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Adoptive transfer of ex vivo generated alloantigen-specific regulatory T cells (Treg) by using CD28/B7 co-stimulatory blockade (CSB) to induce transplant tolerance and minimize the use of immunosuppressants has been successfully used in Phase I/II studies of kidney and hematopoietic stem cell transplantation. In these studies, allo-specific Tregs were expanded by co-culturing responder peripheral blood mononuclear cells (PBMCs) with irradiated stimulator PBMCs in the presence of either humanized anti-CD80 and 86 monoclonal antibodies or belatacept.Since deceased donors are a major source of solid organs for allogeneic transplantation, the collection of stimulator PBMCs from cadaveric donors is problematic and ripe to be addressed by exploring alternative sources of stimulator cells. The objective of this study was to evaluate the use of organ-donor splenocytes as stimulators for the ex vivo expansion of regulatory T cells using CSB.Responder (R) PBMCs obtained from healthy donors by leukapheresis and purified by Ficoll were co-cultured with irradiated stimulator (S) PBMCs (benchmark) or mononuclear cells derived from disaggregated splenocytes from cadaveric donors (1x106 cells/mL & 1R:1S ratio) for 72 hours in the presence of belatacept in T-25 flasks. The ability of splenocytes to stimulate PBMC proliferation was assessed in a primary (1°) mixed lymphocyte reaction (MLR), and their effect on the induction of anergy was measured by CSB-mediated inhibition of secondary (2°) MLR. Expansion of Tregs (frequency of CD4+CD25highCD127- cells) and Treg potency (Foxp3 expression) were evaluated by FACS after the 1° CSB culture and in after a 2° MLR (which models in vivo re-exposure to specific alloantigen in absence of Belatacept). Treg function was assessed using a suppression assay. Culture conditions, including S radiation dose, were optimized to achieve the necessary Treg cell numbers for administration to patients.40 Gy appeared to be the optimal dose to limit splenocyte proliferation while maintaining their ability to stimulate R PBMCs in a 1° MLR. In comparison to PBMC stimulators, the use of splenocytes as stimulators during CSB produced comparable inhibition of proliferation in 1° and 2° MLR, increased Treg frequency, and elevation in intracellular Foxp3. Finally, culture conditions were optimized to scale up Treg expansion by 1) using tissue culture bags, 2) increasing cell density (1.5x106 cells/mL), and 3) modifying R:S ratio (2R:1S). The scale up procedures did not impair cell viability, anergy induction, Treg expansion, or Treg potency.The ex vivo expansion of regulatory T cells using belatacept-mediated CSB is an efficient and relatively brief procedure that can be performed using organ-donor splenocytes as stimulators. Use of splenocytes supports scale up that can achieve the necessary Treg cell numbers for clinical use.Immune Tolerance Network/NIH:UM1AI109565.