Introduction: COH-MC-17 regimen consists of conditioning with low-dose cyclophosphamide (CY), pentostatin (PT), and anti-thymocyte globulin (ATG) and infusion of CD4+ T-depleted hematopoietic graft. Induction of MHC-mismatched mixed chimerism by COH-MC-17 regimen not only reverses autoimmunity but also provides immune tolerance to donor-type organs. Current studies are designed to unravel the molecular mechanisms about how donor CD8+ T cells in transplants augments engraftment without causing graft versus host disease (GVHD) and how residual host-anti-donor CD4+ and CD8+ T cells in the periphery are tolerized.
Method: We applied combined single cell TCR-CDR3-Seq with RNA-Seq to analyze the donor- and host-type CD4+, CD8+, or double negative (DN) T cells from draining lymph node of recipients given CD4+ T-depleted splenic cells with WT or MHCII-/- bone marrow cells 30 days after HCT/skin graft transplantation, with donor T cells before HCT as control. The key data is validated with flow cytometry analysis at protein level.
Results: The donor-type CD8+ T cells were divided into 9 clusters. As compared with CD8+ T before HCT, Cluster 1, 6 and 8 were expanded with dominance of 3 clones, and all the clones showed anergy/exhaustion phenotype with little FoxP3 expression, indicating biased clonal expansion and tolerance of donor-type CD8+ T cells in the absence of donor CD4+ T cells in the mixed chimeras. Host-type CD4+ T cells were divided into 13 clusters. Cluster 7, 9, 11 are defined as pTreg, precursors, and tTreg. As compared with MHCII-/- BM chimeras, WT chimeras had expansion of pTreg cells in cluster 7. Interestingly, we identified 5 TCRβ clones that became pTreg cells in the tolerant WT mixed chimeras but not in the non-tolerant MHCII-/-recipients, indicating that Tcon cells differentiated into pTreg cells in the mixed chimeras in a donor MHCII-dependent manner. The host-type CD8+ T cells were divided into 8 clusters. Cluster 5 in the WT mixed chimeras expressed the highest level of FoxP3, consistent with flow cytometry analysis. Finally, donor-type but not host-type CD4-CD8- double negative (DN) T cells showed activation and expansion with anergy phenotype.
Conclusion: In the MHC-mismatched mixed chimeras established with COH-MC-17 regimen, absence of donor CD4+ T cells allows anti-host donor CD8+ T cells in the transplant to facilitate engraftment early after HCT and then become tolerant without causing GVHD; on the other hand, anti-donor host-type CD4+ and CD8+ T cells in the periphery are driven into FoxP3+ Treg cells in donor APC-dependent manner.