Fas (CD95) is of principal importance to normal functioning of the immune system, as evidenced by the marked dysregulation of autoreactive B cells and accompanying autoantibody formation that characterize Fas-deficient animals. The sensitivity of B cells to Fas-mediated apoptosis is regulated by specific receptor signaling, in which surface immunoglobulin engagement, and IL-4R engagement, produce a state of Fas-resistance. Modulation of susceptibility to Fas killing suggests a system that protects B cells during critical interactions with FasL-bearing, activated T cells, but that can contribute to the survival of autoreactive B cells when activated inappropriately. The broad, long term objective of this work is to understand the role of inducible Fas-resistance in normal immune responses and in the genesis of autoimmunity. Three anti-apoptotic gene products are implicated as proximate mediators for resistance to Fas killing:
F AIM, FLIP, and Bcl-xL. The specific goal of this work is to illuminate the capability of these molecules to enhance serological immune responses and to alter the behavior of autoreactive B cells, separate from any other collateral effects of sIg or IL-4R engagement. This will be accomplished through 4 specific aims. 1. Determine the FAIM-dependency of normal immune responses by constructing and evaluating FAIM-deficient (knock-out) mice. 2. Evaluate the level, timing, and cellular origin of upregulated FAIM, FLIP, and Bcl-XL expression in lymphoid tissue during in vivo immune responses. 3. Compare the relative potency of FAIM, FLIP, and Bcl-xL in producing Fas-resistance in primary B cells by retroviral transduction in vitro and in promoting normal immune responses in vivo by constructing mixed chimeras in which bone marrow donors overexpress anti-apoptotic molecules alone and together. 4. Test the individual influence of FAIM, FLIP, and Bcl-xL, on the behavior of autoreactive B cells in two well-defined models: a) Ig receptor transgenic mice expressing VH3H9 anti-dsDNA in which B cells normally fail to enter germinal centers but do so on a Fas-deficient background; and, b) doubly transgenic mice expressing anti-HEL B cell receptors and soluble HEL in which B cells are normally short-lived and tolerant but break tolerance in the presence of IL-4, B7.2 expression, or Fas-deficiency. The results of these studies will test the hypothesis that inducible Fas-resistance promotes normal immune responses, and the hypothesis that aberrant Fas-resistance contributes to a breakdown in autoreactive B cell tolerance, and will thereby enhance understanding of both immunization strategy and the origin of autoimmunity.
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