The overall goals of this research are directed toward elucidating the minimum requirements for the stimulation of T cells by Ia molecules on allogeneic-stimulator cells in the mixed lymphocyte reaction (MLR). The objectives and specific aims for the coming year are as follows: (1) To continue the evaluation of the capacity of resting B cells and B cells at various stages of activation to present allo-Ia-determinants for a MLR as well as to stimulate alloreactive T cell hybridomas. Primary emphasis of these studies will be directed towards the question of Ia-antigen density and stimulating capacity and also towards evaluating possible structural differences (e.g., carbohydrate composition) that may relate to stimulating capacity. (2) To determine whether Ia allostimulation can be defined in molecular terms by the use of a cell-free stimulating system comprised of purified Ia antigens and soluble cytokines. Several alloreactive T-cell hybridomas have been produced over the last year. We will evaluate the ability of plasma membrane fractions, detergent-solubilized reconstituted membrane vesicles from an Ia?+? B-cell lymphoma plasma membrane fraction, and liposomes containing affinity purified I-A and I-E antigens to serve as the alloantigen stimulus for these alloreactive T-cell hybridomas. (3) To isolate and characterize the T-cell receptor for Ia alloantigen. The alloreactive T-cell hybridomas will serve as a clonal source for the isolation of receptor material. A monoclonal antibody, KJ16.133, produced at this institution, which reacts with T-cell antigen receptors on some but not all T-cell populations, is available for this study. One or our alloreactive T-cell hybridomas, DlGlO, is reactive with this monoclonal reagent. This monoclonal antibody will be used as an affinity matrix for the isolation and characterization of the D1G10 T-cell receptors for allo-Ia. In addition, we have recently prepared a xenogeneic antiserum (rabbit) against murine T-cell receptors which appears to be pan-specific. This antiserum detects T-cell receptors on both KJ16.133-positive and KJ16.133-negative T-cell hybridomas, as well as normal T cells. (LB)
