was found that the vaccine was capable of inducing prophylactic immunity mediated mainly by CD4+
T cells with no requirement for CD8+
T cells 1516
. Also, this cell-based vaccine by itself was not effective in a therapeutic setting, and depigmentation was not reported even when prophylactic immunity had been successfully obtained.
We now report an extensive examination of the mechanisms involved in the generation of immunity to B16 using the combination therapy. We have found that neither CD4+ T cells, nor antibody responses are required for treatment effect, as tumor rejection and depigmentation occur in B cell–deficient mice. Both the Fas and perforin pathways are required, but TNF-α is dispensable. T cells responding to the melanocyte differentiation antigen tyrosinase-related protein 2 (TRP-2) antigen were found in spleen cell cultures and in peripheral blood from depigmented mice. Depletion studies confirm that CD8+ T cells are required for both the antitumor effect and depigmentation in a therapeutic setting. In the setting of prophylaxis, CD8+ T cells are not required for tumor immunity, but are required for depigmentation. These findings demonstrate that the cellular mechanisms involved in protection against subsequent tumor challenge are different from those observed in a therapeutic setting. This underscores the added value of studying tumor immunity in tumor-bearing subjects.