This proposal addresses the central hypothesis that significant antitumor responses can be achieved through the use of fever- range hyperthermia to stimulate L-selectin and alpha4beta7 integrin-dependent recruitment of immune effector cells to malignant tissues and tumor-draining lymph nodes. The hypothesis is formulated on the basis of new information demonstrating that fever range, long duration whole body hyperthermia (WBH) stimulates expression of endothelial ligands for two leukocyte homing receptors, L-selectin and alpha4beta7 integrin, on tumor vessels in the RIP-Tag5 transgenic mouse model. This finding is correlated with increased lymphocyte infiltration into beta islet tumors. Notably, pancreatic tumors that develop as a result of transgene expression of the SV40 T antigen (Tag) in RIP-Tag5 mice are normally devoid of infiltrating lymphocytes. Three independent approaches are proposed to address the central hypothesis: (1) Strategies are designed to examine the direct role of L-selectin and alpha4beta7 integrin in targeting immune effector cells to pancreatic beta islet tumors in response to fever-range WBH in RIP-Tag5 single transgenic mice and in RIP- Tag5 double transgenic mice that have a high frequency of tumor- reactive cytotoxic T lymphocytes (CTL) as a result of coexpression of the B7.1 costimulatory molecule or a Tag-specific T cell receptor. The specific role of adhesion molecules in lymphocyte recruitment will be delineated by antibody blockade studies and using cell lines that restrictively bind to endothelium via L-selectin or alpha4beta7 integrin. (2) The molecular mechanisms underlying hyperthermia control of lymphocyte-endothelial adhesion will be investigated in cell lines using in vitro kinase assays and dominant negative approaches to examine the involvement of specific signal transduction pathways (i.e., MAPK, HSP90, JAK/STATs, Src kinases, and IL-6). (3) Studies are designed to determine if fever-range WBH, in combination with cytokines (IFN-alpha, IL-2), inhibits tumor progression in RIP-Tag transgenic mice by enhancing L- selectin and/or alpha4beta7 integrin-dependent accumulation of CTL in beta islet tumors. WBH effects on intratumoral localization of immune cells that produce Th1-cytokines (e.g., IFN-gamma) will also be examined since these cytokines are fundamental to the generation of cell-mediated tumor immunity. The proposed studies are expected to provide a framework for future evaluation of the efficacy of fever-range WBH as an adjuvant in the treatment of cancer in combination with immunotherapies designed to enhance the generation of tumor- specific CTL.
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