T-bet and CD11c expression in B cells is linked with IgG2c isotype switching, virus-specific immune responses, and humoral autoimmunity. However, the activation requisites and regulatory cues governing T-bet and CD11c expression in B cells remain poorly defined. In this article, we reveal a relationship among TLR engagement, IL-4, IL-21, and IFN-γ that regulates T-bet expression in B cells. We find that IL-21 or IFN-γ directly promote T-bet expression in the context of TLR engagement. Further, IL-4 antagonizes T-bet induction. Finally, IL-21, but not IFN-γ, promotes CD11c expression independent of T-bet. Using influenza virus and Heligmosomoides polygyrus infections, we show that these interactions function in vivo to determine whether T-bet+ and CD11c+ B cells are formed. These findings suggest that T-bet+ B cells seen in health and disease share the common initiating features of TLR-driven activation within this circumscribed cytokine milieu.

Although initially implicated in CD4 T cell differentiation, T-bet is a key transcriptional regulator in many immune cells. Thus, as shown in the companion report (1), B cell–intrinsic T-bet expression is required to control chronic viral infections and fosters switching to IgG2a (24), an isotype associated with both TH1-driven Ab responses and humoral autoimmunity (5, 6). Moreover, T-bet is required for the generation of age-associated B cells, which are transcriptionally distinct from other B cell subsets and have also been associated with both viral clearance and humoral autoimmunity (79). Finally, many T-bet+ B cells express CD11c, a phenotype associated with viral or bacterial infections, autoimmunity, and neoplasia (8, 1013). Despite growing appreciation for the importance of T-bet–expressing B cell subsets, the signals that yield B lineage effectors characterized by T-bet expression, as well as how these regulate appropriate versus pathogenic outcomes, remain poorly defined. Candidates include cell-intrinsic cues from adaptive and innate receptors, including the BCR and TLRs, as well as signals from T follicular helper (TFH) cells. In this regard, several TH1 cytokines, including IL-12, IL-18, and IFN-γ, can induce T-bet in activated B cells (5, 6). Nonetheless, the roles and interactions of canonical TFH cell cytokines, IL-21, IL-4, and IFN-γ, in regulating T-bet expression have not been systematically interrogated (1416).

In this article, we show that mouse and human B cells integrate signals from IL-4, IL-21, and IFN-γ to regulate T-bet expression. In the context of TLR engagement, both IL-21 and IFN-γ directly drive follicular (FO) B cells to express T-bet in vitro. However, IL-4 antagonizes IL-21–driven T-bet upregulation, but enhances IFN-γ–induced T-bet expression. Moreover, IL-21, but not IFN-γ, promotes CD11c expression. Consistent with these in vitro results, the in vivo frequencies of germinal center (GC) and memory B (BMEM) cells expressing T-bet or CD11c vary based on the prevailing cytokine milieu. Finally, using viral and helminthic infections in single- and double-cytokine knockout mice, we show that the relative abundance of these cytokines determines whether GC and BMEM cells generated during ongoing immune responses express T-bet and CD11c. Together, these findings reveal a previously unappreciated interplay of IL-4, IL-21, and IFN-γ that, in concert with innate sensors, controls T-bet and CD11c expression in B cells.

Tbx21−/−, Stat6−/−, Tbx21f/fCd19Cre/+, C57BL/6 (B6), and BALB/c mice were maintained and used in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee guidelines. The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal experiments. Il4−/− mice were a gift from Dr. Paula Oliver. Ifng−/− mice were a gift from Dr. Edward Behrens. Il4−/−Ifng−/− double-deficient mice were bred in-house. Il21r−/− and Il21tg spleens and sera were shipped overnight on ice from Dr. Warren Leonard. All mice were 2–6 mo of age.

Mice were infected by oral gavage with 200 infectious larvae of Heligmosomoides polygyrus as previously described (17). Mice were infected by intranasal infection with 30 tissue culture infectious dose50 of influenza strain A/Puerto Rico/8/1934 (PR8) (American Type Culture Collection).

Mouse CD23+ splenic B cells were enriched by magnetic positive selection (Miltenyi Biotec), labeled with either Violet Cell Trace (VCT; Invitrogen) or CFSE (eBioscience [eBio]), and cultured as previously described (18). Human PBMCs were isolated from blood samples obtained from healthy donors that expressed written informed consent and after ethical approval by the University of Pennsylvania Institutional Review Board. All investigations were conducted according to the principles expressed in the Declaration of Helsinki. Human B cells were enriched by CD27 microbead negative selection followed by CD19 microbead positive selection (Miltenyi Biotec), labeled with CFSE, and cultured with indicated stimuli for 5 d. Mouse or human IL-21, IL-4, and IFN-γ were used at 25, 10, and 10 ng/ml, respectively (Shenandoah). ODN2006 was used at 1 μM (Invivogen).

FACS reagents were purchased from BioLegend (BL), BD Biosciences, or eBio: T-bet (4B10; BL), CD11c (N418; BL), IgM (R6-60.2; BD Biosciences), CD38 (90; eBio), CD138 (281-2; BL), IgD (11–26c.2a; BL), CD4 (RM4-5; BL), B220 (RA3-6B2; BL), CD62L (MEL-14; eBio), TCR-β (H57-597; BL), CD19 (6D5; BL), CXCR5 (L138D7; BL); PD-1 (RMP1-30; BL); CD8 (53-6.7; eBio), CD4 (H129.19; BL); F4/80 (BM8; eBio); Ly-6G/GR1 (RB6-8C5; eBio); CD43 (S7; BD Biosciences); CD21/CD35 (CR2/CR1; BL); CD23 (B3B4; eBio); CD93 (AA4.1; BL); peanut agglutinin–FITC (Sigma); Zombie Aqua (BL). FACS analyses were performed as described previously (18).

ELISAs were performed as previously described (18) using anti-mouse IgG2a, IgG2b, IgG2c, or IgG1 HRP Abs (Southern Biotech).

Quantitative PCR experiments were performed as previously published (18) using the following probes: Il4 (Mm00445260_m1), Ifng (Mm00801778_m1), Il21 (Mm00517640_m1), Tbx21 (Mm00450960_m1), Aicda (Mm00507774_m1). Transcriptional profiling data were generated as previously described (19) and have been deposited in the Gene Expression Omnibus database for public access (accession no. GSE77145; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77145).

Student t test was used to generate all p values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as box and whisker plots with mean depicted with plus sign (+).

In preliminary in vitro studies, we established that IL-21 drives T-bet expression in mouse FO B cells responding to TLR9, but not BCR or CD40 signals (Fig. 1A). To explore these interactions further, we cultured FO B cells with IL-4, IL-21, or IFN-γ in the presence of TLR7 or TLR9 agonists. Both Tbx21 transcripts and T-bet protein increased markedly in FO B cells cultured with IL-21 or IFN-γ, but IL-4 influenced these outcomes differently. IL-4 blocked IL-21–driven T-bet upregulation, but enhanced IFN-γ–mediated T-bet upregulation (Fig. 1B, Supplemental Fig. 1A).

FIGURE 1.

IL-4 and IL-21 act in a cell-intrinsic manner to regulate T-bet expression in vitro. Magnetically enriched CD23+ splenic B cells were cultured in vitro with various combinations of anti–Ig-μ (IgM), anti-CD40 (40), IL-4 (4), IL-21 (21), and IFN-γ (γ). Mouse data are representative of three independent experiments. (A) WT or Cd19cre/+Tbx21f/f B cells treated for 48 h and assessed for T-bet mean fluorescent intensity (ΔMFI = WT − mutant). (B) Tbx21 mRNA levels in WT cells treated for 20 h. (C) WT, Il21r−/−, or Stat6−/− B cells were labeled with either CFSE (green plots) or VCT (purple plots), treated with ODN1826 and indicated cytokines for 48 h, and then stained for CD11c and T-bet. (D) Magnetically enriched CD27CD19+ human B cells were labeled with CFSE, treated for 108 h, and probed for T-bet on live CFSE cells. (E) Frequency of T-bet+ B cells from each treatment across six healthy adult donors. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

IL-4 and IL-21 act in a cell-intrinsic manner to regulate T-bet expression in vitro. Magnetically enriched CD23+ splenic B cells were cultured in vitro with various combinations of anti–Ig-μ (IgM), anti-CD40 (40), IL-4 (4), IL-21 (21), and IFN-γ (γ). Mouse data are representative of three independent experiments. (A) WT or Cd19cre/+Tbx21f/f B cells treated for 48 h and assessed for T-bet mean fluorescent intensity (ΔMFI = WT − mutant). (B) Tbx21 mRNA levels in WT cells treated for 20 h. (C) WT, Il21r−/−, or Stat6−/− B cells were labeled with either CFSE (green plots) or VCT (purple plots), treated with ODN1826 and indicated cytokines for 48 h, and then stained for CD11c and T-bet. (D) Magnetically enriched CD27CD19+ human B cells were labeled with CFSE, treated for 108 h, and probed for T-bet on live CFSE cells. (E) Frequency of T-bet+ B cells from each treatment across six healthy adult donors. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To determine whether IL-21 and IL-4 directly regulate T-bet in B cells, either Il21r−/− or Stat6−/− B cells were cocultured with wild type (WT) B cells and stimulated as described earlier. Because IL-21R is required for IL-21 signaling and STAT6 is the key signal transducer of IL-4 (20, 21), we reasoned that coculturing these mutants with WT cells would reveal any secondary trans effects. To track both cell origin and division, we labeled WT or knockout cells with VCT or CFSE, respectively (Supplemental Fig. 1B). Whereas IL-21 induced T-bet expression in WT B cells, the cocultured Il21r−/− B cells remained T-bet (Fig. 1C, top row). Analogously, although IL-21–driven T-bet upregulation in WT B cells was reversed by IL-4, cocultured Stat6−/− cells were refractory to this negative effect (Fig. 1C, bottom row). Similar results were obtained using the TLR7 agonist, CL097 (data not shown). Importantly, in all cases, IFN-γ treatment induced T-bet irrespective of Il21r or Stat6 deficiency (Fig. 1C). To assess whether similar relationships exist in human B cells, we cultured CD27CD19+ PBMCs as described earlier. TLR9 stimulation alone upregulated T-bet in these cultures. It is not clear whether intrinsic effects of TLR signaling or trans effects induced by these signals underlie this observation. Nonetheless, IFN-γ significantly increased T-bet expression, and IL-4 completely blocked T-bet in all cultures except those with IFN-γ (Fig. 1D, 1E). In toto, these results show that in the context of TLR signaling, IL-4, IL-21, and IFN-γ interact to regulate T-bet expression in both mouse and human B cells.

The converse effect of IL-4 on IFN-γ– versus IL-21–induced T-bet expression suggests that unique, T-bet–associated programs are facilitated by each cytokine. We interrogated this possibility in several ways. First, because previous studies have linked T-bet with CD11c expression (8), we asked whether IFN-γ or IL-21 influence CD11c differently. The results show that IL-21 drives CD11c expression, but IFN-γ does not (Fig. 1C). Further, as with T-bet, IL-4 blocks IL-21–induced CD11c expression. Finally, IFN-γ drives T-bet expression and is not appreciably influenced by either IL-4 or IL-21 (Supplemental Fig. 1C). These findings indicate that IL-21 and IFN-γ drive T-bet and CD11c expression through distinct mediators, and that T-bet expression per se is insufficient for CD11c induction. To further interrogate differential T-bet expression driven by IL-21 versus IFN-γ, as well as to distinguish T-bet–dependent and -independent effects of each cytokine, we performed genome-wide transcriptional profiling on WT or Tbx21−/− B cells stimulated with either IFN-γ or IL-21. Principal components analysis shows that 82.7% of variance in these data was explained by the cytokine used, whereas Tbx21 genotype accounted for 6.3% of the variance (Supplemental Fig. 1D). Further, each cytokine induces a unique transcriptional profile, including some T-bet–dependent shifts in gene expression (Supplemental Fig. 1E, Supplemental Table I). Thus, IFN-γ and IL-21 drive similar but distinct T-bet–associated phenotypes in B cells.

Together, these results show that in the context of TLR engagement, the aggregate of IFN-γ, IL-21, and IL-4 signals determines whether B cells express T-bet. TLR engagement, but not BCR cross-linking (Fig. 1A), appears necessary to position B cells for T-bet expression upon subsequent IFN-γ or IL-21 signaling. We obtained similar results with the TLR2/4 ligand LPS (not shown), suggesting pathways common to most TLRs, and perhaps other innate receptors, provide these key initial signals. We speculate that these signals alter gene loci accessibility for subsequent cytokine cues. Indeed, prior reports that CD11c+ or T-bet+ B cells emerge in response to a variety of viral and bacterial infections are consistent with this idea (7, 10). Moreover, the differential effects of IL-4 on IL-21 versus IFN-γ suggest a complex interplay of STAT-dependent transcriptional regulation. The clear dose–response relationship of IL-4–mediated effects is consistent with the idea that competitive relationships are involved (Supplemental Fig. 1F). Although IL-4 and IL-21 both require common γ-chain receptor to initiate STAT signal transduction (22), our Stat6−/− coculture data (Fig. 1C) indicate that competition for membrane proximal receptor components is unlikely to explain these findings. If this were the case, then Stat6−/− cells would also be subject to the repressive effects of IL-4. Instead, downstream events are more likely candidates, including differential occupation of transcriptional regulatory sites and altered stoichiometric relationships among the JAK-STAT proteins involved.

Our in vitro findings suggest that IFN-γ, IL-4, and IL-21 interact to modulate T-bet and CD11c expression in B cells. As an initial assessment of whether this relationship exists in vivo, we surveyed GC B and BMEM cells for T-bet expression in B6 versus BALB/c mice (Supplemental Fig. 1G), because these strains display inherent TH1 versus TH2 skewing, respectively (23). We reasoned that if T-bet expression is promoted by milieus rich in IFN-γ, but repressed in those with plentiful IL-4 and little IFN-γ, then the frequencies of T-bet+ B cells in these two strains should differ. In agreement with this prediction, whereas most GC B cells in B6 mice are T-bet+, BALB/c have a lower frequency of T-bet+ GC B cells (Fig. 2A). Importantly, CD11c protein expression was restricted to B6 BMEM cells (Fig. 2B) and not GC B cells (Supplemental Fig. 1H). These findings are consistent with the notion that IFN-γ and IL-4 levels regulate T-bet expression in GC B cells. To probe the impact of IL-21 on this overall relationship, we next asked whether extraphysiological levels of IL-21 would foster accumulation of T-bet+CD11c+ B cells. Profound increases in both T-bet and CD11c expression were seen in all splenic B cells in Il21tg mice (Fig. 2C), which is consistent with our in vitro results suggesting that IL-21 drives both T-bet and CD11c expression. Although the partially activated state of B cells in these mice confounds conventional phenotyping strategies, nearly all mature B cells in the Il21tg bear a CD23CD21 phenotype (Supplemental Fig. 1I) identical to the T-bet–dependent age-associated B cell subset (18, 24). Finally, consistent with the role of T-bet in fostering class-switch recombination to IgG2a/c, we observed a marked increase of IgG2a/c, but not IgG1, serum Ab titers in Il21tg compared with WT mice (Fig. 2D).

FIGURE 2.

T-bet+CD11c+ cells delineate a BMEM cell subset and accumulate in Il21tg mice. (A and B) GC B and BMEM cells were analyzed for T-bet and CD11c expression by FACS. GC B and BMEM cell gating strategies are in Supplemental Fig. 1G. All panels are representative of three independent experiments with ≥3 mice per strain. (A) T-bet staining on GC B cells from B6 (n = 14) or BALB/c (n = 23) mice with frequency enumeration. (B) T-bet and CD11c staining on BMEM cells from B6 mice. (C) T-bet and CD11c staining on splenic B-2 cells from WT and Il21tg mice. (D) Serum IgG1 or IgG2a/c (IgG2a + IgG2c) levels in WT and Il21tg mice were determined by ELISA. Values are means ± SEM from five WT and seven Il21tg mice. **p < 0.01, ****p < 0.0001.

FIGURE 2.

T-bet+CD11c+ cells delineate a BMEM cell subset and accumulate in Il21tg mice. (A and B) GC B and BMEM cells were analyzed for T-bet and CD11c expression by FACS. GC B and BMEM cell gating strategies are in Supplemental Fig. 1G. All panels are representative of three independent experiments with ≥3 mice per strain. (A) T-bet staining on GC B cells from B6 (n = 14) or BALB/c (n = 23) mice with frequency enumeration. (B) T-bet and CD11c staining on BMEM cells from B6 mice. (C) T-bet and CD11c staining on splenic B-2 cells from WT and Il21tg mice. (D) Serum IgG1 or IgG2a/c (IgG2a + IgG2c) levels in WT and Il21tg mice were determined by ELISA. Values are means ± SEM from five WT and seven Il21tg mice. **p < 0.01, ****p < 0.0001.

Close modal

Together, our in vitro and in vivo observations prompt a model in which the relative availability of IL-4, IL-21, and IFN-γ governs the likelihood of establishing BMEM cells expressing T-bet and CD11c. Further, they suggest that abundant IFN-γ will drive a T-bet+CD11c phenotype regardless of IL-4 or IL-21 levels, but that in the absence of IFN-γ, the T-bet+CD11c+ phenotype is reciprocally regulated by IL-21 versus IL-4. We therefore evaluated these predictions by tracking the immune responses to either influenza virus or H. polygyrus in mice where cytokine availability could be experimentally manipulated.

Influenza virus infection yields a well-characterized T-dependent and TH1-skewed response, in which responding TFH cells produce copious IFN-γ, as well as IL-21 and IL-4 (14). Thus, we reasoned that IFN-γ would induce T-bet expression in GC B and BMEM cells, but in the absence of IFN-γ, IL-4 would prevent T-bet expression. Accordingly, WT or Ifng−/− mice were infected with the influenza virus strain PR8. As expected, WT animals mounted a robust GC B cell response to PR8 (Fig. 3A), and these GC B cells expressed T-bet (Fig. 3B; sort strategy and Tbx21 expression, Supplemental Fig. 1J, 1K). In contrast, GC B cells in Ifng−/− mice failed to express T-bet even though the magnitude of the GC B cell response was similar to WT. Assuming that TFH cells are the major source of cytokine, we confirmed that both WT and Ifng−/− mice made substantial numbers of TFH cells (Supplemental Fig. 1J, 1L), and their capacity to make IL-4 and IL-21 was unperturbed (Fig. 3C, 3D). These results are consistent with the idea that, in the absence of IFN-γ, IL-4 blocks T-bet expression in response to IL-21. To directly test this, we infected Il4−/−Ifng−/− double-deficient mice with PR8. Although Il4−/−Ifng−/− mice mounted a blunted GC B cell response (Fig. 3A), these cells nonetheless express T-bet (Fig. 3B, Supplemental Fig. 1K).

FIGURE 3.

Influenza virus infection drives T-bet+CD11c+ BMEM cell formation in the absence of both IFN-γ and IL-4. Splenocytes were harvested from noninfected (−) or day 10 after intranasal 30 tissue culture infectious dose50 PR8 infection (+) WT (n = 21, black bars), Ifng−/− (n = 10, white bars), or Il4−/−Ifng−/− (n = 13, gray bars) mice across 3–7 experiments with ≥3 mice per group. GC B, BMEM, and TFH cell gating strategies are in Supplemental Fig. 1G and 1J. (A) Enumeration of GC B cells. (B) T-bet staining on GC B cells. (C) Il4 and (D) Il21 mRNA levels from sorted naive CD62L+ CD4 T (TN, n = 9) or TFH cells. (E) Proportions and (F) numbers of T-bet+CD11c+ BMEM cells. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Influenza virus infection drives T-bet+CD11c+ BMEM cell formation in the absence of both IFN-γ and IL-4. Splenocytes were harvested from noninfected (−) or day 10 after intranasal 30 tissue culture infectious dose50 PR8 infection (+) WT (n = 21, black bars), Ifng−/− (n = 10, white bars), or Il4−/−Ifng−/− (n = 13, gray bars) mice across 3–7 experiments with ≥3 mice per group. GC B, BMEM, and TFH cell gating strategies are in Supplemental Fig. 1G and 1J. (A) Enumeration of GC B cells. (B) T-bet staining on GC B cells. (C) Il4 and (D) Il21 mRNA levels from sorted naive CD62L+ CD4 T (TN, n = 9) or TFH cells. (E) Proportions and (F) numbers of T-bet+CD11c+ BMEM cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Although the splenic plasma cell numbers were reduced in Ifng−/− mice, BMEM cell numbers remained intact across genotypes (Supplemental Fig. 1M, 1N). However, the composition of the BMEM cell pool differed according to genotype (Fig. 3E, 3F). Whereas WT mice generated some T-bet+CD11c+ BMEM cells, Ifng−/− mice produced few, if any, above noninfected control animals, likely reflecting the dominance of IL-4 in the absence of IFN-γ. Consistent with this interpretation, Il4−/−Ifng−/− mice generated the most T-bet+CD11c+ BMEM cells. Lastly, CD11c expression was restricted to BMEM cells and not GC B cells (Supplemental Fig. 1O). Overall, these findings confirm and extend our in vitro findings, because the same interplay of cytokines directs T-bet expression among B effectors in vivo. Further, our observations suggest that T-bet+CD11c+ BMEM cells will be fostered in immune responses where IL-4 is limited.

Results with influenza virus infection are consistent with the notion that IFN-γ drives T-bet expression irrespective of concomitant IL-4 or IL-21, and that eliminating IFN-γ creates a situation where the relative levels of IL-4 and IL-21 govern the T-bet+CD11c+ phenotype. However, this subtractive approach does not necessarily show that, in responses where IFN-γ is normally absent, the sole determinant of T-bet expression is IL-4 availability. Accordingly, we asked whether IL-4 deficiency is sufficient to permit T-bet expression in GC B and BMEM cells during a TH2 response, using H. polygyrus. This intestinal helminth induces IL-4 and IL-21 production by TFH cells, which drives a robust IgG1 response (15). Thus, we hypothesized that, in the absence of IL-4, IL-21 would be sufficient to induce T-bet expression in GC B and BMEM cells. To test this idea, we infected WT or Il4−/− mice with H. polygyrus and probed GC B cells for T-bet. As expected, WT mice mounted a GC B cell response that lacked T-bet expression, which correlated with increased serum IgG1 titers. Conversely, although blunted in magnitude, Il4−/− mice initiated a T-bet+ GC B cell response with decreased serum IgG1 titers compared with WT (Fig. 4A–C, Supplemental Fig. 1J, 1P). To eliminate the possibility that excess IFN-γ in Il4−/− mice explains these phenotypes, we infected Il4−/−Ifng−/− mice with H. polygyrus. The GC B cell response in Il4−/−Ifng−/− mice was similar to WT levels (Fig. 4A) but maintained T-bet expression independently of IFN-γ (Fig. 4B, Supplemental Fig. 1J, 1P). Isotype representation varied with T-bet expression: whereas WT mice produced >95% IgG1, more than half of the serum Abs in Il4−/−Ifng−/− and Il4−/− mice were IgG2b and IgG2c (Fig. 4C). Further, whereas Il4−/−Ifng−/− mice mounted a higher TFH cell response (Supplemental Fig. 1Q), both Il4−/− and Il4−/−Ifng−/− mice produced less IL-21 (Fig. 4D). Regardless, the magnitude of the plasma cell and BMEM cell response remained intact across genotypes (Supplemental Fig. 1R, 1S). However, we again observed alterations in the BMEM pool according to cytokine availability. Whereas H. polygyrus–infected WT mice did not generate T-bet+CD11c+ BMEM cells, both Il4−/− and Il4−/−Ifng−/− mice did, again suggesting IL-21 drives a unique T-bet+ phenotype (Fig. 4E, 4F). Whereas prior reports showed CD11c mRNA in GC B cells defined by CD95 and peanut agglutinin (25), we observed CD11c protein expression only in BMEM cells (Supplemental Fig. 1T). This seeming disparity may indicate that CD11c transcripts in GC B cells go untranslated, as well as the further resolution of GC and BMEM by CD38 in our gating strategy. Overall, the H. polygyrus infection data support our model, inasmuch as in the absence of IFN-γ we observe both T-bet and CD11c expression that is modulated by IL-4. Further, the consistent relationships observed in both types of infection argue that this is a feature common to most humoral immune responses.

FIGURE 4.

Activated B cells express T-bet independent of IFN-γ in IL-4 limiting conditions. Splenocytes and sera were harvested from noninfected (−) or day 14 after oral gavage (+) of 200 H. polygyrus in WT (n = 20, black bars), Il4−/− (n = 24, white bars), or Il4−/−Ifng−/− (n = 11, gray bars) mice across 3–6 experiments with ≥3 mice per group. GC B, BMEM, and TFH cell gating strategies are in Supplemental Fig. 1G and 1J. (A) Enumeration of GC B cells. (B) T-bet staining on GC B cells. (C) Serum concentrations of IgG1 and IgG2c + IgG2b. (D) Il21 mRNA levels from sorted TFH cells. (E) Proportions and (F) numbers of T-bet+CD11c+ BMEM cells. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 4.

Activated B cells express T-bet independent of IFN-γ in IL-4 limiting conditions. Splenocytes and sera were harvested from noninfected (−) or day 14 after oral gavage (+) of 200 H. polygyrus in WT (n = 20, black bars), Il4−/− (n = 24, white bars), or Il4−/−Ifng−/− (n = 11, gray bars) mice across 3–6 experiments with ≥3 mice per group. GC B, BMEM, and TFH cell gating strategies are in Supplemental Fig. 1G and 1J. (A) Enumeration of GC B cells. (B) T-bet staining on GC B cells. (C) Serum concentrations of IgG1 and IgG2c + IgG2b. (D) Il21 mRNA levels from sorted TFH cells. (E) Proportions and (F) numbers of T-bet+CD11c+ BMEM cells. *p < 0.05, **p < 0.01, ****p < 0.0001.

Close modal

In toto, our findings reveal a novel cytokine network that governs T-bet expression in the context of TLR stimulation. In the absence of IFN-γ, IL-4 and IL-21 reciprocally regulate T-bet and CD11c expression both in vitro and in vivo. Because immune responses are rarely monolithic with regard to these three cytokines (14, 26), distinct or multifunctional TFH cells likely generate a diverse set of B effectors. Consequently, altering the cytokine milieu affects the isotypes generated (Fig. 4C) and the composition of the BMEM pools (Figs. 3F, 4F) while maintaining the magnitude of the response.

It is tempting to speculate that the T-bet+CD11c+ B cells reported in autoimmunity, viral infections, and aging share a common underlying origin involving TLR engagement coupled with either copious IFN-γ or abundant IL-21 with little IL-4. Indeed, both TLR7 and IL-21 deficiencies ameliorate disease in humoral autoimmunity models (27, 28), and poor IL-4 production has been observed in TFH cells from aged mice (29). Thus, understanding this interplay among IL-4, IL-21, and IFN-γ might better define the etiology of humoral autoimmune syndromes where such cells are implicated (8, 13, 30). Lastly, although it is clear that IFN-γ and IL-21 differentially induce CD11c expression (Fig. 1C), the functional consequences of expressing this integrin remain elusive. Importantly, the restriction of CD11c expression to BMEM cells is consistent with prior BMEM subsetting studies in human tonsils and may thus define a tissue-homing population (31). Accordingly, further studies are needed to assess the role of these different T-bet+ BMEM cells in both health and disease.

We thank Burton Barnett and Gretchen Harms Pritchard for Cd19Cre/+Tbx21f/f and Tbx21−/− mice, respectively.

This work was supported by National Institutes of Health Grants T32AI055428, T32CA009171, R01AI113047, and R01AI108686 and by Department of Defense Grant PR130769. R.S. and W.J.L. were supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health. B.B. was supported by German Research Foundation Fellowship BE5496/1-1.

The transcriptional profiling data presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77145) under accession number GSE77145.

The online version of this article contains supplemental material.

Abbreviations used in this article:

B6

C57BL/6

BL

BioLegend

BMEM

memory B

eBio

eBioscience

FO

follicular

GC

germinal center

PR8

A/Puerto Rico/8/1934

TFH

T follicular helper

VCT

Violet Cell Trace

WT

wild type.

1
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The authors have no financial conflicts of interest.

Supplementary data