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 4

At the time of rejection of skin allografts, TIM-3+ Tregs in dLNs are proliferating, express surface PD-1, are programmed for cell death, and are galectin-9 sensitive.

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At the time of rejection of skin allografts, TIM-3+ Tregs in dLNs are pr...
(A) Cells were analyzed from C57BL/6-KI recipients grafted with full-thickness body skin of BALB/c mice on day 7 after transplantation. Proliferation of TIM-3+ Tregs was compared with that of TIM-3– Tregs in dLNs on day 7, as deduced via staining for BrdU (injected i.p. 24 and 12 hours prior to harvesting of cells). Representative plots of n = 4 animals are depicted. (B) The proportion of PD-1–expressing TIM-3+ Tregs in comparison with that of TIM-3– Tregs and TIM-3+ Teffs in dLNs. In the histogram, 1 represents fluorescence minus one for PD-1 staining; 2 represents FoxP3+TIM-3– cells; 3 represents FoxP3+TIM-3+ cells; and 4 represents FoxP3–TIM-3+ cells. Data are representative of n = 5 animals. (C) TIM-3+ Tregs were analyzed in comparison with TIM-3– Tregs and TIM-3+ Teffs from dLNs for their ability to stain cell surface phosphatidyl serine with annexin V and exclude the LIVE/DEAD blue viability dye. Representative plots of n ≥ 8 animals are shown. (D) Susceptibility of TIM-3+ Tregs to galectin-9–induced cell death was tested on GFP+FoxP3+ cells obtained by sorting from spleens of mice grafted with allogeneic skin on day 7. Cells were cultured with 0.5 μM galectin-9/PBS for 4 hours ex vivo. TIM-3+ Tregs were assessed for staining with annexin V and LDB by flow cytometric analysis. Numbers represent the percentage of TIM-3+ or TIM-3– Tregs or TIM-3+FoxP3– cells as indicated in each panel. Data are representative of n = 3 experiments.