Microbial Immunity and Vaccines

The Transcription Factor NFAT1 Participates in the Induction of CD4+ T Cell Functional Exhaustion during Plasmodium yoelii Infection

Rachel Y. Ames, Li-Min Ting, Inessa Gendlina, Kami Kim, Fernando Macian
John H. Adams, Editor
DOI: 10.1128/IAI.00364-17

ABSTRACT

Repeated stimulation of T cells that occurs in the context of chronic infection results in progressively reduced responsiveness of T cells to pathogen-derived antigens. This phenotype, known as T cell exhaustion, occurs during chronic infections caused by a variety of pathogens, from persistent viruses to parasites. Unlike the memory cells that typically form after successful pathogen clearance following an acute infection, exhausted T cells secrete lower levels of effector cytokines, proliferate less in response to cognate antigen, and upregulate cell surface inhibitory molecules such as PD-1 and LAG-3. The molecular events that lead to the induction of this phenotype have, however, not been fully characterized. In T cells, members of the NFAT family of transcription factors not only are responsible for the expression of many activation-induced genes but also are crucial for the induction of transcriptional programs that inhibit T cell activation and maintain tolerance. Here we show that NFAT1-deficient CD4+ T cells maintain higher proliferative capacity and expression of effector cytokines following Plasmodium yoelii infection and are therefore 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 absence of NFAT1 compared with wild-type exhausted T cells. Furthermore, adoptive transfer of NFAT1-deficient CD4+ T cells into mice infected with P. yoelii results in increased production of antibodies to cognate antigen. Our results support the idea that NFAT1 is necessary to fully suppress effector responses during Plasmodium-induced CD4+ T cell exhaustion.

INTRODUCTION

Both CD8+ and CD4+ T cells experience a significant loss of function after chronic stimulation 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 productive memory 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 exhaustion is the increased expression of coinhibitory surface molecules on exhausted T cells, and the exhaustion phenotype can be reversed through the blockade of those receptors (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 that underlie the transcriptional regulation of the many exhaustion molecules and phenotypes have not been fully elucidated.

Nuclear factor of activated T cell (NFAT) proteins constitute a family of transcription 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 to T cell receptor (TCR)-triggered calcium signaling, NFAT proteins are activated by the calcineurin-mediated removal of several phosphate groups located in the N-terminal regulatory region. Dephosphorylation of NFAT leads to nuclear translocation and the activation of NFAT-dependent gene expression (10, 11). Though NFAT proteins were originally described as regulators of activation-induced cytokine expression, the functions regulated by NFAT in T cells now include not only positive but also negative programs that hinder T cell activation (12). For instance, the transcription factor NFAT1 controls the induction of programs of gene expression that allow for the regulatory T cell (Treg)-mediated suppression of CD4+ T cell activation and mediate the induction of CD4+ T cell anergy, a state of functional inactivation that bears phenotypic similarity with T cell exhaustion (13–15). Clonal T cell anergy occurs as a consequence of suboptimal stimulation, and it is characterized by a unique transcriptional program driven, at least in part, by NFAT1 (9, 12, 16). Exhausted T cells feature a distinct transcriptional program that is not identical to but has some overlap with the T cell anergy program (17, 18), suggesting that NFAT1 might also contribute to T cell exhaustion and participate in the induction of the transcriptional program activated following chronic engagement of the T cell receptor.

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

RESULTS

NFAT1-deficient CD4+ T cells maintain proliferative responses in a model of T cell exhaustion induced by Plasmodium yoelii infection.NFAT1 participates in the regulation of different programs of T cell inactivation, including T cell anergy and regulatory T cell-mediated suppression of CD4+ T helper cells (13–15). Similar to anergic cells, exhausted T cells show reduced responses to antigen stimulation. To determine if NFAT1 could also play a role in controlling the exhaustion of T cells, we infected wild-type and Nfat1−/− mice with Plasmodium yoelii 17XNL. Infection with this parasite 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 isolated from spleens. CD11ahigh CD49d+ staining has been shown to delineate previously activated CD4+ T cells from naive cells in Plasmodium-infected mice (4, 19) and therefore represent T cells that are likely to respond to a variety of Plasmodium antigens. We compared the responses and phenotypes of the CD4+ CD11ahigh CD49d+ T cell populations from wild-type and Nfat1−/− naive mice with those from mice infected with P. yoelii 17XNL. We could detect similar levels of initial expansion of the CD4+ CD11ahigh CD49d+ compartment following infection in wild-type and NFAT1-deficient mice (Fig. 1A). However, we found that Nfat1−/− CD4+ T cells were able to maintain greater proliferative ability than their wild-type counterparts after exhaustion following P. yoelii infection (Fig. 1B). As expected, T cells from mice infected with P. yoelii showed diminished proliferation following subsequent stimulation compared with T cells from uninfected mice (Fig. 1B) (4). Though Nfat1−/− T cells also lost some proliferative ability following P. yoelii exposure, the decrease in proliferative capacity was 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 receptors, though there was a small but significant difference in the increase of PD-1 expression in Nfat1−/− cells (Fig. 1C). The decreased response of wild-type CD4+ T cells isolated from infected mice was not due to an increase in cell death, as similar levels of apoptosis were induced by restimulation under all conditions tested in T cells from either wild-type or NFAT1-deficient mice (Fig. 1D). We followed parasitemia of infected mice but did not observe significant differences in the course of infection of our wild-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 typically able to effectively clear the infection in less than 4 weeks.

FIG 1
FIG 1

NFAT1-deficient mice are less susceptible to exhaustion induced by Plasmodium yoelii infection in the CD4+ T cell population. (A) Gating strategy and quantification (mean + SEM) of the frequency of CD49d+ CD11ahigh CD4+ T cells 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 show means + 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 mice infected with Plasmodium yoelii. Graphs show means + SEM from 6 to 10 mice per group from three independent experiments. *, P < 0.05; ****, P < 0.0001; ns, not significant (ANOVA). (D) Percentages of the populations of cells analyzed in panel A that were apoptotic following restimulation ex vivo (annexin V+ LIVE/DEAD−) were measured by flow cytometry. Bars show means from 4 or 5 mice from two independent experiments. (E) Parasitemia in Nfat1+/+ and Nfat1−/− mice infected with Plasmodium yoelii. The graph shows average numbers of parasites ± SEM from 6 different mice from two independent experiments.

To assess exhaustion of a specific TCR-antigen combination and to control for possible effects of NFAT1 deficiency in other cell types, including B cells, in the full-body NFAT1-defiicent mice (20), we performed adoptive transfers of OT-II+ wild-type or Nfat1−/− CD4+ TH1-polarized cells into C57BL6/J hosts infected with a Plasmodium yoelii 17XNL strain that had been genetically engineered to express ovalbumin (P. yoelii OVA). For experiments measuring effector functions (cytokine secretion and proliferation), we used TH1-polarized cells in order to observe any decreases in function upon further 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 differentiation that might occur in NFAT1-deficient T cells (21). Differentiation bias has been attributed to differences in the ability of wild-type and NFAT1-deficient CD4+ T cells to sustain interleukin 4 (IL-4) expression, but can be overcome by in vitro differentiation in the presence of polarizing cytokines. Using that approach, we confirmed that in vitro-differentiated Nfat1+/+ and Nfat1−/− TH1 cells showed equivalent abilities to produce IL-2 and gamma interferon (IFN-γ) following reactivation (see Fig. S1 in the supplemental 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 infected with P. yoelii (Fig. S2). However, when we analyzed T cells 21 days postinfection by ex vivo 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-γ was also significantly reduced in OT-II cells isolated from infected wild-type mice, while Nfat1−/− cells maintained the same levels of expression of this effector cytokine as the nonexhausted controls (Fig. 2B). We also observed a similar trend in IL-2 expression, though the difference in IL-2 production between wild-type control and wild-type exhausted CD4+ T cells was not statistically significant (Fig. 2B). No differences in the extent of cell death were observed in any of the various experimental groups (Fig. 2C). Despite a better functional response in T cells isolated from infected mice that received Nfat1−/− OT-II cells, levels of parasitemia in the first 3 weeks of infection were comparable in mice that received wild-type and NFAT1-deficient CD4+ T cells (Fig. 2D). Similar levels of upregulation of PD-1 and LAG-3, CTLA-4, or Tim-3 surface expression also occurred in Nfat1+/+ and Nfat1−/− OT-II cells (Fig. 2E; see also Fig. S2). Interestingly, following repeated stimulation of OT-II CD4+ T cells in vitro via the TCR, using splenocytes presenting OVA peptide, we also saw less downregulation of T cell function (measured by IL-2 secretion), without differences in the upregulation of LAG-3 and PD-1 (Fig. S3), similar to our observations during T cell exhaustion induced by P. yoelii, lending support to the idea that modulation of the response to chronic antigen-specific stimulation of the TCR is central to the role of NFAT1 in the regulation of the T cell exhaustion phenotype (22, 23).

FIG 2
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-γ or IL-2 expression (B) (measured as percentage of cytokine-positive cells analyzed by intracellular staining) were determined 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 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, not significant (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 independent experiments. (D) Parasitemia in mice infected with Plasmodium OVA receiving Nfat1+/+ or Nfat1−/− OT-II CD4+ T cells. The graph shows the average numbers of 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-3 and the levels of CTLA-4 expression were determined by flow cytometry in T cells isolated 21 days postinfection from control or Plasmodium OVA-infected mice receiving 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.

Increased protection against lethal P. yoelii OVA infection is conferred by adoptive transfer of Nfat1−/− CD4+ T cells.The P. yoelii OVA line demonstrated a more virulent phenotype than that of its parental strain, P. yoelii 17XNL, causing lethal infection in some experiments. Though the acute courses of parasitemia were not different between mice receiving wild-type and NFAT1-deficient OT-II cells during these lethal infections (Fig. 3A), we observed a significant delay in death due to P. yoelii OVA in mice adoptively transferred with OT-II Nfat1−/− CD4+ T cells compared with OT-II Nfat1+/+ (Fig. 3B) during infections that were lethal to the cohort. Mice that received Nfat1+/+ CD4+ T cells had a median survival time of 12 days, versus 18 days for the group that received Nfat1−/− OT-II T cells.

FIG 3
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 P. yoelii OVA infection. The graph shows the average numbers of parasites ± SEM in 4 mice per group from a representative infection. (B) Survival of C57BL/6 mice receiving either Nfat1+/+ or Nfat1−/− OT-II+ CD4+ T cells and lethally infected with Plasmodium OVA. Data are from 10 mice per group from two independent experiments. **, P < 0.05 (Mantel-Cox). (C to F) Levels of anti-OVA (C and D) and anti-MSP-1 (E and F) IgG were measured by ELISA in serum from uninfected mice or mice infected with P. yoelii OVA after receiving naive or TH1 Nfat1+/+ or Nfat1−/− OT-II+ CD4+ T cells as indicated. Graphs show means + SEM from 5 to 7 mice per group from two independent experiments. *, P < 0.05; ***, P < 0.001; ns, not significant (ANOVA). (G) Geometric mean fluorescence intensity (gMFI) of ICOS expression on adoptively transferred naive wild-type and Nfat1−/− CD4+ T cells measured by flow cytometry. (H) Geometric mean fluorescence intensity of ICOS expression on Bcl-6+ and Bcl-6− adoptively transferred naive wild-type and Nfat1−/− CD4+ T cells measured by flow cytometry. (I) Frequency of CD4+ CXCR5+ Bcl-6+ Foxp3− TFH was quantified 21 days postinfection by flow cytometry in control 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 vivo stimulation with APCs plus OVA in OT-II cells obtained from C57BL/6 control or Plasmodium yoelii OVA-infected mice that received either naive wild-type or NFAT1-deficient OT-II cells. *, P < 0.05 (t test).

We also observed higher titers of antibodies to ovalbumin in serum from mice infected 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 endogenous Plasmodium antigen MSP-142 between mice receiving wild-type and Nfat1−/− OT-II T cells, which would rely on the activity of the host's polyclonal CD4+ T cell population rather than the transferred T cells (Fig. 3D and F). Additionally, adoptively transferred, naive Nfat1−/− CD4+ T cells featured higher expression of the costimulatory receptor ICOS 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 was more pronounced in Bcl-6+ CD4+ T cells (Fig. 3H and I). Interestingly, Plasmodium infection led to increased production of IL-21 in ex vivo-restimulated cells only in Nfat1−/− OT-II cells obtained from infected mice and not in cells from infected mice that received Nfat1+/+ OT-II cells (Fig. 3J).

NFAT1 is a transcription factor, and therefore, we sought to determine what downstream program of gene expression could be affected by the absence or NFAT1 that may drive the less exhausted phenotype that we had observed in NFAT1-deficient T cells. We purified CD11ahigh CD49d+ CD4+ T cells from spleens of both wild-type and Nfat1−/− mice following 3 weeks of P. yoelii 17XNL infection, as well as from uninfected controls, and isolated RNA for microarray analysis. Analysis of the microarray data indicated that 51 genes were significantly upregulated and 3 downregulated 2-fold or higher in the absence of NFAT1 (Fig. 4A; see also Fig. S5 and Table S1). By comparing the set of genes upregulated in the Nfat1−/− T cells with a database of gene sets generated from published studies of immune cell phenotypes (24) (ImmuneSigDB, Broad Institute, MIT), we found that five of the top six gene sets that overlapped with our list of significantly upregulated genes in Nfat1−/−P. yoelii-exhausted T cells over wild-type were gene sets representing effector T cell states, in agreement with the increased functional phenotypes we have observed (Fig. 4B). We were also interested to know whether Plasmodium-induced exhaustion would have a similar gene profile in T cells to the well-characterized lymphocytic choriomeningitis virus (LCMV)-induced exhaustion, in addition to the previously characterized overlap in exhaustion phenotypes and selected surface marker expression (4). We compared our results with a 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 cells following Plasmodium infection, compared with uninfected controls, there were 13 genes 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 there are likely core mechanisms that drive T cell exhaustion, specific programs of genes expression may also participate in the regulation of T cell exhaustion in response to distinct pathogens.

FIG 4
FIG 4

Microarray analysis of wild-type versus Nfat1−/− CD4+ T cells following Plasmodium-induced exhaustion. (A) Heat map of significantly upregulated or downregulated genes in Nfat1−/− versus wild-type exhausted, CD4+ CD11ahigh CD49d+ T cells following P. yoelii infection as measured by microarray analysis. Significance was measured by one-way between-subject ANOVA (unpaired) (P < 0.05) and the gene list filtered to include genes up- or downregulated >2-fold. Scale bar values indicate relative intensities normalized for each gene. (B) Top six gene sets with the highest overlap with set of genes significantly upregulated in Nfat1−/− CD4+ T cells following P. yoelii infection (see Table S1) in the ImmuneSigDB (Broad Institute, MIT). Filled boxes indicate the presence of indicated gene in the corresponding gene set. (C) Genes upregulated in both Plasmodium-induced CD4+ exhausted T cells versus uninfected controls and in LCMV-induced CD4+ T cell exhaustion (as reported by Crawford et al. [17]).

DISCUSSION

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

The need for chronic antigen stimulation to induce and maintain T cell exhaustion supports a central role for TCR-mediated signaling in this process. At least two mediators of TCR signals, SPRY2 and NFAT, have been identified as factors involved in supporting the establishment of exhaustion in CD8+ T cells (25, 26). Our data indicate that 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 the TCR in vitro (Fig. S3), lending further support to the idea that NFAT1 may play a central role in the control of the response of T cells to repeated TCR engagement during a chronic infection and regulate the establishment of T cell exhaustion.

Although ex vivo activation of CD4+ T cells from mice infected with Plasmodium yoelii showed that NFAT1-deficient cells preserved responses to antigen significantly better than wild-type T cells, we did not observe major differences in the courses of parasitemia. This could be a consequence of the fact that although Plasmodium infection 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 T cells 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 some models to require the continuous presence of the pathogen for several weeks (27). We isolated CD4+ T cells after 3 weeks to ensure that exhaustion had been established, yet wild-type mice were able to control infection with this strain in many cases in 4 weeks or less. However, transfer of NFAT1-deficient CD4+ OT-II T cells increased survival time during lethal infections that occurred sporadically when a more virulent Plasmodium OVA parasite was used to infect mice. The effectiveness of transfer of CD4+ T cells on disease outcomes was likely dependent on the better preservation of CD4+ T cell function in the context of this infection in NFAT1-deficient T cells. Furthermore, we detected a significant increase in the production of anti-OVA antibodies in mice infected with P. yoelii that received NFAT1-deficient OT-II cells, suggesting that the NFAT1-deficient CD4+ T cells we transferred may show increased ability to provide help to B cells compared with that of their more exhausted wild-type counterparts. This increased help may have been mediated at least in part by the higher ICOS expression we observed on the transferred Nfat1−/− T cells. While NFAT2 has been shown to be important for the development of T follicular helper cells and the expression of costimulatory molecules such as ICOS during acute LCMV infection, ICOS expression was shown to be increased in Nfat1−/− T cells compared with controls (28), in agreement with our data following P. yoelii infection. This may suggest a common suppressive role for NFAT1 in helper T cells during an infection. Furthermore, it is possible that the increased ICOS expression and concomitant increase in antibody production that we observed also contributed to delay of death in the lethal infections, as antibody production in response to Plasmodium plays a critical role in the control of malaria (29, 30).

Several recent studies have reported characterization the patterns of gene expression that define exhaustion in CD4+ and CD8+ T cells. These studies have identified genes that comprise a specific gene expression program that shows marked differences from 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, and genes involved in the regulation of different aspects of T cell biology, including apoptosis, cell metabolism, control of cytokine expression, and cell-to-cell communication. In addition, several transcription factors, including Blimp1, Eomes, and T-bet, are also differentially expressed in exhausted T cells (17, 18, 31).

Despite differences in effector function following P. yoelii-induced exhaustion of Nfat1+/+ versus Nfat1−/− CD4+ T cells, expression of PD-1 and LAG-3 proteins were still upregulated to similar degrees in both cases. However, we have observed that expression of PD-1 and LAG-3 is increased in T cells in response to ionomycin treatment and that this effect is inhibited by cyclosporine (data not shown). These data suggest that NFAT proteins other than NFAT1 may also participate in the regulation of the expression of these exhaustion-associated genes. Indeed, NFAT2 has been shown to regulate PD-1 expression in activated T cells and to cooperate with NFAT1 in the expression of PD-1 and LAG-3 in CD8+ T cells exhausted by LCMV infection (26, 32). However, the disconnect between the continued expression of these two molecules and the decreased exhausted phenotype seen in CD4+ T cells may support the idea that Plasmodium infection-induced expression of PD-1 and LAG-3 may be required (4) but not sufficient to induce a full exhausted phenotype. While some of the functions of NFAT proteins during CD4+ T cell exhaustion may be redundant, NFAT1 appears to exert more specific control upon the expression of a subset of genes rather than the entire T cell exhaustion program. Furthermore, our data suggest that some of the processes that dampen effector T cell responses during exhaustion may occur independently of the upregulation of PD-1 and LAG-3.

The functions controlled by NFAT in T helper cells range from the regulation of activation to the induction of tolerance (9). The ability of NFAT proteins to modulate such contrasting programs is due to their capacity to function as signal integrators through the formation of distinct transcriptional cooperative complexes with other transcription factors. During acute activation of T cells, concomitant induction of Fos and Jun in response to the activation of MAPK-regulated pathways results in the expression of many activation-induced genes that present NFAT/AP-1 composite binding sites in the regulatory regions (33, 34). However, in response to suboptimal activation, inefficient activation of AP-1 results in the expression of anergy-associated genes, 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 during exhaustion of CD4+ T cells. However, expression of a mutant, constitutively active NFAT1 protein unable to interact with AP-1 has been recently shown to induce the expression of many genes associated with the exhausted phenotype in in vitro-cultured CD4+ and CD8+ T cells (26). Given that NFAT1 monomers have limited transcriptional activity (15), it is tempting to speculate that exhaustion-specific complexes containing NFAT1 and other transcription factors might be responsible for the expression of a distinct program in exhausted T cells. Several other transcription factors have been reported to contribute to the expression of exhaustion-associated genes, including Eomes 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 gene expression in exhausted T cells.

Our microarray analysis of CD4+ CD11ahigh CD49d+ cells following P. yoelii infection showed that a subset of genes were differentially expressed between Nfat1+/+ and Nfat1−/− T cells following exposure to P. yoelii. Most of the genes that are significantly differentially expressed in Nfat1−/− T cells compared with controls are genes that support an effector T cell phenotype, including transcripts encoding cytokines and cytokine receptors (e.g., Il21, Il2ra, and Il12rb2) and cyclins and a cyclin-dependent kinase (Ccna2, Cdk1, and Ccnb2). These data are consistent with the increased effector activity that we observed in these cells in terms of cytokine production and proliferation. NFAT1, therefore, promotes downregulation of effector functions in CD4+ T cells during P. yoelii-induced exhaustion by either directly or indirectly suppressing expression of genes supporting effector T cell function. It is possible that NFAT1 may drive expression of suppressive factors that control expression of these genes as a secondary event during the establishment of T cell exhaustion or that NFAT1 may cooperate with transcriptional repressors to directly inhibit the expression of those genes. Determining the specific promoters that NFAT1 binds during T cell exhaustion and the transcriptional complexes it may form will require further study.

Our data show that NFAT1 plays an important role in the induction of exhaustion by Plasmodium infection in CD4+ T cells. The control of the expression of a subset of genes by NFAT1, whose expression is necessary to fully induce an exhausted phenotype in T helper cells, supports the idea that NFAT1 is a novel regulator of the T cell exhaustion transcriptional program in CD4+ T cells that could be targeted to prevent decreased T cell function during chronic infection and boost antipathogen responses.

MATERIALS AND METHODS

Mice.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) with OT-II+ mice. Mice were maintained under selected pathogen-free conditions except during Plasmodium infections, at which time they were housed in a conventional nonbarrier facility. Transgenic and control mice were bred and housed together prior to and during experiments to ensure that differences in environment did not confound experimental results. All animal work was carried out according to the guidelines 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 stimulated with plate-coated anti-CD3ε and anti-CD28 antibodies (0.25 μg/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 μg/ml; 11B11 clone), and recombinant human IL-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 bovine serum (FBS), 2 mM l-glutamine, 50 μM β-mercaptoethanol, essential vitamins, 550 nM l-Arg, 240 nM l-Asn, and 14 nM folic acid.

Plasmodium infections.Infection with Plasmodium yoelii 17XNL or Plasmodium yoelii 17XNL-OVA was 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 with methanol, and stained with Giemsa reagent (Sigma-Aldrich, St. Louis, MO). We examined at least 1,000 erythrocytes per sample and calculated the percentage of infected erythrocytes (parasitemia). Images of the 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 was described previously (38). In brief, a gene construct containing full-length chicken ovalbumin and the signal 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-infected erythrocytes were transfected by electroporation and were selected in Swiss Webster mice (female, 6 to 8 weeks old; Charles River) using pyrimethamine in the drinking water. The surviving parasites were passaged to new mice under pyrimethamine selection, and drug-resistant P. yoelii parasites expressing ovalbumin (Plasmodium OVA) were used in experiments.

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

Enzyme-linked immunosorbent assay (ELISA).For detection of anti-OVA and anti-MSP-1 antibodies, serum was harvested from mice following euthanasia of mice at day 21 postinfection by cardiac puncture. For measurement, Maxisorp microtiter plates (Nunc) were coated with chicken ovalbumin at 0.2 mg/ml in carbonate buffer (1.57% Na2CO3, 2.93% NaHCO3 [pH 9.7]) or MSP-142 recombinant protein in phosphate-buffered saline (PBS) and then blocked with 5% nonfat milk. Serum was then plated at a 1:100 dilution in PBS. Following extensive washing, bound anti-OVA or anti-MSP-1 antibodies were detected using horseradish peroxidase (HRP)-coupled anti-mouse IgG antibodies and developed by incubation 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 antibodies as described above. IL-2 concentrations in culture supernatants were measured using a sandwich ELISA with 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 Vα2 TCR (B20.1), OT-II Vβ 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 with CD3 and CD28 antibodies or 1 × 105 CD4+ T cells were stimulated OVA323–339-loaded splenocytes (for OT-II cell experiments). Bromodeoxyuridine (BrdU) was added 24 h poststimulation for 12 h. Cells were then surface stained, processed by the manufacturer's instructions (BrdU flow cytometry kit; BD Biosciences), and analyzed by flow cytometry as described above.

Apoptosis assay.Apoptosis following restimulation in vitro was assessed by staining cells with annexin 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 V positive and LIVE/DEAD stain negative.

Intracellular cytokine staining.Cells were stimulated with either ionomycin (1 μM) and phorbol myristate 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 μg/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-γ antibodies (clones JES6-5H4 and XMG1.2, respectively; eBiosciences), and analyzed by flow cytometry as described above.

qPCR and primers.RNA was isolated using the Qiagen RNeasy kit, and cDNA was synthesized using the qScript Supermix reagent (Quanta Biosciences, Gaithersburg, MD). Quantitative PCR (qPCR) was performed using a StepOnePlus real-time PCR system (Applied Biosystems). Expression of each gene was normalized 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 RNeasy minikit (Qiagen). Three biological replicates per condition from two separate infections were collected for this analysis. RNA quality and integrity were determined utilizing an Agilent Bioanalyzer 2100. Microarray hybridization was performed using the mouse GeneChip ST2.0 array with WT Pico preparation from Affymetrix 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 as specified 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 Analysis Console (Affymetrix), with filtering of results by P values of ≤0.05 and by a change in expression between groups of interest 2-fold or greater.

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

ACKNOWLEDGMENTS

We thank James Burns, Drexel University School of Medicine, for the gift of recombinant 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.

FOOTNOTES

    • Received 17 May 2017.
    • Accepted 7 June 2017.
    • Accepted manuscript posted online 19 June 2017.

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

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KEYWORDS

CD4-Positive T-Lymphocytes
host-pathogen interactions
Immune Tolerance
malaria
NFATC Transcription Factors
Plasmodium yoelii
exhaustion
NFAT
Plasmodium
T cells

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