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We have selected a well-characterized human renal cell carcinoma (RCC) line as the basis for development of a genetically engineered tumor cell vaccine to be applied in an allogeneic setting. This cell line was genetically modified by retroviral transduction to express B7.1 costimulatory molecules. The unmodified tumor cells and B7.1-expressing tumor cells were compared for their ability to induce tumor-associated responses in allogeneic peripheral blood mononuclear cells (PBMC) of two normal control donors having single MHC class I allele matches with the tumor cells. PBMC primed using B7.1-modified tumor cells showed a preponderance of CD3+CD8+ cytotoxic T lymphocytes (CTL) that proliferated over extended periods of time in mixed lymphocyte tumor cell (MLTC) cultures. Strong cytolytic activity developed in the primed populations and included allospecific CTL with specificity for mismatched HLA-A, -B and -C molecules. Nevertheless, it was possible to isolate CTL clones that were able to lyse tumor cells but not lymphoblastoid cells that expressed all the corresponding allospecificities. Thus, induction of complex allospecific responses did not hinder the development of tumor-associated CTL in vitro. These results support the use of this genetically modified allogeneic tumor cell line for vaccination of partial-MHC matched RCC patients.

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Renal cell carcinomas (RCCs) are thought to be immunogenic, because cytokine-induced and even spontaneous tumor regression has been observed in a significant number of patients. However, little is known about the nature of immune responses that might lead to tumor regression. We studied naturally arising human T-cell responses against RCC by combining molecular analyses of T-cell receptor (TCR) usage in primary tumors in situ with functional analyses of tumor-infiltrating lymphocytes (TILs) in vitro. TILs of patient 26 that were cultured in vitro showed a human leukocyte antigen (HLA-A*0201)-restricted cytotoxic activity specific for autologous tumor cells. These tumor-derived lymphocytes were dominated by a family of T cells expressing V alpha20- and V beta22-positive TCRs. Their specificity-conferring third complementarity-determining regions were highly homologous with respect to the loop length and selection of particular amino acids in both TCR chains. These characteristics are similar to those reported for antigen-selected murine T cells recognizing immunodominant epitopes of non-self proteins. To evaluate the biological significance of these CTLs in vivo, we analyzed the corresponding TCR transcripts in the cryopreserved tumor material of patient 26 and in a second HLA-A*0201-positive RCC patient whose tumor cells were also lysed by TIL-26. The in situ TIL populations of both patients used related families of highly homologous TCRs, supporting the contention that immunodominant responses directed against a shared tumor-associated antigen occurred in both individuals in vivo. Furthermore, in the absence of overt metastatic disease, the tumor antigen-specific CTLs of patient 26 were shown to persist in the periphery 4 years after removal of the primary tumor. These results demonstrate that antigen-driven T-cell responses specific for spontaneously arising carcinomas developed in these patients and showed long-term persistence, even in the absence of immunotherapy.

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Renal cell carcinomas belong to the small group of tumors that are able to induce antitumor responses. Here we describe two general types of cytotoxic effector lymphocytes that can eliminate autologous tumor cells and discuss the role that major histocompatibility complex encoded molecules play in governing their specificities. Improved understanding of the cellular and molecular basis of renal cell carcinoma recognition opens new avenues of research with the potential to develop better immunotherapies for patients with metastatic disease.

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The nature of alloantigens seen by T lymphocytes, in particular the role of peptides in allorecognition, has been studied intensively whereas knowledge about the in vivo emergence, diversity, and the structural basis of specificity of alloreactive T cells is very limited. Here we describe human T cell clones that recognize HLA-B35 alloantigens in a peptide-dependent manner. TCR sequence analysis revealed that several of these allospecific clones utilize homologous TCR: they all express TCRAV2S3J36C1 and TCRBV4S1J2S7C2 chains with highly related CDR3 sequences. Thus peptide-specific alloreactivity is reflected in homologous CDR3 sequences in a manner similar to that described for T cells that recognize nominal peptide/self-MHC complexes. The in vivo frequency of this TCR specificity was studied in unstimulated PBL of the responding cell donor who was not sensitized against HLA-B35. The vast majority (approximately 75%) of the VA2S3J36 junctional regions obtained from two samples of PBL, isolated at a 9-yr interval, encode CDR3 identical or homologous to those of the functionally characterized HLA-B35 allospecific T cells. These data are most easily explained by a model of alloreactivity in which persistent or recurrent exposure to a foreign peptide/self-MHC complex led to the in vivo expansion and long-term maintenance of specific T cells that show fortuitous crossrecognition of an HLA-B35/peptide complex and dominate the alloresponse against HLA-B35.

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