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 5

Preexisting donor-specific Tregs expand in tolerance.

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Preexisting donor-specific Tregs expand in tolerance. (A) Tregs detected...
(A) Tregs detected with activated donor B cells among the total CD4 repertoire as a fraction of all clones (Clone Fraction) and by cumulative frequency. Clone fraction and cumulative frequency are defined as the number of unique TCRβ sequences and the number of reads corresponding to the method in the total CD4 sample, respectively, divided by the total number of unique sequences and the total number of reads at the given time point, respectively. Labeled n corresponds to the number of unique TCRβ sequences identified as donor-specific Tregs in the subject. *P < 0.05, **P < 0.01, ***P < 0.001, reduction or increase in clone fraction compared with before transplant (2-sided Fisher’s exact test). (B) Fold change in the clone fraction and cumulative frequency relative to before transplant. Open symbols, statistically significant reduction or increase by clone fraction compared with before transplant (P < 0.05, 2-sided Fisher’s exact test). (C) Sequences in the circulation from the repertoire of Tregs expanded with activated donor B cells that map to the pre-transplant unstimulated Tregs (left) and pre-transplant unstimulated non-Tregs (right) as a proportion of the cumulative frequency of all sequences mapping to this method in the circulation at the indicated time after transplant. (D) Fold expansion of preexisting donor-specific Tregs (repertoire identified via culture of Tregs with activated donor B cells that maps to pre-transplant unstimulated Treg repertoire) among CD4 repertoire by clone fraction and cumulative frequency compared with fold expansion of pre-transplant-identified unstimulated Tregs at 6 months after transplant relative to before transplant.