TIM-3, initially described as a Th1-specific marker belonging to the TIM family of proteins, fosters the specific elimination of TIM-3+
Teffs, e.g., Th1 and Th17, on interaction with its ligand galectin-9 (7
). Often coexpressed with TIM-3, PD-1 is also a marker expressed upon underperforming dysfunctional CD4+
T cells (18
). Based on these studies, we hypothesized that TIM-3 expression on Tregs would identify a senescent or exhausted subpopulation of Tregs.
In a fully allogeneic mouse skin transplant model, we have identified a subset of graft-infiltrating Tregs that expresses TIM-3 (ca. 40% of all graft-infiltrating Tregs as rejection becomes apparent), of which, about half coexpress PD-1 (data not shown). The number and frequency of CD4+FoxP3+TIM-3+ cells increased in recipient lymphoid tissues, peaked at the time of rejection, and fell to basal levels thereafter, likely due to a combination of cell death and the clearance of antigen that drives proliferation. The physiological proinflammatory and proliferative cytokine milieu during surgical intervention and healing process may contribute to the observed increase in TIM-3+ Tregs in dLNs of syngeneic graft recipients on day 7, which is significantly lower than that for allogeneic graft recipients. Indeed, TIM-3+ Tregs comprise a major proportion of skin graft-infiltrating Tregs. The TIM-3+ Treg subset is enriched for donor reactivity as (a) after DST, expansion of a donor-directed CD4+FoxP3+TIM-3+ Vβ6-TCR, but not Vβ8-TCR, Treg subset was observed in a tolerance-promoting model, wherein DBA/2 splenocytes (H-2d, Mlsa) transfused into BALB/c mice (H-2d, Mlsb) stimulated expansion of donor-reactive Mlsa-specific Vβ6+, but not Mlsa unresponsive Vβ8+, CD4+ T cells; (b) CD4+FoxP3+TIM-3+ T cells were particularly prominent, ca. 40% of graft-infiltrating Tregs, as clinically apparent rejection begins; (c) during the allograft response, TIM-3+ Tregs proliferated more vigorously than TIM-3– Tregs; and (d) contraction of the TIM-3+PD-1+ Tregs after transplantation was more profound than contraction of TIM-3– Tregs after graft rejection.
Both in vitro and in vivo studies demonstrated that TIM-3+ Tregs emerge from the TIM-3– Treg subset. Anti-CD3/CD28–mediated activation in vitro of Tregs stimulates emergence of a TIM-3+ Treg population. TIM-3+ Tregs also arise from CD4+FoxP3+TIM-3– cells in vivo during homeostatic proliferation. Hence, physiological processes leading to Treg expansion in the allograft response drive expression of TIM-3 by Tregs.
An intracellular PD-1 protein pool is detected in resting Tregs, whereas cell surface expression of PD-1 is detected on activated Tregs (24
). Here, we have noted that 75% to 80% of TIM-3+
Tregs express cell surface PD-1 molecules in the dLNs of mice challenged with a skin allograft. Expression of inhibitory receptor PD-1 on Teffs is associated with imminent cell death. Tregs that coexpress TIM-3 in addition to PD-1 stain more intensely for cell surface phosphatidylserine and therefore appear more likely to perish than cells that are TIM-3+
. Senescent and dysfunctional characteristics of TIM-3+
Teffs that coexpress PD-1 have been well appreciated (13
). Signaling through PD-1 plays an important role in the exhaustion of Teffs (15
); however, PD-1 expression on T cells may not always be indicative of T cell dysfunction (32
We tested the hypothesis that CD4+
Tregs, which are often PD-1+
like the CD4+
Teff population, are programmed for cell death and senescent, as detected by annexin V staining (a marker of early stage programmed cell death), or dead, as deduced by their failure to exclude vital dyes. In keeping with our prediction, 70% of CD4+
T cells were stained by annexin V; however, more than 95% were viable, as these cells exclude vital dyes. Thus, TIM-3 expression on Tregs unlike TIM-3+
Teffs may not serve as a marker of senescent Tregs. In fact, TIM-3+
Tregs, unlike TIM-3+
Teffs, proliferate vigorously. While, the blockade of inhibitory signals via PD-1 has been shown to rescue senescent Teffs (33
), PD-1 blockade of CD4+
cells, which are already annexin V+
, with anti–PD-1 in vitro (data not shown), did not influence the viability of TIM-3+
Tregs, as deduced by staining with annexin V and LIVE/DEAD blue vital dye. Subsequently, we analyzed the functional and molecular phenotypes of TIM-3+
Tregs. Unlike the dysfunctional status of TIM-3+
Tregs were better suppressors of Teff proliferation than TIM-3–
Tregs in an in vitro MLR. The increased frequency and expression of CTLA-4, CD39, IL-10, and TGF-β by TIM-3+
Tregs hints at the molecular basis for their increased potency in comparison to TIM-3–
Tregs. Paradoxical to the in vitro suppression assay, TIM-3+
Tregs, which are also annexin V+
, demonstrated less pronounced suppressive effects in an in vivo suppressive assay. The in vivo results do not invalidate the in vitro data, as the in vitro suppression assessment is made by culturing the cells with Teffs immediately, which may not affect the cell potency, and the assay lasts for 4 days. During the in vivo experiment, for over 15 days, the properties, including the viability and potency of this subpopulation, may have changed. It seems likely that many of the TIM-3+
cells die. Moreover, cell surface phosphatidylserine+
Tregs could not be rescued, even in the absence of ligands that trigger cell death, galectin-9, and PDL-1 (data not shown), indicating that these cells are indeed programmed to undergo cell death.
The presence of death molecules TIM-3 and PD-1 on this highly potent Treg subset may serve as a checkpoint, leading to the elimination of the TIM-3+ Treg subset after graft rejection. We asked what may account for the contraction of the TIM-3+ Treg subset after rejection. One factor must be disappearance of donor antigen after rejection. The sensitivity of TIM-3+ Treg to galectin-9 expressed on other graft-infiltrating mononuclear leukocytes may lead to death and contraction of TIM-3+ Tregs. While expression of other Treg effector molecules was increased on the TIM-3+ Treg subset, conspicuously the expression of galectin-9 was reduced drastically. We suspect that survival of the TIM-3+galectinhi Treg subset is infrequent, because the expression of both the death molecules, TIM-3 and PD-1, and their ligands, galectin-9 and PD-L1, likely commit these cells to suicide.
In addition, this highly potent TIM-3+PD-1+galectin-9lo Treg subset arises and expands in a microenvironment, as a result of continued T cell activation. Absence of antigen renders these annexin V+TIM-3+ and PD-1+ cells vulnerable to death, as they are programmed for apoptosis and are sensitive to galectin-9. The antigen-dependent disappearance of such a potent subset of immune-regulatory cells is suggestive of a homeostatic mechanism to deplete a highly potent Treg population that could otherwise hinder Teff immunity in the graft and dLNs.
Several biological effects have been attributed to the galectin-9/TIM-3 interaction (17
); however, in addition to galectin-9, TIM-3 binds to at least one other unidentified ligand (37
). The loss of TIM-3+
cells is attributed to galectin-9–induced apoptosis, based on in vitro studies (13
), but a direct in vivo role has yet to be demonstrated directly. Our in vitro results are consistent with these findings, demonstrating that galectin-9 induces the loss of TIM-3+
Tregs. Su et al. recently demonstrated a dose-dependent, TIM-3–independent role for galectin-9 in the induction of apoptosis and cytokines in T cells. Wild-type and TIM-3KO Th1 cells were equally susceptible to galectin-9–induced apoptosis (38
). Conclusions regarding the biological effects of the galectin-9/TIM-3 axis have been drawn based on galectin-9 administration (39
); in such a situation it is difficult to distinguish between the TIM-3–dependent and –independent effects of galectin-9. TIM-3 is classically believed to be a death molecule, due to its binding to galectin-9; however, a ligand-independent role of TIM-3 has been demonstrated to enhance cytokine production and proliferation by primary T cells after stimulation through CD3 and CD28 (41
Tregs arising during the anti-allograft response are highly proliferative and have enhanced cytokine production, a function that seems to be independent of galectin-9.
Studying the ligand-receptor role of TIM-3+ Tregs by blocking the TIM-3/galectin-9 and PD-1/PDL-1 axes in transplant models suggested a concomitant affect on the TIM-3+ Treg and TIM-3+ Teff compartments. To study the role of galectin-9 and PDL-1 on the function and phenotype of TIM-3+PD-1+ Tregs in vivo, a FoxP3+ Treg lineage-specific TIM-3 and PD-1 double knockout, and perhaps an alloreactive T cell receptor transgene, is necessary. Moreover, a deeper understanding of the role that ligand interactions play in modulating TIM-3+ Treg functions in transplantation also awaits the discovery of the second ligand of TIM-3.
Thus, in this study we describe a highly potent subset of Tregs arising in response to alloantigens and expressing death molecules TIM-3 and PD-1. Contrary to our hypothesis, Tregs expressing these death molecules were not functionally impaired as Tregs. Instead TIM-3+PD-1+galectin-9lo Tregs vigorously proliferated and robustly expressed Treg functional molecules. As compared with TIM-3– Tregs, the TIM-3+ Tregs manifested enhanced immunoregulatory effector properties, but following rejection, i.e., in the absence of donor antigen cells and perhaps owing to expression of the TIM-3 and PD-1 death molecules, the TIM-3+ Tregs did not persist. It is interesting that, as the allograft response progresses, Tregs become more potent and prominent and yet express death molecules. Noted in parallel with phosphatidylserine positivity, TIM-3 and PD-1 expression by Tregs may represent an activation-induced stage in differentiation, manifested by increased potency and a commitment death. In this way local immunoregulation by antigen-driven Tregs is likely heightened and then largely ablated, thereby allowing subsequent local immune reactions to take place without undo and potentially harmful restraint by Tregs. Thus, the activation-induced expression of TIM-3 and PD-1 is not likely crucial to their immunoregulatory effector properties. Instead, the expression of these molecules manifests after several waves of proliferation and is expressed parallel with cell surface phosphatidylserine, possibly reflecting a means by which proliferating, graft-infiltrating antigen-reactive Tregs are cleared from the environment. Thus, the surge of Tregs with increased potency after multiple waves of proliferation is balanced by ensuring their clearance. The apparent result of these events is to produce both potent and short-lived Tregs that are cleared from the microenvironment after rejection. Hence, the immune response to microbes within this microenvironment is not permanently hampered.