Project 3

Anti-CD3 based induction of regulatory CD8+ T cells

Progress in the past few years has shown the feasibility of new approaches to prevent the destruction of islet cells and restore β cell mass in Type 1 diabetes. These approaches have included cellular therapies (e.g. islet transplantation) and immune interventions (e.g. anti-CD3 mAb), but the clinical benefits of these approaches are not permanent, and additional strategies are needed to completely and permanently reverse the disease(1-5). The studies in this proposal are based on observations that were made in the course of our studies of anti-CD3 mAb treatment of patients with new onset T1DM(4; 6). The chronic effects of the anti-CD3 mAb (i.e. clinical effects were seen after T cells in the peripheral blood had fully repopulated) together with preclinical studies in the NOD mouse, suggested that regulatory T cells might play an active role in its efficacy. Indeed, we found that there was a population of CD4+IL-10+ T cells induced in vivo by mAb treatment(7). Moreover, we found that we could identify clinical responders to the drug therapy by an expansion of a CD8+ T cell subpopulation. Further studies that we conducted in vitro, showed that the anti-CD3 mAb expanded a subpopulation of CD8+ T cells with regulatory properties. We postulated that the effect of the anti-CD3 mAb was to induce a population of regulatory T cells, some of which may be CD8+ that mediate the effects of the anti-CD3 mAb in this setting. Therefore, the goal of this Project is to test this hypothesis in order to develop expanded CD8+ T cells for adoptive immunotherapy. In order to accomplish this, the following Aims are proposed:

  1. To test whether anti-CD3 mAb and modified dimeric anti-CD3 mAbs induce regulatory CD8+ T cells in the NOD mouse. The goal of these studies is determine whether the NOD mouse and analogous dimeric anti-CD3 mAbs will be a useful model system in which to study the mechanisms of the anti-CD3 mAb in man. These studies will also allow us to address questions such as antigen specificity that can more easily be done in mice than in humans.
  2. Test the ability of hOKT3γ1(Ala-Ala) to stimulate CD8+ Tregs in vivo and inhibit immune responses in reconstituted NOD/scid common γchain-/- mice . In these studies we will test the induction and function of the CD8+ Tregs in vivo using an immune deficient NOD/scid mouse that has been reconstituted with human immune cells. We will test the direct effects of the mAb on the reconstituted human T cells and the effects of the CD8+ Tregs on GVHD and allograft rejection. These studies will provide important pre-clinical information regarding the efficacy of CD8+ Tregs in vivo.
  3. Characterize the function of CD8+ regulatory T cells and identify the mechanisms of regulation that are used by these cells. We will identify the ligands that are utilized by the Tregs and their mechanism of inhibition. We will determine whether the Tregs are antigen specific and whether their regulatory effect is on all or select subsets of CD4+ T cells.
  4. To develop new means of expanding CD8+ regulatory T cells. We will modify the culture techniques used to expand the regulatory cell population based on the Program. We will test whether antigen specific CD8+ Treg cells can be generated and expanded.
 
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