Our objective is to study and compare the ability of functionally distinct T cell clones to mediate allograft rejection in a new, more completely T cell depleted mouse model. Combining two recent advances, monoclonal antibody-toxin conjugates for more efficient elimination of T cells from bone marrow and T cell cloning to provide functionally distinct, pure T cell subsets for adoptive transfer, it will be possible to examine this problem in a more definitive way. This program will be completed in three overlapping phases. First, T cell depleted (ATXBM) mice will be prepared by adult thymectomy, lethal total body irradiation, and reconstitution with bone marrow depleted of T cells by pretreatment in vitro with monoclonal anti-Thy 1.2 antibody conjugated to the plant toxin ricin with known inhibitory activity and specificity. Depleted bone marrow and ATXBM mice will be rigorously examined for the presence of residual T cells and T cell reactivity in vitro and in vivo. In vitro testing will include examination of depleted bone marrow and bone marrow, lymph node, spleen, and peripheral blood cells from ATXBM mice for the presence of Thy-1.2 positive cells using the flourescence activated cell sorter. The presence and frequency of proliferative and cytolytic T cell precursors will be determined using limiting dilution analysis in alloantigen and mitogen stimulated cultures. In vivo testing will center on the ability of these mice to reject skin allografts and whether an increase in frequency of cytoltic or proliferative T cell precursors can be identified after skin or sponge matrix grafting. Second, cytolytic, helper, and helper-independent cytolytic T cell clones will be derived by limiting dilution and characterized in terms of phenotype, specific cytolytic activity, proliferative response to antigen, requirement for exogenous interleukin-2 for proliferative response to antigen, and production of interleukin-2 in response to antigen or concanavalin A. Third, T cell clones, alone or in various combinations, with or without the addition of lymphokines, will be adoptively transferred into ATXBM mice bearing skin allografts, and migration patterns and graft survival times compared. The ability of clones to alter patterns and magnitude of cell migration and inflammation in ATXBM mice will also be examined. The ability to develop and characterize a more thoroughly T cell-depleted mouse model and to adoptively transfer T cell clones should allow the identification of the role of T cell subsets and their in vivo interaction in mediating allograft rejection. This should also provide the basis for the development of specific monoclonal antibody-toxin conjugates to T cell subsets for modulating allograft rejection experimentally and in the clinical setting.
| Shelton, M W; Walp, L A; Basler, J T et al. (1992) Mediation of skin allograft rejection in scid mice by CD4+ and CD8+ T cells. Transplantation 54:278-86 |
| Berens, E S; Shelton, M W; Walp, L A et al. (1989) Survival of cytotoxic T-cell clones (Tc) in vivo and their ability to effect skin allograft rejection in T-cell depleted mice. Transplant Proc 21:138-9 |
