Tim-3 co-stimulation promotes short-lived effectorT cells, restricts memory precursors, and isdispensable for T cell exhaustionLyndsay Averya,b, Jessica Fildermanc, Andrea L. Szymczak-Workmana, and Lawrence P. Kanea,1

aDepartment of Immunology, University of Pittsburgh, Pittsburgh, PA 15261; bInfectious Disease and Microbiology Graduate Program, University ofPittsburgh, Pittsburgh, PA 15261; and cGraduate Program in Microbiology and Immunology, University of Pittsburgh, Pittsburgh, PA 15261

Edited by Lewis L. Lanier, University of California, San Francisco, CA, and approved January 25, 2018 (received for review July 7, 2017)

Tim-3 is highly expressed on a subset of T cells during T cellexhaustion in settings of chronic viral infection and tumors. Usinglymphocytic choriomeningitis virus (LCMV) Clone 13, a model forchronic infection, we found that Tim-3 was neither necessary norsufficient for the development of T cell exhaustion. Nonetheless,expression of Tim-3 was sufficient to drive resistance to PD-L1 blockade therapy during chronic infection. Strikingly, expres-sion of Tim-3 promoted the development of short-lived effectorT cells, at the expense of memory precursor development, afteracute LCMV infection. These effects were accompanied by in-creased Akt/mTOR signaling in T cells expressing endogenous orectopic Tim-3. Conversely, Akt/mTOR signaling was reduced ineffector T cells from Tim-3–deficient mice. Thus, Tim-3 is essentialfor optimal effector T cell responses, and may also contribute toexhaustion by restricting the development of long-lived memoryT cells. Taken together, our results suggest that Tim-3 is actuallymore similar to costimulatory receptors that are up-regulated afterT cell activation than to a dominant inhibitory protein like PD-1.These findings have significant implications for the developmentof anti–Tim-3 antibodies as therapeutic agents.

T cell exhaustion | LCMV | chronic infection | co-stimulation | mTOR

Tim-3 was first identified as a transmembrane protein ex-pressed by T helper type 1 (Th1) T cells (1), but has since

been described on multiple lymphoid and myeloid cell types.Tim-3 has also been intensively studied as a protein up-regulatedon “exhausted” or dysfunctional T cells (2, 3) in settings ofpersistent antigen (4), including both tumors and chronic viralinfection (5–7). In addition to Tim-3, other checkpoint receptors,such as PD-1, LAG-3, CTLA-4, and TIGIT, are highly expressedon exhausted T cells. However, the intrinsic mechanistic func-tions of Tim-3 on these cells remain largely unknown.Research into Tim-3 function on T cells has yielded conflicting

data and conclusions. Expression of Tim-3 is correlated withpoor viral control and weak recall responses in HIV- and HCV-infected patients (8–10). Tim-3 is also present on the most dys-functional tumor-infiltrating T cells. Antibodies to Tim-3 canenhance T cell function in some settings, but may work best insynergy with PD1/PDL1 blockade (11–13). On the other hand,there is also evidence that Tim-3+ T cells are the most responsivecells in active tuberculosis (14). Similarly, a previous study sug-gested that Tim-3 is necessary for an optimal CD8+ T cell responseto Listeria monocytogenes, although the mechanistic basis for thiswas not described (15). In vitro work from our laboratory hascorroborated a positive role for Tim-3 on T cells, as ectopic ex-pression of Tim-3 resulted in increased phosphorylation of multiplepositive-acting signaling molecules and enhancement of T cell ac-tivation (16). Studies on mast cells and dendritic cells (17–20) arealso consistent with a positive role for Tim-3 in cellular activation.An additional complicating factor is the fact that several distinctligands have been identified for the extracellular domain of Tim-3.Galectin-9, HMGB1, phosphatidylserine, and CEACAM-1 haveall been described to bind to Tim-3 (7); however, none of these

ligands is specific to Tim-3, and it is not yet clear to what degreeeach contributes to Tim-3 function under different conditions.To help address the discrepancies outlined above, we de-

veloped a mouse model that expresses Tim-3 without the needfor chronic antigen stimulation. We also used a recently de-scribed Tim-3 KO mouse model (15). Using these tools, we in-vestigated the intrinsic role of Tim-3 in acute and chronic viralinfection and found a previously undescribed role for Tim-3 inthe formation of effector vs. memory T cells through the mTORpathway. Our findings suggest a more complex role for Tim-3,based in part on an early costimulatory effect of this protein.

ResultsExpression of Tim-3 Is Associated with Enhanced TCR Signaling and TCell Activation. To more precisely assess the relationship betweenTim-3 and T cell activation, we stimulated naïve T cells fromNur77GFP reporter mice (21). Expression of GFP in leukocytesfrom these mice is driven by antigen receptor signaling, pro-portional to the strength of signaling (21). As has been suggestedpreviously (1, 22), Tim-3 expression was detected followingin vitro stimulation of naïve CD8+ T cells. Tim-3 expression in-creased with each round of stimulation and was sustained bymore cells with each stimulation (Fig. 1A and Fig. S1). After tworounds of stimulation, CD8+ T cells expressing the most Tim-3(with or without PD-1 coexpression) had significantly greaterexpression of Nur77GFP. After a third stimulation, CD8+ T cellswere uniformly Tim-3Hi, with high levels of Nur77GFP (Fig. 1A).A similar trend was observed after two rounds of stimulation,although Tim-3 expression was less robust (Fig. S1).

Significance

During a chronic viral infection, prolonged exposure to viralantigens leads to dysfunction or “exhaustion” of T cells specificto the virus, a condition also observed in T cells that infiltratetumors. The exhausted state is associated with poor T cellmemory and expression of specific cell-surface proteins, someof which may inhibit T cell activation. Expression of Tim-3 isassociated with acquisition of T cell exhaustion, although it isalso expressed transiently during acute infection. We havefound that a major function of Tim-3 is to enhance T cell acti-vation during either acute or chronic viral infection, and thatTim-3 is not required for the development of T cell exhaustion.

Author contributions: L.A. and L.P.K. designed research; L.A., J.F., and A.L.S.-W. performedresearch; L.A., J.F., and L.P.K. analyzed data; and L.A., A.L.S.-W., and L.P.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: lkane@pitt.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1712107115/-/DCSupplemental.

Published online February 20, 2018.

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Tim-3 and other coexpressed “checkpoint” receptors areusually associated with chronic T cell activation in persistent viralinfection or tumors. Infection with lymphocytic choriomeningitisvirus (LCMV)-Armstrong (Arm) is known to lead to an increasein the percentage of Tim-3+CD8+ T cells during the acute phase(11). However, some Tim-3+ cells remained after the infectionwas cleared (Fig. 1B). These cells also expressed high levels ofCD44 and KLRG1, moderately high CD127 (IL-7Rα), and lowCD62L (Fig. 1C). At day 30 postinfection, we found an increasein both the percentage of T cells expressing Tim-3 and the levelof Tim-3 on tetramer+ cells (Fig. 1D). Furthermore, on challengewith L. monocytogenes expressing the LMCV GP33 epitope (LM-GP33), tetramer+CD8+ cells were uniformly Tim3+KLRG1+ (Fig.1E). These data suggest that Tim-3 may enhance the activation of

antigen-specific T cells during acute stimulation of naïve T cells andalso within the memory compartment.

Tim-3 Deficiency Results in Impaired CD8+ T Cell Recall Responses.Since Tim-3 is induced by acute LCMV infection, we testedwhether expression of Tim-3 is required to clear this infection.By 8 d postinfection (dpi), LCMV-Arm was effectively clearedfrom both groups of mice, with no detectable virus in the spleen.During the acute response, antigen-specific CD8+ cells in bothKO and WT mice were activated and expanded, and producedcytokines at similar levels (Fig. 2A). Among antigen-experiencedcells, there was similar expression of KLRG1 and CD127 (Fig.2B). We assessed the phenotype of memory T cells at 30 dpi withLCMV-Arm, at which time Tim-3 KO mice had significantlyfewer CD8+CD44+ and LCMV-specific T cells (Fig. 2 C and D).In addition, fewer antigen-experienced CD8+ T cells from Tim-3KO mice expressed KLRG1, a marker of short-lived effectorcells (Fig. 2E). However, antigen-experienced cells had a higherpercentage of CD127+ cells (Fig. 2E), a marker of memory

Fig. 1. Endogenous Tim-3 expression is associated with increased T cellactivation. (A) CD25-depleted T cells from Nur77GFP mice were stimulated for3 d with anti-CD3/CD28 mAbs, followed by a 7-d rest with IL-2. After threerounds of stimulation, cells were analyzed for PD-1 and Tim-3 by flowcytometry. Data are representative of two experiments with five technicalreplicates per experiment. (B) WT mice were infected with LCMV-Arm. After30 d, spleens were harvested for flow cytometry (n = 5 mice, mean ± SD).Data are representative of three independent experiments. (C) Tim-3+ vs.Tim-3− CD8+ cells were further analyzed for the activation and differentia-tion markers shown in the histograms. (D) Splenocytes from the same ex-periments as in B and C were stained with LCMV tetramers plus α−Tim-3 (n =5 mice, mean ± SD). Data are representative of three independent experi-ments. **P < 0.01, two-tailed paired Student’s t test. (E) C57BL/6 mice pre-viously infected with LCMV-Arm (>30 dpi) were challenged with LM-GP33,and Tet− vs. Tet+ CD8+ splenocytes were analyzed for KLRG1 and Tim-3 4 dlater. Data are representative of three independent experiments.

Fig. 2. Tim-3 is required for optimal acute responses to primary and sec-ondary infection. Splenocytes from mice infected with 2 × 105 PFU LCMV-Arm were stained for flow cytometry. (A) %Tetramer+, %CD44+, and%IFNγ+TNFα+ at 8 d (B) Representative plots of KLRG1 × CD127 in WT orTim-3 KO CD8+CD44+CD62L− live cells. (C) %CD44+ cells. (D) Staining forLCMV-specific T cells, using pooled tetramers (GP33, GP276, and NP396).(E) Effector vs. memory populations. (F) Response of splenocytes to stimu-lation with 100 ng/mL pooled LCMV peptides for 5 h. (G) Cytokine pro-duction, degranulation, and pS6 staining of in vitro stimulated T cells fromLM-GP33–challenged mice. All groups are gated on live CD8+ cells. The flowplots are representative of three independent experiments; in the graphs,each point represents an individual mouse with mean ± SD. *P < 0.05; **P <0.01, two-tailed unpaired Student’s t test.

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precursor T cells. In addition, fewer Tim-3 KO CD8+ T cellsresponded to restimulation, based on cytokine production (Fig.2F). Finally, we challenged mice that had cleared LCMV-Armwith LM-GP33. During the acute recall response, T cells lackingTim-3 also responded less robustly than WT cells at the levels ofcytokine production, degranulation, and pS6 staining (Fig. 2F).Thus, in the absence of Tim-3, fewer antigen-specific T cells aremaintained after primary or secondary infection, with less effi-cient activation of Akt/mTOR.

Tim-3 Is Not Required for Acquisition of Functional Exhaustion DuringChronic Infection. Sustained expression of Tim-3 is associated withthe development of T cell exhaustion during infection withLCMV Clone 13 (LCMV-Cl13). To determine whether Tim-3 isnecessary for the development of T cell exhaustion, we infectedWT and Tim-3 KO mice with LCMV-Cl13. The Tim-3 KO micelost significantly more weight and took longer to recover com-pared with WT mice (Fig. 3A). There was a trend toward higherviral titers in the blood of Tim-3 KO mice (Fig. 3B), and therewere significantly fewer antigen-specific T cells in Tim-3 KOmice (Fig. 3C). Although LCMV-Cl13 induces profound T cellexhaustion (23), even fewer Tim-3 KO T cells responded afterCl13 infection, based on the production of IFNγ and TNFα (Fig.3D). When investigating transcription factor expression, wefound similar increases in Tbet and Eomes among PD1+ cells(Fig. 3E); enhanced expression of Eomes over Tbet is associatedwith exhaustion (24). Regardless of Tim-3 expression, there wereno major differences in expression of other checkpoint receptors(Fig. S3A). These findings demonstrate that Tim-3 is not re-quired for the development of T cell exhaustion in the LCMV-Cl13 model.

Tim-3 Expression Modulates the Response to PDL1 Blockade.We nextasked whether modulation of Tim-3 alone would affect theability of anti-PDL1 mAb treatment to enhance the response toLCMV-Cl13 (25). Thus, treatment of infected WT mice withanti-PDL1 led to a significant increase in LCMV-specific T cells(Fig. 4A), and the percentage of CD8+ T cells making cytokinesin response to peptide stimulation (Fig. 4B). However, whileanti-PDL1 partially enhanced expansion and function of Tim-3KO T cells, these effects were not as robust as in WT mice (Fig.4 A and B).Current models for studying Tim-3 on T cells require persis-

tent antigen stimulation or chronic infection, as Tim-3 is notstably expressed on T cells until after several rounds of stimu-lation (22). In addition, other checkpoint receptors (e.g., PD-1,TIGIT, LAG-3) are also induced with Tim-3. Thus, we de-veloped a flox-stop-flox Tim3 (FSF-Tim3) mouse model to driveTim-3 expression in a Cre-dependent manner (Fig. S4). On ex-pression of Cre, the stop cassette is removed, and a Flag-taggedmurine Tim-3 is expressed. When these mice were bred withCD4-Cre mice, Tim-3 was expressed on all αβ T cells, with noovert effects on T cell development in the thymus or periphery(Fig. S3). This was also the case when FSF-Tim3 mice were bredwith E8i-Cre mice, yielding expression of Tim-3 only on CD8+

T cells (Fig. S4). Importantly, when Tim-3 was induced duringLCMV-Cl13 infection, other checkpoint receptors were expressedat roughly the same levels as in control animals (Fig. S2B).Using CD8-specific inducible Tim-3 mice, we tested the ef-

fectiveness of PDL1 blockade during LCMV-Cl13 infection.Treatment of control animals with anti-PDL1 mAb reduced viralburden, consistent with a previous report (25) (Fig. 4C). In-triguingly, ectopic expression of Tim-3 on CD8+ cells seemed toresult in a higher viral load in both isotype control and anti-PDL1–treated animals relative to Cre-only controls, with littleresponse to PDL1 blockade (Fig. 4C). These results are alsoconsistent with data on the function of the CD8+ T cells undersuch conditions, with fewer IFNγ/TNFα-producing CD8+ T cells

in mice with ectopic Tim-3 expression (Fig. 4D). Thus, enforcedexpression of Tim-3 increases T cell dysfunction in the context ofLCMV-Cl13, similar to complete absence of Tim-3 expression,with downstream effects on both T cell phenotypes and viral ti-ters. However, ectopic expression of Tim-3 was eventually as-sociated with higher levels of PD-1 on CD8+ T cells, and adecrease in the fraction of PD-1Int cells that have been impli-cated in the responsiveness to PDL1 blockade (Fig. S5).

Tim-3 Enhances TCR Signaling in a Cell-Intrinsic Fashion.Based on theforegoing results, and our previously published data (16), wefurther investigated the effects of Tim-3 on TCR signaling. Sucheffects may occur at a proximal point, as we observed enhancedinduction of total phosphotyrosine after stimulation of T cellsfrom FSF-Tim3/CD4-Cre mice with anti-CD3/CD28 mAbs (Fig.5A). We observed even more striking effects when we lookedfurther downstream at Akt/mTOR signaling. Thus, both pAkt andpS6 were dramatically enhanced in T cells with ectopic Tim-3

Fig. 3. Tim-3 is not required for the development of functional T cell ex-haustion. (A) Mice were infected with 2 × 105 PFU LCMV-Cl13 and weighedthroughout the course of infection. Uninfected, n = 2; WT, n = 5; Tim-3 KO,n = 6. *P ≤ 0.01, two-tailed unpaired Student’s t test between WT and KO atthat time point (mean ± SD). (B) At day ≥30, mice from A were killed, andvirus in the blood was measured by qPCR using a plasmid standard curve.Data are presented as mean ± SEM. (C) Tetramer+ cells in the spleen werequantified using pooled LCMV tetramers (GP33, GP276, and NP396); eachpoint represents an individual mouse (mean ± SD). Data are representativeof three independent experiments. (D) Splenocytes were stimulated withpooled LCMV peptides. Each point represents an individual mouse (mean ±SD) pooled from three independent experiments. (E) Splenocytes were fixedand permeabilized for staining with Abs to Tbet and Eomes. Each pointrepresents an individual mouse. Data (mean ± SD) are representative ofthree independent experiments. In C and D, *P < 0.05, **P < 0.01, two-tailedunpaired Student’s t test.

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expression (Fig. 5 B and C), consistent with the decreasedpS6 noted above in Tim-3 KO T cells and the increased pS6 inFSF-Tim3 mice after LCMV infection. To rule out confoundingdevelopmental effects of the Tim-3 transgene driven by CD4-Cre, we probed activation of CD8+ T cells from E8i-Cre x FSF-Tim3 mice, where expression is specific to CD8 single positiveT cells (26). Only the CD8+, but not CD4+, T cells in this casedisplayed enhanced pS6 (Fig. 5D). This effect was only modestlyincreased on the addition of Tim-3 ligand galectin-9 or HMGB1(Fig. S6), suggesting that Tim-3 may function independently ofligand or that the cultures contained one or more endogenousTim-3 ligands. Finally, we purified T cells from WT or FSF-Tim3 mice that did not carry a Cre transgene, and treated thesecells overnight with Tat-Cre fusion protein, which induced Tim-3 on a subset of T cells from FSF-Tim3 mice (Fig. 5E). Thesecells were stimulated with CD3/CD28 mAbs and analyzed forpS6 and CD69. Consistent with the above results, T cells withinduced Tim-3 expression responded more robustly to stimulation.

Enforced Expression of Tim-3 Enhances Generation of Short-LivedEffector T Cells in an mTOR-Dependent Manner. Given the en-hanced TCR signaling seen in Tim-3–induced mice, we sought todetermine whether Tim-3 alone might drive T cell exhaustionduring an acute response. We infected control and FSF-Tim3/CD4-Cre mice with LCMV-Arm and monitored viral titer in the spleen.The FSF-Tim3/CD4-Cre mice had significantly more KLRG1+

T cells in the antigen-experienced pool (CD8+CD44+CD62L−)at 30 dpi (Fig. 6A). This was accompanied by a lower per-centage of KLRG1+CD127+ T cells, indicating that Tim-3promotes the formation of more short-lived effector T cells(SLECs), perhaps at the expense of long-lived memory cells.Although memory-precursor KLRG1+CD127+ T cells gener-ally have higher Tbet expression and lower Eomes expression,memory precursor T cells with enforced Tim-3 expression hadan even higher Tbet:Eomes ratio (Fig. 6B). This appears to bethe result of decreased Eomes expression in Tim3-expressing cells,as Tbet remained unchanged (Fig. 6B). When memory T cells

from FSF-Tim3/CD4-Cre or CD4-Cre–only mice were stimulatedex vivo, there was no significant difference in the percentage ofcells producing cytokines (Fig. 6C). However, Tim3-expressingCD8+ T cells had a greater increase in pS6 staining followingstimulation, compared with control cells (Fig. 6D).Inhibition of mTOR with rapamycin can skew CD8+ T cell

differentiation toward a more long-lived memory phenotype(27). To further explore the idea that Tim-3 expression drivesmore SLECs via mTOR, CD8-specific Tim-3–induced mice(FSF-Tim3/E8iCre) were treated with vehicle or low-dose rapa-mycin throughout the course of LCMV-Arm infection. Rapamycintreatment significantly reduced the KLRG1+CD127− SLEC pop-ulation (Fig. 6E). There was an increase (albeit not significant)in the KLRG1−CD127+ memory precursor effector cell (MPEC)population with rapamycin treatment as well. Taken together,these data indicate that Tim-3 signaling through mTOR candrive differentiation of antigen-specific T cells toward a short-lived effector phenotype. Thus, Tim-3 promotes the formation

Fig. 5. Enhanced TCR signaling in primary T cells with enforced Tim-3 ex-pression. (A) Purified splenic T cells from CD4-Cre or FSF-Tim3/CD4-Cre micewere stimulated in vitro with anti-CD3/CD28 mAbs for the indicated times.Cells were lysed and analyzed by Western blot analysis for total phospho-tyrosine. β-actin served as a loading control (Bottom). (B and C) PurifiedT cells were stimulated in vitro and analyzed by Western blot analysis forphosphorylation of Akt (S473) (B) or ribosomal protein S6 (C). Blots werereprobed for Tim-3 (B) and β-actin (B and C), and each is representative ofthree independent experiments. (D) Purified T cells from E8i-Cre or FSF-Tim3/E8i-Cre mice were stimulated in vitro for 4 h, followed by analysis of pS6.(Left) A representative flow cytometry histogram. (Right) Compiled datafrom three experiments (n = 3 mice, mean ± SD). **P < 0.01, two-tailedunpaired Student’s t test. (E) Splenocytes from WT or FSF-Tim3 mice weretreated with Tat-Cre overnight, then stimulated with anti-CD3/CD28 for 4 h;cells were analyzed for Tim-3 expression (gated on CD8+ live cells). pS6 andCD69 were analyzed within the Tim-3Lo and Tim-3Hi groups of the FSF-Tim3 cells. Data are representative of three independent experiments eachwith three biological replicates.

Fig. 4. Expression of Tim-3 modulates the response to PDL1 blockade dur-ing chronic LCMV infection. The indicated strains of mice were infected withLCMV-Cl13 and then treated every 3 d for 2 wk, beginning at 23 dpi, withisotype control or anti-PDL1 mAb. At 2 d after the final treatment (31 dpi),mice were killed, and splenocytes were analyzed. (A) %LCMV-specific cellsmeasured with pooled tetramers. (C) Viral titers in blood at 31 dpi. In A andC, data are mean ± SEM, representative of two independent experiments.(B and D) Quantitation of %IFNγ+TNFα+ CD8+ T cells by flow cytometry inspleens of the indicated groups of mice after in vitro peptide stimulation.Each data point represents one mouse. Data are mean ± SD, representativeof two independent experiments.

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of SLECs, possibly through enhancement of Akt/mTOR sig-naling and suppression of Eomes expression.

DiscussionExpression of Tim-3 on T cells is often associated with acquisi-tion of a dysfunctional phenotype, but accumulating evidenceindicates that Tim-3 is necessary for an optimal effector T cellresponse. Using genetic models of Tim-3 deficiency or enforcedexpression, and infection with LCMV-Arm, we obtained findingsconsistent with a previous study that used L. monocytogenes (15).Here we have linked the effects of Tim-3 on early T cell acti-vation and effector differentiation with subsequent impairmentsin memory T cell formation. The impact of Tim-3 on effectorT cells during acute infection correlates well with the early, albeittransient, expression of Tim-3 after acute infection (11), and ourown data showing enhanced Nur77GFP activity in Tim-3+ cells.

Our findings suggest a novel mechanism by which Tim-3 couldcontribute to T cell exhaustion, i.e., by limiting the pool ofmemory T cells, while enhancing initial T cell activation and thegeneration of short-lived effector cells (see the model in Fig. S7).Consistent with this model, either ectopic Tim-3 expression orTim-3 deficiency altered formation of MPECs vs. SLECs.Depletion of the T cell memory pool is observed in multiple

settings of T cell exhaustion, including both during chronic viralinfection and within the tumor microenvironment (23, 28). Thus,enhanced differentiation of T cells to SLECs appears to markthese cells for eventual deletion. The Tim-3/galectin-9 in-teraction has been linked to induction of apoptosis (29), which isa possible target for enhancement of T cell function by Tim-3 blockade. In this study, we did not extensively address thefunction of the various reported ligands for Tim-3 (galectin-9,phosphatidylserine, HMGB1, or CEACAM1) (6, 7, 30). None-theless, we did obtain corroborative evidence that treatment withgal-9 or HMGB1 led to a further modest increase in pS6 inT cells with ectopic expression of Tim-3. As we had observedpreviously in our cell line studies, ectopic expression of Tim-3 wasitself sufficient to enhance T cell activation, including induction ofpS6 (16), suggesting either a ligand-independent function of Tim-3 or constitutive expression of one or more Tim-3 ligands. Inaddition, exhausted T cells express low levels of receptors for thehomeostatic cytokines IL-15 and IL-7, and without chronic anti-gen exposure, these cells will die (10, 11). If Tim-3 enhances thisdifferentiation during acute infection, it could have similar effectsduring chronic infection. However, coexpression of negative reg-ulators like PD-1 during chronic infection may suppress theproactivation function of Tim-3 in this setting.During acute infection, expression of Tim-3 and other

checkpoint receptors can be observed, but is normally only tran-sient (11). We hypothesized that enforced expression of Tim-3 may cause persistence of an acute infection, although this turnedout to not be the case. WT and Tim-3–expressing mice clearedLCMV-Arm with similar kinetics, at least at the doses of virus weexamined, consistent with extremely robust responses to this virusin BL/6 mice. However, enforced expression of Tim-3 did cause anexpansion of KLRG1+ SLECs, at the apparent expense of long-lived CD127+ memory precursor T cells. In the transitional pop-ulation of KLRG1+CD127+ CD8+ T cells, we found that T cellswith enforced expression of Tim-3 had a higher Tbet-to-Eomesratio, which is thought to impact T cell fate decisions like SLEC vs.MPEC formation (24). We also observed consistent enhancementof pS6 when Tim-3 expression was induced. Treatment of FSF-Tim3 mice with rapamycin throughout the course of LCMV-Arminfection reduced the KLRG1+CD127− SLEC population, as wasshown previously in normal mice, confirming that Tim-3 acts atleast in part through mTOR (27, 31).Manipulation of Tim-3 expression during acute LCMV in-

fection had no overt effects on pathology, at least at the doses ofvirus used here. However, ectopic expression or KO of Tim-3 resulted in clear phenotypes in LCMV-Cl13 infection. Thus,Tim-3 KO mice showed exacerbated weight loss, which is usuallyascribed to immune-mediated pathology (32). However, we ac-tually observed fewer antigen-specific T cells and higher viraltiters in these animals. Because this is a germ line Tim-3 KOmodel, expression of Tim-3 on another cell type may contributeto the weight loss and/or viral titer phenotype (17–20). We arecurrently developing an inducible Tim-3 KO model to betteraddress this possibility. We also noted higher viral titers in Cl13-infected mice with ectopic Tim-3 expression only on T cells.Thus, our findings suggest that Tim-3 does not simply function asa digital “switch” toward activation or exhaustion, but rather maymodulate T cell responses in a subtler fashion, depending on thetiming and/or strength of its expression and signaling.A previous study found that combining a Tim-3 Ig fusion protein

with PDL1 blockade further enhanced the function of exhausted

Fig. 6. Tim-3 promotes the formation of terminal effector phenotype CD8+

T cells and enhances early T cell activation. Splenocytes from mice infectedwith 2 × 105 PFU LCMV-Arm were harvested at ≥30 dpi. (A, Left) Repre-sentative flow plots of KLRG1 and CD127 from CD4-Cre or FSF-Tim3/CD4-Cremice were examined in the live antigen-experienced (CD8+CD44+CD62L−)population. (A, Right) Summary data. (B) The ratio of Tbet:Eomes (Right)was plotted based on the mean fluorescence intensity (MFI) of Tbet andEomes (Left) in the CD8+CD44+CD62L−KLRG1+CD127+ population. (C and D)Splenocytes were stimulated with pooled LCMV peptides for 5 h and thenanalyzed for IFNγ/TNFα (C ) or pS6 (D). (E ) Infected mice were treatedwith rapamycin i.p. daily starting on day −1 preinfection and continuing today 30 postinfection and processed as in A. Data (mean ± SD) are repre-sentative of three independent experiments; each symbol represents anindividual mouse. *P < 0.05; **P < 0.01, two-tailed unpaired Student’st test.

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T cells (11), supporting the possible efficacy of combinationcheckpoint blockade. Based in part on studies in which expressionof Tim-3 was associated with reduced effector T cell function insettings of exhaustion, it has been suggested that Tim-3 is a dom-inant negative regulator of T cell function. On closer inspection,the effects of Tim-3 on T cell responses appear to be much morecomplex. It is possible that the function of Tim-3 on T cells iscontext-dependent (acute vs. chronic). Thus, in HIV-infected in-dividuals, Tim-3+ T cells show a reduced response to stimulation,but display enhanced baseline phosphorylation of multiple proteins(33). This is consistent with our finding that T cells with enforcedTim-3 expression also have a higher baseline activation, based ontotal phosphotyrosine levels. In addition, inducible phosphoryla-tion, particularly pS6 and pAkt, was enhanced on TCR stimulationof Tim3-expressing T cells. Kaech et al. (34) found that constitu-tively active Akt resulted in enhanced KLRG1 expression and re-duced memory T cell formation. A similar role for PI3K/Aktsignaling in promoting the production of SLECs has been de-scribed by Cantrell et al. (35). Finally, it has been reported thatmTOR, a downstream target of PI3K/Akt signaling, might restrictformation of memory precursor T cells (31).Our work suggests that Tim-3 plays multiple roles in regulating

effector and memory T cell responses, in addition to T cell ex-haustion. A better understanding of how Tim-3 contributes toT cell responses in these scenarios will help guide the develop-ment of novel therapies to improve T cell function under oth-erwise suboptimal conditions.

Materials and MethodsAll animal procedures were conducted in accordance with the National In-stitutes of Health and the University of Pittsburgh’s Institutional Animal Care

and Use Committee guidelines. LCMV-Cl13 and LCMV-Arm strains wereobtained from Rafi Ahmed, Emory University, Atlanta, and propagated andtitered as described previously (36). For acute LCMV-Arm infections, micewere infected with 2 × 105 PFU i.p. For chronic LCMV-Cl13 infections, micewere infected with 2 × 106 PFU i.v. Infections occurred on day 0 and necropsyon day ≥30 postinfection in age- and sex-matched groups. Viral titer wasmeasured from infected tissue after RNA extraction using TRIzol LS reagent,followed by qPCR for the GP protein. CT values were compared with aplasmid standard curve, as previously described, to calculate viral copies (37).L. monocytogenes-GP33 (LM-GP33) was obtained from Susan Kaech, YaleUniversity, New Haven, CT, and propagated using brain-heart infusion brothas described previously (38). Mice challenged with LM-GP33 received 2 × 106

CFU i.v. Anti-PDL1 or isotype treatment began on day 23 after LCMV-Cl13 infection. Mice were randomized to treatment or isotype group, given200 μg of Ab i.p. every 3 d for 2 wk, and harvested at 2 d after the lasttreatment. The researcher was not blinded to mouse group.

Rapamycin (LC Laboratories) was dissolved in ethanol then further dilutedin Tween-80/PEG-400 (10:80) and sterile-filtered. For rapamycin treatment,mice were injected every day with 2.5 μg of rapamycin i.p. starting on the daybefore LCMV-Arm infection and extending until the day of necropsy.

Additional information is provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Rafi Ahmed (Emory University) for provid-ing viral stocks and protocols, Susan Kaech (Yale University) for providing LM-GP33 stocks, Katherine Davioli (University of Pittsburgh) for propagating theLCMV virus, and Juan C. de la Torre (The Scripps Research Institute) for theLCMV plasmids used to generate standard curves for viral titer analysis. Wealso thank T. Hand, L. P. Andrews (University of Pittsburgh), and members ofthe L.P.K. laboratory for critical feedback and discussion, as well as commentson the manuscript. This work was supported by National Institutes of HealthGrants AI109605 and CA206517 (to L.P.K.), an American Association of Immu-nologists Careers in Immunology Fellowship (to L.A. and L.P.K.), and NIH GrantEY08098 to support Katherine Davioli in the virology and histology core.

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