Expansion of Tregs for the treatment of autoimmune diabetes

Regulatory T cells (Treg) are a subset of T cells that maintain the balance between autoimmunity and tolerance. Functional defects in Tregs in mouse model systems of diabetes and other autoimmune diseases suggest that abnormalities in the Tregs of T1D patients may contribute to the pathogenesis of the disease. Recent studies by our group have demonstrated that treatment with the humanized anti-CD3 antibody, hOKT3γ1 (Ala-Ala) halted the progression of disease in a manner that was associated with increased numbers and/or function of Tregs. The overall goal of this proposal is to isolate and maximize the expansion of both polyclonal and antigen-specific Tregs isolated from normal and anti-CD3-treated T1D patients with the goal of both furthering the basic understanding of Treg activity in this patient population and developing a protocol for adaptation in this clinical setting. In order to accomplish these goals the following specific aims are proposed:

1. To characterize Treg expression/function in T1D patients versus patients treated with the immunomodulatory drug hOKT3γ1 (Ala-Ala). We will compare cell numbers and function of isolated Tregs in normal healthy donors, T1D patients and T1D patients participating in the therapeutic trials of hOKT3γ1 (Ala-Ala), a drug that alters the immune system and slows the progression of β cell destruction in new onset T1D patients. Isolated Tregs from treated and untreated T1D patients will be compared to controls for their ability to regulate in vitro proliferation and cytokine secretion.

2. To expand and analyze Tregs from T1D and anti-CD3-treated T1D patients. In order to overcome the limitation of low Treg numbers in peripheral blood, we propose to expand Tregs from T1D patients treated with a non-mitogenic anti-CD3 mAb that has been shown to increase the number of Tregs in humans. We will examine their phenotype and function in comparison with normal healthy donor populations, maximize the expansion protocol for in vivo therapy, and determine the Treg function activity both in vitro and in a model being developed by Kevan Herold in Project 3.

3. To expand and analyze antigen-specific Tregs from T1D patients. We propose to expand antigenspecific Tregs in both T1D and anti-CD3-treated participants. We will utilize MHC-peptide tetramers containing specific peptides derived from autoantigens and infectious agents.

The combination of the study of ex vivo expansion and the study of patients treated with the immunomodulatory drug hOKT3γ1 (Ala-Ala) will provide critical validation of the hypothesis that Tregs are essential and potentially therapeutic for the treatment of T1D.