P038 “Donor”-specific regulatory T cells for clinical transplant tolerance: Optimization of ex vivo expansion and characterization

Aim CD4 + CD127 − CD25 + FOXP3+ Regulatory T cells (Tregs) are shown to be enhanced in tolerant transplant patients and after ex vivo expansion to prolong graft survival in animal models without immunosuppression. Therefore, a number of centers have initiated clinical trials with expanded Tregs. The purpose of the present study was to further optimize the ex vivo expansion conditions and to characterize the expanded Tregs so as to transfer the technology to the clinic. Methods and results B cells to be used as stimulators in Treg cultures were expanded from the peripheral blood mononuclear cells (PBMC) of healthy “donors” using multimeric CD40L (Ultra-CD40L) ( n = 12). The resultant B cells expanded up to 1000 fold by day 14 and displayed increased expression of CD80, CD86, and HLA-DR, the hallmark of mature APC. These expanded “donor” B cells, were irradiated and used to stimulate purified CD4 + CD127 − CD25 + Tregs isolated from PBMC of an allogeneic volunteer (“recipient”) under Treg-promoting conditions with exogenous IL-2 and sirolimus(SRL). Initial cultures were for 28 days with weekly antigenic restimulations. The Tregs expanded up to 13-fold when SRL was removed after 21 days ( n = 8) and up to 28-fold when SRL was removed after 14 days ( n = 4). In the final optimized protocol, the Tregs were stimulated “donor”-specifically on days 0 and 7 and then polyclonally with anti-CD3/CD28 on day 14 without SRL, and were harvested on day 21 ( n = 3). This resulted in fold expansion into the thousands, as only + CD127 − CD25 + phenotype with >60% being FOXP3+ . More importantly, they inhibited MLRs of autologous responders in an antigen-specific manner ( p n = 8). These Tregs also demonstrated antigen-specific infectious tolerance by generating new Tregs in CFSE-labeled autologous responder cells cultured with the specific allostimulator ( n = 6). Conclusions We have optimized the protocol for ex vivo expansion of potent “donor”-specific Tregs. Therefore, after further scale-up in the cGMP and obtaining an IND from the FDA, we will be ready to utilize such Tregs clinically to induce transplant tolerance. R.S. Kornbluth: Other (Identify); Company/Organization; President and Chief Scientific Officer; Multimeric Biotherapeutics Inc. J.R. Leventhal: Other (Identify); Company/Organization; Co-Founder; TRACT Therapeutics, Inc.