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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: J Immunol. 2014 Jan 31;192(5):2156–2166. doi: 10.4049/jimmunol.1300675

Type I Interferon induces binding of STAT1 to Bcl6: Divergent Roles of STAT-family transcription factors in the TFH cell genetic program2

Shingo Nakayamada *,1,3, Amanda C Poholek *,1, Kristina T Lu #, Hayato Takahashi *,4, Masanari Kato *, Shigeru Iwata *, Kiyoshi Hirahara *,5, Jennifer L Cannons #, Pamela L Schwartzberg #, Golnaz Vahedi *, Hong-wei Sun †, Yuka Kanno *, John J O’Shea *
PMCID: PMC3967131  NIHMSID: NIHMS554118  PMID: 24489092

Abstract

CD4+ T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. The factors that contribute to the differentiation of this helper cell subset are incompletely understood, although several cytokines including IL-6, IL-21 and IL-12 can promote TFH cell formation. Yet, none of these factors, nor their downstream cognate STATs, have emerged as non-redundant, essential drivers of TFH cells. This suggests a model in which multiple factors can contribute to the phenotypic characteristics of TFH cells. As type I interferons (IFNs) are often generated in immune responses, we set out to investigate if these factors are relevant to TFH cell differentiation. Type I IFNs promote Th1 responses, thus one possibility was these factors antagonized TFH-expressed genes. However, we show that type I IFNs (IFN-α/β) induced Bcl6 expression, the master regulator transcription factor for TFH cells, and CXCR5 and PD-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by STAT1, which bound to the Bcl6, Cxcr5 and Pdcd1 loci. These data suggest that type I IFNs (IFN-α/β) and STAT1 can contribute to some features of TFH cells but are inadequate in inducing complete programming of this subset.

INTRODUCTION

In response to microbial pathogens, CD4+ T cells have the capacity to differentiate into multiple, distinct effector subsets, each with a specific and unique role in the adaptive immune response. One of the oldest described functions of CD4+ T cells is to mediate help to B cells and influence the antibody response to infection or immunization (1–3). More recently this activity has been attributed to a defined subset of cells termed T follicular helper cells (TFH), whose primary task is to drive the formation of B cell responses and provide helper function (4). TFH cells are commonly identified by high surface expression of the chemokine receptor, CXCR5, and the inhibitory receptor programmed cell death 1 (PD-1)(5–8). CXCR5 expression allows TFH cells to migrate from the T cell zone to the B cell follicle where they localize to the germinal center (GC), and mediate B cell help via cell-cell contact using the co-stimulatory molecules CD40-Ligand and ICOS (5, 9), and secretion of the cytokines IL-21 and IL-4 (10–15) (16). In addition, the signaling lymphocytic activation molecule– associated protein (SAP) is critical for T cell–B cell interaction (17–19). GCs are the site of high-affinity antigen specific antibody production, memory B cell formation, and long-lived plasma cell differentiation. Deficiencies in TFH cell function in the absence of ICOS or SAP, or the absence of CD40-Ligand, or double deficiency in IL-21 and IL-4, all result in severely diminished or absent B cell responses including reduced total antigen specific antibody and skewed isotype responses (20–27).

The master regulator transcription factor required for TFH cell formation is the transcriptional repressor B cell lymphoma 6 (Bcl6)(28–30). In the absence of Bcl6, TFH cells are unable to form, and subsequently GCs are not present (28). Like other master regulators, overexpression of Bcl6 not only enforces TFH cell differentiation but also attenuates differentiation to other fates by repressing the expression of master transcription factors for other CD4+ T cell subsets, including T-bet, GATA3, and Rorγ-t (29, 31).

CD4+ T cell subset differentiation is mediated in large part by exposure to various cytokines. For example, Th1 cells develop in the presence of IL-12 and IFN-γ, whereas Th2 cells form after exposure to IL-4 (1). For TFH cells, several cytokines have been reported to effect differentiation. In vitro, exposure of CD4+ T cells to the cytokines IL-6 and IL-21 drives a TFH-like phenotype (30, 32–35), but in vivo, deletion or neutralization of IL-6 and IL-21 does not completely ablate TFH cell generation and GC formation (36–41). This suggests that although these factors may be sufficient to achieve differentiation, they are not necessary, implying that other cytokines can contribute to TFH cell differentiation.

Many cytokines that drive helper T cell specification bind Type I/II cytokine receptors, which activate Janus kinases and STAT family transcription factors to translate the cytokine signal into specific programs of gene transcription that mediate effector differentiation (42, 43). Both IL-6 and IL-21 act through STAT3 (44, 45) and in circumstances where STAT3 signaling is impaired, TFH cell formation is reduced (34, 46). Although in vivo STAT3 is not absolutely critical for TFH cell formation, STAT3 clearly plays a positive role in promoting the TFH cell program. These data add to the argument that signals other than IL-6, IL-21 and STAT3 can contribute to TFH cell induction.

Human CD4+ T cells exposed to IL-12 acquire an increased capacity to help B cells in vitro and express many TFH cell signature genes (47). Studies have differed looking at patients with IL-12Rβ1 mutations. While one study reported reduced circulating memory TFH cell numbers, another found normal numbers (46, 48). Regardless, naïve T cells from these patients are impaired in their ability to develop into functional TFH cells after exposure to IL-12 (46). In addition, murine CD4+ T cells cultured with IL-12 acquire TFH cell characteristics early in a STAT4-dependent manner, yet consistent exposure to IL-12 increases expression of T-bet and promotes Th1 cell differentiation (49). Like STAT3 or IL-6 and IL-21 deficiency, the absence of IL-12 and STAT4 in murine models has only a modest effect on TFH cell development in vivo (49). These data further support the contention that there is redundancy in the cytokines and STATs that control TFH cell formation and further argue for the role of additional factors in TFH cell differentiation.

While it appears that multiple cytokines can promote TFH differentiation, IL-2 interferes with TFH cell formation (50–53). Two non-mutually exclusive mechanisms have been proposed: in the first, IL-2 acting via STAT5 induces the transcriptional repressor Blimp-1, which serves to repress Bcl6 and TFH cell formation (51, 52). Additionally, active STAT5 can displace STAT3 binding from the Bcl6 promoter (50). Thus, many cytokines can influence TFH cell development, suggesting in vivo that the balance of signals a CD4+ T cell receives during differentiation plays a critical role in driving this effector program.

Type I interferons (IFNs, IFN-α/β) are ubiquitous cytokines produced by innate immune cells early in infection (54). These critical antiviral cytokines also have key immunoregulatory roles. During chronic viral infection, IFNs have paradoxical roles, both promoting control of viral replication and mediating immunosuppresive pathways that limit viral control (55–57). In T cells, IFN-α/β activates STAT1 and to a lesser extent STAT4, inducing T-bet and IFN-γ (58–60). In vivo, IFN-α/β contributes to T cell survival and clonal expansion during viral infection, and it can support Th1 differentiation by synergizing with IL-12 and IFN-γ (61, 62). Yet when IFN-α/β is compared to IL-12, the classic cytokine for Th1 differentiation, it is not sufficient to induce full Th1 cell development (63). In addition to its effects on Th1 cells, an indirect role for type I interferon in TFH cell differentiation has been demonstrated in vivo. Reduced IL-6 levels in the absence of type I IFN signaling in dendritic cells (DCs) resulted in impaired TFH cell formation (64). In addition, interferon signaling through DCs promotes both primary and long-lived antibody responses (65). These findings suggested the importance of type I interferons in humoral immune responses. However, the direct effects of type I IFNs in TFH cell differentiation have not been carefully explored; given its ability to induce T-bet, it might be anticipated that it would antagonize expression of TFH-associated genes.

In this study, we set out to examine the role of type I IFNs on TFH cell differentiation. We found that type I IFNs induced Bcl6 and promoted CXCR5 and PD-1 expression, but unlike IL-6 and IL-12, IFN-α/β did not induce IL-21 secretion. In conjunction with IL-6, however, IFN-α/β enhanced IL-21 production. The ability of IFN-α/β to drive these TFH cell features was entirely STAT1-dependent and STAT1 bound directly to the Bcl6 locus. This suggests type I IFNs and STAT1 promote some, but not all aspects of TFH cell development. Together, these findings help explain some of the apparent redundancy in the requirements for factors that induce TFH cell responses.

MATERIALS AND METHODS

Mice, Cell Isolation, and Cell Culture

C57BL/6J were purchased from the Jackson Laboratory (Bar Harbour, ME, USA). Stat4−/− mice were provided by Dr. Mark Kaplan (Indiana University). Stat1−/− (66) and Stat3−/− (Cd4 Cre; Stat3fl/fl) mice were generated as previously described and provided by D.E. Levy (New York University) (67). All mice were handled in accordance with the guidelines of the NIH Animal Care and Use Committee. Splenic and lymph node T cells were obtained by disrupting organs of 8 to 12 week-old mice. All cell cultures were performed in RPMI supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 10 mM Hepes, 1 mM sodium pyruvate, and 2.5 µM β-mercaptoethanol. T cells were enriched with a CD4+ T cell kit and AutoMacs isolator (Miltenyi Biotec). Naïve CD4+ T cells were stained with antibodies to CD4, CD62L, CD44, and CD25 (BDBiosciences and ebioscience), and isolated by flow cytometry cell sorting using a BDBiosciences FACSAria IIIu. Isolated cells were cultured with plate-bound anti-CD3 and anti-CD28 (10 µg/mL each) under neutral (no cytokines), IL-6 (20 ng/mL), IL-12 (10 ng/mL), IFN-α (5000 U/mL), or IFN-β (5000 U/mL) conditions for 3 days and then cultured further in IL-2 (50 U/mL) alone (neutral condition) or in combination with IL-6, IL-12, IFN-α, or IFN-β and grown an additional 2 days. For T-B co-culture, naïve B cells were sorted and cultured with T cells that had been cultured for 5 days as above at a 1:3 T:B ratio without stimulation or on plates coated with anti-CD3 and anti-CD28 (stimulated) for 4 days. Cytokines were from R&D Systems. Anti-CD3 and anti-CD28 were from eBioscience.

Intracellular Staining and Flow Cytometry

For intracellular cytokine staining, cells were restimulated for 2 h with 50 ng/ml PMA and 1 µg/ml ionomycin with the addition of brefeldin A (GolgiPlug; BD), then fixed and permeabilized with Cytofix/Cytoperm solution (BD). Intracellular IL-21 staining was performed using soluble IL-21R/Fc (R&D Systems), followed by PE-labeled affinity-purified F(ab’)2 fragment of goat anti-human Fcγ (Jackson ImmunoResearch Laboratories) (68), then further stained with PerCP-Cy5.5 anti-CD4 and APC anti-IFN-γ (BD). The following antibodies were used for cell surface or intracellular staining: PE anti-PD-1, PE anti-ICOS, PerCP-Cy5.5 anti-CD4, PE anti-Bcl6, and Alexa Fluor-647 anti-T-bet (eBioscience). CXCR5 staining was done using biotinylated anti-CXCR5 for 1 h, followed by APC-labeled streptavidin (BD). For phosphorylated STAT staining (p-STAT), freshly isolated naïve T cells were incubated for 30 minutes alone (no stim) or with IL-6 (20 ng/mL), IFN-α (5000 U/mL), or IFN-β (5000 U/mL). Cells were fixed in Lyse/Fix Buffer (BD) and permeabilized in Perm Buffer III (BD), then stained with the following antibodies, FITC anti-p-STAT1, PE anti-p-STAT4, APC anti-p-STAT3 (BD). Stained cells were analyzed on a flow cytometer (FACSVerse; BD). Events were collected and analyzed with FlowJo software (Tree Star).

Quantitative Real-time PCR

Total RNA was isolated with the use of the mirVana miRNA kit (Applied Biosystems/ Ambion); cDNA was synthesized with the Taqman Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed with an ABI PRISM 7500 sequence detection system with site-specific primers and probes (Applied Biosystems). The comparative threshold cycle method and an internal control (β-actin) were used to normalize the expression of the target genes. List of primers and probes from Applied Biosystems: mouse ACTB, 4352341E; Bcl6, Mm01342164_m1; Tbx21, Mm00450960_m1; Maf, Mm02581355-s1; Irf-4, Mm00516431-m1; Batf, Mm00479410-m1.

Chromatin immunoprecipitation-sequencing and chromatin immunoprecipitation-quantitative PCR

Chromatin immunoprecipitation (ChIP)�sequencing (seq) experiments and data processing were performed as previously described (69, 70) using anti-STAT1 antibody (Santa Cruz: sc-592). Briefly around 10 ng of DNA was recovered from 10 million cells by ChIP and processed into a library. Illumina HiSeq 2000 was used for sequencing and non-redundant reads of 18772477 were obtained and processed for analysis (SICER). The data is deposited to Gene Expression Omnibus (GEO) database with the accession number GSE51531 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51531). ChIP-quantitative PCR (qPCR) was performed from chromatin prepared similarly as ChIP-seq using anti-p300 antibody (Santa Cruz: sc-585) and anti-STAT1 antibody (sc-592). SYBR green-based qPCR was performed with custom designed primers as follows. Bcl6 forward: CTA GCG TCT GAC CAG GAT CCA, Bcl6 reverse: AGG TCA CGC TCA AGG TTT GC, Pdcd1 forward: CAG CAC AGA AAT GGA AAA AGA GTG T, Pdcd1 reverse: CAG GCC CCA TGC TGA GAC T, Cxcr5 forward: GGA TCA GGT GTT TAT CGG ATG AG, Cxcr5 reverse: TGC CCC ACG TTG TCT CTT C.

Statistical Analysis

Statistical significance was determined by ANOVA using Prism software (GraphPad). A P value less than 0.05 denoted the presence of a statistically significant difference.

RESULTS

Type I IFNs promote sustained expression of Bcl6

To assess whether type I IFNs promote or inhibit TFH cell differentiation we first tested the ability of these cytokines to induce Bcl6, the key transcription factor that influences specification of this subset of helper T cells. As shown in figure 1, naïve CD4+ T cells activated by anti-CD3 and anti-CD28 and cultured in the absence of exogenous cytokines for 5 days expressed modest levels of Bcl6 and no T-bet, as judged by intracellular staining (Figure 1A) or measurement of mRNA (Figure 1B). Both IL-6 and IL-12 induced higher levels of expression of Bcl6, but the latter, as previously shown, also induced T-bet (49, 71). Interestingly, IFN-α and IFN-β both induced Bcl6 expression (Figure 1A, 1B). Of note, there was no further induction of Bcl6 obtained when type I IFNs were combined with IL-6 or IL-12 (Figure 1C, 1D). IFN-α and IFN-β both induced T-bet expression, but neither was as effective as IL-12 in upregulating this transcription factor (Figure 1A, 1B). Other transcription factors have been shown to play a role in TFH differentiation, including Maf, Irf-4, and Batf (72–75). While IL-12 induced both Maf and Irf-4, IL-6 induced only Maf. In contrast, IFN-α and IFN-β had no effect on any of these transcription factors (Figure 1B). Thus, type I IFNs promote Bcl6 expression, a key factor for TFH cell function, and induce only low levels of T-bet.

Figure 1. Type I IFNs promote Bcl6 expression.

Figure 1

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(A, C, D) Bcl6 and T-bet staining in CD4+ T cells cultured in the presence of the indicated cytokines for 5 days. (B) Relative mRNA expression of Bcl6, T-bet, Maf, Irf-4, and Batf was evaluated by qPCR. The data (mean ± SD) are representative of three independent experiments. (E, F) Bcl6 and T-bet staining in CD4+ T cells cultured with increasing doses of IFN-α or IFN-β for 5 days (E) or in neutral conditions or IFN-β for the indicated number of days (F). Data (mean ± SD) are from duplicate cultures of three independent experiments (n=6). The plots are representative of three independent experiments. *, p<0.05; **, p<0.01.

Given that type I IFNs can drive both T-bet and Bcl6, and that these factors can be mutually antagonistic (49), we wondered if varying the dose of IFN-α or IFN-β would modulate the relative expression of these factors. Again, IFN-α and IFN-β induced T-bet expression in dose dependent manner (Figure 1E). Surprisingly, Bcl6 expression also continued to rise when cells were stimulated with IFN-α and IFN-β. However, the induction of Bcl6 was much greater than that of T-bet (approximately 80% vs 40%). Therefore, unlike IL-12, type I IFNs preferentially induced Bcl6 rather than T-bet.

Initially, IL-12 induces both Bcl6 and T-bet, but with time T-bet expression overrides Bcl6 expression (49). To better understand the kinetics of these two factors in response to type I IFNs, we assessed their expression in the absence of exogenous cytokines or in the presence of IFN-β. Consistent with our previous findings, T cell receptor (TCR)-and CD28-mediated signals were sufficient to induce expression of Bcl6 in the majority of activated CD4+ T cells (Figure 1F), but expression was transient and declined by day 5. In contrast, in the presence of IFN-β, Bcl6 expression was sustained. Thus, type I IFNs appeared to be positive regulators of TFH cell differentiation, via induction of the master regulator responsible for this functionality.

Type I IFNs induce key TFH cell surface molecules

Having shown that type I IFNs induce the expression of a key transcription factor for TFH cells, we next set out to determine if these cytokines would induce other aspects of the genetic program of these cells. CXCR5 and PD-1 are key surface molecules expressed on TFH cells that not only define this subset of cells but also are functionally relevant (8, 22–24, 76). We assessed whether all or some of these molecules were upregulated after treatment with type I IFNs. In the absence of exogenous cytokines, activated CD4+ T cells expressed little PD-1 and CXCR5 (Figure 2A). With IL-6 treatment, very few cells became CXCR5+ PD-1+, and as previously shown, IL-12 was more effective in inducing expression of these molecules. The effects of IFN-α or IFN-β were more pronounced than either IL-6 or IL-12, as they induced a roughly 5 fold increase in the percentage of cells expressing CXCR5 and PD-1 compared to cultures in which no cytokine was added (Figure 2A). Like Bcl6, increasing doses of IFN-α or IFN-β resulted in more cells expressing CXCR5 and PD-1 (Figure 2B). We also assessed the time course of CXCR5 and PD-1 induction in the absence of cytokines, and after treatment with IFN-β. Not surprisingly, at day 3, the percent of cells expressing CXCR5 and PD-1 was similar in both conditions, as these molecules are induced by general T cell stimulation (77, 78). By day 5, only cells treated with IFN-β continued to express these molecules (Figure 2C), suggesting type I IFN drives and maintains both Bcl6 and the defining TFH cell surface markers CXCR5 and PD-1.

Figure 2. Type I IFNs induce CXCR5 and PD-1.

Figure 2

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(A-E) CXCR5 and PD-1 staining in CD4+ T cells cultured in the presence of the indicated cytokines for 5 days (A&D), with increasing doses of IFN-α or IFN-β for 5 days (B) or in neutral conditions or IFN-β for the indicated number of days (C). Location of the gates was chosen at each time point based on a negative control sample. Data (mean ± SD) are from duplicate cultures of three independent experiments (n=6). The plots are representative of three independent experiments. *, p<0.05; **, p<0.01. (F) ICOS staining in CD4+ T cells cultured in the presence of the indicated cytokines for 5 days.

Unlike its effects on Bcl6 expression, addition of IL-6 or IL-12 to IFN-α/β had an inhibitory effect on the percentage of cells expressing CXCR5 and PD-1 (Figure 2D). Bcl6 upregulation typically parallels an increase in CXCR5 and PD-1, as was seen with IFN-α/β treatment alone; however, the addition of IL-6 or IL-12 to IFN-α or IFN-β resulted in sustained levels of Bcl6, but reduced expression of CXCR5 and PD-1.

ICOS is another key surface molecule for TFH cells. It is required for TFH cell development, and sustained expression of ICOS is an important feature of TFH cells (4). We therefore also explored the effects of type I IFNs on the expression of ICOS. Unlike CXCR5 and PD-1, ICOS levels were highly expressed in all conditions (Figure 2F). This suggests the regulation of ICOS is independent from the regulation of Bcl6, CXCR5 and PD-1.

Type I IFNs fail to induce IL-21

IL-21 is the “signature” cytokine for TFH cells; although, the expression of this cytokine is not limited to TFH cells and TFH cells also make other cytokines (4). Given that IFN-α or IFN-β induced other key features of TFH cells, we next asked if treatment with IFN-α or IFN-β could promote IL-21 production. As expected, IL-6 was a very potent inducer of IL-21 (32, 33, 79)(Figure 3A and C). Consistent with previous work, IL-12 also induced IL-21, but was less effective than IL-6 (49) (Figure 3A and C). Despite their ability to induce Bcl6, CXCR5 and PD-1, IFN-α or IFN-β failed to robustly induce IL-21 at any point after culture with IFN-β, regardless of the dose used (Figure 3A, C, D, E), or when subsequently co-cultured with B cells (Figure 3F, G) (80, 81). Higher doses of IFN-α/β increased the levels of IFN-γ production (Figure 3D), paralleling its ability to induce T-bet (Figure 1E). Thus, IFN-α/β did not efficiently induce IL-21, despite the induction of other TFH cell features, namely Bcl6, CXCR5 and PD-1. This was consistent with the inability of IFN-α/β alone to induce Maf, an important transcription factor involved in IL-21 production (Figure 1B)(72). Of particular interest though, type I IFNs did synergize with IL-6 to induce IL-21; however, this was not the case for IL-12 (Figure 3B and C). Collectively, these data support the notion that type I IFNs can promote some aspects of the TFH cell program, but are not sufficient to induce the entire TFH cell phenotype.

Figure 3. Type I IFNs do not induce secretion of IL-21.

Figure 3

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(A-E) IL-21 and IFN-γ staining in CD4+ T cells cultured in the presence of the indicated cytokines for 5 days (A&B), with increasing doses of IFN-α or IFN-β for 5 days (D) or in neutral conditions or IFN-β for the indicated number of days (E). Data (mean ± SD) are from duplicate cultures of three independent experiments (n=6). The plots are representative of three independent experiments. *, p<0.05; **, p<0.01. (C-E). (F,G) Intracellular cytokine staining of IL-21 and IFN-γ in T cells co-cultured with naïve B cells for 4 days either with (stimulated) or without (unstimulated) anti-CD3 and anti-CD28 stimulation.

Divergent STAT requirements for Bcl6 and T-bet regulation

STAT3 is a contributing factor in TFH cells but unlike other subsets, its function appears to be partly redundant. STAT4 also promotes TFH cell differentiation mediated by IL-12. Type I IFNs activate STAT1, but can also activate STAT3 and STAT4 (54, 58). To address the mechanism by which IFN-α or IFN-β support TFH cell programs, we first assessed STAT activation in naïve CD4+ T cells after treatment with type I IFNs. Whereas IL-6 induced phosphorylation of both STAT3 and STAT1, type I IFNs predominantly induced STAT1 activation (Figure 4A and B). Under these circumstances, there was little STAT4 phosphorylation induced by any of these cytokines. Naïve T cells do not express IL-12 receptors, so the effects of this cytokine were not examined (82).

Figure 4. STAT1, but not STAT3 or STAT4 is required for type I IFN mediated induction of key TFH cell molecules.

Figure 4

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(A, B) p-STAT3, p-STAT4, and p-STAT1 staining in CD4+ T cells cultured in the presence of the indicated cytokines without TCR stimulation for 30 minutes. Graphs (means ± SD) are from duplicate cultures and are representative of three independent experiments. (C-E) Bcl6 and T-bet (C), IL-21 and IFN-γ (D), or CXCR5 and PD-1 (E) staining in CD4+ T cells cultured in the presence of the indicated cytokines for 5 days from WT, Stat1−/−, Stat3−/−, or Stat4−/− mice. Data (mean ± SD) are from duplicate cultures of three independent experiments (n=6). The plots are representative of three independent experiments. *, p<0.05; **, p<0.01.

To determine which STAT, if any, was responsible for driving IFN-α- or IFN-β-induced TFH cell features, we isolated naïve CD4 T cells from wildtype, STAT1-, STAT3- or STAT4-deficient mice and assessed the ability of IFN-α or IFN-β to induce TFH cell characteristics. As expected, expression of Bcl6 increased after culture with IFN-α or IFN-β (Figure 4C). As type I IFNs did not induce STAT3 activation, this transcription factor was not necessary for Bcl6 induction. Similarly, in the absence of STAT4 the regulation of Bcl6 was also unaffected (Figure 4C). In contrast, the induction of Bcl6 was markedly reduced in cells lacking STAT1 compared to cells expressing this factor, arguing that STAT1 is a major factor that drives Bcl6 levels in response to type I IFNs (Figure 4C).

Interestingly, we found a different STAT is important for regulating T-bet. Expression of T-bet was minimal in cells lacking STAT4, despite stimulation with type I IFNs (Figure 4C). IFN-α/β also failed to induce IFN-γ production in STAT4-deficient cells (Figure 4D). Thus, type I IFNs promote Bcl6 via STAT1, while promoting T-bet and IFN-γ via STAT4.

We also asked if the regulation of CXCR5 and PD-1 by type I IFNs had similar requirements to Bcl6. Like Bcl6, upregulation of CXCR5 and PD-1 was dependent on STAT1, but not STAT3 or STAT4 (Figure 4E). Collectively, these data suggest that type I IFNs largely exert their effect on Bcl6, CXCR5, and PD-1 via STAT1.

STAT1 directly regulates key TFH cell genes

Since our data indicated that the ability of type I IFNs to regulate critical TFH cell genes was STAT1 dependent, we next investigated whether this factor bound these genes directly. To this end, we performed STAT1 Chip-seq analysis after treatment of naïve CD4+ T cells with IFN-β and assessed the genome-wide binding of this factor. A total of 17635 STAT1 binding peaks were identified after IFN-β treatment. Consistent with the importance of STAT1 in regulating Bcl6 expression, we identified a strong area of STAT1 binding in the promoter region of Bcl6 (Figure 5A). In addition, we found multiple sites of STAT1 binding upstream of the Bcl6 gene. As there are no other genes in proximity, it is possible that these are enhancer elements and may be relevant for Bcl6 expression. Collectively, these data suggest that STAT1 might control Bcl6 by acting on its promoter but also distal enhancers. In addition to Bcl6, we also found binding of STAT1 upstream of Cxcr5 and Pdcd1 loci (Figure 5A). The binding of the acetyltransferase p300 is a mark of active enhancers (83). We have previously shown that associated binding of p300 and STAT proteins is indicative of genomic regulatory enhancer elements generated by STATs, which can be associated with transcriptional activity (84). To determine if STAT1 binding detected on TFH genes is functionally relevant, we performed ChIP-qPCR for p300 (Figure 5B) and STAT1 (Figure 5C) at selected sites where STAT1 binding was identified by Chip-seq. We found co-localization of p300 and STAT1 on Bcl6, Pdcd1, and Cxcr5 loci, and the amount of p300 was significantly reduced in STAT1 deficient cells, suggesting that STAT1 positively regulates those TFH-expressed genes by enhancing p300 recruitment. Together, these data suggest STAT1 has a direct role in controlling the expression of several genes involved in the TFH cell signature.

Figure 5. STAT1 binds to TFH cell signature genes.

Figure 5

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(A) STAT1 binding was mapped by ChIP-seq in IFN-β treated CD4+T cells. Naïve CD4+ T cells were cultured for 3 days with plate bound anti-CD3 and anti-CD28 (10 ug/ml of each) and IFN-β (5000 U/ml). The genome browser views for Bcl6 (chr16:23,961,956-24,227,286), Cxcr5 (chr9:44,319,494-44,365,190 ), Pdcd1 (chr1:95,927,980-95,963,539), and Il21 (chr3:37,092,096-37,201,024) loci are shown. Y axis depicts the normalized tag number, defined as the tag count per one million. (B,C) p300 (B) and STAT1 (C) binding to selected regions of the Bcl6, Pdcd1, and Cxcr5 genes was determined by ChIP-qPCR. The amount of precipitated DNA was calculated as % input. The results are representative of two independent experiments, qPCR was done in triplicate (mean ± SEM). Unpaired t test was perfermed to calculate p values; *<0.1, **<0.01, ***<0.001.

DISCUSSION

Type I interferons are highly expressed early during infections, but the role these factors play on CD4+ T cell differentiation remains unclear. In this study we tested the role of IFNα/β on TFH cell differentiation by exploring cardinal TFH cell features. IFNα/β promoted several key TFH features, such as Bcl6, CXCR5 and PD-1 expression, and this effect was STAT1-dependent. Using ChIP-seq analysis, we found several STAT1 binding sites in the Bcl6, Cxcr5 and Pdcd1 loc. At the Bcl6 loci, we found binding at both at the promoter and potential enhancer regions, and these sites co-localized with p300 binding, a mark of active enhancers. These findings identify a new role for type I IFNs and extend our knowledge of the role of STAT transcription factors in TFH cell differentiation.

During an in vivo infection or immunization, multiple cytokines are induced and each cytokine can activate more than one STAT transcription factor. Previous work from our group and others demonstrated that both STAT3 and STAT4 can promote TFH cell development, but in vivo neither of these factors is exclusively required, suggesting redundancy in the pathway to TFH cell generation (32–34, 49). Our results from this study demonstrate that STAT1, in addition to STAT3 and STAT4 can promote TFH cells. Our work is supported by recent evidence from Choi et al that STAT1 activation via IL-6 plays an important role during early TFH cell formation in a viral infection (85). In contrast, STAT5 antagonizes TFH cell development (50–52, 86). Collectively, this indicates that the regulation of TFH cell development in vivo is, unsurprisingly, complex with multiple modes of positive and negative regulation.

Our data highlight this complexity even in the context of a single cytokine. Type I IFNs induce expression of Bcl6 and other important features of TFH cells, but they do not induce the expression of the signature cytokine IL-21. Together with in vivo data indicating a redundant role for cytokines in TFH cell differentiation, our data argue for a model in which cytokines work in concert to fine-tune TFH cell phenotypes. In other words, rather than invoking conventional instructive models analogous to the role of IL-12 and Th1 differentiation, TFH cell differentiation appears to be the product of a multiplicity of factors that can induce different aspects of the TFH cell program to different extents. This perspective is more similar to newer views of the combined actions of cytokines on T helper cell differentiation, including IL-2 on Th1 and Th2 differentiation (87, 88). Rather than T helper cell differentiation being an all or none event in response to cytokines, cytokines act in concert to modulate the degree of specification. Supporting this concept, IFNAR-deficient CD4 T cells were able to differentiate to TFH cells in vivo (data not shown). Type I IFN signaling is required in vivo for T cell survival, as IFNAR-deficient T cells are reduced in number compared to IFNAR-intact T cells after viral infection (61). Despite this, we found that transfer of TCR transgenic OT-II IFNAR-deficient T cells still differentiated to TFH cells in normal percentages after Ova and polyIC immunization. Thus, the picture that emerges is a more nuanced view of T helper cell differentiation in response to the cytokine milieu. Furthermore, it is logical that TFH cells would form under a variety of cytokine conditions. Germinal center derived high-affinity antibody is a property of all infections, including Th1 type viral and bacterial infections, and Th2 type helminth infections. Thus, TFH cell differentiation must occur in multiple cytokine environments such that T-dependent antibody responses exist under Th1, Th2 and Th17 conditions(87).

Adding to this complexity is the recognition that type I IFNs, like other cytokines, activate multiple STATs, including STAT1, STAT3, and STAT4. While these all promote Bcl6 expression, they also promote expression of other master transcription factors, such as T-bet and Rorγ-t. We explored the potentially antagonistic effect of type I IFNs to other cytokines, as it is now well recognized that T-bet and Bcl6 can co-exist under certain conditions (89). We found that type I IFNs induce Bcl6 expression via STAT1, and T-bet expression via STAT4. The co-expression of Bcl6 and T-bet has an interesting functional role, as these two factors can form a complex. In Th1 cells, T-bet on its own acts as an activator, but in complex with Bcl6, it has the ability to become a repressor for some genes, such as Th2 related genes (50). Thus, the balance of Bcl6 and T-bet is critical, as Bcl6 has the ability on its own to mediate the TFH cell gene program. Our study has uncovered another scenario where Bcl6 and T-bet co-exist; in this case, type I IFNs generate cells that have a partial TFH cell phenotype.

As type I IFNs are expressed during both viral and bacterial infections, our data argue that type I IFNs may play an important role in shaping the T cell effector response to infection. This could be important for vaccine design, but also in autoimmune disease. For example, systemic lupus erythmatosus (SLE) is an autoimmune disease characterized by high-titer autoantibodies, and TFH cells are likely to be a critical element in driving those auto-antibody responses. In addition, patients with SLE and a variety of other autoimmune diseases have a type I IFN signature that correlates with disease activity, suggesting constant exposure to these cytokines is a driver of disease (90). Our data suggest that IFN-α/β may be capable of supporting TFH cells, and this support could play an important role in auto-antibody driven autoimmune diseases.

Our observation that STAT1 binds to both promoter and potential enhancer elements of the Bcl6 loci suggest that STATS may play a critical and direct role in regulating Bcl6 expression. As the binding motif for STATS is the same for all the STAT members except STAT6, we hypothesize that many of these potential enhancer sites can be occupied by STAT1, STAT3, STAT4 or STAT5. During an infection where multiple cytokines activate multiple STATS, it is easy to understand why several pathways can contribute to TFH cell differentiation both in vitro and in vivo. In the future, it will be important to carefully map all these sites throughout the extended Bcl6 locus and clarify their functional relevance. Clearly understanding the positive and negative roles of the STAT family and other transcription factors and their effects on the extended Bcl6 gene will be of considerable interest.

ACKNOWLEDGEMENTS

The authors thank G. Gutierrez-Cruz (Biodata Mining Core Facility, NIAMS), J. Simone, and J. Lay (Flow Cytometry Section, NIAMS) for their technical support.

Footnotes

2

This work was supported by the Intramural Research Programs of NIAMS and NHGRI, the Postdoctoral Research Associate Program, NIGMS, NIH (A.C.P. & K.T.L.), and the JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH (K.H.).

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