CTL-based Immunotherapy in an Organotypic Me

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Infiltration of primary melanomas with T lymphocytes is associated with a favorable clinical outcome. This suggests that T cell-based immunotherapies may be beneficial for melanoma patients. However, adoptive cytolytic T lymphocyte (CTL) immunotherapy of melanoma patients and active immunotherapy with CTL-based antigen (Ag)/peptide vaccines have not held promise. These limitations may rest in the selection for immunotherapy of CTL in vitro that do not reflect the Ag specificity of the CTL in situ. CTL traditionally are selected in two-dimensional mixed lymphocyte-tumor culture (MLTC) directly on plastic surfaces. We have developed the three-dimensional human melanoma organolypic culture model (melanoma reconstruct) in which CTL are cultured under tissue-like condition. Our preliminary studies suggest that the T cell receptor (TCR) repertoire in tumor reconstructs more closely resembles the repertoire of the T cells in situ as compared to the TCR repertoire in MLTC. However, the two culture systems are complementary. Thus, the chances of generating T cells with in situ (tissue)-Iike TCR are enhanced when T cells from both culture systems are cornbined, compared to either culture alone. Our major goal is to develop, including the melanoma reconstruct as a culture system, improved CTL-based adoptive and active immunotherapies against melanoma. Specifically we will: 1. Compare the phenotypic (CD markers, TCR, adhesion and apoptosis-related molecules, HLA, chemokines, chemokine receptors) and functional characteristics (proliferative and lytic activity; cytokine production) of CTL derived from fresh tissues in the reconstruct or MLTC. 2. Clone Ag(s) recognized by CTL that are highly cytolytic and express the original TCR found in situ. These Ags have vaccine potential for melanoma patients and may boost pre-existing immune responses. 3. Identify chemokines and chemokine receptors that are involved in CTL migration toward tumor cells in the reconstruct. This involves: i) Selection of migrating CTL and ii) blocking of chemokines and their receptors to determine their role in CTL migration. Chemokines relevant in CTL migration in the reconstruct may be used to enhance anti-tumor effects of adoptive and active immunotherapy. The proposed studies open new approaches to CTL-based adoptive and active immunotherapies, which may be initiated during the third year of the SPORE.