HLA-mismatched allograft tolerance was intentionally induced via combined kidney and bone marrow transplant (CKBMT) in two ITN protocols (NKDO3 and ITN036ST), but the mechanisms of tolerance are incompletely understood. We previously developed and validated a technique for identifying alloreactive T cells pre-transplant via deep sequencing of the T cell receptor (TCR) β chain hypervariable (CDR3) region and tracking them post-transplantation. Here we describe TCR sequencing of sorted T regulatory (Treg) clones (CD3+CD4+ CD25+++ CD127-) pre- and post-transplant in three tolerant CKBMT patients. There were 7430, 9593, and 116,350 total Treg clones from each patient, respectively. Donor-specific Treg clones were defined by both: 1) presence in a sorted Treg sample; and 2) at least 5-fold expansion following donor stimulation in a pre-transplant CFSE-mixed lymphocyte reaction, with frequency ≥10^-5 in the sorted CFSE-low sample. For the three patients, 37, 11, and 122 unique donor-specific Treg clones were identified, respectively. At 6 months post-transplant, all three patients showed ≥5-fold expansion of donor-specific Tregs relative to pre-transplant in total CD4 unstimulated populations, and in two of the patients, ≥2-fold expansion persisted until 18 and 24 months post-transplant, respectively, the last time points examined. Furthermore, in two of the patients, donor-specific Tregs were enriched post-transplant ≥3-fold relative to pre-transplant in the CD4+Treg compartment, suggesting that the donor-specific Treg expansion was at least partially driven by graft antigens. Finally, donor-specific induced Tregs — pre-transplant sorted non-Treg clones that were found post-transplant only in sorted Treg samples — were detected post-transplant in all three patients. These findings suggest that both expansion of pre-existing donor-specific Tregs and induction of Tregs from donor-reactive conventional CD4 cells contribute to the marked enrichment of donor-specific Tregs, which may contribute to the immunologically quiescent post-transplant environment of the tolerated allograft. Ongoing Treg TCR sequencing of a non-tolerant CKBMT recipient should add further insight into the role of Tregs in tolerance in CKBMT recipients.

CITATION INFORMATION: Savage T, Dewolf S, Obradovic A, Lau S, Zuber J, Turka L, Shen Y, Sykes M. Clonal Expansion of Donor-Specific Tregs Following Combined Kidney and Bone Marrow Transplantation. Am J Transplant. 2016;16 (suppl 3).

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