Early expansion of donor-specific Tregs in tolerant kidney transplant recipients
Thomas M. Savage, Brittany A. Shonts, Aleksandar Obradovic, Susan Dewolf, Saiping Lau, Julien Zuber, Michael T. Simpson, Erik Berglund, Jianing Fu, Suxiao Yang, Siu-Hong Ho, Qizhi Tang, Laurence A. Turka, Yufeng Shen, Megan Sykes
Thomas M. Savage, Brittany A. Shonts, Aleksandar Obradovic, Susan Dewolf, Saiping Lau, Julien Zuber, Michael T. Simpson, Erik Berglund, Jianing Fu, Suxiao Yang, Siu-Hong Ho, Qizhi Tang, Laurence A. Turka, Yufeng Shen, Megan Sykes
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Research Article Immunology Transplantation

Early expansion of donor-specific Tregs in tolerant kidney transplant recipients

Abstract

Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.

Authors

Thomas M. Savage, Brittany A. Shonts, Aleksandar Obradovic, Susan Dewolf, Saiping Lau, Julien Zuber, Michael T. Simpson, Erik Berglund, Jianing Fu, Suxiao Yang, Siu-Hong Ho, Qizhi Tang, Laurence A. Turka, Yufeng Shen, Megan Sykes

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Figure 3

Tregs cultured with activated donor B cells have higher Foxp3 expression and are more suppressive of donor-specific responses in vitro.

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Tregs cultured with activated donor B cells have higher Foxp3 expression...
(A) Representative Foxp3 stain of an unstimulated Treg sample prior to FACS sorting, gated on CD3+CD4+CD8– cells. (B) CD25+CD127– percentage among CD4+ cells and Foxp3 expression among CD4+CD25+CD127– cells from 3 independent healthy controls at the end of each assay assessed by flow cytometry. (C) With either donor or HLA-mismatched third-party T cell–depleted PBMC stimulation, inhibition of CFSE-labeled fourth-party CD3+ T cell proliferation by sorted CD4+CD25+CD127– cells from healthy control 1 at the end of each expansion. (D) Comparison of potency of anti-donor suppression as in C, with unstimulated Treg data summarizing 3 experiments, including from healthy control 1. (C and D) Mean ± SEM. Experiment performed in triplicate. (D) *P < 0.05, **P < 0.01, ***P < 0.001 for comparison of Tregs with activated (w/ act.) donor B cells to restimulated CTLA4Ig MLR (2-tailed t test).