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The adoptive transfer of tumor-reactive CD8+ T cells into tumor-bearing hosts provides an attractive alternative to vaccination-based active immunotherapy of melanoma. The development of techniques that result in the preferential expansion of tumor-reactive T cells is therefore of great importance. In this study, we report the generation of HLA-A*0201-restricted CD8+ T cell populations that recognize either tyrosinase369–376 or gp100209–217 from tolerant human class I MHC-transgenic mice by using single amino acid-substituted variant peptides. Low peptide concentration or restimulation with the parent peptide was used to enhance the functional avidity, defined by stimulation of IFN-γ accumulation, and cross-reactivity of the resulting T cell populations. We found a direct correlation between the ability of a T cell population to respond in vitro to low concentrations of the precise peptide expressed on the tumor and its ability to delay the outgrowth of B16 melanoma after adoptive transfer. Surprisingly, we found that some T cells that exhibited high functional avidity and were effective in controlling tumor outgrowth exhibited low structural avidity, as judged by MHC-tetramer staining. Our results establish strategies for the development and selection of CD8+ T cell populations that persist despite peripheral tolerance, and that can control melanoma outgrowth. Furthermore, they support the use of human MHC class I-transgenic mice as a preclinical model for developing effective immunotherapies that can be rapidly extended into therapeutic settings.

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201+ melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD+tyrosinase+ mice after activation under certain conditions. At least some of these residual CTLs in AAD+tyrosinase+ mice were of high avidity and induced vitiligo upon adoptive transfer into AAD+tyrosinase+ hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.

Several HLA-A*0201-restricted peptide epitopes that can be used as targets for active immunotherapy have been identified within melanocyte differentiation proteins. However, uncertainty exists as to the most effective way to elicit CD8+ T cells with these epitopes in vivo. We report the use of transgenic mice expressing a derivative of HLA-A*0201, and dendritic cells, to enhance the activation of CD8+ T cells that recognize peptide epitopes derived from human tyrosinase and glycoprotein 100. We find that by altering the cell surface density of the immunizing peptide on the dendritic cells, either by pulsing with higher concentrations of peptide, or by changing the MHC-peptide-binding affinity by generating variants of the parent peptides, the size of the activated CD8+ T cell populations can be modulated in vivo. Significantly, the density of peptide that produced the largest response was less than the maximum density achievable through short-term peptide pulsing. We have also found, however, that while some variant peptides are effective at eliciting both primary and recall CD8+ T cell responses that can recognize the parental epitope, other variant epitopes lead to the outgrowth of CD8+ T cells that only recognize the variant. HLA-A*0201 transgenic mice provide an important model to define which peptide variants are most likely to stimulate CD8+ T cell populations that recognize the parental, melanoma-specific peptide.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Self-tolerance to melanocyte differentiation Ags limits the ability to generate therapeutic antimelanoma responses. However, the mechanisms responsible for CD8 T cell tolerance to these Ags are unknown. We have used a newly generated TCR-transgenic mouse to establish the basis of tolerance to one such Ag from tyrosinase. Despite expression of tyrosinase transcripts in the thymus, central deletion does not shape the tyrosinase-specific CD8 T cell repertoire. We demonstrate that this endogenously expressed melanocyte Ag is constitutively presented in both peripheral and mesenteric lymph nodes, leading to abortive activation and deletion of tyrosinase-specific CD8 T cells. Importantly, this Ag is not presented by either radio-sensitive dendritic cells, or by radio-resistant Langerhans cells. Thus, for this endogenous Ag, cross-tolerization does not appear to be an operative mechanism. Instead, we find radioresistant tyrosinase mRNA expression in lymphoid compartments where CD8 T cell deletion occurs. This suggests that direct presentation of tyrosinase by radio-resistant lymph node resident cells is entirely responsible for tolerance to this endogenous melanocyte differentiation Ag.

Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.