The present proposal seeks to determine the mechanism(s) by which non- mitogenic anti-T cell receptor (TCR) mAbs inhibit in vivo T helper cell responses and to apply this tot he treatment of murine mAbs induce a long-term inhibition of IL-2 secretion from CD4+T helper cells, induce a differentiation of these cells to a memory phenotype, and can inhibit of a variety of autoimmune diseases. At least one mechanism of action of these mAbs appears to involve induction of T cell anergy. However, other factors may also contribute tot he observed inhibition. A precise understanding of the mechanisms of action of the mags will allow a better assessment of their applicability to the clinical setting. Comparison will be made between the effects of mitogenic (whole mAb) versus non- mitogenic mags (F(ab)2 fragments) and between mags binding to the CD3 complex versus the TCR alpha beta heterodimer. This proposal has three aims.
In aim #1, the nature of the inhibition will be determined by purifying CD4+, CD8+, and memory subpopulations from mAb-treated and control mice analyzing each population for cytokine secretion and gene transcription following in vitro stimulation with both cognate non- cognate stimuli. It will be determined whether the inhibition of IL-2 secretion is a consequence of a skewing of CD4+ helper cells from a naive to a memory phenotype, a skewing form a Th1 to Th2 phenotype, or a specific inhibition of transcription of the IL-2 gene both CD4+ and CD8+ cells. Potential suppressor activity will be assessed by cell mixing experiments and by adoptive cell transfer. It will be determined whether anergy can be induced in cells previously primed to alloantigen.
In aim #2, the mechanism the mechanism of the inhibition of autoimmunity will be determined by assessing the response of T cell subpopulations to type II collagen (CII). The role of IL-2 in severity of CIA will be determined. Studies will be undertaken to determine whether binding of mags to the TCR complex in the presence of autoantigen can result in antigen-specific tolerance. Long-term effects mAb treatment on the health of the mice will be assessed.
In aim #3, biochemical events involved in anergy induction will be investigated, including the required conditions of exposure to non-mitogenic mags, requirement for intracellular calcium mobilization, and whether signal transduction is altered in anergized cells. Although mitogenic forms of mags against the TCR complex are presently in clinical use, data to be described in this proposal suggest that engagement of the TCR complex offer superior efficacy in inhibiting in vivo T helper cell responses.
