Pre-Transplant Infusion of Donor CD4+ CD25+ T Cells Suppresses Host Anti-Donor MiHA-Specific CD8 T Cells and Facilitates Stable Mixed Chimerism Following MHC-Matched Allogeneic Marrow Transplant.

Abstract

Surmounting the barrier mediated by host T cells against successful engraftment of MHC-matched allogeneic marrow is a crucial objective of hematopoietic stem cell transplant (HCT). We proposed that the ability of unmanipulated donor CD4+ CD25+ regulatory T cells (Tregs) to suppress activation of host effector CD8+ T cells would enhance donor HC engraftment between strains disparate for multiple minor HA (MiHA), and promote stable chimerism. C57BL/6 (H-2b, Ly9.1−) mice conditioned (5.5 Gy TBI) 24 hrs earlier, were transplanted with Tregs and TCD-BM from 129P3/J (H-2, Ly9.1+) mice. By four weeks post-HCT, the mean frequency of circulating donor-derived (total Ly9.1+) cells was 15.7 ± SE 5.9% in recipients of 4 × 106 TCD-BM + Tregs (4.5 × 105) compared to the absence of donor chimerism in recipients of allogeneic HCT only (0.3 ± 0.09%) - a finding also evident from the analysis of circulating donor B220+ cells (8.7 ± 3.0 vs. 0.2 ± 0.06%). Based on these findings, we hypothesized that pre-HCT infusion of Tregs into a lymphopenic, allogeneic host might enhance their facilitating function. Accordingly, Tregs (4 or 1.5 × 105) were transplanted 24hr. post-conditioning either 3 days prior to HCT or co-transplanted with (day 0) donor TCD-BM. The transplant was rejected in recipients not administered donor Tregs. The higher dose of Tregs administered pre-transplant or co-transplanted facilitated equivalent donor cell chimerism. In contrast, administration of 1.5 × 105 Tregs resulted in chimerism only when this donor population was infused 3 days prior to HCT (1.6±0.9%). To test the hypothesis that donor Tregs suppressed host anti-donor specific CD8 T cells, we monitored host CD8+ T cell responses to the immunodominant donor epitope H60 by tetramer staining (LTFNYRNL/Kb). One month post-HCT of TCD-BM alone, the frequency of circulating tetramer+ CD8+ cells in these recipients rejecting the marrow graft was clearly greater compared to the frequency in recipients of TCD-BM + Tregs which engrafted (p<0.0008). In contrast, the host anti-donor CD8 levels were not different between recipients of TCD-BM alone and TCD-BM + D.0 'low dose' Treg administration which failed to engraft (p>0.05). Notably, transplants using unmanipulated host Tregs failed to support engraftment. These findings illustrate that donor Tregs support chimerism via suppression of host anti-donor antigen-specific T cells. Finally, the persistence of donor cells in circulation as well as in lymphoid tissues 3 months post-HCT indicated that long-term, stable chimerism was established by this Treg administration regimen. In total, our results demonstrate that donor Tregs promote MHC-matched allogeneic marrow engraftment under conditions of reduced intensity conditioning and that the strategy of pre-HCT Treg infusion supports the notion that antigen driven expansion of donor anti-host reactive Treg cells prior to stem cell transplants can increase their efficacy to facilitate hematopoietic engraftment. Experiments to study transient vs. long-term engraftment and expansion of donor Treg cells are being examined.