T CELL DIVERSITY AND FUNCTIONAL BIOLOGY

A. Mcmurchy¹, D. Dai², J. Gillies³, C.B. Verchere⁴, M.K. Levings^3
1Department Of Surgery, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver/CANADA, ²Department Of Pathology And Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver/CANADA, ³Department Of Surgery, Vancouver Coastal Health Research Institute and University of British Columbia, Vancouver/CANADA, ⁴Pathology And Laboratory Medicine, Child and Family Research Institute, University of British Columbia, Vancouver/CANADA

Body: Introduction: T regulatory cells (Tregs) suppress inappropriate immune responses, and their potential use as a cellular therapy to prevent graft rejection has been demonstrated in many mouse models. The major barriers preventing the clinical translation of this approach are limiting cell numbers and the lack of pre-clinical models in which the efficacy of human Tregs can be evaluated in vivo. We previously developed a method to generate human Tregs by transducing conventional T cells with lentivirus encoding the transcription factor FOXP3. FOXP3-transduced T cells recapitulate the expected phenotype and in vitro function of ex vivo Treg cells, are a stable and homogeneous population of cells, and can be generated in sufficient numbers for use as a cellular therapy for transplant patients. The purpose of the present study is to test the in vivo suppressive capacity of FOXP3-transduced T cells in a xenogeneic mouse model of human islet allograft rejection. Methods: Clinical grade human islets are transplanted into NOD/SCID mice previously treated with streptozotocin to induce diabetes. Once blood glucose levels have normalized, indicating a functional graft, mice are injected with allogeneic PBMCs to initiate rejection, which is monitored on the basis of blood glucose. The objective is to test the relative ability of co-injected FOXP3-transduced T cells versus ex vivo Tregs to suppress allograft rejection. We first optimized a method to expand ex vivo Tregs while preserving FOXP3 expression. Next, we investigated the abilities of human PBMCs, ex vivo expanded Tregs, and transduced T cells to survive in NOD/SCID mice following IP injection and to suppress islet allograft rejection. Results: We found that sorting naive ex vivo Tregs (defined as CD4⁺CD25^hiCD45RA⁺ cells) resulted in the most homogeneous population of FOXP3⁺ cells. Over 100-fold expansion of CD4⁺CD25⁺CD45RA⁺ Tregs was achieved upon stimulation with artificial antigen presenting cells in OpTmizer T cell Expansion Medium in the presence of 100ng/mL rapamycin. Expanded Tregs remained 90% FOXP3⁺ and did not acquire the capacity to produce inflammatory cytokines. Two weeks after IP injection, human lymphocytes and transduced T cells can be detected in the peripheral blood of NOD/SCID mice. Experiments are on-going to determine the optimal ratio of expanded ex vivo Tregs:PBMCs necessary to prevent allograft rejection so that we can evaluate the relative efficacy of FOXP3-transduced T cells. Conclusions: With an effective expansion protocol developed for ex vivo Tregs and evidence that transduced cells survive in the mouse, we can move forward with testing ex vivo expanded and generated Tregs in our in vivo model. Evidence that human Tregs can prevent rejection of human islets will provide a strong rationale for clinical translation of cellular therapy to prevent allograft rejection.

Disclosure: All authors have declared no conflicts of interest.