Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs
Shipra Gupta, Thomas B. Thornley, Wenda Gao, Rafael Larocca, Laurence A. Turka, Vijay K. Kuchroo, Terry B. Strom
Shipra Gupta, Thomas B. Thornley, Wenda Gao, Rafael Larocca, Laurence A. Turka, Vijay K. Kuchroo, Terry B. Strom
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Research Article Immunology

Allograft rejection is restrained by short-lived TIM-3+PD-1+Foxp3+ Tregs

Abstract

Tregs play a pivotal role in inducing and maintaining donor-specific transplant tolerance. The T cell immunoglobulin and mucin domain-3 protein (TIM-3) is expressed on many fully activated effector T cells. Along with program death 1 (PD-1), TIM-3 is used as a marker for exhausted effector T cells, and interaction with its ligand, galectin-9, leads to selective death of TIM-3+ cells. We report herein the presence of a galectin-9–sensitive CD4+FoxP3+TIM-3+ population of T cells, which arose from CD4+FoxP3+TIM-3– proliferating T cells in vitro and in vivo and were often PD-1+. These cells became very prominent among graft-infiltrating Tregs during allograft response. The frequency and number of TIM-3+ Tregs peaked at the time of graft rejection and declined thereafter. Moreover, these cells also arise in a tolerance-promoting donor-specific transfusion model, representing a pool of proliferating, donor-specific Tregs. Compared with TIM-3– Tregs, TIM-3+ Tregs, which are often PD-1+ as well, exhibited higher in vitro effector function and more robust expression of CD25, CD39, CD73, CTLA-4, IL-10, and TGF-β but not galectin-9. However, these TIM-3+ Tregs did not flourish when passively transferred to newly transplanted hosts. These data suggest that a heretofore unrecognized graft-infiltrating, short-lived subset of Tregs can restrain rejection.

Authors

Shipra Gupta, Thomas B. Thornley, Wenda Gao, Rafael Larocca, Laurence A. Turka, Vijay K. Kuchroo, Terry B. Strom

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Figure 5

CD4+TIM-3+ Tregs are potent suppressors of Teff proliferation in an in vitro MLR culture and robustly express Treg markers.

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CD4+TIM-3+ Tregs are potent suppressors of Teff proliferation in an in v...
(A) In an MLR culture, naive CD4+GFP– Teffs from spleens of BALB/c-KI mice were stimulated with irradiated CD90– splenocytes from BALB/c-KI mice for 3 days in the presence or absence of Tregs. Sorted CD4+GFP+TIM-3+/TIM-3– Tregs were obtained from spleens of BALB/c-KI mice injected with DBA/2 DST 5 days prior to harvesting. T cell proliferation in these cultures was analyzed by the mean values of incorporated thymidine of duplicate/triplicate wells and compared with that of MLR cultures with Teff alone (normalized as 100%). Data are represented as mean ± SEM; n = 3 independent experiments done in duplicate, triplicates, or quadruplicates leading to a total of ≥ 8 independent values for each condition. **P = 0.002; ***P < 0.001. (B) TIM-3+GFP+FoxP3+ and TIM-3–GFP+FoxP3+ cells were sorted from harvested dLNs of 30 to 35 C57BL/6-KI mice grafted with BALB/c skin on day 7. RNA was isolated from sorted TIM-3+GFP+FoxP3+ and TIM-3–GFP+FoxP3+ populations. Gene expression of various markers was assessed by quantitative real-time PCR. Data are representative of 3 independent experiments. (C) CD4+TIM-3+ Tregs and CD4+TIM-3– Tregs harvested from the dLNs on day 7 were stained and analyzed by flow cytometry for various Treg functional markers as shown. Arrows in the histograms depict TIM-3– Tregs (a) and TIM-3+ Tregs (b). Numbers represent the percentage of TIM-3+ and TIM-3– Tregs, respectively. n ≥ 5 mice.