The proposed research will delineate the effects of removing or inhibiting tumor-induced T suppressor (Ts) cells on the in vitro activation of tumor-reactive lymphocytes in mice and humans. Using the P815 tumor model in which we have been studying Ts effects, Ts cells from tumor-bearing hosts (TBH) whose ability to respond to their tumors has begun to decline, will be removed or inhibited by in vivo treatment with cyclophosphamide (CYP), irradiation, or cimetidine or by in vitro treatment with 4- hydroperoxycyclophosphamine (4HpCY) or cimetidine. We will first determine whether these manipulations increase the generation of CTL and subsequently, whether they will increase the in vitro expansion of tumor-reactive cells in interleukin-2- supplemented cultures. We will also test whether depletion of Lyt2+ or L3T4+ T-cell subsets improves the propagation of T cells with helper or lytic functions, respectively, respectively. Murine tumor-reactive T cells expanded in vitro after Ts depletion will then be tested for in vivo efficacy in early TBHs and in our adjuvant model (TBH mice with systemic metastases and in whom Ts activity has become dominant). The adoptive transfer of these T cells will be combined with anti-Ts treatments of the host and exogenous IL-2. In our preliminary studies of the in vitro immune responses of cells from draining lymph nodes (DLN) breast cancer patients, there is considerable variability among DLNs from each patient, and the proliferative response can be significantly increased by removal of Leu-7+ cells. In the proposed research, we will determine the effect of eliminating or inhibiting Ts cells on the in vitro proliferation and expansion of tumor-reactive lymphocytes from breast cancer patients' DLN (obtained at the time of surgery). Ts cells will be removed on the basis of phenotypic markers (Leu-7, OKM1, CD8 antigens and Fc receptors) or inhibited by in vitro incubation with 4-HpCY. Propagation of cytolytic and T helper cells will also be studied. For both the murine and human lymphocytes, we will also determine whether Ts-depleted tumor-reactive T cells can be propagated by stimulation with tumor antigen or phorbol dibutyrate + ionomycin and IL-2, as we have done for normal mouse and human killer lymphocytes. Thus, by combining abrogation of Ts function with activation and expansion of tumor-reactive T cells and testing in vivo efficacy in a relevant mouse model, the research proposed should lead to improved strategies of adoptive immunotherapy for cancer.
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