The goal of this application is to understand the role played by the CD4 and CD8 coreceptors in T cell activation. Furthermore, the effect of HIV-gp 120 binding to CD4 will be studied on T cell activation. Studies from many laboratories have definitively shown that the ligands for CD4 and CD8 are on class II and class I MHC molecules, respectively. Conclusive evidence has been provided that CD4 and CD8 are signaling molecules that require association with the lymphoid specific tyrosine kinase, p56lck, for their function. In many of the studies, these studies have expressed wild-type or mutant human CD4 and CD8 molecules by DNA mediated gene transfer in an antigen reactive murine T cell hybridoma. This model system has allowed the study of the human CD4 and CD8 coreceptors in an antigen reactive hybridoma. BY hybridomas have been selected that lack the murine homologues of CD4 and CD8 and structure/function analyses have been performed by assessing the effect of expression of wild-type or mutant human CD4 or CD8 molecules on T cell responses. This renewal application proposes to continue to study the role of CD4 and CD8 in T cell activation.
Specific Aim 1 proposes to evaluate the signaling pathways in CD4 and CD8 mediated T cell activation. """"""""Double positive"""""""" (CD4+CD8+) T cell hybridomas have been recently generated which suggest that CD4 versus CD8 mediated T cell activation involve different signaling pathways. Experiments are proposed to delineate the signaling molecules used in each of these pathways.
Specific Aim 2 proposes to further delineate the role of p56lck in T cell signaling. The data regarding the signaling activity of lck may not depend on its kinase activity. Experiments in which mutants of lck are expressed in these T cell hybridoma are proposed to explore the biochemical role of lck in signaling.
Specific Aim 3 proposes to define the role played by the cytoplasmic domains of each of the CD3 chains in T cell signaling. Specifically, chimeric molecules of CD16 and the cytoplasmic domains of gamma, delta, epsilon and zeta will be expressed in TcR, CD4+ hybridomas to investigate their role.
Specific Aim 4 proposes to study regulated adhesion of CD4 and CD8. It has now been demonstrated that the avidity of several T cell molecules, including CD8, can be regulated by cellular activation.
Specific Aim 5 proposes to study the in vivo function of human CD4 and CD8 molecules. CD4 and CD8 will be expressed by retroviral mediated transfer in murine stem cells which will in turn be used to reconstitute lethally irradiated mice. These experiments will allow study of the CD4 and CD8 function in normal thymocytes and peripheral T cells.
Specific Aim 6 proposes to delineate mechanism of gp 120 inhibition of T cell activation. Gp 120 can inhibit CD4-dependent T cell activation by competing for the ligand of CD4 on MHC class II molecules. Preliminary data suggest that gp 120 can also suppress T cell activation by inducing a """"""""negative"""""""" signal.
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