Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 112-132).

A side effect of generating an immune system for defense against invading pathogens is the potential to develop destructive cells that recognize self-tissues. Typically, through the "education" of developing immune cells, the organism inactivates potentially self-destructive cells, resulting in what is called self-tolerance. I proposed to explore the molecular mechanisms responsible for the induction and maintenance of tolerance. Our lab has developed a model of induced immune tolerance to skin and islet allografts utilizing a donor-specific transfusion of spleen cells and a brief course of anti-CD40L antibody. Because the difficulty in isolation of tolerant T cells from this system is prohibitive to performing large screens on these cells directly, I have chosen to study an in vitro CD4+Th1 cell line, A.E7, which can be made anergic via stimulation through the T cell receptor in the absence of costimulation. I hypothesized that anergized T cells upregulate genes that are responsible for the induction and maintenance of anergy and therefore exhibit a unique RNA expression profile. I have screened anergic cells using Affymetrix GeneChips and identified a small number of genes that are differentially expressed long-term in the anergic population compared to mock-stimulated and productively activated controls. The results have been confirmed by quantitative RT-PCR for each of the candidates. One of the most promising, the zinc-finger transcription factor Egr-2, was verified to be expressed long-term by western blotting, demonstrating perfect correlation between Egr-2 protein expression and the anergic phenotype. Silencing Egr-2 gene expression by siRNA in A.E7 T cells prior to anergy induction rescues the cells from the inability to phosphorylate ERK-1 and ERK-2 and also results in increased proliferation in response to antigen rechallenge. In this study I report that Egr-2 is specifically expressed long-term in anergic cells, protein expression correlates inversely with responsiveness to antigen rechallenge, and that Egr-2 is required for the full induction of anergy in T cell clones.

The prevalence of diabetes is approaching epidemic proportions worldwide. There is currently no cure for type 1 diabetes, and successful treatment requires constant monitoring of blood sugars and use of exogenous insulin to prevent hyperglycemia. Diabetes will be curable when pancreatic β-islet cells can be transplanted into diabetes patients without requiring long-term immunosuppression. This will require learning more about the induction of functional tolerance, a state that maintains the competence of the immune system to most antigens but protects graft-specific antigens from immune rejection, permitting transplantation. One known mechanism of peripheral tolerance is T cell anergy, a phenotype of hypo-reponsiveness in CD4+ T cells. The focus of this thesis is a description of factors shown to be specific to the induction and maintenance of T cell anergy, whose loss reverses the anergic phenotype, restoring the ability of the cells to proliferate in response to antigen. The first of these is Egr-2, a zinc-finger transcription factor, whose presence is required for the induction of anergy induced in T cell clones by TCR stimulation in the absence of costimulation. Egr-2 is shown to be important to anergy induction but not anergy maintenance. In contrast, a negative costimulation receptor, PD-1, is shown to be necessary for the maintenance of anergy. It is possible that learning more about the genetic factors that orchestrate T cell anergy will prove useful in the development of tolerance-based protocols for organ and tissue transplantation without the use of long-term immunosuppression.