BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.


Introduction
CD4 T helper cells play a central role in adaptive immunity by providing essential help to both antibody-producing B cells and cytotoxic CD8 T cells in addition to eliciting their own effector functions.Depending on the context of infection, CD4 T cells can differentiate into a variety of Th subsets (Th1/Th2/Th17/pTreg/Tfh) following antigen-specific priming by dendritic cells [1].Mucosal infection by the obligate intracellular bacterium Chlamydia induces a strong type 1 immune response characterized by robust IFN-γ production by both CD4 and CD8 T cells [2].Although it is well-documented that protective immunity to Chlamydia relies on CD4 cells via both IFN-γ-dependent and -independent mechanisms in the female reproductive tract (FRT), the IFN-γ-independent CD4 T cell functions are largely undefined [3][4][5][6][7][8].Moreover, the key molecules that drive the differentiation of protective CD4 T cells in Chlamydia infection remain to be identified.
The protein basic helix-loop-helix family, member e40 (BHLHE40) is a widely expressed transcription factor that regulates a broad range of biological processes including circadian rhythm, lipid metabolism, neurogenesis and host immunity [9,10].At steady state, Bhlhe40 is expressed by several myeloid cell populations including neutrophils, macrophages and dendritic cells [11].In alveolar and large peritoneal macrophages, Bhlhe40 expression is required for proliferation, self-renewal and their responses to helminth infections [12,13].
Although not detected in naïve T cells, Bhlhe40 expression is quickly induced upon T cell activation and maintained in a CD28-dependent manner [14].Recent studies describe Bhlhe40 as a "molecular switch" between proinflammatory and anti-inflammatory responses in CD4 T cells for its dynamic regulation of cytokine production [15].Given that immune-mediated protection requires a fine balance between pro-inflammatory response that limits pathogen replication and anti-inflammatory response that prevents immunopathology, Bhlhe40 emerges as a key player in host immune responses to infections, autoimmune disorders and cancer [10,16].In Mycobacterium tuberculosis and Toxoplasma gondii infections, Bhlhe40 -/-mice manifest increased pathogen burdens and reduced survival, owing to an increase in IL-10 production by CD4 T cells and reduced IFN-γ responses in both models [15,17].On the contrary, Bhlhe40 deficiency ameliorates immunopathology in autoimmune conditions such as colitis and experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis [11,14,15,18].Mechanistically, BHLHE40 suppresses IL-10 production by co-binding to a regulatory region with c-Maf in the Il10 locus in T cells and myeloid cells, which in turn promotes proinflammatory cytokine production during intracellular parasite infections [13,17].Moreover, Bhlhe40 can also function as a transcription activator to promote Csf2 transcription for GM-CSF production, a hallmark cytokine for pathologic CD4 T cells in EAE [11,18].Nevertheless, a role for Bhlhe40 appears to be context dependent, as in other intracellular infection models such as Listeria monocytogenes, Bhlhe40 is largely dispensable for host resistance [17].
To date, a role for Bhlhe40 in the FRT mucosa has not been demonstrated.Using the mouse model of Chlamydia muridarum intravaginal infection, we showed that loss of Bhlhe40, either in the germline or specifically in T cells, resulted in higher bacterial shedding and significant delay in Chlamydia clearance from the FRT.The defects of Bhlhe40 -/-mice were results of impaired CD4 T cell responses, including increased IL-10-producing Tr1 differentiation and reduced Th1, Th17 and ThGM populations and cytokine production.Moreover, single-cell analysis revealed a fundamental defect in CD4 T cell differentiation in Bhlhe40 -/-FRT manifested by increased stemness-and decreased effector function signatures in the Bhlhe40 -/-CD4 T cells.These results established Bhlhe40 as a novel regulator of CD4 T cell differentiation and protective immune responses in the FRT against Chlamydia.

Bhlhe40 expression by T cells is required for anti-Chlamydia immunity
To investigate whether Bhlhe40 plays a role in protective immunity against Chlamydia in the female reproductive tract (FRT), we infected WT and Bhlhe40 -/-mice intravaginally with Chlamydia muridarum and monitored bacterial shedding from the lower FRT by vaginal swabs.As expected, WT B6 mice showed effective bacterial control and naturally resolved the infection around day 35 (Fig 1A and 1B).In contrast, Bhlhe40 -/-mice exhibited persistent high bacterial

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Role of Bhlhe40 in Chlamydia infection shedding from the FRT during the first 3 weeks of infection.Although bacterial control was eventually achieved in Bhlhe40 -/-mice, clearance was significantly delayed (Fig 1A and 1B).The differences in bacterial burden between WT and Bhlhe40 -/-mice were most prominent between days 14 and 35, with 2-5 magnitudes higher inclusion forming units (IFUs) detected in Bhlhe40 -/-mice than in their WT counterparts, indicating a defective adaptive immune response in these mice.On the contrary, no difference in serum antibody titers was detected on both day 21 and day 70 post infection (S1A Fig) .In addition, long-term FRT tissue pathology was not affected by Bhlhe40 deficiency, as WT and Bhlhe40 -/-mice had similar pathology scores in uterine horns and oviducts at 140-150 days post infection (dpi) (S1B Fig) .These data suggest that Bhlhe40 regulates host susceptibility to Chlamydia infection in the FRT, likely in a T cell-dependent manner.
To determine whether T cell-intrinsic expression of Bhlhe40 is required for anti-Chlamydia immunity, we next infected Bhlhe40 fl/fl -Cd4-Cre mice with C. muridarum and compared bacterial burdens to Bhlhe40 fl/fl controls.Much like the Bhlhe40 -/-mice, Bhlhe40 fl/f/ -Cd4-Cre mice exhibited heightened bacterial shedding and delayed clearance.Moreover, the kinetics of bacterial shedding in Bhlhe40 fl/f/ -Cd4-Cre mice were similar to Bhlhe40 -/-mice (Fig 1C), indicating that T cell-intrinsic Bhlhe40 expression accounts for the defects in bacterial control observed in Bhlhe40 -/-mice.

IL-10-producing Tr1 cells are increased in the FRT of Bhlhe40 -/-mice
The T cell-intrinsic requirement of Bhlhe40 prompted us to identify the essential CD4 T cell effector functions controlled by Bhlhe40 during Chlamydia infection.Previous studies have reported a disturbed balance between proinflammatory IFN-γ and anti-inflammatory IL-10 in Bhlhe40 -/-mice during intracellular parasite infections [15,17].We therefore measured CD4 T cell cytokine production in WT and Bhlhe40 -/-mice at day 14 post C. muridarum intravaginal infection.As expected, a strong Th1 response was observed in WT mice, demonstrated by robust IFN-γ production by activated CD4 T cells (CD44 hi ) in the spleen, draining iliac lymph nodes (DLNs) and FRT (Fig 2A).Although similar numbers of total and activated CD44 hi CD4 T cells were present in WT and Bhlhe40 -/-mice (not depicted), lower frequencies and total numbers of IFN-γ-producing cells were found in the Bhlhe40 -/-FRT (Fig 2B).Moreover, less IFN-γ was produced on a per cell basis in IFN-γ + CD4 T cells in Bhlhe40 -/-mice compared to WT (Fig 2C).Consistent with previous reports, we observed an increase in the frequencies of IL-10-producing cells in Bhlhe40-deficient mice, and IFN-γ and IL-10 double-producing CD4 T cells were also more abundant in the spleen, DLNs, and FRT of Bhlhe40 -/-mice (Fig 2B).It was evident that the increased IFN-γ + IL-10 + CD4 T cells in Bhlhe40 -/-mice were T regulatory 1 (Tr1) cells rather than regulatory T cells (Tregs), as these cells do not co-express the transcription factor Foxp3 (S2 Fig) .These findings confirmed that Bhlhe40 promotes the production of pro-inflammatory Th1 cells while inhibiting the differentiation of immunosuppressive Tr1 cells in the FRT.

Bhlhe40 suppresses antigen-specific Tr1 cell differentiation in a CD4 T cell intrinsic manner
We next sought to determine whether the skew towards anti-inflammatory Tr1 response in Bhlhe40 -/-mice was due to cell-intrinsic regulation of CD4 T cell differentiation by Bhlhe40.
To do this, we co-transferred WT (CD45.1 + ) and Bhlhe40-deficient (CD45.2+ ) naïve CD4 T cells into TCRβ -/-mice at a 1:1 ratio and infected the recipients intravaginally with C. muridarum (Fig 2D).At 14 dpi, higher percentages of Bhlhe40 -/-CD4 T cells were observed in the host DLNs (normalized ratio of Bhlhe40 -/-: WT = 1.62 ± 0.26) (Fig 2E), possibly due to enhanced T follicular helper cell proliferation within the Bhlhe40-deficient CD4 T cell compartment as reported previously [19].In contrast, comparable numbers of WT and Bhlhe40 -/- CD4 T cells were found in the FRT, indicating that Bhlhe40 -/-CD4 T cells had no defect in tissue homing or accumulation in this mucosal site.When quantifying cytokine producing cells within each donor compartment, we found that the frequencies of IFN-γ + cells were slightly lower in the Bhlhe40 -/-T cell compartment in the FRT, and IFN-γ production on a per cell basis was significantly lower in Bhlhe40-deficient CD4 T cells in both DLNs and FRT compared to WT (Fig 2F -2H).Consistent with our findings in Bhlhe40 -/-mice, the frequencies of IL-10 + CD4 T cells and IFN-γ + IL-10 + Tr1 cells were significantly higher in the Bhlhe40 -/-CD4 T cell compartment in the DLNs and FRT (Fig 2G).These results demonstrated that although Bhlhe40 is widely expressed in many leukocyte lineages, it suppresses Tr1 differentiation in a CD4 T cell-intrinsic manner independent of other cell types or environment cytokine milieu.
Chlamydia infection in the FRT drives a potent CD4 T cell expansion in the secondary lymphoid organs and recruits both antigen-specific and bystander CD4 T cells to the site of infection [20,21].These two populations contribute differently to host protective immunity and development of immunopathology [22,23].To interrogate the role of Bhlhe40 in antigen-specific CD4 T cells, we crossed the Chlamydia-specific TCR transgenic mice (TP1) onto the Bhlhe40-deficient background and conducted co-transfer experiments with WT (CD45.2+ CD90.1 + ) and Bhlhe40-deficient (CD45.2+ CD90.2 + ) TP1 cells (Fig 2I) [24].Using CD45.1 + B6 mice as recipients, we were able to detect WT TP1, Bhlhe40 -/-TP1 and endogenous CD4 T cells within the same host (Fig 2J).At 14 dpi, WT TP1 cells were predominantly IFN-γ + cells with minimum IL-10 + CD4 T cells or IFN-γ + IL-10 + Tr1 cells detected (Fig 2J and  2K).Endogenous CD4 T cells contained comparable frequencies of IFN-γ + cells to WT TP1, with a small but evident Tr1 cell population also detected in this population.Notably, Bhlhe40 -/-TP1 cells contained the highest percentages of Tr1 cells with lower frequencies of IFN-γ single-producers compared to WT TP1 (Fig 2J and 2K).Thus, we conclude that Bhlhe40 is essential for driving Tr1 differentiation in antigen-specific CD4 T cells during Chlamydia FRT infection.

Increased IL-10-producing Tr1 cells dampen protective CD4 T cell responses in Bhlhe40 -/-mice
We next aimed to test whether elevated IL-10 production by increased immunosuppressive Tr1 cells in Bhlhe40 -/-mice accounts for the defects in bacterial control in these mice.WT and Bhlhe40 -/-mice were treated with either anti-IL-10R or isotype control antibody starting 1 day prior to C. muridarum intravaginal infection.As shown in Fig 3 , IL-10R blockade significantly reduced bacterial shedding in both WT and Bhlhe40 -/-mice compared to isotype-treated groups, confirming the immunosuppressive function of IL-10 in both mouse models.Moreover, anti-IL-10R treated WT mice mirrored the phenotype of Il10 -/-mice, indicating that antibody-mediated IL-10R blockade has similar biological effects as IL-10 cytokine knockout in this infection model.Importantly, whereas anti-IL-10R treatment in Bhlhe40 -/-mice reduced bacterial burdens to comparable levels as WT isotype-treated group, it did not attain the accelerated bacterial control as in WT anti-IL-10R group or Il10 -/-mice.These results indicated that in addition to restricting Tr1 differentiation and IL-10 production, Bhlhe40 modulates other CD4 T cell effector functions to facilitate Chlamydia clearance from the FRT.experiments with 6 samples per group.Each data point represents a pooled FRT sample from 4 mice.Error bars represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant by unpaired t test in (B-C), and paired t test in (E-K).https://doi.org/10.1371/journal.ppat.1011983.g002

Bhlhe40 promotes the differentiation of protective polyfunctional CD4 T cells
To expand our investigation of how Bhlhe40 regulates CD4 T cell effector functions, we performed a multiplex cytokine array of ex vivo cultures of DLNs and FRT cells isolated from C. muridarum infected WT and Bhlhe40 -/-mice.At 10 dpi, major Th1 cytokines, including IFN-γ and TNF-α were significantly lower in the Bhlhe40 -/-FRT compared to WT; a trend of decrease in IL-2 was also observed, but not statistically significant (Fig 4A and S1 Table ).Moreover, IL-17A and GM-CSF, signature cytokines produced by Th17 and ThGM cells, respectively, were also drastically reduced in the absence of Bhlhe40.These observations were reinforced by intracellular cytokine staining in which lower percentages of IL-17A + and GM-CSF + CD4 T cells were detected in Bhlhe40 -/-mice (Fig 4B), suggesting that Bhlhe40 regulates a broader range of CD4 T cell effector functions beyond Th1 and Tr1 differentiation.It is worth noting that although IL-10-producing Tr1 cells were increased in Bhlhe40 -/-mice, we did not detect a difference in IL-10 levels in this ex vivo cell culture setting (Fig 4A ), one possibility being reduced fitness and survival of Bhlhe40 -/-T cells in tissue culture [14,16].We next sought to quantify CD4 T cells capable of producing more than one pro-inflammatory cytokine.SPICE analysis showed that WT mice contained higher proportions of CD4 T cells capable of producing 2 or 3 cytokines than Bhlhe40 -/-mice (Fig 4C).Within WT polyfunctional CD4 T cells, IFN-γ + GM-CSF + cells were the most abundant, followed by IFN-γ + IL-17A + and IFN-γ + IL-17A + GM-CSF + cells (Fig 4D).To ask whether this phenotype recapitulates in Chlamydia-specific CD4 T cells, we utilized the TP1 mixed transfer strategy described above (Fig 2I ) and measured cytokine productions from both TCR Tg and endogenous CD4 T cells.Interestingly, unlike polyclonal CD4 T cells in Bhlhe40 -/-mice, Bhlhe40 -/-TP1 cells contained higher frequencies of CD4 T cells capable of producing IL-17A, as percentages of both IL-17A + and IFN-γ + IL-17A + cells were considerably higher in Bhlhe40 -/-TP1 compared to WT TP1 or endogenous CD4 T cells (Fig 4E and 4F).In contrast, lower frequencies of IFNγ + GM-CSF + cells were detected in Bhlhe40 -/-TP1 cells.The overall CD4 T cell polyfunctionality was comparable between WT and Bhlhe40 -/-TP1 cells (not depicted).Together, these data  suggest that Bhlhe40 positively regulates T cell GM-CSF production, and potentiates polyfunctional CD4 T cell differentiation via a combined T cell-intrinsic and -extrinsic mechanism.
It is possible that the discrepant T cell cytokine profiles in Bhlhe40-deficient mice and Bhlhe40 -/-TP1 cells were due to the immunosuppressive environment created by increased Tr1 cell and IL-10 production in Bhlhe40 -/-mice.To answer whether the Tr1/IL-10 axis curtails T cell polyfunctionality, we crossed Bhlhe40 -/-mice to Il10 -/-mice and infected these double knockout mice with C. muridarum.As expected, Bhlhe40 -/-x Il10 -/-and Il10 -/-mice had higher percentages of polyfunctional CD4 T cells compare to Bhlhe40 -/-and WT mice, respectively (Fig 4B and 4C).Importantly, although IL-10 deficiency led to an increase in IL-17Aproducing polyfunctional CD4 T cells in both WT and Bhlhe40 -/-mice, it did not impose a detectable effect on IFN-γ, IL-17A, or GM-CSF single-producing T cells, and had a marginal effect on polyfunctional CD4 T cells that produce GM-CSF.These results suggest that the Tr1/ IL-10 axis partially accounts for the limited polyfunctional CD4 T cell differentiation in Bhlhe40 -/-mice.
Polyfunctional CD4 and CD8 T cells are associated with enhanced protective immunity during infections and vaccinations [25][26][27].We therefore interrogated the relationship between CD4 T cell polyfunctionality and protective immunity against Chlamydia.As shown in Fig 4G , CD4 T cell polyfunctionality index was lowest in Bhlhe40 -/-mice and highest in Il10 -/-mice, and these indices reversely correlated with bacterial shedding over the course of C. muridarum primary infection [28].Thus, we conclude that CD4 T cell polyfunctionality is positively associated with protective immunity against Chlamydia.

Single-cell RNA sequencing reveal an increase in CD4 T cell stemness in Bhlhe40 -/-mice
To further expand our understanding of how Bhlhe40 regulates anti-Chlamydia immunity, we performed 5' single-cell RNA sequencing (scRNAseq) and TCR profiling on activated CD4 T cells (CD44 hi ) sorted from WT and Bhlhe40 -/-mouse FRT 14 days post intravaginal infection.Unsupervised clustering revealed 14 clusters from a combined population of 5,782 WT and 3,732 Bhlhe40 -/-CD4 T cells (Fig 5A).Following cell number normalization, we found that WT CD4 T cells were more enriched in clusters 0, 4, and 8 whereas Bhlhe40 -/- CD4 T cells were dominant in clusters 3, 6 and 10 (>80% of total, Fig 5B).As expected, CD4 T cells in the largest WT cluster 0 expressed high levels of Bhlhe40, along with a panel of genes downstream of TCR signaling including Ifitm1, Ifitm2, and Nr4a1 (Fig 5C) [29,30].In contrast, the predominant Bhlhe40 -/-CD4 T cells clusters 3 expressed genes that resembled T cell stemness, such as Tcf7, Ccr7, Slamf6 and Sell [31,32].Gene expression pattern in cluster 10 closely resembled that of cluster 3, with significantly more ribosomal genes upregulated in this cluster (e.g.Rps 29, Rpl12), indicating increased protein translation.Analysis of T helper lineage specific genes revealed that majority of the clusters expressed the Th1-specific Tbx21 and Ifng genes, with the lowest expression detected in Bhlhe40 -/-dominant clusters 3 and 10 (Fig 5D).On top of the strong type 1 response, Th17-specific transcripts Rorc and Il17a were detected at low levels in the WT-dominant cluster 4, and this cluster also uniquely expressed Th17-related genes, including Tmem176b, Ltb4r1, Il17re, and Ramp1 [33][34][35], revealing its identity as polyfunctional Th1/Th17 cells.Additionally, WT cluster 8 expressed a panel of genes associated with cytotoxicity and terminal differentiation, such as Gzma, Klrg1, Cx3cr1 and Zeb2 [36], indicating their cellular identify of cytotoxic CD4 T helpers.Lastly, Bhlhe40 -/-cluster 6 presented a terminal-differentiated Tr1 phenotype by expressing Lag3, Maf, Il10ra and Havcr2, supporting our previous observations that Tr1 cells were increased in these mice [37,38].To delineate the cellular differentiation trajectory within these T cell clusters, we conducted pseudotime trajectory analysis using the stem-like cluster 3 T cells as origin.Most of Bhlhe40 -/- T cells scored low on the differentiation trajectory, including the Tr1 cluster 6 despite the expression of several exhaustion markers by these T cells (Fig 6A).In contrast, WT enriched clusters 0, 4 and 8 were among the clusters with highest differentiation scores.Clusters with intermediate differentiation scores, including clusters 2 (migratory T) and 7 (ISG-enriched T), were also over-represented by WT CD4 T cells (Figs 6A and 5B).To determine whether T cell clonal expansion correlates with differentiation, we analyzed TCR clonotypes in WT and Bhlhe40 -/-T cells and projected the top 25% abundant clones ("expanders") onto the T cell clusters.Similar profiles of clonal expansion were detected in WT and Bhlhe40 -/-CD4 T cells, with the top 25% cell population falling between clonal abundance of 3 to 164 (Fig 6B).The most differentiated clusters 0 and 8 contained highly expanded CD4 T cells, indicating that these T cells were the most dynamic populations during the immune response.Curiously, although cluster 6 (Tr1) had a less differentiated profile on the trajectory, it contained high frequencies of "expanders" in both WT and Bhlhe40 -/-T cells.Altogether, these data demonstrated that Bhlhe40 is essential for driving CD4 T cell differentiation from stem-like CD4 T cell progenitors into multi-functional effectors.

Discussion
The prevailing paradigm in protective adaptive immunity to the intracellular bacterium Chlamydia is centered on IFN-γ production by CD4 Th1 cells.Nevertheless, mice lacking either Th1-specific transcription factor T-bet or IFN-γ-producing CD4 T cells were both capable of restricting Chlamydia replication in the female reproductive tract [7,8].Moreover, increasing evidence suggest that multiple Th lineages and T cell effector functions, including Th2, Th17, IL-13 production and CD4 T cell-dependent cytotoxicity, may participate in host defense against Chlamydia [20,[39][40][41][42]. Additionally, our evolving understanding of CD4 T cell heterogeneity over recent years indicate that the functional state of CD4 T cells is likely a continuum shaped by the microbes and the tissue microenvironment rather than a defined Th subtype [43,44].In this current study, we provide new evidence supporting this scenario by showing that protective immunity to Chlamydia relies on highly differentiated polyfunctional CD4 T cells driven by the transcription factor Bhlhe40 rather than a single Th subset or cytokine in the FRT mucosa.
Although Bhlhe40 is widely expressed in many cell types and tissues, our data demonstrated that T cell-intrinsic expression of Bhlhe40 is essential for Chlamydia control in the FRT, as Bhlhe40 -/-and Bhlhe40 fl/fl -Cd4-Cre mice exhibit almost identical kinetics in bacterial clearance.Previous studies have shown that Bhlhe40 deficiency resulted in reduced IL-2 production by T cells, and consequently decreased T cell proliferation and survival in an autocrine manner [14].Nevertheless, we did not observe a reduction in either total or activated CD4 T cell numbers in the Bhlhe40 -/-FRT, albeit a trend of decrease in IL-2 production was observed in in vitro culture assay.CD4 T cell accumulation in the FRT was also unaffected by Bhlhe40 deficiency in a competitive setting where WT and Bhlhe40 -/-CD4 T cells were co-transferred into T cell deficient TCRβ -/-mice.These results collectively suggest that T cell effector functions, rather than T cell homing or accumulation at the FRT mucosa, accounts for the defects in bacterial control in the Bhlhe40-deficient mice during Chlamydia infection.
Previous studies have established a role for Bhlhe40 in host resistance to several intracellular pathogens including M. tuberculosis, T. gondii, and influenza [15][16][17].Our results add to that by showing the importance for Bhlhe40 for anti-Chlamydia immunity at the FRT mucosa.The defects in pathogen control in Bhlhe40 -/-mice were caused, at least in part, by increased antiinflammatory IL-10 production from CD4 + Tr1 cells, as the phenotype can be partially reversed by either antibody-mediated IL-10R blockade or genetic ablation of IL-10.Curiously, although IL-10-producing cells were increased in Bhlhe40 -/-mice, we did not observe any difference in long-term pathological outcomes between WT and Bhlhe40 -/-FRT, indicating that the IL-10-producing Tr1 cells may not directly contribute to the dampening of tissue pathology.Given that long-term FRT pathology reflects an accumulated effect, it is also possible that IL-10-mediated anti-inflammatory responses from both Tr1 cells other cell types were counterbalanced by increased chronic inflammation caused by longer durations of bacterial shedding in Bhlhe40 -/-mice.The direct contribution of cytokine-producing CD4 T cells to tissue pathology deserves further investigation.
In both lung and FRT Chlamydia infection models, IL-10-deficiency leads to accelerated bacterial clearance [45,46].Our study recapitulates these findings and further demonstrates that IL-10 functions as a roadblock in polyfunctional CD4 T cell differentiation, as IL-10 ablation in both WT and Bhlhe40 -/-mice increases T cell polyfunctionality index.The importance of multifunctional Th1 cells in protective immunity to Chlamydia has been demonstrated

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previously.Intranasal immunization with live Chlamydia elementary body (EB) renders better protection against Chlamydia FRT challenge than immunization with dead EB, and the IFNγ + TNF-α + IL-2 + triple-and IFN-γ + TNF-α + double-positive CD4 T cell profiles correlate with protection [47].Using TCR transgenic mice, Poston et al. showed that Chlamydia-specific CD4 T cells preferentially adopt a polyfunctional Th1 phenotype and confer better protection at low cell numbers compared to polyclonal T cells [24].Here we expand the scope beyond the Th1 spectrum and illustrate that the frequency of polyfunctional CD4 T cells producing multiple proinflammatory cytokines, including IFN-γ, IL-17A and GM-CSF, correlates with better protective immunity to Chlamydia.We propose two potential interpretations of these observations: First, synergistic effects of these cytokines may be essential for protection.It has been known for some time that mice deficient in a single cytokine, such as IFN-γ or IL-17A had only minor defects in Chlamydia control in the FRT mucosa [3,39,48,49].The observations that Bhlhe40 -/-mice had comparable numbers of IFN-γ single-producing CD4 T cells to WT, but failed to control Chlamydia effectively further reinforce the notion that IFN-γ alone does not account in full for anti-Chlamydia immunity in the FRT.Csf2 -/-mice are susceptible to several intracellular bacterial and viral infections in the lung [50][51][52], but a role for GM-CSF in CD4 T cell-mediated immunity to Chlamydia is yet to be established.Previous studies have identified Chlamydia-specific IFN-γ + IL-17A + CD4 T cells in the FRT following Chlamydia infection or vaccination [20,53].It is somewhat surprising that we detected IFN-γ + GM-CSF + cells as the most abundant polyfunctional CD4 T cells in the FRT.Given that IFN-γ-producing Th1 cells can gain GM-CSF signature, IL-17A + CD4 T cells can differentiate into IFNγ + GM-CSF + pathologic Th17 cells upon IL-23 stimulation, and a separate ThGM subset has also been proposed, the origins of IFN-γ + GM-CSF + double-and IFN-γ + IL-17A + GM-CSF + triple-producing cells in Chlamydia infection remain unknown [54][55][56].Future studies will be needed to identify the cell fate and determine the effector functions of these populations.It is worth noting that in our model both IFN-γ + IL-10 + Tr1 and GM-CSF-producing CD4 T cells are regulated by Bhlhe40 in a CD4 T cell-intrinsic manner, likely at the transcription level as reported before.In contrast, the reduction of IL-17A + and IFN-γ + IL-17A + CD4 T cells in Bhlhe40 -/-mice is attributed to a T cell-extrinsic factor, as the defects were not observed when Bhlhe40-deficient TP1 cells were primed in the WT B6 host (Fig 4E).It would be of interest to identify the contributing cell types and/or the environmental milieu that block the differentiation of IL-17A-producing CD4 T cells in Bhlhe40 -/-mice.A second and perhaps more appealing interpretation would be that the polyfunctionality of CD4 T cells co-producing 2 or 3 cytokines simply reflects a state of differentiation required for protective immunity.This argument is supported by our scRNAseq data that Bhlhe40 -/-CD4 T cells are enriched in clusters expressing more "stem-like" T cell markers, whereas WT CD4 T cells are further down on the differentiation trajectory with diverse effector functions, along with a more robust clonal expansion profile.In line with our findings, recent work from Wherry and colleagues reported that during chronic LCMV infection, Bhlhe40 drives exhausted CD8 T cells (T ex ) from stemlike T ex into terminal differentiated T ex , demonstrating its role in regulating CD8 T cell differentiation [57].Although it is difficult to precisely project each polyfunctional CD4 T cell population identified by flow cytometry to specific T cell clusters in scRNAseq due to the low transcript numbers of Il17a and Csf2, and potential discrepancy between cytokine transcription and translation, the fact that all WT clusters contain Ifng transcripts while co-expressing Th17 or cytotoxic T cell signature genes reveals polyfunctionality of these cells.The cytotoxic CD4 T cell cluster is of particular interest, as previous studies by Johnson and colleagues have showed that CD4 T cell clones had direct killing capability of infected epithelium cells in vitro [58].The emerging roles of cytotoxic CD4 T cells have been demonstrated in a variety of infection models, autoimmune diseases and cancer [59,60].Experiments are currently underway to evaluate whether Chlamydia infection induces cytotoxic function in the polyfunctional CD4 T cells in vivo and whether these cells contribute to protective immunity.
In summary, our study provides compelling evidence that a multifaceted CD4 T cell response in Chlamydia infection is essential for protective immunity.Moreover, we identify the transcription factor Bhlhe40 as a novel regulator of CD4 T cell differentiation in the FRT.Understanding the protective features and identifying the key regulators of CD4 T cell responses provide important guidelines for future Chlamydia vaccine design.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The University of Arkansas for Medical Sciences is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).All animal experiments were approved by University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee (IACUC) (Animal Use Protocol number 4108).

Chlamydia strain, mouse infection and bacteria enumeration
Chlamydia muridarum strain Nigg II was originally purchased from ATCC (VR-123; Manassas, VA). C. muridarum was propagated in McCoy cells, elementary bodies (EBs) purified by discontinuous density gradient centrifugation and titrated on HeLa 229 cells as previously described [20].Mice were synchronized for estrous by subcutaneous injection of 2.5 mg medroxyprogesterone (Depo-Provera, Greenstone, NJ) 5-7 days prior to intravaginal infection.For intravaginal (i.vag.)infection, 1x10 5 C. muridarum in SPG buffer was deposited directly into the vaginal vault using a pipet tip.To enumerate bacterial shedding from the lower FRT, vaginal swabs were collected, suspended in SPG buffer, and disrupted with glass beads.Inclusion forming units (IFUs) were determined by plating serial dilutions of swab samples on HeLa 229 cells, staining with anti-MOMP mAb (clone Mo33b), and counting under a microscope.

Histopathology
Female reproductive tracts from WT and Bhlhe40 -/-mice were harvested between 140-150 days post infection and fixed in 4% paraformaldehyde overnight.The tissues were then embedded in paraffin blocks, longitudinal sections prepared and stained with hematoxylin and eosin.The sections were scored using a previously elaborated semiquantitative scoring system by a board-certified pathologist who was blind to the experimental design [62,63].The following parameters were evaluated on a scale of 0 (normal) to 4 (severe lesion): acute inflammation, chronic inflammation, plasma cells, epithelial erosion, and fibrosis.Left and right sides of the uterine horns and oviducts were evaluated individually.

Chlamydia-specific serum Ab ELISA
Mice were bled via the submandibular vein at 21 days post infection to isolate serum.Heatkilled C. muridarum elementary bodies (HKEBs) were prepared by heating EBs at 56˚C for 30 min.High protein binding ELISA plates (Costar) were coated with 1×10 6 HKEBs and blocked before serial dilutions of serum samples were added to the plates.Chlamydia-specific Abs were detected using HRP-based SBA Clonotyping System (Southern Biotech).

Multiplex cytokine/chemokine array
At 10 days post C. muridarum intravaginal infection, five million cells from DLNs and total cells from the FRT were cultured ex vivo with 1×10 6 /mL HKEBs for 72 hours.Culture supernatants were collected and analyzed using the Mouse Cytokine/Chemokine 31-Plex Discovery Assay Array (Eve Technologies, Canada).

T cell adoptive transfer
CD4 T cells were isolated from the spleens of donor mice and purified using the mouse CD4 T Cell Isolation Kit (Miltenyi Biotec).The purity and cell count of donor CD4 T cells were assessed using flow cytometry.For experiments using WT and Bhlhe40 -/-mice as donors, 3×10 6 CD4 T cells from each donor were mixed at a 1:1 ratio and transferred intravenously into TCRβ -/-recipient mice.For TP1 adoptive transfer, 5×10 4 WT TP1 cells (CD90.1+) and 5-7.5×10 4 Bhlhe40 -/-TP1 cells (CD90.2+)were isolated, mixed and transferred intravenously into CD45.1 recipient mice.All recipient mice were challenged intravaginally with C. muridarum one day after adoptive transfer.

Single-cell RNA sequencing and TCR profiling
FRT cells from WT and Bhlhe40 -/-mice were isolated 14 days post C. muridarum intravaginal infection and stained with surface markers.Live CD11b -F4/80 -B220 -CD90.2+ CD4 + CD44 hi CD4 T cells were sorted using FACSAria III (BD Biosciences), partitioned using a Chromium Controller (10x Genomics), and libraries prepared using Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (10x Genomics).GEX and VDJ libraries were sequenced on Illumina NovaSeq 6000 at the sequencing depths of >25,000 reads/cell and >5,000 reads/cell, respectively.Data analysis was conducted using Seurat v.4.3 [65] and Monocle 3 [66] under R v4.3.1 environment.Contaminating non-CD4 T cells were cleaned up by filtering out cells without VDJ tags and/or with the following features: Cd8a, Ncr1.To avoid clustering based on T cell clonotypes, "Trav" and "Trbv" were removed from features when conducting unsupervised clustering using UMAP.Data generated from this study are available through the NCBI Gene Expression Omnibus (GEO) under accession number GSE253394.

Statistical analysis
Statistical analysis was performed using Prism 10 (GraphPad Software).An unpaired t test or a paired t test was used for normally distributed continuous-variable comparisons.A Mann-Whitney U test was used for nonparametric comparisons.For comparison of multiple groups, the ANOVA tests were used followed by multiple comparisons of means.A p value < 0.05 was considered statistically significant.

Fig 5 .
Fig 5. Single-cell RNA sequencing detects stem-like CD4 T cells in Bhlhe40 -/-FRT.WT and Bhlhe40 -/-mice were infected intravaginally with 1×10 5 C. muridarum.Activated CD4 T cells (live CD90.2 + CD4 + CD44 hi ) in the FRT were sorted for scRNAseq using 10x genomics 5' GEX.(A) UMAP depicting 14 clusters of CD4 T cells from merged WT and Bhlhe40 -/-CD4 T cells (left); deconvolution of the composite UMAP into WT and Bhlhe40 -/-components.(B) Percentages of WT and Bhlhe40 -/-CD4 T cells in each cluster following normalization of the two populations into the same cell numbers.(C) Heatmap showing top 30 most upregulated genes in each cluster, and representative genes specifically expressed within each of the top 3 clusters in WT and Bhlhe40 -/-CD4 T cells are shown on the sides with violin plots depicting expression levels in each cluster.(D) Dot plots depicting Th lineage-specific cytokines and transcription factors.https://doi.org/10.1371/journal.ppat.1011983.g005