T cell exhaustion is thought to be a natural mechanism for limiting immune pathology, although it may be desirable to circumvent this mechanism to help eliminate viral reservoirs or tumors. Although there are no definitive markers, a fingerprint for exhausted T cells has been described that includes the transmembrane proteins PD-1, LAG3, and Tim-3. However, apart from the recruitment of tyrosine phosphatases to PD-1, little is known about the biochemical mechanisms by which these proteins contribute to the development or maintenance of exhaustion. Tim-3 contains no known motifs for the recruitment of inhibitory phosphatases, but it may actually increase signaling downstream of TCR/CD3, at least under acute conditions. Other studies showed that T cell exhaustion results from chronic stimulation that extends the effector phase of T cell activation, at the expense of T cell memory. We suggest that Tim-3 may contribute to T cell exhaustion by enhancing TCR-signaling pathways.

T cell activation, including development of a robust memory response, is critical for the development of an efficient immune response to viral infection and can also be instrumental in mounting an immune response to solid tumors. However, overly vigorous or sustained immune responses can cause immune-mediated pathology that is detrimental to the host. Such a problem is particularly evident with viruses that cause chronic infections (1). In these cases, the sustained presence of viral Ags appears to drive the formation of a state of Ag-specific T cell exhaustion. Although this has the beneficial effect of limiting immune pathology, it can result in the establishment of a viral reservoir that may become reactivated under conditions of physiological stress. T cell exhaustion can also be detrimental when it impairs the ability of an adaptive immune response to eliminate a tumor.

Functionally, the development of T cell exhaustion is characterized by the gradual loss of expression of various cytokines and effector molecules, with IL-2, cytotoxicity and proliferation among the earliest affected functions and IFN-γ among the latest (1, 2). Exhausted T cells also may become “addicted” to AgR signals and lose responsiveness to the homeostatic cytokine IL-7, with the latter due in part to loss of CD127 (IL-7r α-chain) expression (2). Importantly, for possible therapeutic reversal, exhausted T cells also gain high-level and persistent, as opposed to transient, expression of several proteins, including the transcription factor BLIMP-1 and the transmembrane proteins PD-1, Tim-3, and LAG3 (1, 2). The latter proteins, so-called “checkpoint” receptors, have attracted attention as possible dominant mediators of T cell exhaustion because Abs to these proteins or their ligands can sometimes rescue the function of exhausted T cells (24). Because this topic has been covered extensively in other relatively recent reviews (1, 2), we focus mainly on recent studies of Tim-3, which has attracted substantial preclinical attention of late as a novel therapeutic target for reversal of T cell exhaustion. We also review what is known regarding signal transduction pathways implicated in Tim-3 function. Finally, we discuss the role of TCR signaling in driving the development of exhaustion and how this might be influenced by Tim-3.

The tumor microenvironment is known to be immunosuppressive as a result of inhibitory signals from cell surface and soluble mediators (5), although the precise strategies used by different tumors can vary by tissue and even from patient to patient. Although T cells specific to tumor Ags can be readily isolated from solid tumors of patients and in mouse models, these cells often respond poorly to ex vivo stimulation. This T cell dysfunction is thought to result, at least in part, from exhaustion (or over-stimulation) of effector tumor-infiltrating lymphocytes. T cell exhaustion is caused by chronic antigenic stimulation and expression of inhibitory coreceptors and cytokines, among other factors (6). Based on the recent success of CTLA-4 Ab therapy (7), as well as accumulating data from preclinical models, there is now considerable excitement surrounding molecules whose targeting may allow for broad enhancement of T cell responses against tumors. Solid tumor–infiltrating T cells often express high levels of one or more inhibitory or exhaustion-associated receptors, including PD-1, LAG3, and/or Tim-3. Indeed, consistent with Ag acting as a driver of exhaustion, a recent study on melanoma patients demonstrated that PD-1 can be used to prospectively distinguish tumor-specific T cells at the tumor site (8). Tim-3 expression on T cells is also seen in the context of nonsolid tumors. For example, upregulation of Tim-3 (possibly driven by IL-12) on effector T cells of patients with follicular B cell non-Hodgkin lymphoma was associated with poor outcomes (9).

PD-1 has been extensively studied as a potential therapeutic target, and recent clinical trial data suggest that mAbs to PD-1 or one of its ligands, PD-L1, are clinically effective against certain solid tumors, including melanoma, as well as non-small cell lung cancer, generally regarded as a nonimmunogenic tumor (1012). mAbs specific for Tim-3 also were shown to promote rejection of solid tumors in murine models (13, 14), and mAbs to human Tim-3 can rescue the ex vivo function of apparently exhausted T cells from tumor-bearing patients (15). In the former case, the efficacy of Tim-3 mAb therapy appeared to result, at least in part, from effects on regulatory T cells (Tregs), which also can express Tim-3 (16). Strikingly, these Tim-3+ Tregs appear to be among the most potent at inhibiting effector T cell function and express greater levels of IL-10. In this study, monotherapy with Tim-3 Ab was not sufficient to augment antitumor immunity in vivo, but rather it cooperated with primary anti–PD-L1 treatment (13). High levels of Tim-3 also have been observed on tumor-infiltrating Tregs from human patients with non-small cell lung cancer, a finding that correlated with poor clinical outcomes (17). Thus, in the case of tumors, it clearly will be important to parse out the effects of Tim-3 manipulation on effector/exhausted T cells from the effects on Tregs. In addition, the precise role played by Tim-3 may vary, depending on tumor type.

T cell exhaustion has been observed in mouse models (discussed above), and in human disease, mainly in the context of various chronic viral infections (1825). For instance, a population of Tim-3+CD8+ T cells is readily identified in HIV patients (26), and the expression of Tim-3 correlates positively with progression to AIDS and inversely with viral control (27). Ex vivo stimulation of these HIV-associated, exhausted, CD8+ T cells in the presence of Tim-3 mAb can at least partially rescue their function (26), whereas antiretroviral therapy may reduce Tim-3 expression (28). Similarly, study of hepatitis C virus (HCV)-infected patients revealed the presence of dysfunctional CD4+ and CD8+ T cells with high-level Tim-3 expression (29, 30). In both HIV and HCV, upregulation of Tim-3 was associated with the accumulation of central memory (CD45RACCR7+) T cells (26, 29). Thus, a large body of evidence now supports a net negative impact of Tim-3 expression on T cell–dependent antiviral immune responses.

The role of Tim-3 in tuberculosis (TB) has proven to be quite complex and points out that effects of Tim-3 expression (or its therapeutic targeting) on infectious disease outcomes may be disease and/or context specific. An elegant study by Behar and colleagues (31) revealed a novel and unexpected function for Tim-3 in a murine model of TB. Thus, the investigators found that interaction of Tim-3 with one of its ligands—galectin-9 (gal9), expressed on macrophages—stimulated the production of the cytokine IL-1b, enhancing bacterial killing. This effect was mimicked by administration of Tim-3–Ig fusion protein, through its interaction with gal9. A similar phenomenon was observed in human macrophages (32). At this point, the nature of the signal transmitted by gal9 after Tim-3 binding is unknown. Still, this finding is of possible relevance for the interpretation of multiple other studies, because a soluble Tim-3–Ig fusion protein is often used as a blocking reagent in other settings. Returning to T cells and human patients, active TB, as opposed to latent infection, is associated with the upregulation of Tim-3 on both CD4+ and CD8+ T cells (33). Surprisingly, however, these Tim-3+ T cells display more potent anti-TB responses, contrary to what has been observed in chronic viral infection. Although the reasons for these differences are not clear, the investigators speculated that they may be due to the somewhat divergent phenotypes of Tim-3+ TB-specific T cells compared with Tim-3+ T cells found in HIV and HCV patients. For example, Tim-3+ T cells in TB patients were found to express CD127 (IL-7r α-chain) (33), whereas this marker is well known to be lost in exhausted T cells (including those that are Tim-3+) during chronic viral infections of humans and mice (1, 2).

A recently published study using a newly generated Tim-3–knockout mouse model provides further support for a context-dependent positive role for Tim-3 in vivo, in this case in acute bacterial infection. Colgan and colleagues (34) first infected wild-type (WT) mice with Listeria monocytogenes and followed the expression of Tim-3. Tim-3 was robustly expressed on CD8+ T cells during this infection, with particularly high expression on effector T cells. In mice with a germline deletion of Tim-3, both primary and secondary T cell responses to L. monocytogenes were significantly impaired. A similar, although less severe, defect was also seen when Tim-3–deficient T cells were transferred to WT hosts. Finally, evidence was provided that the decreased T cell responses in the absence of Tim-3 were due in part to the compromised survival of the knockout T cells. It should be noted that the Tim-3–deficient mice used in this study were derived from 129 strain embryonic stem cells and then backcrossed to C57BL/6 mice for 10 generations. Because the Tim locus is polymorphic between 129 and C57BL/6, the carryover of polymorphisms in Havcr1 (encoding Tim-1) and in other neighboring genes should be taken into account as potential confounding variables in this study. Nonetheless, this report provides additional compelling evidence that the effects of Tim-3 expression during infection are complex and may lead to enhanced, rather than diminished, T cell responses.

Surprisingly, at this point there is relatively sparse evidence to support the model that Tim-3 directly mediates suppression of T cell activation or cytokine secretion in a manner similar to PD-1. Rather, despite evidence that Tim-3 manipulation can ameliorate T cell exhaustion, there are a number of observations that do not fit with a simple narrative of direct inhibition of T cell activation by Tim-3. First is the fact that Tim-3+ T cells from HIV-infected patients display defective stimulation-induced phosphorylation of Stat5, ERK1/2, and p38, but actually possess higher basal phosphorylation of all of the same pathways (26). This is consistent with our own data obtained with ectopic expression of Tim-3 in T cell lines and primary T cells. Thus, ectopic expression of Tim-3 actually enhanced T cell activation, leading to increased activation of NFAT/AP-1 and NF-κB transcriptional reporters, and it even enhanced cytokine production from both T cell lines and primary T cells (35).

When considering how Tim-3 might signal to regulate the development or activation of exhausted T cells, it may be instructive to consider the reported functions of Tim-3 in myeloid lineage cells. Ligation of Tim-3 with specific Abs or one of the reported Tim-3 ligands can (under some circumstances) enhance the activation and function of various myeloid lineage cell types (36). This is consistent with induction of the transcription factor NF-κB after Tim-3 mAb treatment of dendritic cells (37). In addition, Ab ligation of Tim-3 on murine bone marrow–derived mast cells was shown to augment IgE/Ag-induced cytokine production (38), a finding that we reproduced with multiple Abs to Tim-3 (B. Phong and L.P. Kane, unpublished observations). Another recently described function for Tim-3 in dendritic cells is to inhibit an innate response to nucleic acids, an effect that involves interaction of Tim-3 with the endogenous danger signal HMGB1 (39). This unusual pathway seems to prevent the trafficking of exogenously acquired DNA into endosomes. The precise mechanism underlying this effect is unclear (e.g., whether it is mediated by active Tim-3 signaling or by passive blockade of HMGB1 function), but it may have relevance for the effects of Tim-3 in T cell responses against viral and/or tumor Ags. It should be noted that this is one of the few published studies to directly demonstrate that a particular Tim-3 Ab could block interaction with the ligand under study (in this case HMGB1). Several studies also suggested a negative regulatory role for Tim-3 in myeloid cells (4042). Thus, as in the case of T cells, it appears that the precise effects of Tim-3 on myeloid cells depend upon the context in which Tim-3 ligation occurs.

Administration of certain Tim-3 Abs can rescue the function of exhausted T cells in both in vivo mouse models (13, 23) and in vitro experiments with cells from patients with chronic viral infections or tumors (15, 26). However, particularly in the case of the aforementioned in vivo experiments, it is not clear whether the effects of the Tim-3 Abs are actually the direct result of blocking the interaction of Tim-3 with one or more of its ligands (e.g., gal9 and/or HMGB1 or other yet-to-be discovered ligands). The presence of Tim-3 on other cells, including APCs and Tregs, might also explain the function of at least some Tim-3 Abs in certain disease settings. In such cases, one might imagine that either agonistic or antagonistic activities of such Abs could eventually lead to the observed downstream functional effects. However, specific agonistic or antagonistic activity has not been directly ascribed yet to the most commonly used Tim-3 Abs. One approach to this problem would be to compare the activities of Tim-3 mAbs in their native forms versus the effects of F(ab)′ fragments of the same Abs. This will be an important point to clarify in future studies.

When we began studying signaling pathways linked to Tim-3, we were intrigued by the presence of multiple tyrosine residues in the cytoplasmic tails of both murine and human Tim-3 (Fig. 1A). However, the sequences around these tyrosines do not conform to any known inhibitory signaling motifs (e.g., ITIM, ITSM), such as those found in PD-1. Analysis of the cytoplasmic tail of murine Tim-3 with the Scansite algorithm (http://scansite3.mit.edu) revealed that several of these tyrosines conformed well to putative sites of phosphorylation by multiple tyrosine kinases, particularly those of the Src family (Fig. 1B). Importantly, the analogous tyrosines in human Tim-3 also scored as possible sites of phosphorylation by the same classes of kinases (data not shown). We (35) and other investigators (43, 44) demonstrated that the cytoplasmic tail of Tim-3 can be phosphorylated on multiple tyrosine residues. Although the identity of the kinases that phosphorylate the Tim-3 cytoplasmic tail have not been definitively shown in vivo, our data are consistent with such phosphorylation being carried out by the Src family kinases Fyn and/or Lck, at least in T cells (35). However, it should be pointed out that the Tec family kinase Itk also has been implicated in the phosphorylation of Tim-3 (43). Tyrosine phosphorylation often functions to recruit downstream signaling proteins, particularly those containing Src homology 2 (SH2) domains. Thus, we found that the SH2 domain of Fyn and one of the SH2 domains of the PI3K adaptor protein p85 could bind to a phosphorylated peptide corresponding to the region around Y256 and Y263 of murine Tim-3 (35). Consistent with these findings, we also found that ectopic expression of Tim-3 enhanced the phosphorylation of both PLC-γ1 (which is dependent on Src and Syk family kinases) and ribosomal protein S6, which lies downstream of the PI3K/Akt/mTOR pathway (35). Some of these experiments were performed with the workhorse Jurkat T cell model (45), which does have some limitations, including lack of PTEN expression. Importantly, however, key findings of these studies were reproduced in the murine T cell clone D10, as well as in primary murine T cells (35). Thus, at the levels of both signal transduction and function (cytokine production), acute upregulation of Tim-3 expression can enhance T cell activation. A current challenge is deciphering how these observations fit into the current paradigm of Tim-3 function in vivo.

FIGURE 1.

Amino acid sequence of the Tim-3 cytoplasmic tail and possible sites of phosphorylation. Alignment of the murine and human Tim-3 cytoplasmic tail sequences (upper panel). Predicted sites of tyrosine phosphorylation are numbered, and other tyrosines are in bold type. Conserved charged residues (suggesting favorable sites for phosphorylation) upstream of Y256 and Y263 are underlined. Results of a Scansite search using the murine Tim-3 cytoplasmic domain and restricting results to cytoplasmic tyrosine kinases (lower panel). Analogous tyrosines in human Tim-3 also were predicted to be phosphorylated by the same kinases (data not shown).

FIGURE 1.

Amino acid sequence of the Tim-3 cytoplasmic tail and possible sites of phosphorylation. Alignment of the murine and human Tim-3 cytoplasmic tail sequences (upper panel). Predicted sites of tyrosine phosphorylation are numbered, and other tyrosines are in bold type. Conserved charged residues (suggesting favorable sites for phosphorylation) upstream of Y256 and Y263 are underlined. Results of a Scansite search using the murine Tim-3 cytoplasmic domain and restricting results to cytoplasmic tyrosine kinases (lower panel). Analogous tyrosines in human Tim-3 also were predicted to be phosphorylated by the same kinases (data not shown).

Close modal

A recent report suggested that interaction of Tim-3 with a chaperone protein known variously as Bat3, Bag6, or Scythe regulates suppression of T cell responses by Tim-3 (46). One of the more intriguing findings reported in this article was that knockdown of Bat3 led to a dramatic upregulation of Tim-3 and other (although not all) phenotypic and functional markers of T cell exhaustion. The investigators also identified an interaction between Bat3 and a pool of Lck tyrosine kinase that was phosphorylated on its activation loop tyrosine (Y394), but not the inhibitory tyrosine near the C terminus (Y505), suggesting a preferential interaction of Bat3 with active Lck. Furthermore, this interaction appeared to be inhibited by Ab ligation of Tim-3, which itself was also seen to interact with Lck (46). The latter finding is consistent with our own observation that Tim-3 could interact with Lck, as well as with the related Src family tyrosine kinase Fyn (35). Importantly, we confirmed that the T cell lines used in our signaling experiments express Bat3 (L.P. Kane, unpublished observations). Whereas these results suggest a possible mechanism for inhibition of proximal TCR signaling by Tim-3 and Bat3, this study did not address the effects of TCR signaling on the Tim-3/Bat3 interaction. Thus, further investigation will be necessary to determine the precise relationship between these different pools of Tim-3 and Src family kinases, as well as the consequences on downstream-signaling pathways, in exhausted T cells.

Another recent study examined the interaction of Tim-3 with transmembrane proteins expressed on T cells and reported that Tim-3 can associate with the transmembrane phosphatases CD45 and CD148, which the investigators proposed might promote dephosphorylation of downstream mediators of T cell activation (44). Furthermore, Tim-3, CD45, and CD148 were found to be recruited into the immunological synapse. Such a mechanism could help to explain the observed negative effects of Tim-3 on T cell activation during exhaustion. It remains to be seen to what extent these various interactions affect Tim-3 function, including whether the interaction of these phosphatases with Tim-3 modulates its interaction with Bat3 or Lck, as discussed above.

T cell exhaustion appears to be maintained by transcriptional reprogramming (e.g., through BATF) and/or active negative signaling through receptors like PD-1 (2). Indeed, tyrosine phosphatases like SHP1/2 were shown to mediate dominant suppression of TCR signaling after ligation of PD-1 (47). However, it is quite clear that development of T cell exhaustion is driven, at least in part, by high levels—and/or the sustained presence—of cognate Ag (48, 49). Evidence (much of it still indirect) has begun to emerge regarding how this process may be controlled by TCR-signaling pathways. Several studies have implicated Akt- and/or mTOR-dependent signaling in this checkpoint. Thus, a study from Kaech and colleagues (50) demonstrated that ectopic expression of Akt led to reduced memory T cell formation. In addition, Cantrell and colleagues (51) showed that although inhibition of Akt impairs the function of CTL effector cells, it actually promotes the development of memory cells. Similar conclusions were reached in a study by Suresh and colleagues (52). Among the many downstream effects of Akt signaling is the activation of an mTOR-containing complex known as mTOR complex 1 (mTORC1) (53). Consistent with the above data, several groups demonstrated that limiting the activation of mTOR enhances the development of T cell memory. This was shown through the use of the relatively specific mTORC1 inhibitor rapamycin (54, 55) or by reducing expression of the Raptor gene, which encodes a critical subunit of the mTORC1 complex (54).

These previous studies on Akt and mTOR in T cell memory are also intriguing in light of a recent report describing a human immunodeficiency associated with activating mutations of the PI3K catalytic protein p110δ, with consequent hyperactivation of downstream Akt and mTOR signaling (56). Of relevance for the discussion of T cell exhaustion, patients with these mutations present with an accumulation of terminally differentiated T cells. These T cells are refractory to stimulation with mitogen and display a deficient recall response to tetanus toxoid, consistent with a defect in generation of stable memory T cell responses. It should be noted that patients with the activating p110δ mutations have a combined immunodeficiency because B cells are also affected. Strikingly, treatment of one of the affected patients with the mTORC1 inhibitor rapamycin partially restored the T cell compartment and improved clinical outcomes (56).

The studies discussed above provide compelling evidence that excessive and/or sustained activation of one or more signaling pathways is critical for driving the development of T cell exhaustion. However, it is still not known whether such signals are initiated solely from the TCR itself or whether other molecules might also contribute. Given what is known about the multifactorial regulation of T cell activation by costimulatory receptors, the latter seems more likely. Based on our previous findings demonstrating the ability of Tim-3 to enhance TCR signaling under acute conditions (35), we propose that Tim-3 might function, at least in part, to help drive T cell exhaustion by enhancing TCR/CD28-dependent signaling (Fig. 2). In such a scenario, upregulation of Tim-3 during an extended effector phase of T cell activation would act in a feed-forward loop to enhance T cell activation signals and drive T cells even more toward an exhausted phenotype, at the expense of T cell memory (Fig. 2A). An additional nonexclusive possibility is that positive signals from Tim-3 augment the (suppressive) function of Tregs, contributing to the overall immune-activating effects of Tim-3 “blocking” Abs. With respect to the specific signaling pathways downstream of Tim-3, the work described above suggests that at least one such pathway is the PI3K/Akt/mTOR pathway, with upstream involvement of Src or Tec kinases and downstream involvement of transcription factors, like NFAT or NF-κB (Fig. 2B). Coming back to the role of signaling in driving T cell exhaustion (discussed above), we propose that an accessory receptor like Tim-3 could help to drive this process, in part through enhanced PI3K/Akt/mTOR signaling.

FIGURE 2.

Speculative model for the development of T cell exhaustion. (A) Acute infection results in elimination of a pathogen (and its Ags) and allows for the formation of a pool of memory T cells (upper portion). This response may be accompanied by transient and/or low-level expression of Tim-3. Establishment of a chronic infection (or significant tumor burden) promotes the development of T cell exhaustion, marked in part by upregulation of Tim-3 (lower portion). This process may be accelerated by Tim-3–derived signals that enhance TCR/CD3 signaling in the short-term. In addition, these positive signals might contribute to the reversal of exhaustion by some Tim-3 mAbs. (B) Tim-3–derived positive signals that may contribute to the induction of T cell exhaustion or activation. These positive signals could contribute to the reversal of exhaustion by some Tim-3 Abs, although the latter could be the result of qualitatively different signals or blocking of an undefined inhibitory coreceptor.

FIGURE 2.

Speculative model for the development of T cell exhaustion. (A) Acute infection results in elimination of a pathogen (and its Ags) and allows for the formation of a pool of memory T cells (upper portion). This response may be accompanied by transient and/or low-level expression of Tim-3. Establishment of a chronic infection (or significant tumor burden) promotes the development of T cell exhaustion, marked in part by upregulation of Tim-3 (lower portion). This process may be accelerated by Tim-3–derived signals that enhance TCR/CD3 signaling in the short-term. In addition, these positive signals might contribute to the reversal of exhaustion by some Tim-3 mAbs. (B) Tim-3–derived positive signals that may contribute to the induction of T cell exhaustion or activation. These positive signals could contribute to the reversal of exhaustion by some Tim-3 Abs, although the latter could be the result of qualitatively different signals or blocking of an undefined inhibitory coreceptor.

Close modal

As discussed above, it will be critical to more thoroughly define the biophysical and biochemical properties of the various Tim-3 Abs being used in both mouse and human studies. Thus, it is still a distinct possibility that some Abs act to enhance signaling in T cells (or other cell types). In this regard, it is interesting to note that the efficacy of mAbs targeting CTLA-4 for immunotherapy of melanoma, at least in mouse models, was just recently attributed to FcR-dependent depletion of Treg (57, 58), despite two decades of study of CTLA-4 biology. Finally, the unintended consequences of Ab manipulation of CD28 offer a cautionary tale with regard to the clinical translation of a target that regulates T cell activation (5962).

Recent investigation into Tim-3 function has clearly elucidated its importance, in part due to its frequent upregulation during antiviral T cell responses or in the tumor microenvironment. Preclinical studies in mouse models are also encouraging. However, these studies are generally restricted by a lack of suitable reagents to clarify the significance and function of a putatively stimulatory signal on a population of exhausted T cells. These gaps in the field are slowly being rectified, but this must be accelerated because therapeutic manipulation in patients is being seriously contemplated.

This work was supported by National Institutes of Health Grants DE019727 and CA097190 (to R.L.F.), CA167229 and CA097190 (to B.L.), and AI109605 and AI073748 (to L.P.K.).

Abbreviations used in this article:

gal9

galectin-9

HCV

hepatitis C virus

mTORC1

mTOR complex 1

SH2

Src homology 2

TB

tuberculosis

Treg

regulatory T cell

WT

wild-type.

1
Virgin
H. W.
,
Wherry
E. J.
,
Ahmed
R.
.
2009
.
Redefining chronic viral infection.
Cell
138
:
30
50
.
2
Wherry
E. J.
2011
.
T cell exhaustion.
Nat. Immunol.
12
:
492
499
.
3
Odorizzi
P. M.
,
Wherry
E. J.
.
2012
.
Inhibitory receptors on lymphocytes: insights from infections.
J. Immunol.
188
:
2957
2965
.
4
Sakuishi
K.
,
Jayaraman
P.
,
Behar
S. M.
,
Anderson
A. C.
,
Kuchroo
V. K.
.
2011
.
Emerging Tim-3 functions in antimicrobial and tumor immunity.
Trends Immunol.
32
:
345
349
.
5
Drake
C. G.
,
Jaffee
E.
,
Pardoll
D. M.
.
2006
.
Mechanisms of immune evasion by tumors.
Adv. Immunol.
90
:
51
81
.
6
Schietinger
A.
,
Greenberg
P. D.
.
2014
.
Tolerance and exhaustion: defining mechanisms of T cell dysfunction.
Trends Immunol.
35
:
51
60
.
7
Lipson, E. J., and C. G. Drake. 2011. Ipilimumab: an anti-CTLA-4 antibody for metastatic melanoma. Clin. Cancer Res. 17: 6958–6962
.
8
Gros
A.
,
Robbins
P. F.
,
Yao
X.
,
Li
Y. F.
,
Turcotte
S.
,
Tran
E.
,
Wunderlich
J. R.
,
Mixon
A.
,
Farid
S.
,
Dudley
M. E.
, et al
.
2014
.
PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors.
J. Clin. Invest.
124
:
2246
2259
.
9
Yang
Z. Z.
,
Grote
D. M.
,
Ziesmer
S. C.
,
Niki
T.
,
Hirashima
M.
,
Novak
A. J.
,
Witzig
T. E.
,
Ansell
S. M.
.
2012
.
IL-12 upregulates TIM-3 expression and induces T cell exhaustion in patients with follicular B cell non-Hodgkin lymphoma.
J. Clin. Invest.
122
:
1271
1282
.
10
Topalian
S. L.
,
Hodi
F. S.
,
Brahmer
J. R.
,
Gettinger
S. N.
,
Smith
D. C.
,
McDermott
D. F.
,
Powderly
J. D.
,
Carvajal
R. D.
,
Sosman
J. A.
,
Atkins
M. B.
, et al
.
2012
.
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
N. Engl. J. Med.
366
:
2443
2454
.
11
Brahmer
J. R.
,
Tykodi
S. S.
,
Chow
L. Q.
,
Hwu
W. J.
,
Topalian
S. L.
,
Hwu
P.
,
Drake
C. G.
,
Camacho
L. H.
,
Kauh
J.
,
Odunsi
K.
, et al
.
2012
.
Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.
N. Engl. J. Med.
366
:
2455
2465
.
12
Hamid
O.
,
Robert
C.
,
Daud
A.
,
Hodi
F. S.
,
Hwu
W. J.
,
Kefford
R.
,
Wolchok
J. D.
,
Hersey
P.
,
Joseph
R. W.
,
Weber
J. S.
, et al
.
2013
.
Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma.
N. Engl. J. Med.
369
:
134
144
.
13
Sakuishi
K.
,
Apetoh
L.
,
Sullivan
J. M.
,
Blazar
B. R.
,
Kuchroo
V. K.
,
Anderson
A. C.
.
2010
.
Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity.
J. Exp. Med.
207
:
2187
2194
.
14
Ngiow
S. F.
,
von Scheidt
B.
,
Akiba
H.
,
Yagita
H.
,
Teng
M. W.
,
Smyth
M. J.
.
2011
.
Anti-TIM3 antibody promotes T cell IFN-γ-mediated antitumor immunity and suppresses established tumors.
Cancer Res.
71
:
3540
3551
.
15
Fourcade
J.
,
Sun
Z.
,
Benallaoua
M.
,
Guillaume
P.
,
Luescher
I. F.
,
Sander
C.
,
Kirkwood
J. M.
,
Kuchroo
V.
,
Zarour
H. M.
.
2010
.
Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.
J. Exp. Med.
207
:
2175
2186
.
16
Sakuishi
K.
,
Ngiow
S. F.
,
Sullivan
J. M.
,
Teng
M. W.
,
Kuchroo
V. K.
,
Smyth
M. J.
,
Anderson
A. C.
.
2013
.
TIM3(+)FOXP3(+) regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer.
OncoImmunology
2
:
e23849
.
17
Gao
X.
,
Zhu
Y.
,
Li
G.
,
Huang
H.
,
Zhang
G.
,
Wang
F.
,
Sun
J.
,
Yang
Q.
,
Zhang
X.
,
Lu
B.
.
2012
.
TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression.
PLoS ONE
7
:
e30676
.
18
Allen
S. J.
,
Hamrah
P.
,
Gate
D.
,
Mott
K. R.
,
Mantopoulos
D.
,
Zheng
L.
,
Town
T.
,
Jones
C.
,
von Andrian
U. H.
,
Freeman
G. J.
, et al
.
2011
.
The role of LAT in increased CD8+ T cell exhaustion in trigeminal ganglia of mice latently infected with herpes simplex virus 1.
J. Virol.
85
:
4184
4197
.
19
Angelosanto
J. M.
,
Wherry
E. J.
.
2010
.
Transcription factor regulation of CD8+ T-cell memory and exhaustion.
Immunol. Rev.
236
:
167
175
.
20
Barber
D. L.
,
Wherry
E. J.
,
Masopust
D.
,
Zhu
B.
,
Allison
J. P.
,
Sharpe
A. H.
,
Freeman
G. J.
,
Ahmed
R.
.
2006
.
Restoring function in exhausted CD8 T cells during chronic viral infection.
Nature
439
:
682
687
.
21
Blackburn
S. D.
,
Shin
H.
,
Haining
W. N.
,
Zou
T.
,
Workman
C. J.
,
Polley
A.
,
Betts
M. R.
,
Freeman
G. J.
,
Vignali
D. A.
,
Wherry
E. J.
.
2009
.
Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection.
Nat. Immunol.
10
:
29
37
.
22
Day
C. L.
,
Kaufmann
D. E.
,
Kiepiela
P.
,
Brown
J. A.
,
Moodley
E. S.
,
Reddy
S.
,
Mackey
E. W.
,
Miller
J. D.
,
Leslie
A. J.
,
DePierres
C.
, et al
.
2006
.
PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression.
Nature
443
:
350
354
.
23
Jin
H. T.
,
Anderson
A. C.
,
Tan
W. G.
,
West
E. E.
,
Ha
S. J.
,
Araki
K.
,
Freeman
G. J.
,
Kuchroo
V. K.
,
Ahmed
R.
.
2010
.
Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection.
Proc. Natl. Acad. Sci. USA
107
:
14733
14738
.
24
Nakamoto
N.
,
Cho
H.
,
Shaked
A.
,
Olthoff
K.
,
Valiga
M. E.
,
Kaminski
M.
,
Gostick
E.
,
Price
D. A.
,
Freeman
G. J.
,
Wherry
E. J.
,
Chang
K. M.
.
2009
.
Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.
PLoS Pathog.
5
:
e1000313
.
25
Shin
H.
,
Wherry
E. J.
.
2007
.
CD8 T cell dysfunction during chronic viral infection.
Curr. Opin. Immunol.
19
:
408
415
.
26
Jones
R. B.
,
Ndhlovu
L. C.
,
Barbour
J. D.
,
Sheth
P. M.
,
Jha
A. R.
,
Long
B. R.
,
Wong
J. C.
,
Satkunarajah
M.
,
Schweneker
M.
,
Chapman
J. M.
, et al
.
2008
.
Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection.
J. Exp. Med.
205
:
2763
2779
.
27
Kassu
A.
,
Marcus
R. A.
,
D’Souza
M. B.
,
Kelly-McKnight
E. A.
,
Golden-Mason
L.
,
Akkina
R.
,
Fontenot
A. P.
,
Wilson
C. C.
,
Palmer
B. E.
.
2010
.
Regulation of virus-specific CD4+ T cell function by multiple costimulatory receptors during chronic HIV infection.
J. Immunol.
185
:
3007
3018
.
28
Kassu
A.
,
Marcus
R. A.
,
D’Souza
M. B.
,
Kelly-McKnight
E. A.
,
Palmer
B. E.
.
2011
.
Suppression of HIV replication by antiretroviral therapy reduces TIM-3 expression on HIV-specific CD8(+) T cells.
AIDS Res. Hum. Retroviruses
27
:
1
3
.
29
Golden-Mason
L.
,
Palmer
B. E.
,
Kassam
N.
,
Townshend-Bulson
L.
,
Livingston
S.
,
McMahon
B. J.
,
Castelblanco
N.
,
Kuchroo
V.
,
Gretch
D. R.
,
Rosen
H. R.
.
2009
.
Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells.
J. Virol.
83
:
9122
9130
.
30
McMahan
R. H.
,
Golden-Mason
L.
,
Nishimura
M. I.
,
McMahon
B. J.
,
Kemper
M.
,
Allen
T. M.
,
Gretch
D. R.
,
Rosen
H. R.
.
2010
.
Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity.
J. Clin. Invest.
120
:
4546
4557
.
31
Jayaraman
P.
,
Sada-Ovalle
I.
,
Beladi
S.
,
Anderson
A. C.
,
Dardalhon
V.
,
Hotta
C.
,
Kuchroo
V. K.
,
Behar
S. M.
.
2010
.
Tim3 binding to galectin-9 stimulates antimicrobial immunity.
J. Exp. Med.
207
:
2343
2354
.
32
Sada-Ovalle
I.
,
Chávez-Galán
L.
,
Torre-Bouscoulet
L.
,
Nava-Gamiño
L.
,
Barrera
L.
,
Jayaraman
P.
,
Torres-Rojas
M.
,
Salazar-Lezama
M. A.
,
Behar
S. M.
.
2012
.
The Tim3-galectin 9 pathway induces antibacterial activity in human macrophages infected with Mycobacterium tuberculosis.
J. Immunol.
189
:
5896
5902
.
33
Qiu
Y.
,
Chen
J.
,
Liao
H.
,
Zhang
Y.
,
Wang
H.
,
Li
S.
,
Luo
Y.
,
Fang
D.
,
Li
G.
,
Zhou
B.
, et al
.
2012
.
Tim-3-expressing CD4+ and CD8+ T cells in human tuberculosis (TB) exhibit polarized effector memory phenotypes and stronger anti-TB effector functions.
PLoS Pathog.
8
:
e1002984
.
34
Gorman
J. V.
,
Starbeck-Miller
G.
,
Pham
N. L.
,
Traver
G. L.
,
Rothman
P. B.
,
Harty
J. T.
,
Colgan
J. D.
.
2014
.
Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection.
J. Immunol.
192
:
3133
3142
.
35
Lee
J.
,
Su
E. W.
,
Zhu
C.
,
Hainline
S.
,
Phuah
J.
,
Moroco
J. A.
,
Smithgall
T. E.
,
Kuchroo
V. K.
,
Kane
L. P.
.
2011
.
Phosphotyrosine-dependent coupling of Tim-3 to T-cell receptor signaling pathways.
Mol. Cell. Biol.
31
:
3963
3974
.
36
Han
G.
,
Chen
G.
,
Shen
B.
,
Li
Y.
.
2013
.
Tim-3: An Activation Marker and Activation Limiter of Innate Immune Cells.
Front. Immunol.
4
:
449
.
37
Anderson
A. C.
,
Anderson
D. E.
,
Bregoli
L.
,
Hastings
W. D.
,
Kassam
N.
,
Lei
C.
,
Chandwaskar
R.
,
Karman
J.
,
Su
E. W.
,
Hirashima
M.
, et al
.
2007
.
Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells.
Science
318
:
1141
1143
.
38
Nakae
S.
,
Iikura
M.
,
Suto
H.
,
Akiba
H.
,
Umetsu
D. T.
,
Dekruyff
R. H.
,
Saito
H.
,
Galli
S. J.
.
2007
.
TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells.
Blood
110
:
2565
2568
.
39
Chiba
S.
,
Baghdadi
M.
,
Akiba
H.
,
Yoshiyama
H.
,
Kinoshita
I.
,
Dosaka-Akita
H.
,
Fujioka
Y.
,
Ohba
Y.
,
Gorman
J. V.
,
Colgan
J. D.
, et al
.
2012
.
Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1.
Nat. Immunol.
13
:
832
842
.
40
Zhang
Y.
,
Ma
C. J.
,
Wang
J. M.
,
Ji
X. J.
,
Wu
X. Y.
,
Jia
Z. S.
,
Moorman
J. P.
,
Yao
Z. Q.
.
2011
.
Tim-3 negatively regulates IL-12 expression by monocytes in HCV infection.
PLoS ONE
6
:
e19664
.
41
Zhang
Y.
,
Ma
C. J.
,
Wang
J. M.
,
Ji
X. J.
,
Wu
X. Y.
,
Moorman
J. P.
,
Yao
Z. Q.
.
2012
.
Tim-3 regulates pro- and anti-inflammatory cytokine expression in human CD14+ monocytes.
J. Leukoc. Biol.
91
:
189
196
.
42
Yang
X.
,
Jiang
X.
,
Chen
G.
,
Xiao
Y.
,
Geng
S.
,
Kang
C.
,
Zhou
T.
,
Li
Y.
,
Guo
X.
,
Xiao
H.
, et al
.
2013
.
T cell Ig mucin-3 promotes homeostasis of sepsis by negatively regulating the TLR response.
J. Immunol.
190
:
2068
2079
.
43
van de Weyer
P. S.
,
Muehlfeit
M.
,
Klose
C.
,
Bonventre
J. V.
,
Walz
G.
,
Kuehn
E. W.
.
2006
.
A highly conserved tyrosine of Tim-3 is phosphorylated upon stimulation by its ligand galectin-9.
Biochem. Biophys. Res. Commun.
351
:
571
576
.
44
Clayton
K. L.
,
Haaland
M. S.
,
Douglas-Vail
M. B.
,
Mujib
S.
,
Chew
G. M.
,
Ndhlovu
L. C.
,
Ostrowski
M. A.
.
2014
.
T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases.
J. Immunol.
192
:
782
791
.
45
Abraham
R. T.
,
Weiss
A.
.
2004
.
Jurkat T cells and development of the T-cell receptor signalling paradigm.
Nat. Rev. Immunol.
4
:
301
308
.
46
Rangachari
M.
,
Zhu
C.
,
Sakuishi
K.
,
Xiao
S.
,
Karman
J.
,
Chen
A.
,
Angin
M.
,
Wakeham
A.
,
Greenfield
E. A.
,
Sobel
R. A.
, et al
.
2012
.
Bat3 promotes T cell responses and autoimmunity by repressing Tim-3–mediated cell death and exhaustion.
Nat. Med.
18
:
1394
1400
.
47
Chemnitz
J. M.
,
Parry
R. V.
,
Nichols
K. E.
,
June
C. H.
,
Riley
J. L.
.
2004
.
SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation.
J. Immunol.
173
:
945
954
.
48
Richter
K.
,
Brocker
T.
,
Oxenius
A.
.
2012
.
Antigen amount dictates CD8+ T-cell exhaustion during chronic viral infection irrespective of the type of antigen presenting cell.
Eur. J. Immunol.
42
:
2290
2304
.
49
Mueller
S. N.
,
Ahmed
R.
.
2009
.
High antigen levels are the cause of T cell exhaustion during chronic viral infection.
Proc. Natl. Acad. Sci. USA
106
:
8623
8628
.
50
Hand
T. W.
,
Cui
W.
,
Jung
Y. W.
,
Sefik
E.
,
Joshi
N. S.
,
Chandele
A.
,
Liu
Y.
,
Kaech
S. M.
.
2010
.
Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival.
Proc. Natl. Acad. Sci. USA
107
:
16601
16606
.
51
Macintyre
A. N.
,
Finlay
D.
,
Preston
G.
,
Sinclair
L. V.
,
Waugh
C. M.
,
Tamas
P.
,
Feijoo
C.
,
Okkenhaug
K.
,
Cantrell
D. A.
.
2011
.
Protein kinase B controls transcriptional programs that direct cytotoxic T cell fate but is dispensable for T cell metabolism.
Immunity
34
:
224
236
.
52
Kim
E. H.
,
Sullivan
J. A.
,
Plisch
E. H.
,
Tejera
M. M.
,
Jatzek
A.
,
Choi
K. Y.
,
Suresh
M.
.
2012
.
Signal integration by Akt regulates CD8 T cell effector and memory differentiation.
J. Immunol.
188
:
4305
4314
.
53
Laplante
M.
,
Sabatini
D. M.
.
2012
.
mTOR signaling in growth control and disease.
Cell
149
:
274
293
.
54
Araki
K.
,
Turner
A. P.
,
Shaffer
V. O.
,
Gangappa
S.
,
Keller
S. A.
,
Bachmann
M. F.
,
Larsen
C. P.
,
Ahmed
R.
.
2009
.
mTOR regulates memory CD8 T-cell differentiation.
Nature
460
:
108
112
.
55
Rao
R. R.
,
Li
Q.
,
Odunsi
K.
,
Shrikant
P. A.
.
2010
.
The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin.
Immunity
32
:
67
78
.
56
Lucas
C. L.
,
Kuehn
H. S.
,
Zhao
F.
,
Niemela
J. E.
,
Deenick
E. K.
,
Palendira
U.
,
Avery
D. T.
,
Moens
L.
,
Cannons
J. L.
,
Biancalana
M.
, et al
.
2014
.
Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency.
Nat. Immunol.
15
:
88
97
.
57
Simpson
T. R.
,
Li
F.
,
Montalvo-Ortiz
W.
,
Sepulveda
M. A.
,
Bergerhoff
K.
,
Arce
F.
,
Roddie
C.
,
Henry
J. Y.
,
Yagita
H.
,
Wolchok
J. D.
, et al
.
2013
.
Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.
J. Exp. Med.
210
:
1695
1710
.
58
Bulliard
Y.
,
Jolicoeur
R.
,
Windman
M.
,
Rue
S. M.
,
Ettenberg
S.
,
Knee
D. A.
,
Wilson
N. S.
,
Dranoff
G.
,
Brogdon
J. L.
.
2013
.
Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies.
J. Exp. Med.
210
:
1685
1693
.
59
Eastwood
D.
,
Findlay
L.
,
Poole
S.
,
Bird
C.
,
Wadhwa
M.
,
Moore
M.
,
Burns
C.
,
Thorpe
R.
,
Stebbings
R.
.
2010
.
Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells.
Br. J. Pharmacol.
161
:
512
526
.
60
Horvath
C.
,
Andrews
L.
,
Baumann
A.
,
Black
L.
,
Blanset
D.
,
Cavagnaro
J.
,
Hastings
K. L.
,
Hutto
D. L.
,
MacLachlan
T. K.
,
Milton
M.
, et al
.
2012
.
Storm forecasting: additional lessons from the CD28 superagonist TGN1412 trial.
Nat. Rev. Immunol.
12
:
740
, author reply 740
.
61
Hünig
T.
2012
.
The storm has cleared: lessons from the CD28 superagonist TGN1412 trial.
Nat. Rev. Immunol.
12
:
317
318
.
62
Suntharalingam
G.
,
Perry
M. R.
,
Ward
S.
,
Brett
S. J.
,
Castello-Cortes
A.
,
Brunner
M. D.
,
Panoskaltsis
N.
.
2006
.
Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412.
N. Engl. J. Med.
355
:
1018
1028
.

R.L.F. has received research funding from Bristol-Myers Squibb (related to Tim-3), and consulted for Bristol-Myers Squibb and Astra-Zeneca/MedImmune. L.P.K. has consulted for Janssen Pharmaceuticals and Jounce Therapeutics on Tim-3.