Tolerance Induced by B-Cell Gene Therapy: Expansion and Increased Activity of Antigen-Specific Tregs.

Abstract 2129

Poster Board II-106

Patients with hemophilia A treated with coagulation co-factor VIII (FVIII) may mount a response against this therapeutic protein, thus forming antibodies that block FVIII function (inhibitors). To treat those currently forming inhibitors (or to prevent inhibitor formation), a B-cell based gene therapy has been used that employs a chimeric antigen-Ig tolerogenic construct comprised of FVIII C2 and A2 domains. We have shown that hemophilic mice receiving B cells transduced with these Ig fusion proteins have diminished Bethesda titers, even after further FVIII treatment. Tolerogenic B-cell therapy requires the presence of CD25+ regulatory T cells (Tregs). We have recently shown that tolerogenic B-cell treatment results in the increase of antigen-specific Treg cells and a selective loss of antigen-specific responder cells. To extend these studies, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using CFSE dilution as a measure of T-cell responsiveness to FVIII, we now show that Tregs from treated mice are more suppressive than those from control treated animals. In order to follow antigen-specific Tregs, we repeated these studies in DO11.10 T-cell receptor transgenic mice that recognize an OVA peptide. Tregs from DO11.10 mice treated with an OVA-Ig construct are also more suppressive than polyclonal Tregs when stimulated with OVA peptide. Although functionally superior, there appear to be no discernible phenotypic differences in these Treg populations. These changes in the lymphocyte population shift the balance away from effector function towards a tolerogenic phenotype and may provide the basis for future therapies in hemophilia A patients with inhibitors. (Supported by NIH RO1 HL061883, NIH T32 HL007698, and AHA Fellowship 0815219E)