Multivirus-Specific CTl for Adoptive Transfer Using in Vitro Pepmix Stimulation

Immunotherapy with both adaptive and innate regulatory T cells facilitates induction of transplant tolerance, including the prevention and treatment of GVHD. Current strategies to expand regulatory Tr1 cells from CD3CD4CD25 conventional T cells include culture with IL-10 and APCs (Groux, et al., Nature, 1997; Gregori et al., Methods Mol. Biol., 2007) and a non-APC dependent protocol utilizing anti-CD3 and IL-2 combined with antibody cross-linking of the innate complement inhibitor I cofactor protein (CD46) (Kemper et al., Nature, 2003). Optimizing a robust APC-independent means of Tr1 cell generation is of particular utility to hematopoietic cell transplant (HCT) immunotherapy, as it offers potential for large-scale Tr1 cell expansion from autologous or allogeneic cell sources without need for re-separation of Tr1 cells from host or donor APCs prior to infusion. To determine the optimal methods and cellular products for nonAPC-dependent expansion of Tr1 suppressors, we performed anti-CD46-mediated expansion of Tr1 cells for 21-28 days from various potential cellular therapy sources. CD3CD4CD25 cells were FACS-isolated at . 98% purity and underwent parallel ex vivo expansions from adult peripheral blood apheresis units (PBTr1), adult bone marrow (BM-Tr1) and cord blood (CB-Tr1) using low-dose IL-2, anti-CD3, and antibody-crosslinking of CD46, without addition of IL-10 or APCs, to derive a uniform population of CD3CD4CD25Foxp3 cells (. 95% conversion of CD25Foxp3 to CD25Foxp3 cells, regardless of cellular product type). Differential levels of expansion of Tr1 cells occurred according to the source of cell product, with greatest foldexpansion in PB-Tr1 [mean absolute numbers at day 21 from y10 starting CD3CD4CD25 cells: PB-Tr1 (n 5 3): 2.5 6 0.8 10; BM-Tr1 (n 5 2): 7.2 10; CB-Tr1 (n 5 4): 5.6 6 0.3 10]. By day 14-21 of expansion, PB-Tr1 cells secrete IL-10 and IL-5, very low levels of IL-2 and IFN-g, and no IL-4 or IL-17 by anti-CD2/CD3/CD28 bead stimulation and Luminex supernatant assay, and they demonstrate suppressor function against sorted autologous CFSE-labeled CD3CD4CD25 responders in 72-hour MLR using irradiated allogeneic APC stimulators (Table 1). We are currently optimizing similar protocols for expansion of BM-Tr1 and CB-Tr1 to allow application in tolerance induction and immunotherapy after HCT from each of these cellular therapy sources.