The Transcription Factor NFAT1Participates in the Induction of CD4�

T Cell Functional Exhaustion duringPlasmodium yoelii Infection

Rachel Y. Ames,a Li-Min Ting,b Inessa Gendlina,b Kami Kim,b

Fernando Maciana

Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USAa; Departments ofMedicine, Pathology, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NewYork, USAb

ABSTRACT Repeated stimulation of T cells that occurs in the context of chronic in-fection results in progressively reduced responsiveness of T cells to pathogen-derivedantigens. This phenotype, known as T cell exhaustion, occurs during chronic infec-tions caused by a variety of pathogens, from persistent viruses to parasites. Unlikethe memory cells that typically form after successful pathogen clearance followingan acute infection, exhausted T cells secrete lower levels of effector cytokines, prolif-erate less in response to cognate antigen, and upregulate cell surface inhibitorymolecules such as PD-1 and LAG-3. The molecular events that lead to the inductionof this phenotype have, however, not been fully characterized. In T cells, membersof the NFAT family of transcription factors not only are responsible for the expres-sion of many activation-induced genes but also are crucial for the induction of tran-scriptional programs that inhibit T cell activation and maintain tolerance. Here weshow that NFAT1-deficient CD4� T cells maintain higher proliferative capacity andexpression of effector cytokines following Plasmodium yoelii infection and are there-fore more resistant to P. yoelii-induced exhaustion than their wild-type counterparts.Consequently, gene expression microarray analysis of CD4� T cells following P. yoelii-induced exhaustion shows upregulation of effector T cell-associated genes in the ab-sence of NFAT1 compared with wild-type exhausted T cells. Furthermore, adoptivetransfer of NFAT1-deficient CD4� T cells into mice infected with P. yoelii results inincreased production of antibodies to cognate antigen. Our results support the ideathat NFAT1 is necessary to fully suppress effector responses during Plasmodium-induced CD4� T cell exhaustion.

KEYWORDS exhaustion, NFAT, Plasmodium, T cells

Both CD8� and CD4� T cells experience a significant loss of function after chronicstimulation driven by a variety of pathogens as well as in response to tumor

antigens (1). CD4� T cells are critical for the formation and maintenance of productivememory CD8� T cells, but they also contribute to the changes that occur in CD8�

memory T cell populations during chronic infection (2, 3). A hallmark of T cell exhaus-tion is the increased expression of coinhibitory surface molecules on exhausted T cells,and the exhaustion phenotype can be reversed through the blockade of those recep-tors (4–6). The involvement of several transcription factors, such as Blimp-1 and T-bet,in the induction of T cell exhaustion has been reported (7, 8), but the mechanisms thatunderlie the transcriptional regulation of the many exhaustion molecules and pheno-types have not been fully elucidated.

Nuclear factor of activated T cell (NFAT) proteins constitute a family of transcription

Received 17 May 2017 Accepted 7 June 2017

Accepted manuscript posted online 19June 2017

Citation Ames RY, Ting L-M, Gendlina I, Kim K,Macian F. 2017. The transcription factor NFAT1participates in the induction of CD4+ T cellfunctional exhaustion during Plasmodium yoeliiinfection. Infect Immun 85:e00364-17. https://doi.org/10.1128/IAI.00364-17.

Editor John H. Adams, University of SouthFlorida

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Fernando Macian,fernando.macian@einstein.yu.edu.

MICROBIAL IMMUNITY AND VACCINES

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factors that play central roles in the regulation of T cell activation and differentiation (9).In T cells, NFAT proteins remain heavily phosphorylated in the cytosol. In response toT cell receptor (TCR)-triggered calcium signaling, NFAT proteins are activated by thecalcineurin-mediated removal of several phosphate groups located in the N-terminalregulatory region. Dephosphorylation of NFAT leads to nuclear translocation and theactivation of NFAT-dependent gene expression (10, 11). Though NFAT proteins wereoriginally described as regulators of activation-induced cytokine expression, thefunctions regulated by NFAT in T cells now include not only positive but alsonegative programs that hinder T cell activation (12). For instance, the transcriptionfactor NFAT1 controls the induction of programs of gene expression that allow forthe regulatory T cell (Treg)-mediated suppression of CD4� T cell activation andmediate the induction of CD4� T cell anergy, a state of functional inactivation thatbears phenotypic similarity with T cell exhaustion (13–15). Clonal T cell anergyoccurs as a consequence of suboptimal stimulation, and it is characterized by aunique transcriptional program driven, at least in part, by NFAT1 (9, 12, 16).Exhausted T cells feature a distinct transcriptional program that is not identical tobut has some overlap with the T cell anergy program (17, 18), suggesting thatNFAT1 might also contribute to T cell exhaustion and participate in the inductionof the transcriptional program activated following chronic engagement of the T cellreceptor.

In this study, using a model of CD4� T cell exhaustion caused by Plasmodium yoeliiinfection (4), we found that NFAT1 is necessary for full inactivation of CD4� T cells.Furthermore, we have elucidated transcriptional control of chronically stimulated T cellsby NFAT1 by performing microarray analysis on P. yoelii-exhausted CD4� T cells andfound differential regulation of a subset of genes in the absence of this transcriptionfactor. We conclude that NFAT1 contributes to the regulation of a novel transcriptionalprogram in chronically stimulated CD4� T cells to promote suppression of T cellfunction.

RESULTSNFAT1-deficient CD4� T cells maintain proliferative responses in a model of T

cell exhaustion induced by Plasmodium yoelii infection. NFAT1 participates in theregulation of different programs of T cell inactivation, including T cell anergy andregulatory T cell-mediated suppression of CD4� T helper cells (13–15). Similar toanergic cells, exhausted T cells show reduced responses to antigen stimulation. Todetermine if NFAT1 could also play a role in controlling the exhaustion of T cells, weinfected wild-type and Nfat1�/� mice with Plasmodium yoelii 17XNL. Infection with thisparasite had been previously shown to induce potent exhaustion of CD4� T cells (4).Following 3 weeks of infection, mice were sacrificed and CD4� T cells were isolatedfrom spleens. CD11ahigh CD49d� staining has been shown to delineate previouslyactivated CD4� T cells from naive cells in Plasmodium-infected mice (4, 19) andtherefore represent T cells that are likely to respond to a variety of Plasmodiumantigens. We compared the responses and phenotypes of the CD4� CD11ahigh CD49d�

T cell populations from wild-type and Nfat1�/� naive mice with those from miceinfected with P. yoelii 17XNL. We could detect similar levels of initial expansion of theCD4� CD11ahigh CD49d� compartment following infection in wild-type and NFAT1-deficient mice (Fig. 1A). However, we found that Nfat1�/� CD4� T cells were able tomaintain greater proliferative ability than their wild-type counterparts after exhaustionfollowing P. yoelii infection (Fig. 1B). As expected, T cells from mice infected with P.yoelii showed diminished proliferation following subsequent stimulation comparedwith T cells from uninfected mice (Fig. 1B) (4). Though Nfat1�/� T cells also lost someproliferative ability following P. yoelii exposure, the decrease in proliferative capacitywas significantly more pronounced in wild-type T cells than in NFAT1-deficient cells(Fig. 1B). Both PD-1 and LAG-3 were upregulated in the wild-type cells (Fig. 1C).Nfat1�/� exhausted T cells also upregulated the expression of both inhibitory recep-tors, though there was a small but significant difference in the increase of PD-1

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expression in Nfat1�/� cells (Fig. 1C). The decreased response of wild-type CD4� T cellsisolated from infected mice was not due to an increase in cell death, as similar levelsof apoptosis were induced by restimulation under all conditions tested in T cells fromeither wild-type or NFAT1-deficient mice (Fig. 1D). We followed parasitemia of infectedmice but did not observe significant differences in the course of infection of ourwild-type or Nfat1�/� mice during the 3 weeks of infection before mice were sacrificed(Fig. 1E). In this model, both wild-type mice and NFAT1-deficient mice were typicallyable to effectively clear the infection in less than 4 weeks.

To assess exhaustion of a specific TCR-antigen combination and to control forpossible effects of NFAT1 deficiency in other cell types, including B cells, in the full-bodyNFAT1-defiicent mice (20), we performed adoptive transfers of OT-II� wild-type orNfat1�/� CD4� TH1-polarized cells into C57BL6/J hosts infected with a Plasmodiumyoelii 17XNL strain that had been genetically engineered to express ovalbumin (P. yoeliiOVA). For experiments measuring effector functions (cytokine secretion and prolifera-tion), we used TH1-polarized cells in order to observe any decreases in function uponfurther stimulation in the T helper subtype that is mainly responsible for the anti-Plasmodium T cell response and to bypass any in vivo bias in T helper differentiationthat might occur in NFAT1-deficient T cells (21). Differentiation bias has been attributedto differences in the ability of wild-type and NFAT1-deficient CD4� T cells to sustaininterleukin 4 (IL-4) expression, but can be overcome by in vitro differentiation in thepresence of polarizing cytokines. Using that approach, we confirmed that in vitro-differentiated Nfat1�/� and Nfat1�/� TH1 cells showed equivalent abilities to produceIL-2 and gamma interferon (IFN-�) following reactivation (see Fig. S1 in the supple-mental material). Similar to what we saw in the Nfat1�/� mouse experiments, Nfat1�/�

and Nfat1�/� OT-II cells showed similar levels of expansion when mice were infectedwith P. yoelii (Fig. S2). However, when we analyzed T cells 21 days postinfection by exvivo restimulation with antigen-presenting cells (APCs) loaded with OVA323–339 peptide,we observed a significant decrease in the proliferative ability of OT-II� wild-type CD4�

T cells from mice infected with P. yoelii OVA that was not seen in OT-II� Nfat1�/� cells(Fig. 2A). Furthermore, the production of IFN-� was also significantly reduced in OT-IIcells isolated from infected wild-type mice, while Nfat1�/� cells maintained the samelevels of expression of this effector cytokine as the nonexhausted controls (Fig. 2B). Wealso observed a similar trend in IL-2 expression, though the difference in IL-2 produc-tion between wild-type control and wild-type exhausted CD4� T cells was not statis-tically significant (Fig. 2B). No differences in the extent of cell death were observed inany of the various experimental groups (Fig. 2C). Despite a better functional responsein T cells isolated from infected mice that received Nfat1�/� OT-II cells, levels ofparasitemia in the first 3 weeks of infection were comparable in mice that receivedwild-type and NFAT1-deficient CD4� T cells (Fig. 2D). Similar levels of upregulation ofPD-1 and LAG-3, CTLA-4, or Tim-3 surface expression also occurred in Nfat1�/� andNfat1�/� OT-II cells (Fig. 2E; see also Fig. S2). Interestingly, following repeated stimu-lation of OT-II CD4� T cells in vitro via the TCR, using splenocytes presenting OVApeptide, we also saw less downregulation of T cell function (measured by IL-2 secre-

FIG 1 NFAT1-deficient mice are less susceptible to exhaustion induced by Plasmodium yoelii infection in the CD4� Tcell population. (A) Gating strategy and quantification (mean � SEM) of the frequency of CD49d� CD11ahigh CD4� Tcells in control uninfected and Plasmodium yoelii-infected wild-type or NFAT1-deficient mice (n � 4). (B) Activation-induced proliferation ex vivo, measured as BrdU incorporation by flow cytometry, was determined in CD4� CD49d�

CD11ahigh T cells from Nfat1�/� and Nfat1�/� control mice or mice infected with Plasmodium yoelii. Graphs showmeans � SEM from 4 to 6 mice per group from two independent experiments. **, P � 0.01; ***, P � 0.001; ****, P �0.0001 (ANOVA). (C) Representative flow cytometry histograms and quantification of the percentage of CD4� CD49d�

CD11ahigh T cells expressing PD-1 or LAG-3 in CD4� T cells isolated from Nfat1�/� and Nfat1�/� control mice or miceinfected with Plasmodium yoelii. Graphs show means � SEM from 6 to 10 mice per group from three independentexperiments. *, P � 0.05; ****, P � 0.0001; ns, not significant (ANOVA). (D) Percentages of the populations of cellsanalyzed in panel A that were apoptotic following restimulation ex vivo (annexin V� LIVE/DEAD�) were measured byflow cytometry. Bars show means from 4 or 5 mice from two independent experiments. (E) Parasitemia in Nfat1�/� andNfat1�/� mice infected with Plasmodium yoelii. The graph shows average numbers of parasites � SEM from 6 differentmice from two independent experiments.

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FIG 2 A lack of NFAT1 makes CD4� T cells less susceptible to exhaustion induced in an antigen-specific manner. (A and B) Activation-induced proliferation(measured as BrdU incorporation) (A) and IFN-� or IL-2 expression (B) (measured as percentage of cytokine-positive cells analyzed by intracellular staining) weredetermined by flow cytometry in OT-II T cells isolated 21 days after infection from control or Plasmodium OVA-infected mice that had received Nfat1�/� or

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tion), without differences in the upregulation of LAG-3 and PD-1 (Fig. S3), similar to ourobservations during T cell exhaustion induced by P. yoelii, lending support to the ideathat modulation of the response to chronic antigen-specific stimulation of the TCRis central to the role of NFAT1 in the regulation of the T cell exhaustion phenotype(22, 23).

Increased protection against lethal P. yoelii OVA infection is conferred byadoptive transfer of Nfat1�/� CD4� T cells. The P. yoelii OVA line demonstrated a morevirulent phenotype than that of its parental strain, P. yoelii 17XNL, causing lethalinfection in some experiments. Though the acute courses of parasitemia were notdifferent between mice receiving wild-type and NFAT1-deficient OT-II cells during theselethal infections (Fig. 3A), we observed a significant delay in death due to P. yoelii OVAin mice adoptively transferred with OT-II Nfat1�/� CD4� T cells compared with OT-IINfat1�/� (Fig. 3B) during infections that were lethal to the cohort. Mice that receivedNfat1�/� CD4� T cells had a median survival time of 12 days, versus 18 days for thegroup that received Nfat1�/� OT-II T cells.

We also observed higher titers of antibodies to ovalbumin in serum from miceinfected with P. yoelii OVA that had received Nfat1�/� CD4� OT-II naive or TH1 T cells(Fig. 3C and E). This effect was specific to the OT-II interaction with cognate antigen(OVA), as we saw no differences in antibody levels specific for the endogenousPlasmodium antigen MSP-142 between mice receiving wild-type and Nfat1�/� OT-II Tcells, which would rely on the activity of the host’s polyclonal CD4� T cell populationrather than the transferred T cells (Fig. 3D and F). Additionally, adoptively transferred,naive Nfat1�/� CD4� T cells featured higher expression of the costimulatory receptorICOS than wild-type cells following exposure to P. yoelii OVA (Fig. 3G; see also Fig. S4).This increase in ICOS expression in mice receiving naive NFAT1-deficient T cells wasmore pronounced in Bcl-6� CD4� T cells (Fig. 3H and I). Interestingly, Plasmodiuminfection led to increased production of IL-21 in ex vivo-restimulated cells only inNfat1�/� OT-II cells obtained from infected mice and not in cells from infected micethat received Nfat1�/� OT-II cells (Fig. 3J).

NFAT1 is a transcription factor, and therefore, we sought to determine whatdownstream program of gene expression could be affected by the absence or NFAT1that may drive the less exhausted phenotype that we had observed in NFAT1-deficientT cells. We purified CD11ahigh CD49d� CD4� T cells from spleens of both wild-type andNfat1�/� mice following 3 weeks of P. yoelii 17XNL infection, as well as from uninfectedcontrols, and isolated RNA for microarray analysis. Analysis of the microarray dataindicated that 51 genes were significantly upregulated and 3 downregulated 2-fold orhigher in the absence of NFAT1 (Fig. 4A; see also Fig. S5 and Table S1). By comparingthe set of genes upregulated in the Nfat1�/� T cells with a database of gene setsgenerated from published studies of immune cell phenotypes (24) (ImmuneSigDB,Broad Institute, MIT), we found that five of the top six gene sets that overlapped withour list of significantly upregulated genes in Nfat1�/� P. yoelii-exhausted T cells overwild-type were gene sets representing effector T cell states, in agreement with theincreased functional phenotypes we have observed (Fig. 4B). We were also interestedto know whether Plasmodium-induced exhaustion would have a similar gene profile inT cells to the well-characterized lymphocytic choriomeningitis virus (LCMV)-inducedexhaustion, in addition to the previously characterized overlap in exhaustion pheno-types and selected surface marker expression (4). We compared our results with a

FIG 2 Legend (Continued)Nfat1�/� OT-II CD4� TH1 cells by adoptive transfer. Graphs show means from 5 to 8 mice per group from two independent experiments. *, P � 0.05; ns, notsignificant (two-tailed t test). SSC, side scatter. (C) Percentages of the populations of cells analyzed in panels A and B undergoing early apoptosis (annexin V�

LIVE/DEAD�) following restimulation ex vivo were measured by flow cytometry. Bars show means from 4 to 6 mice per group from two independentexperiments. (D) Parasitemia in mice infected with Plasmodium OVA receiving Nfat1�/� or Nfat1�/� OT-II CD4� T cells. The graph shows the average numbersof parasites � SEM in 3 mice per group from a representative infection. RBCs, erythrocytes. (E) The percentages of OT-II T cells expressing PD-1, LAG-3, or TIM-3and the levels of CTLA-4 expression were determined by flow cytometry in T cells isolated 21 days postinfection from control or Plasmodium OVA-infected micereceiving Nfat1�/� and Nfat1�/� OT-II T cells. Graphs show means � SEM from 4 or 5 mice per group from two independent experiments. *, P � 0.05; ***, P �0.001; ns, not significant (ANOVA). gMFI, geometric mean fluorescence intensity.

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FIG 3 Adoptive transfer of NFAT1-deficient CD4� T cells increases antibody production in response to cognate antigen.(A) Course of parasitemia in C57BL/6 mice adoptively transferred with Nfat1�/� or Nfat1�/� OT-II CD4� T cells during lethal

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previously published study of gene expression in CD4� T cells following LCMV infection(17) and found that of genes significantly upregulated in wild-type CD4� T cellsfollowing Plasmodium infection, compared with uninfected controls, there were 13genes shared by these two data sets, including two well-characterized exhaustion-associated genes, Lag-3 and Maf (Fig. 4C). This suggests the possibility that while thereare likely core mechanisms that drive T cell exhaustion, specific programs of genesexpression may also participate in the regulation of T cell exhaustion in response todistinct pathogens.

DISCUSSION

Although an exhausted phenotype has been described for both CD8� and CD4� Tcell populations, the molecular mechanisms that are responsible for the induction of Tcell exhaustion in response to chronic antigen stimulation remain not fully character-ized. In this study, we showed that NFAT1 is required for full exhaustion of CD4� T cellsinduced by Plasmodium yoelii infection in mice. The absence of NFAT1 lengthenedsurvival in a lethal infection, which correlated with increased antibody production inresponse to cognate antigen. In addition, NFAT1 suppressed expression of effector Tcell-associated genes following 3 weeks infection with P. yoelii.

The need for chronic antigen stimulation to induce and maintain T cell exhaustionsupports a central role for TCR-mediated signaling in this process. At least two medi-ators of TCR signals, SPRY2 and NFAT, have been identified as factors involved insupporting the establishment of exhaustion in CD8� T cells (25, 26). Our data indicatethat NFAT1 is also a central regulator of the induction of exhaustion in CD4� T cells.Interestingly, we observed similar differences in downregulation of function of wild-type versus NFAT1-deficient CD4� T cells in response to repeated stimulation of theTCR in vitro (Fig. S3), lending further support to the idea that NFAT1 may play a centralrole in the control of the response of T cells to repeated TCR engagement during achronic infection and regulate the establishment of T cell exhaustion.

Although ex vivo activation of CD4� T cells from mice infected with Plasmodiumyoelii showed that NFAT1-deficient cells preserved responses to antigen significantlybetter than wild-type T cells, we did not observe major differences in the courses ofparasitemia. This could be a consequence of the fact that although Plasmodiuminfection was not able to downregulate CD4� T cell responses in NFAT1-deficient cells,we observed that levels of effector cytokines were lower in control NFAT1-deficient Tcells than in wild-type cells, possibly due to the overall reduction in total NFAT protein,as has been previously reported (13). Exhaustion of T cells has been shown in somemodels to require the continuous presence of the pathogen for several weeks (27). Weisolated CD4� T cells after 3 weeks to ensure that exhaustion had been established, yetwild-type mice were able to control infection with this strain in many cases in 4 weeksor less. However, transfer of NFAT1-deficient CD4� OT-II T cells increased survival timeduring lethal infections that occurred sporadically when a more virulent PlasmodiumOVA parasite was used to infect mice. The effectiveness of transfer of CD4� T cells ondisease outcomes was likely dependent on the better preservation of CD4� T cell

FIG 3 Legend (Continued)P. yoelii OVA infection. The graph shows the average numbers of parasites � SEM in 4 mice per group from a representativeinfection. (B) Survival of C57BL/6 mice receiving either Nfat1�/� or Nfat1�/� OT-II� CD4� T cells and lethally infected withPlasmodium OVA. Data are from 10 mice per group from two independent experiments. **, P � 0.05 (Mantel-Cox). (C toF) Levels of anti-OVA (C and D) and anti-MSP-1 (E and F) IgG were measured by ELISA in serum from uninfected mice ormice infected with P. yoelii OVA after receiving naive or TH1 Nfat1�/� or Nfat1�/� OT-II� CD4� T cells as indicated. Graphsshow means � SEM from 5 to 7 mice per group from two independent experiments. *, P � 0.05; ***, P � 0.001; ns, notsignificant (ANOVA). (G) Geometric mean fluorescence intensity (gMFI) of ICOS expression on adoptively transferred naivewild-type and Nfat1�/� CD4� T cells measured by flow cytometry. (H) Geometric mean fluorescence intensity of ICOSexpression on Bcl-6� and Bcl-6� adoptively transferred naive wild-type and Nfat1�/� CD4� T cells measured by flowcytometry. (I) Frequency of CD4� CXCR5� Bcl-6� Foxp3� TFH was quantified 21 days postinfection by flow cytometry incontrol uninfected and Plasmodium yoelii OVA-infected C57Bl6 mice that received either naive wild-type or NFAT1-deficient OT-II cells (n � 4 to 6). (J) IL-21 production was quantified by intracellular cytokine staining after ex vivostimulation with APCs plus OVA in OT-II cells obtained from C57BL/6 control or Plasmodium yoelii OVA-infected mice thatreceived either naive wild-type or NFAT1-deficient OT-II cells. *, P � 0.05 (t test).

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FIG 4 Microarray analysis of wild-type versus Nfat1�/� CD4� T cells following Plasmodium-induced exhaustion. (A) Heat map of significantly upregulatedor downregulated genes in Nfat1�/� versus wild-type exhausted, CD4� CD11ahigh CD49d� T cells following P. yoelii infection as measured by microarray

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function in the context of this infection in NFAT1-deficient T cells. Furthermore, wedetected a significant increase in the production of anti-OVA antibodies in miceinfected with P. yoelii that received NFAT1-deficient OT-II cells, suggesting that theNFAT1-deficient CD4� T cells we transferred may show increased ability to provide helpto B cells compared with that of their more exhausted wild-type counterparts. Thisincreased help may have been mediated at least in part by the higher ICOS expressionwe observed on the transferred Nfat1�/� T cells. While NFAT2 has been shown to beimportant for the development of T follicular helper cells and the expression ofcostimulatory molecules such as ICOS during acute LCMV infection, ICOS expressionwas shown to be increased in Nfat1�/� T cells compared with controls (28), in agreementwith our data following P. yoelii infection. This may suggest a common suppressive role forNFAT1 in helper T cells during an infection. Furthermore, it is possible that the increasedICOS expression and concomitant increase in antibody production that we observed alsocontributed to delay of death in the lethal infections, as antibody production in responseto Plasmodium plays a critical role in the control of malaria (29, 30).

Several recent studies have reported characterization the patterns of gene expres-sion that define exhaustion in CD4� and CD8� T cells. These studies have identifiedgenes that comprise a specific gene expression program that shows marked differencesfrom genes activated in other processes of T cell inactivation, such as T cell anergy (17,18). These genes encode inhibitory receptors, such as PD-1, LAG-3, or CTLA-4, andgenes involved in the regulation of different aspects of T cell biology, includingapoptosis, cell metabolism, control of cytokine expression, and cell-to-cell communi-cation. In addition, several transcription factors, including Blimp1, Eomes, and T-bet, arealso differentially expressed in exhausted T cells (17, 18, 31).

Despite differences in effector function following P. yoelii-induced exhaustion ofNfat1�/� versus Nfat1�/� CD4� T cells, expression of PD-1 and LAG-3 proteins were stillupregulated to similar degrees in both cases. However, we have observed that expres-sion of PD-1 and LAG-3 is increased in T cells in response to ionomycin treatment andthat this effect is inhibited by cyclosporine (data not shown). These data suggest thatNFAT proteins other than NFAT1 may also participate in the regulation of the expres-sion of these exhaustion-associated genes. Indeed, NFAT2 has been shown to regulatePD-1 expression in activated T cells and to cooperate with NFAT1 in the expression ofPD-1 and LAG-3 in CD8� T cells exhausted by LCMV infection (26, 32). However, thedisconnect between the continued expression of these two molecules and the de-creased exhausted phenotype seen in CD4� T cells may support the idea that Plasmo-dium infection-induced expression of PD-1 and LAG-3 may be required (4) but notsufficient to induce a full exhausted phenotype. While some of the functions of NFATproteins during CD4� T cell exhaustion may be redundant, NFAT1 appears to exertmore specific control upon the expression of a subset of genes rather than the entireT cell exhaustion program. Furthermore, our data suggest that some of the processesthat dampen effector T cell responses during exhaustion may occur independently ofthe upregulation of PD-1 and LAG-3.

The functions controlled by NFAT in T helper cells range from the regulation ofactivation to the induction of tolerance (9). The ability of NFAT proteins to modulatesuch contrasting programs is due to their capacity to function as signal integratorsthrough the formation of distinct transcriptional cooperative complexes with othertranscription factors. During acute activation of T cells, concomitant induction of Fosand Jun in response to the activation of MAPK-regulated pathways results in theexpression of many activation-induced genes that present NFAT/AP-1 composite bind-

FIG 4 Legend (Continued)analysis. Significance was measured by one-way between-subject ANOVA (unpaired) (P � 0.05) and the gene list filtered to include genes up- ordownregulated �2-fold. Scale bar values indicate relative intensities normalized for each gene. (B) Top six gene sets with the highest overlap with set ofgenes significantly upregulated in Nfat1�/� CD4� T cells following P. yoelii infection (see Table S1) in the ImmuneSigDB (Broad Institute, MIT). Filled boxesindicate the presence of indicated gene in the corresponding gene set. (C) Genes upregulated in both Plasmodium-induced CD4� exhausted T cells versusuninfected controls and in LCMV-induced CD4� T cell exhaustion (as reported by Crawford et al. [17]).

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ing sites in the regulatory regions (33, 34). However, in response to suboptimalactivation, inefficient activation of AP-1 results in the expression of anergy-associatedgenes, which may feature sites that bind NFAT1 dimer in their promoters or enhancers(15). We still do not know specifically which genes NFAT1 may directly control duringexhaustion of CD4� T cells. However, expression of a mutant, constitutively activeNFAT1 protein unable to interact with AP-1 has been recently shown to induce theexpression of many genes associated with the exhausted phenotype in in vitro-culturedCD4� and CD8� T cells (26). Given that NFAT1 monomers have limited transcriptionalactivity (15), it is tempting to speculate that exhaustion-specific complexes containingNFAT1 and other transcription factors might be responsible for the expression of adistinct program in exhausted T cells. Several other transcription factors have beenreported to contribute to the expression of exhaustion-associated genes, includingEomes and Blimp-1 (8, 31, 35, 36). NFAT may cooperate with those proteins or other,yet-to-be-identified factors to induce the expression of a specific program of geneexpression in exhausted T cells.

Our microarray analysis of CD4� CD11ahigh CD49d� cells following P. yoelii infectionshowed that a subset of genes were differentially expressed between Nfat1�/� andNfat1�/� T cells following exposure to P. yoelii. Most of the genes that are significantlydifferentially expressed in Nfat1�/� T cells compared with controls are genes thatsupport an effector T cell phenotype, including transcripts encoding cytokines andcytokine receptors (e.g., Il21, Il2ra, and Il12rb2) and cyclins and a cyclin-dependentkinase (Ccna2, Cdk1, and Ccnb2). These data are consistent with the increased effectoractivity that we observed in these cells in terms of cytokine production and prolifera-tion. NFAT1, therefore, promotes downregulation of effector functions in CD4� T cellsduring P. yoelii-induced exhaustion by either directly or indirectly suppressing expres-sion of genes supporting effector T cell function. It is possible that NFAT1 may driveexpression of suppressive factors that control expression of these genes as a secondaryevent during the establishment of T cell exhaustion or that NFAT1 may cooperate withtranscriptional repressors to directly inhibit the expression of those genes. Determiningthe specific promoters that NFAT1 binds during T cell exhaustion and the transcrip-tional complexes it may form will require further study.

Our data show that NFAT1 plays an important role in the induction of exhaustionby Plasmodium infection in CD4� T cells. The control of the expression of a subsetof genes by NFAT1, whose expression is necessary to fully induce an exhaustedphenotype in T helper cells, supports the idea that NFAT1 is a novel regulator of theT cell exhaustion transcriptional program in CD4� T cells that could be targeted toprevent decreased T cell function during chronic infection and boost antipathogenresponses.

MATERIALS AND METHODSMice. C57BL/6 and B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mice were purchased from The Jackson

Laboratory (Bar Harbor, ME). Nfat1�/� OT-II� mice were generated by crossing B6.Nfat1�/� (37) withOT-II� mice. Mice were maintained under selected pathogen-free conditions except during Plasmodiuminfections, at which time they were housed in a conventional nonbarrier facility. Transgenic and controlmice were bred and housed together prior to and during experiments to ensure that differences inenvironment did not confound experimental results. All animal work was carried out according to theguidelines of the Institutional Animal Care Committee at the Albert Einstein College of Medicine.

Primary T cell isolation and culture. Murine CD4� T cells were positively selected using CD4-Dynabeads (Invitrogen, Carlsbad, CA). For in vitro expansion and differentiation, cells were stimulatedwith plate-coated anti-CD3� and anti-CD28 antibodies (0.25 �g/ml each; BD Biosciences, Carlsbad, CA).To differentiate into TH1 cells, CD4� cells were cultured in the presence of mouse IL-12 (10 ng/ml;eBioscience, San Diego, CA), anti-mouse IL-4 antibody (10 �g/ml; 11B11 clone), and recombinant humanIL-2 (10 U/ml; Biological Resources Branch of the National Cancer Institute, Frederick, MD) for 5 to 6 days.Cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovineserum (FBS), 2 mM L-glutamine, 50 �M �-mercaptoethanol, essential vitamins, 550 nM L-Arg, 240 nML-Asn, and 14 nM folic acid.

Plasmodium infections. Infection with Plasmodium yoelii 17XNL or Plasmodium yoelii 17XNL-OVAwas performed by injecting 2 � 104 blood-stage parasites intravenously into wild-type or Nfat1�/�

C57BL/6J mice. Every 48 to 72 h over the course of 21 days, blood smears were prepared, fixed withmethanol, and stained with Giemsa reagent (Sigma-Aldrich, St. Louis, MO). We examined at least 1,000

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erythrocytes per sample and calculated the percentage of infected erythrocytes (parasitemia). Images ofthe smears were collected in the Analytical Imaging Facility of the Albert Einstein College of Medicine.

Generation of Plasmodium yoelii OVA. Generation of a transgenic P. yoelii 17XNL parasite line wasdescribed previously (38). In brief, a gene construct containing full-length chicken ovalbumin and thesignal peptide sequence of Pbama1 under the control of the apical membrane antigen 1 (ama1)promoter was digested with ApaI in the d-ssu-rrna gene to linearize the vector. P. yoelii-infectederythrocytes were transfected by electroporation and were selected in Swiss Webster mice (female, 6 to8 weeks old; Charles River) using pyrimethamine in the drinking water. The surviving parasites werepassaged to new mice under pyrimethamine selection, and drug-resistant P. yoelii parasites expressingovalbumin (Plasmodium OVA) were used in experiments.

Adoptive transfer. Three days following Plasmodium yoelii OVA infection, 1 million Nfat1�/� orNfat1�/� OT-II CD4� naive T cells or in vitro-differentiated TH1 cells were injected intravenously into bothinfected and control uninfected C57BL/6J mice by retro-orbital injection. At day 21 after infection, micewere sacrificed and T cells isolated using CD4�-Dynabeads for analysis.

Enzyme-linked immunosorbent assay (ELISA). For detection of anti-OVA and anti-MSP-1 antibod-ies, serum was harvested from mice following euthanasia of mice at day 21 postinfection by cardiacpuncture. For measurement, Maxisorp microtiter plates (Nunc) were coated with chicken ovalbumin at0.2 mg/ml in carbonate buffer (1.57% Na2CO3, 2.93% NaHCO3 [pH 9.7]) or MSP-142 recombinant proteinin phosphate-buffered saline (PBS) and then blocked with 5% nonfat milk. Serum was then plated at a1:100 dilution in PBS. Following extensive washing, bound anti-OVA or anti-MSP-1 antibodies weredetected using horseradish peroxidase (HRP)-coupled anti-mouse IgG antibodies and developed byincubation with TMB� one-step substrate system (Agilent).

A total of 5 � 104 cells were activated for 24 h using plate-bound anti-CD3 and anti-CD28 antibodiesas described above. IL-2 concentrations in culture supernatants were measured using a sandwich ELISAwith the following antibodies: anti-IL-2 (JES6) and biotinylated anti-IL-2 (30-H12) (eBioscience).

Flow cytometry. Antibodies against the following were used for flow cytometric analysis: CD11a(M17/4), CD49d (R1-2), CD4 (GK1.5), CD44 (IM7), CD62L (MEL-14), LAG3 (eBioC9B7W), PD-1 (RMP1-30),OT-II V�2 TCR (B20.1), OT-II V� 5.1/5.2 (MR9-4), and ICOS (7E.17G9), all from eBioscience, and Bcl-6(K112-91) from BD Biosciences. Stained cells were analyzed by flow cytometry with an LSR II cytometer(BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Ashland, OR).

BrdU incorporation. To measure proliferation of T cells, 3 � 105 CD4� T cells were stimulated withCD3 and CD28 antibodies or 1 � 105 CD4� T cells were stimulated OVA323–339-loaded splenocytes (forOT-II cell experiments). Bromodeoxyuridine (BrdU) was added 24 h poststimulation for 12 h. Cells werethen surface stained, processed by the manufacturer’s instructions (BrdU flow cytometry kit; BD Biosci-ences), and analyzed by flow cytometry as described above.

Apoptosis assay. Apoptosis following restimulation in vitro was assessed by staining cells withannexin V-fluorescein isothiocyanate (FITC) (eBioscience) and LIVE/DEAD fixable blue stain (Invitrogen)and analyzed by flow cytometry as described above. Early apoptotic cells were defined as annexin Vpositive and LIVE/DEAD stain negative.

Intracellular cytokine staining. Cells were stimulated with either ionomycin (1 �M) and phorbolmyristate acetate (PMA; 50 nM) for 4 h or APCs with OVA peptide for 16 h (for OT-II cell experiments),then incubated in the presence of brefeldin A (10 �g/ml) for 4 additional hours, and then fixed with 4%paraformaldehyde. Cells were then permeabilized with saponin, stained with anti-IL-2 or anti-IFN-�antibodies (clones JES6-5H4 and XMG1.2, respectively; eBiosciences), and analyzed by flow cytometry asdescribed above.

qPCR and primers. RNA was isolated using the Qiagen RNeasy kit, and cDNA was synthesized usingthe qScript Supermix reagent (Quanta Biosciences, Gaithersburg, MD). Quantitative PCR (qPCR) wasperformed using a StepOnePlus real-time PCR system (Applied Biosystems). Expression of each gene wasnormalized to that of the actin gene. The following primers were used: Actin forward, 5=-CGTCGACAACGGCTCCGGCATG-3=; Actin reverse, 5=-CCACCATCACACCCTGGTGCCTAGG-3=; Lag3 forward, 5=-TTGGGAAGCTCCAGTTGTGT-3=; Lag3 reverse, 5=-AACCCCTCCTCTTCGTAGAAA-3=; Pdcd1 forward, 5=-GGTTTCAAGGCATGGTCATT-3=; and Pdcd1 reverse, 5=-GCTCCTCCTTCAGAGTGTCG.

Gene expression analysis. Following 21 days of Plasmodium yoelii infection, CD49d� CD11ahigh

CD4� T cells were isolated by cell sorting on a FACSAria and total RNA was isolated using the RNeasyminikit (Qiagen). Three biological replicates per condition from two separate infections were collected forthis analysis. RNA quality and integrity were determined utilizing an Agilent Bioanalyzer 2100. Microarrayhybridization was performed using the mouse GeneChip ST2.0 array with WT Pico preparation fromAffymetrix and was carried out in the Albert Einstein College of Medicine Genomics Core.

Statistical analysis. Statistical analysis was carried out using GraphPad Prism software (GraphPad,Carlsbad, CA). P values were calculated by one-way analysis of variance (ANOVA) or Student’s t test asspecified in the figure legends. Survival curves were tested for significance using the log rank (Mantel-Cox) test. One-way ANOVA of microarray results was performed using the Transcriptome AnalysisConsole (Affymetrix), with filtering of results by P values of �0.05 and by a change in expression betweengroups of interest 2-fold or greater.

Accession number(s). Results from this analysis are deposited the GEO repository under accessionnumber GSE85896.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00364-17.

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SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.SUPPLEMENTAL FILE 2, PDF file, 0.2 MB.SUPPLEMENTAL FILE 3, PDF file, 0.1 MB.SUPPLEMENTAL FILE 4, PDF file, 0.1 MB.SUPPLEMENTAL FILE 5, PDF file, 0.1 MB.SUPPLEMENTAL FILE 6, PDF file, 0.1 MB.

ACKNOWLEDGMENTSWe thank James Burns, Drexel University School of Medicine, for the gift of recom-

binant MSP-1.This work was funded by NIH grants AI059738 (to F.M.) and T32AI070117 (I.G.).We declare that we have no conflicting financial interests related to this study.

REFERENCES1. Wherry EJ, Kurachi M. 2015. Molecular and cellular insights into T cell

exhaustion. Nat Rev Immunol 15:486 – 499. https://doi.org/10.1038/nri3862.

2. Christensen JP, Marker O, Thomsen AR. 1994. The role of CD4� T cells incell-mediated immunity to LCMV: studies in MHC class I and class IIdeficient mice. Scand J Immunol 40:373–382. https://doi.org/10.1111/j.1365-3083.1994.tb03477.x.

3. Matloubian M, Concepcion RJ, Ahmed R. 1994. CD4� T cells are requiredto sustain CD8� cytotoxic T-cell responses during chronic viral infection.J Virol 68:8056 – 8063.

4. Butler NS, Moebius J, Pewe LL, Traore B, Doumbo OK, Tygrett LT,Waldschmidt TJ, Crompton PD, Harty JT. 2012. Therapeutic blockade ofPD-L1 and LAG-3 rapidly clears established blood-stage Plasmodiuminfection. Nat Immunol 13:188 –195. https://doi.org/10.1038/ni.2180.

5. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S,Mackey EW, Miller JD, Leslie AJ, DePierres C, Mncube Z, Duraiswamy J,Zhu B, Eichbaum Q, Altfeld M, Wherry EJ, Coovadia HM, Goulder PJ,Klenerman P, Ahmed R, Freeman GJ, Walker BD. 2006. PD-1 expressionon HIV-specific T cells is associated with T-cell exhaustion and diseaseprogression. Nature 443:350 –354. https://doi.org/10.1038/nature05115.

6. Nakamoto N, Cho H, Shaked A, Olthoff K, Valiga ME, Kaminski M, GostickE, Price DA, Freeman GJ, Wherry EJ, Chang KM. 2009. Synergistic reversalof intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade. PLoS Pathog 5:e1000313. https://doi.org/10.1371/journal.ppat.1000313.

7. Kao C, Oestreich KJ, Paley MA, Crawford A, Angelosanto JM, Ali MA,Intlekofer AM, Boss JM, Reiner SL, Weinmann AS, Wherry EJ. 2011.Transcription factor T-bet represses expression of the inhibitory receptorPD-1 and sustains virus-specific CD8� T cell responses during chronicinfection. Nat Immunol 12:663– 671. https://doi.org/10.1038/ni.2046.

8. Shin H, Blackburn SD, Intlekofer AM, Kao C, Angelosanto JM, Reiner SL,Wherry EJ. 2009. A role for the transcriptional repressor Blimp-1 inCD8(�) T cell exhaustion during chronic viral infection. Immunity 31:309 –320. https://doi.org/10.1016/j.immuni.2009.06.019.

9. Macian F. 2005. NFAT proteins: key regulators of T-cell development andfunction. Nat Rev Immunol 5:472– 484. https://doi.org/10.1038/nri1632.

10. Loh C, Carew JA, Kim J, Hogan PG, Rao A. 1996. T-cell receptor stimu-lation elicits an early phase of activation and a later phase of deactiva-tion of the transcription factor NFAT1. Mol Cell Biol 16:3945–3954.https://doi.org/10.1128/MCB.16.7.3945.

11. Okamura H, Aramburu J, Garcia-Rodriguez C, Viola JP, Raghavan A,Tahiliani M, Zhang X, Qin J, Hogan PG, Rao A. 2000. Concerted dephos-phorylation of the transcription factor NFAT1 induces a conformationalswitch that regulates transcriptional activity. Mol Cell 6:539 –550. https://doi.org/10.1016/S1097-2765(00)00053-8.

12. Baine I, Abe BT, Macian F. 2009. Regulation of T-cell tolerance bycalcium/NFAT signaling. Immunol Rev 231:225–240. https://doi.org/10.1111/j.1600-065X.2009.00817.x.

13. Macián F, Garcia-Cozar F, Im SH, Horton HF, Byrne MC, Rao A. 2002.Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109:719 –731. https://doi.org/10.1016/S0092-8674(02)00767-5.

14. Shin DS, Jordan A, Basu S, Thomas RM, Bandyopadhyay S, de Zoeten EF,Wells AD, Macian F. 2014. Regulatory T cells suppress CD4� T cells

through NFAT-dependent transcriptional mechanisms. EMBO Rep 15:991–999. https://doi.org/10.15252/embr.201338233.

15. Soto-Nieves N, Puga I, Abe BT, Bandyopadhyay S, Baine I, Rao A, MacianF. 2009. Transcriptional complexes formed by NFAT dimers regulate theinduction of T cell tolerance. J Exp Med 206:867– 876. https://doi.org/10.1084/jem.20082731.

16. Schwartz RH. 2003. T cell anergy. Annu Rev Immunol 21:305–334.https://doi.org/10.1146/annurev.immunol.21.120601.141110.

17. Crawford A, Angelosanto JM, Kao C, Doering TA, Odorizzi PM, Barnett BE,Wherry EJ. 2014. Molecular and transcriptional basis of CD4(�) T celldysfunction during chronic infection. Immunity 40:289 –302. https://doi.org/10.1016/j.immuni.2014.01.005.

18. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, SubramaniamS, Blattman JN, Barber DL, Ahmed R. 2007. Molecular signature of CD8�T cell exhaustion during chronic viral infection. Immunity 27:670 – 684.https://doi.org/10.1016/j.immuni.2007.09.006.

19. McDermott DS, Varga SM. 2011. Quantifying antigen-specific CD4 T cellsduring a viral infection: CD4 T cell responses are larger than we think. JImmunol 187:5568 –5576. https://doi.org/10.4049/jimmunol.1102104.

20. Peng SL, Gerth AJ, Ranger AM, Glimcher LH. 2001. NFATc1 and NFATc2together control both T and B cell activation and differentiation. Immu-nity 14:13–20. https://doi.org/10.1016/S1074-7613(01)00085-1.

21. Kiani A, Viola JP, Lichtman AH, Rao A. 1997. Down-regulation of IL-4gene transcription and control of Th2 cell differentiation by a mecha-nism involving NFAT1. Immunity 7:849 – 860. https://doi.org/10.1016/S1074-7613(00)80403-3.

22. Han S, Asoyan A, Rabenstein H, Nakano N, Obst R. 2010. Role ofantigen persistence and dose for CD4� T-cell exhaustion and recov-ery. Proc Natl Acad Sci U S A 107:20453–20458. https://doi.org/10.1073/pnas.1008437107.

23. Mueller SN, Ahmed R. 2009. High antigen levels are the cause of T cellexhaustion during chronic viral infection. Proc Natl Acad Sci U S A106:8623– 8628. https://doi.org/10.1073/pnas.0809818106.

24. Godec J, Tan Y, Liberzon A, Tamayo P, Bhattacharya S, Butte AJ, MesirovJP, Haining WN. 2016. Compendium of immune signatures identifiesconserved and species-specific biology in response to inflammation.Immunity 44:194 –206. https://doi.org/10.1016/j.immuni.2015.12.006.

25. Chiu YL, Shan L, Huang H, Haupt C, Bessell C, Canaday DH, Zhang H, HoYC, Powell JD, Oelke M, Margolick JB, Blankson JN, Griffin DE, Schneck JP.2014. Sprouty-2 regulates HIV-specific T cell polyfunctionality. J ClinInvest 124:198 –208. https://doi.org/10.1172/JCI70510.

26. Martinez GJ, Pereira RM, Aijo T, Kim EY, Marangoni F, Pipkin ME, TogherS, Heissmeyer V, Zhang YC, Crotty S, Lamperti ED, Ansel KM, Mempel TR,Lahdesmaki H, Hogan PG, Rao A. 2015. The transcription factor NFATpromotes exhaustion of activated CD8(�) T cells. Immunity 42:265–278.https://doi.org/10.1016/j.immuni.2015.01.006.

27. Angelosanto JM, Blackburn SD, Crawford A, Wherry EJ. 2012. Progressiveloss of memory T cell potential and commitment to exhaustion duringchronic viral infection. J Virol 86:8161– 8170. https://doi.org/10.1128/JVI.00889-12.

28. Martinez GJ, Hu JK, Pereira RM, Crampton JS, Togher S, Bild N, Crotty S,Rao A. 2016. Cutting edge: NFAT transcription factors promote thegeneration of follicular helper T cells in response to acute viral infection.J Immunol 196:2015–2019. https://doi.org/10.4049/jimmunol.1501841.

NFAT1 and Plasmodium-Induced CD4� T Cell Exhaustion Infection and Immunity

September 2017 Volume 85 Issue 9 e00364-17 iai.asm.org 13

on August 23, 2020 by guest

http://iai.asm.org/

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nloaded from

29. Diggs CL, Osler AG. 1969. Humoral immunity in rodent malaria. II.Inhibition of parasitemia by serum antibody. J Immunol 102:298 –305.

30. Cohen S, McGregor IA, Carrington S. 1961. Gamma-globulin and ac-quired immunity to human malaria. Nature 192:733–737. https://doi.org/10.1038/192733a0.

31. Doering TA, Crawford A, Angelosanto JM, Paley MA, Ziegler CG, WherryEJ. 2012. Network analysis reveals centrally connected genes and path-ways involved in CD8� T cell exhaustion versus memory. Immunity37:1130 –1144. https://doi.org/10.1016/j.immuni.2012.08.021.

32. Oestreich KJ, Yoon H, Ahmed R, Boss JM. 2008. NFATc1 regulates PD-1expression upon T cell activation. J Immunol 181:4832– 4839. https://doi.org/10.4049/jimmunol.181.7.4832.

33. Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN, Verdine GL,Curran T, Rao A. 1993. The T-cell transcription factor NFATp is a substratefor calcineurin and interacts with Fos and Jun. Nature 365:352–355.https://doi.org/10.1038/365352a0.

34. Macián F, Garcia-Rodriguez C, Rao A. 2000. Gene expression elicitedby NFAT in the presence or absence of cooperative recruitment of

Fos and Jun. EMBO J 19:4783– 4795. https://doi.org/10.1093/emboj/19.17.4783.

35. Buggert M, Tauriainen J, Yamamoto T, Frederiksen J, Ivarsson MA, Mi-chaelsson J, Lund O, Hejdeman B, Jansson M, Sonnerborg A, Koup RA,Betts MR, Karlsson AC. 2014. T-bet and Eomes are differentially linked tothe exhausted phenotype of CD8� T cells in HIV infection. PLoS Pathog10:e1004251. https://doi.org/10.1371/journal.ppat.1004251.

36. Paley MA, Kroy DC, Odorizzi PM, Johnnidis JB, Dolfi DV, Barnett BE, Bikoff EK,Robertson EJ, Lauer GM, Reiner SL, Wherry EJ. 2012. Progenitor and terminalsubsets of CD8� T cells cooperate to contain chronic viral infection. Science338:1220–1225. https://doi.org/10.1126/science.1229620.

37. Xanthoudakis S, Viola JP, Shaw KT, Luo C, Wallace JD, Bozza PT, Luk DC,Curran T, Rao A. 1996. An enhanced immune response in mice lackingthe transcription factor NFAT1. Science 272:892– 895. https://doi.org/10.1126/science.272.5263.892.

38. Ting LM, Gissot M, Coppi A, Sinnis P, Kim K. 2008. Attenuated Plasmo-dium yoelii lacking purine nucleoside phosphorylase confer protectiveimmunity. Nat Med 14:954 –958. https://doi.org/10.1038/nm.1867.

Ames et al. Infection and Immunity

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