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Early expansion of donor-specific Tregs in tolerant kidney transplant recipients
Thomas M. Savage, … , Yufeng Shen, Megan Sykes
Thomas M. Savage, … , Yufeng Shen, Megan Sykes
Published November 15, 2018
Citation Information: JCI Insight. 2018;3(22):e124086. https://doi.org/10.1172/jci.insight.124086.
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Categories: Research Article Immunology Transplantation

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

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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 1

Methods to identify the donor-specific Treg repertoire.

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Methods to identify the donor-specific Treg repertoire.
(A) TCRβ sequenc...
(A) TCRβ sequencing was performed on unstimulated Tregs and CD4+ non-Tregs from PBMCs to establish the unstimulated Treg and non-Treg repertoires. (B) Bulk CFSE-MLRs were performed to identify the CD4 donor-reactive repertoire — defined as unique TCRβ sequences expanded ≥5-fold from unstimulated CD4 samples and with frequency ≥10–4 in the CFSElo sample — which was compared with the unstimulated Treg repertoire from A to determine the “CFSE-MLR Treg” repertoire. (C) Activated (act.) donor B cells were generated, with representative flow cytometry staining demonstrating activation, then irradiated to culture with sorted Tregs. (D) Primary and restimulated CTLA4Ig MLRs were performed as shown.
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