Donor Tr1 cells potently expand in vivo and prevent GVHD after TLI/ATS non-myeloablative conditioning and allogeneic bone marrow transplantation (145.17)

Abstract

Abstract Murine total lymphoid irradiation (TLI) + anti-thymocyte serum (ATS) has been well-translated to human transplantation (Lowsky et al, NEJM 2005). Though host TLI/ATS treatment allows post-transplant donor Foxp3+ Treg expansion, >90% of donor CD4+CD25+ cells are Foxp3neg. We now show that the latter are Tr1-type regulatory T cells. BALB/c (H-2d) hosts receiving TLI/ATS + bone marrow + splenocytes (BMT) from C57BL/6 (H-2b) donors had 90% 60-day survival without GVHD (n=10). BALB/c hosts given 800 cGy or 400 cGy total body irradiation (TBI)/ATS+BMT had 100% 30-day GVHD death (n=10). TLI/ATS+BMT hosts had a 10-fold increase in H-2Kb+CD4+CD25+ cell absolute number in day 6 host spleen versus TBI/ATS controls (p< 0.05). Using Foxp3-GFP transgenic donors, >90% of the H-2Kb+CD4+CD25+ cells after TLI/ATS+BMT are Foxp3(GFP)neg. Supernatant assay on day 6 sorted TLI/ATS+BMT H-2Kb+CD4+CD25+, 72-hour PMA/ionomycin stimulated cells revealed potent IL-10, low IL-2, IL-5, and IFN-γ, and no IL-4 or TNF-α (p< 0.05) secretion. Day 6 TLI/ATS+BMT Tr1 donor cells were potent suppressors of donor-host MLR. Donor CD25 cell depletion pre-TLI/ATS+BMT caused day 6 donor CD8+ T cell-mediated GVHD and loss of donor Tr1 cells, indicating that Tr1 cell expansion protects from GVHD post-TLI/ATS+BMT. We are characterizing a host APC-dependent mechanism for this Tr1 expansion. Our findings delineate a novel mechanism and strategy for in vivo Tr1 expansion for tolerance induction and adoptive immunotherapy.