A COMPARISON OF MART-1 STIMULATED PERIPHERAL BLOOD LYMPHOCYTES AND TUMOR-INFILTRATING LYMPHOCYTES IN MELANOMA PATIENTS

Abstract

T-cells can be isolated and administered to patients as immunotherapy and are capable of mediating a decrease in tumor mass. Tumor-infiltrating lymphocytes (TILs) derived from melanoma can be an effective form of immunotherapy in melanoma patients; however, TILs are relatively difficult and expensive to grow, they require a long time to grow, and the outcome of therapy is difficult to predict. Therefore, improved methods of immunotherapy should be investigated that provide a more effective and predictable outcome. TILs have been helpful in the discovery of a number of tumor-associated antigens (TAA) specific to melanoma. MART-1 is a melanoma-associated antigen, and its epitope MART-1(₂₇₋₃₅) is specifically recognized by a majority of melanoma TIL cultures. MART-1(₂₇₋₃₅) may provide a reagent for vaccination in melanoma patients but would require the stimulation of the peripheral blood lymphocytes (PBLs) and the entrance of the PBLs into the tumor site to have an effective cytotoxic response. The potential to elicit an effective immune response from PBLs specific for melanoma using MART-1 (₂₇₋₃₅) exists, but the response of PBLs to stimulation has not been well characterized. In this study, we characterized PBLs isolated from melanoma patients that have been stimulated with MART-1 (₂₇₋₃₅) in vitro by assessing their ability to lyse cells in cytotoxicity assays and by assessing their TCR gene expression. These results were compared to TILs from the same patients that were also stimulated with MART -1(₂₇₋₃₅). The results demonstrated PBL and TIL cultures stimulated with MART- 1(₂₇₋₃₅) were both cytotoxic and possessed a relatively diverse repertoire of TCR β genes. In general, TILs had fewer TCR B variable subfamilies present and had a larger number of clones in the dominant TCR β variable subfamily. We also identified identical TCR genes present in both the TILs and PBLs of one patient. This demonstrates that one T-cell can divide and be present in both the tumor site and in the peripheral blood. In addition, TILs and PBLs are not mutually exclusive to the tumor and the peripheral blood, but rather an interchange between compartments can occur allowing TILs to enter the peripheral blood and for PBLs to enter the tumor. Overall, these results demonstrate PBLs can become stimulated to have an effective cytotoxic response against MART-1 (₂₇₋₃₅) specifically and have the potential to enter the tumor site, thus supporting the use of MART-1(₂₇₋₃₅) based vaccine therapy.