Anergy, as defined by the long-lived inability to secrete IL-2 and proliferate in response to antigenic stimulation, can be induced in IL-2- producing murine T cell clones. Susceptible cells include CD4+ cells of the T helper-1 (Th1) subset and helper-independent CD8+ cytolytic T lymphocytes (CTL). Anergy is induced by the stimulation of the T cell receptor (TCR) for antigen in the absence of """"""""co-stimulatory"""""""" signals; concanavalin A (Con A), immobilized anti-TCR monoclonal antibodies (mAb), antigen-pulsed antigen-presenting cells that do not express co- stimulatory molecules (either because the APC have been fixed or they fail to express such molecules), and immobilized class II major histocompatibility complex (MHC) pulsed with antigen provide such stimulation. However, anergy also can be introduced by treatment with calcium ionophores in the absence of TCR stimulation. Anergy apparently cannot be induced in Th2 of conventional CTL; these cell subsets do not secrete IL-2. Neither the essential biochemical events in CD4+ T cells that lead to the induction of anergy nor the signalling defects that account for the failure of anergic cells to produce IL-2 have been characterized adequately. Also, the essential events induced by co- stimulatory molecules that prevent the induction of anergy have not been fully defined. These will be explored using existing Th1, Th2, and Th0 clones as well as clones derived from mice which do not express p59fyn and from mice that express transgenic TCR. In Project 2, we will concentrate mainly on proximal signalling events including protein phosphorylation and changes in intracellular calcium ([Ca2+]) but will interact with Project 1 in investigating the more distal signaling events in anergy. Although induction of anergy in a CD8+ murine T cell clone has been described, anergy in CD8+ T cells has not been investigated thoroughly. Conventional CTL (in which anergy apparently cannot be induced) and Th1 cells (which can be anergized) appear to share at least some signaling pathways. We have derived a number of CD8+ murine T cell clones that secrete IL-2 or IL-4 as well as clones that secrete neither of these lymphokines. The susceptibility of these clones and other clones that are being derived from mice that express a transgenic TCR will be determined, and the biochemical events associated with induction of anergy in CD8+ T cells will be compared with those found to be important in induction or maintenance of the anergic state in CD4+ T cells. Transgenic and """"""""knockout"""""""" mice produced in Project 3 and transfected tumor and transformed cell lines expressing various cell surface molecules produced in Project 1 have been studied most extensively using Th1 clones that have been derived from conventional mice. However, situations associated with anergy in vivo may involve induction of anergy in naive T cells. T cells from mice expressing transgenic TCR do not display functional activity constitutively; they require stimulation in order to secrete lymphokines, proliferate, and express cytolytic activity. The conditions necessary to induce anergy in cloned T cells will be compared with those needed to induce anergy in naive CD4+ and CD8+ T cells. Characteristics of cells that escape induction of anergy will be determined and the relationship between such cells established CD4+ and CD8+ T cell subsets will be determined. These studies will be coordinated with Project 4 which is investigating anergy induction in vivo.
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