Transcription Factor IRF4 Regulates Germinal Center Cell Formation through a B Cell–Intrinsic Mechanism

Simon N. Willis, Kim L. Good-Jacobson, Joan Curtis, Amanda Light, Julie Tellier, Wei Shi, Gordon K. Smyth, David M. Tarlinton, Gabrielle T. Belz, Lynn M. Corcoran, Axel Kallies and Stephen L. Nutt
J Immunol April 1, 2014, 192 (7) 3200-3206; DOI: https://doi.org/10.4049/jimmunol.1303216

Abstract

In response to antigenic stimulation, mature B cells interact with follicular helper T cells in specialized structures called germinal centers (GCs), which leads to the development of memory B cells and Ab-secreting plasma cells. The transcription factor IFN regulatory factor 4 (IRF4) is essential for the formation of follicular helper T cells and thus GCs, although whether IRF4 plays a distinct role in GC B cells remains contentious. RNAseq analysis on ex vivo-derived mouse B cell populations showed that Irf4 was lowly expressed in naive B cells, highly expressed in plasma cells, but absent from GC B cells. In this study, we used conditional deletion of Irf4 in mature B cells as well as wild-type and Irf4-deficient mixed bone marrow chimeric mice to investigate how and where IRF4 plays its essential role in GC formation. Strikingly, GC formation was severely impaired in mice in which Irf4 was conditionally deleted in mature B cells, after immunization with protein Ags or infection with Leishmania major. This effect was evident as early as day 5 following immunization, before the development of GCs, indicating that Irf4 was required for the development of early GC B cells. This defect was B cell intrinsic because Irf4-deficient B cells in chimeric mice failed to participate in the GC in response to L. major or influenza virus infection. Taken together, these data demonstrate a B cell–intrinsic requirement for IRF4 for not only the development of Ab secreting plasma cells but also for GC formation.

Introduction

Humoral immunity represents an essential arm of the adaptive immune response that functions to target and eliminate foreign Ags. In response to their cognate Ag, B cells can differentiate into plasma cells that produce Abs that bind tightly and selectively to the inducing agent and memory B cells that differentiate into plasma cells following re-exposure to the same Ag (1).

The production of Ag affinity–selected B cells that can develop into plasma cells and memory B cells occurs in specialized structures called germinal centers (GCs). These structures form transiently following exposure to T cell–dependent Ags and are composed predominantly of B cells with smaller numbers of CD4+ T cells, follicular dendritic cells, and macrophages. Within GCs, B cells interact with a specialized subset of CD4+ T cells called follicular T helper cells (TFH). This interaction is necessary for both GC formation and the subsequent somatic mutation of the Ig v genes, which results in the generation of B cells with improved affinity for the target Ag (2–4).

To date, a small number of transcription factors have been implicated in the GC reaction and subsequent development of Ab-secreting plasma cells (1). These factors can be grouped into those that promote the B cell fate (including BCL6, PAX5, and BACH2) and those that drive the production of Ab-secreting plasma cells (including IFN regulatory factor [IRF]4 and BLIMP1). BCL6 is required for the appearance of GC B cells and TFH cells. It also directly inhibits the DNA damage response, allowing extensive somatic hypermutation and affinity maturation to take place in GC B cells (5–7). PAX5 activates the expression of Bach2 in B cells, which in turn represses Blimp1 expression, thus preventing premature exit from the GC reaction and differentiation into plasma cells (8–10). Loss of other factors including Oct2/Obf1, Batf, Myc, and SpiB also have both been shown to severely diminish GC formation, although in most cases the exact mechanisms remain to be fully determined (11–16).

IRF4 belongs to the IRF family of transcription factors that comprises nine members (17). Unlike most IRF family members, the expression of IRF4 is largely restricted to cells of the immune system. Within the B cell lineage, previous studies have demonstrated that IRF4 is essential for many aspects of the humoral immune response, including the anatomical positioning of mature B cells, Ig class-switch recombination, and plasma cell differentiation (18–21). In regard to the latter, IRF4 overexpression in human B cell lines has been shown to repress BCL6 expression providing a potential mechanism for BCL6 downregulation, which is required for post GC plasma cell formation (22). In addition, IRF4 deficiency has been shown to impact on the differentiation of multiple Th subtypes, including TFH cells that are required for GC formation (23–26).

Given the dual role that IRF4 plays in both B cells and TFH cells, a recent study by Bollig et al. (27) attempted to delineate the importance of IRF4 expression in these two cell types for GC formation in response to Leishmania major infection. That study reported that IRF4 was essential for TFH differentiation but dispensable for GC B cells, which could form in Irf4-deficient mice that had been injected with wild-type CD4+ T cells. Although this finding is consistent with the observation made by Klein et al. (18), who showed that IRF4 deletion in GC B cells did not impact on GC formation, it is at odds with a recent study by Ochiai et al. (28) that reported that IRF4 deletion in mature B cells prevented subsequent GC formation.

In an attempt to resolve this issue and to address whether IRF4 expression in B cells is required for the GC response, we have used a genetic approach and examined the GC response in mice in which Irf4 was conditionally deleted in mature B cells and in mixed bone marrow chimeras containing both wild-type and IRF4-deficient B and T cells. We report, using multiple immunization and infection models, that IRF4 expression by B cells is in fact intrinsically required for the generation of early GC B cells, GCs, and the resulting Ab-secreting plasma cells.

Materials and Methods

Mice

Irf4fl/fl (18), Irf4fl/+, Irf4fl/-, Irf4−/− (19), Bcl6−/− (5), CD23-Cre (29), and Blimp1/eGFP (30) mice were maintained on a C57BL/6 background. Mixed bone marrow chimeras were generated from lethally irradiated (2× 5.5 Gy) Ly5.1 mice reconstituted with bone marrow cells as indicated. All mice were maintained, and experiments were carried out according to the guidelines of the Walter and Eliza Hall animal ethics committee.

RNAseq

RNA was isolated from ex vivo-derived follicular B cells (small size, B220+CD23+), GC B cells (B220+Fas+PNA+), and spleen and bone marrow plasma cells (CD138+Blimp1/eGFP+). RNA also was isolated from in vitro B cell cultures where resting B cells (derived from wild-type and Irf4−/− mice) were activated in culture medium containing CD40 ligand and the cytokine IL-4 for 48 h as described previously (30). Two biological replicates were generated and sequenced for each sample. For all samples, 5 μg RNA was subjected to transcriptome resequencing using either 90- to 100-bp paired end (in vivo samples) or 100-bp single end (in vitro cultures) sequencing on an Illumina HiSeq2000 at the Australian Genome Research Facility (Melbourne, VIC, Australia). Between 12 and 190 million reads were analyzed per sample. Reads were aligned to the NCBI37/mm9 build of the Mus musculus genome using the Subread aligner (31). Genewise counts were obtained using featureCounts (32), and log2-reads per kilobase per million reads were averaged over replicates using voom (33). The entire RNAseq dataset will be described in detail elsewhere.

Immunization

Mice were immunized at 6–14 wk of age with 4(hydroxy-3-nitrophenyl)acetyl (NP)-keyhole limpet hemocyanin (KLH) made at a molar ratio of 17:1 (NP/KLH). Ag was precipitated in alum at a concentration of 1 mg/ml and delivered by i.p. injection (100 μg). Mice were sacrificed 5, 7, and 14 d postimmunization, and single-cell suspensions were made from the spleen and bone marrow for analysis.

Influenza infections

Mice were inoculated with 104 PFU of the HKx31 (H3N2) influenza virus as described previously (34, 35). Mice were sacrificed 10 d postinfection, and GC formation in the lung-draining mediastinal lymph node was assessed by flow cytometry as described previously (36, 37).

L. major infections

Six- to 10-wk-old mice (Irf4fl/fl CD23-Cre mice and Irf4+/+ CD23-Cre mice) were infected with L. major as described previously (38). Chimeric mice were infected 6–8 wk after reconstitution. The relative contribution to GC formation in the spleen and the inguinal lymph node was determined by flow cytometric analysis 14 and 42 d postinfection.

Flow cytometry

Single-cell suspensions were stained to the following surface markers: CD19 (1D3; BD Pharmingen), IgD (11-26C), CD21 (7G6), Gr-1 (RB6-8C5), IgG1 (X56; BD Pharmingen), PNA (FL-1071), FcγR (2.4G2), CD138 (281.2), Ly5.2 (104; BD Pharmingen), Ly5.1 (A20; BD Pharmingen), Fas/CD95 (Jo2; BD Pharmingen), GL7 (eBioscience), CD38 (NIMR-5), B220 (RA3-6B2; BD Pharmingen), and CD23 (B3B4; BD Pharmingen). NP binding was detected as described previously (39). Intracellular staining for BCL6 (7D1) was performed as described previously (36). All Abs were produced in-house unless otherwise indicated. Populations from Irf4fl/fl CD23-Cre mice were gated on eGFP expression (indicating Irf4 deletion).

Results

IRF4 is absent from GC B cells

We began our investigation into the role of IRF4 in GC B cells by examining the expression of Irf4 from ex vivo-purified B cell populations. RNAseq data were generated from sorted follicular B (B220+CD23+) and GC B cells (B220+Fas+PNA+) as well as plasma cells from the spleen and the bone marrow (CD138+Blimp1/eGFP+) that were sorted from Blimp1/eGFP reporter mice (30). As shown in Fig. 1, Irf4 was expressed at low levels in follicular B cells and was upregulated significantly in plasma cells derived from both the spleen and bone marrow. However, Irf4 expression was absent from GC B cells, which was consistent with previously published observations using human B cells where it was shown that IRF4 is not expressed by the vast majority of GC B cells (40, 41).

FIGURE 1.
FIGURE 1.

Irf4 expression during late B cell differentiation. Expression of Irf4 as determined by RNA sequencing of follicular (B220+CD23+), germinal center (B220+Fas+PNA+), and plasma cells (CD138+Blimp1/eGFP+) derived from spleen or bone marrow. The read coverage of Irf4 is shown mapped to the mouse genome. Data are representative of two experiments.

Conditional deletion of Irf4 in mature B cells abrogates GC formation in response to T-dependent immunization

The absence of Irf4 expression from GC B cells was compatible with the possibility that GC B cells may not require this factor for either their formation or maintenance. To explore this possibility further, we conditionally deleted loxP-flanked alleles of Irf4 in B cells using a transgene encoded Cre recombinase. In this study, Cre is expressed in mature B cells concurrent with the low-affinity receptor for IgE (CD23) (29). The Irf4fl/fl mice, which have been described previously, allow tracking of Irf4-deficient cells, because deletion of the floxed allele results in eGFP expression (18). Consistent with previously published observations (19), follicular B cells formed in the absence of IRF4, with absolute numbers being reduced by 50%, relative to wild-type controls, whereas the effect on immature and marginal zone B cells was less pronounced (Supplemental Fig. 1). Thus, removal of IRF4 at the mature B cell stage has only a modest impact on B cell numbers and subset composition.

To explore whether IRF4 was required for GC formation, Irf4fl/flCD23-Cre mice and Irf4+/+ CD23-Cre mice were immunized with a T cell–dependent Ag composed of NP hapten–conjugated to KLH and GC formation examined 7 and 14 d later. Irf4 deletion in B cells (identified as eGFP+ cells) severely reduced the frequency of NP-reactive GC B cells (CD19+IgD−Fas+NP+) and those that had class-switched to IgG1 (NP+IgG1+), compared with immunized wild-type controls (Fig. 2). Furthermore, the frequency of NP-specific IgG1 Ab–secreting cells also was significantly impaired in Irf4-deleted mice, suggesting that IRF4 expression in B cells was intrinsically required for both GC formation and the plasma cell response (data not shown).

FIGURE 2.
FIGURE 2.

Conditional deletion of Irf4 in B cells abrogates the germinal center response and development of NP-specific IgG1 B cells. Flow cytometric analysis of splenocytes 7 and 14 d after i.p. immunization of Irf4fl/fl CD23-Cre mice and Irf4+/+ CD23-Cre mice with NP-KLH in alum. Unimmunized mice were included as a comparison. Frequencies of total NP–reactive B cells (CD19+IgD−Gr1−Fas+) and NP-reactive IgG1-switched B cells are shown relative to total B cells (CD19+IgD−Gr1−) or relative to eGFP+ B cells (for Irf4fl/fl CD23-Cre). Frequencies are graphed as mean ± SD from three to four mice per group. p Values compare the indicated samples.

Conditional deletion of Irf4 in mature B cells abrogates GC formation in response to L. major infection

Our observation that IRF4 expression in B cells was required for GC formation in response to NP-KLH immunization agrees with the findings of Ochiai et al. (28) but is at odds with the results of Bollig et al. (27), who showed that IRF4 expression in B cells might be dispensable for GC formation in response to L. major infection. To explore whether these differences might depend on the type of immunizing Ag, we infected Irf4fl/fl CD23-Cre mice and Irf4+/+ CD23-Cre mice with L. major and analyzed the draining lymph nodes and spleens 14 and 42 d postinfection. As shown in Fig. 3, GC B cells (CD19+Fas+GL7+) were readily detectable in the draining lymph nodes of Irf4+/+ CD23-Cre mice at both time points, with GCs also seen in the spleens at 42 d postinfection. However, GC formation was severely impaired in the absence of IRF4 at both time points, suggesting that the dependence of IRF4 for GC formation was Ag independent. Consistent with this observation, the draining lymph nodes of Irf4+/+ CD23-Cre mice had a robust population of plasma cells (CD138+), whereas Irf4fl/fl CD23-Cre mice showed a dramatic reduction (Fig. 3).

FIGURE 3.
FIGURE 3.

Loss of Irf4 in B cells abrogates the germinal center response in L. major–infected mice. Flow cytometric analysis of the inguinal lymph node (LN) and spleen 14 and 42 d after s.c. infection of Irf4fl/fl CD23-Cre mice and Irf4+/+ CD23-Cre mice with L. major. (A) Frequencies of GC B cells (CD19+Fas+GL7+) are shown relative to total (CD19+) or eGFP+ B cells (for Irf4fl/fl CD23-Cre). (B) Frequencies of plasma cells (CD138+) are shown relative to total (CD19+) or eGFP+ B cells (for Irf4fl/fl CD23-Cre) B cells. Frequencies are graphed as mean ± SD from three to four mice per group. p Values compare the indicated samples.

IRF4 regulates GC cell formation through a B cell–intrinsic mechanism

Although GC formation was severely impaired in Irf4fl/fl CD23-Cre mice in response to both NP-KLH immunization and infection with L. major, it remained possible that the absence of IRF4 in B cells prevented GC formation by impacting on TFH differentiation, as these two cell types are known to be codependent (4). To understand the impact of IRF4 on B cells and TFH cells, a series of chimeric mice were generated by reconstituting irradiated mice with mixtures of donor bone marrow from Irf4+/+ (Ly5.1) and Irf4−/− (Ly5.2) mice. In these chimeras, the presence of Irf4+/+ TFH and GC B cells supports the formation of GC structures. They can then be interrogated for the presence and frequency of IRF4-deficient cells in either GC compartment. Eight weeks after reconstitution, mice were infected with L. major, and GC formation was again assessed in the draining lymph nodes and spleens 14 and 42 d postinfection. Consistent with the experiments performed with Irf4fl/fl CD23-Cre mice, GC B cells (CD19+Fas+GL7+) derived from Irf4+/+ (Ly5.1) bone marrow were readily detectable in both the lymph nodes and spleens at both time points, whereas Irf4−/− (Ly5.2) B cells did not contribute to the GC compartment (Fig. 4A). As expected, the Irf4−/− bone marrow contributed efficiently to the naive B and CD4+ T cell compartments but was unable to differentiate into TFH cells in these chimeras (data not shown).

FIGURE 4.
FIGURE 4.

Mixed bone marrow chimeric mice demonstrate a requirement for IRF4 expression by B cells for the GC response in response to L. major or influenza virus infection. (A) Irradiated Ly5.1 mice were reconstituted with mixtures of bone marrows from Irf4+/+ (Ly5.1) and Irf4−/− (Ly5.2) mice and subsequently infected with L. major. Frequencies of GC B cells (CD19+Fas+GL7+) derived from each genotype 14 and 42 d postinfection are indicated relative to CD19+ B cells. (B) Irradiated mice were reconstituted with mixtures of bone marrows from Ly5.1 mice and Irf4+/+ (Ly5.2), Irf4−/− (Ly5.2), or Bcl6−/− (Ly5.2) mice and subsequently infected with influenza virus. Frequencies of GC B cells (Ly5.2+CD19+CD38lowFas+) are shown relative to total CD19+ B cells. Graphed values shown are the mean ± SD from three to four mice per group. p Values compare the indicated samples.

Having demonstrated that IRF4 was required by B cells for GC formation in response to L. major infection, we then extended our analysis to examine the requirement for IRF4 in B cells in response to viral infection. In a second series of experiments, chimeric mice were generated as described above. To compare the effects of IRF4 on GC B cells with that of a transcription factor known to be intrinsically required for GC B cell development (5–7), we also reconstituted irradiated mice with a mixture of donor bone marrow cells from wild-type (Ly5.1) and Bcl6−/− (Ly5.2) mice. Eight weeks after reconstitution, mice were infected with influenza virus and GC formation was assessed in the lung draining mediastinal lymph node 10 d postinfection. As shown in Fig. 4B, although wild-type GC B cells were readily detectable, no GC B cells formed in the absence of IRF4 or BCL6. This was despite the presence of relatively equal reconstitution of the mature cell compartment by each genotype (Fig. 4B, upper panels). Taken together, we demonstrate using multiple immunization and infection models that B cells intrinsically require IRF4 for GC formation.

IRF4 is required for the generation of early GC B cells

Although IRF4 appeared to be required by B cells for GC formation, it remained possible that GCs form early in the absence of IRF4 and then dissolve quickly. To address this, Irf4fl/+ CD23-Cre mice and Irf4fl/− CD23-Cre mice were immunized with NP-KLH, and the formation of early GC B cells was examined 5 d later, a time point when early GC B cells can first be detected in wild-type mice, yet the GC-structure with the characteristic dark and light zone is not present (11, 12, 42). In these experiments, we compared Irf4fl/− CD23-Cre (null) and control Irf4 fl/+ CD23-Cre (heterozygous) B cells because this allowed the unambiguous identification of IRF4-deficient B cells using eGFP and revealed that the extent of IRF4 deletion in the NP-reactive B cells was comparable between the genotypes (Supplemental Fig. 2). In response to the immunization the frequency of NP-reactive early GC B cells, increased 4-fold in the presence of one copy of wild-type Irf4. Within this pool of NP-reactive B cells, a small percentage had undergone class-switching to IgG1, and a proportion expressed the GC markers Bcl6 and Fas. In contract, Irf4fl/− CD23-Cre mice displayed no increase in the percentage of NP-reactive B cells following immunization, and no GC markers were detected in the NP-reactive B cells (Fig. 5). Taken together, these results demonstrate that IRF4 is absolutely required by B cells for the earliest stages of the GC response.

FIGURE 5.
FIGURE 5.

Conditional deletion of Irf4 in B cells abrogates the formation of early GC B cells. Flow cytometric analysis of splenocytes 5 d after i.p. immunization of Irf4fl/+ CD23-Cre mice and Irf4fl/− CD23-Cre mice with NP-KLH in alum. Unimmunized mice are included for comparison. (A) Frequencies of total NP-reactive B cells (B220+) are shown relative to total B cells (B220+), whereas those that have switched to IgG1, or express Bcl6 or Fas are shown relative to NP-reactive B cells (NP+B220+). (B) Frequencies are graphed as mean ± SD from five mice per group. p Values compare the indicated samples.

IRF4 does not regulate the expression of the factors reported to participate in the GC reaction in in vitro-activated B cells

Having established B cells required IRF4 for the earliest stages of GC formation, we then examined whether loss of IRF4 impacted on the expression of other transcription factor targets reported to participate in the GC reaction. Because of absence of early GC B cells in cells that lack IRF4, RNA sequencing was performed on resting B cells derived from wild-type and IRF4-deficient mice following a short period of activation (48 h) with CD40 ligand and IL-4. As shown in Supplemental Fig. 3, although the loss of IRF4 impacted on the expression of the known IRF4 target, Aicda, no effect was seen on other regulators of the GC response, including Batf, Obf1, Myc, Spib, Yy1, Bcl11a, Ebf1, and E2a, suggesting that these genes are not obligate targets of IRF4 in activated B cells. Bcl6 expression was modestly upregulated in the absence of IRF4 (Supplemental Fig. 3A), a finding that agrees with a repressive function of IRF4 on the Bcl6 gene proposed by Saito et al. (22) but contrasts with the findings of Ochiai et al. (28) and the BCL6 protein data presented in this paper and may reflect the known dose-dependent function of IRF4 in B cells (20, 28, 43).

Discussion

Previous studies have established that IRF4 is required for multiple aspects of the B cell response including Ig class-switch recombination and plasma cell development. However, its role in GC formation was less clear. In a recent study, Bollig et al. (27) examined the requirement for IRF4 expression in B and T cells for GC formation. Using a model of L. major infection, they demonstrated that IRF4 is required for TFH cell differentiation but dispensable for the development of GC B cells, because GC formation could be rescued in Irf4-deficient mice by transfer of wild-type CD4+ T cells. This observation was consistent with our RNAseq analysis, which demonstrated a lack of Irf4 expression in GC B cells and suggested that IRF4 may be dispensable for the development and persistence of GC B cells. This conclusion also was supported by the finding that deletion of Irf4 in already-formed GC B cells using Cγ1-Cre did not impact on GC formation (18). In contrast, our data clearly show that the generation of GC B cells, elicited by immunization with protein Ags or infection with L. major, was severely impaired in mice in which Irf4 was conditionally deleted in mature B cells. This conclusion agrees with a recent report by Ochiai et al. (28), which demonstrated that IRF4 expression by B cells also was required for GC formation in response to a different T cell–dependent Ag, sheep RBCs. Given the absence of IRF4 expression in GC B cells, we reasoned that IRF4 must play an essential role in early GC B cells during an early phase of the immune response. Indeed, IRF4-deficient mature B cells are unable to expand in number or acquire characteristics of early GC B cells such as BCL6 expression in response to protein immunization (Fig. 5).

Although IRF4 is known to have an essential role in TFH cell differentiation (27, 29), the approach adopted in the current study establishes that mature B cells also have an intrinsic requirement for IRF4 for GC formation. Thus, IRF4 joins BCL6 in being independently required for both arms of the GC response. Unlike BCL6, which is expressed broadly in both populations (44), our results show that the function of IRF4 is restricted to controlling the development of Ag-specific early GC B cells. In keeping with this, previous studies have shown that IRF4 is rapidly induced in activated B cells in a manner proportional to the intensity of the engagement of the BCR (43), with IRF4 then functioning in a dose-dependent manner with low concentrations correlating with GC formation and high levels activating BLIMP1 and promoting plasma cell development (20). The precise signals that regulate and integrate IRF4 and BCL6 expression remain unclear, with recent evidence suggesting that the expression both BCL6 and OBF1 are activated by IRF4 in early GC B cells (28). This regulation is also likely to be context or dose dependent as our experiments show that IRF4 deficiency resulted in a modest increase in Bcl6 expression after short-term in vitro activation of mature B cells, whereas the expression of Obf1 was IRF4-independent in those cultures. Because the expression of a suite of other transcription factors that are important in GC B cells was also unchanged in the absence of IRF4, further work is required to determine exactly how IRF4 promotes BCL6 expression and early GC B cell formation.

Once the GC B cell is formed, the repression of Irf4 in GC B cells also appears to be important as ectopic expression of IRF4 in activated B cells favors plasma cell differentiation over a prolonged proliferative phase in the GC (28). Although our RNAseq data clearly show that the repression occurs on a transcriptional level, the mechanism by which this occurs remains to the determined. Similarly, it is also unclear how Irf4 expression is re-established because B cells leave the GC and differentiate into memory B cells or plasma cells. The genetic approaches outlined in this study, which clearly show a cell intrinsic function for IRF4 in GC B cell formation, will greatly facilitate future studies to address these important questions.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Tak Mak (Princess Margaret Cancer Centre, Toronto, ON, Canada), Ulf Klein (Columbia University), Louis Staudt (National Cancer Institute, National Institutes of Health, Bethesda, MD), and Meinrad Busslinger (Research Institute of Molecular Pathology, Vienna, Austria) for mice and Jamie Leahy for animal husbandry.

Footnotes

  • This work was supported by a program grant from the National Health and Medical Research Council of Australia (575500), a Multiple Myeloma Research Foundation senior award, the Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support scheme. S.N.W. and K.L.G.-J. are supported by a National Health and Medical Research Council C.J. Martin fellowship; A.K., G.T.B., and S.L.N. by Australian Research Council Future fellowships; and G.K.S., D.M.T., and L.M.C. by National Health and Medical Research Council fellowships.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    GC
    germinal center
    IRF
    IFN regulatory factor
    KLH
    keyhole limpet hemocyanin
    NP
    4(hydroxy-3-nitrophenyl)acetyl
    TFH
    follicular helper T cell.

  • Received December 2, 2013.
  • Accepted January 25, 2014.

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