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J Exp Med. 2010 May 10; 207(5): 933–942.
PMCID: PMC2867277

Batf coordinates multiple aspects of B and T cell function required for normal antibody responses

Abstract

Batf belongs to the activator protein 1 superfamily of basic leucine zipper transcription factors that includes Fos, Jun, and Atf proteins. Batf is expressed in mouse T and B lymphocytes, although the importance of Batf to the function of these lineages has not been fully investigated. We generated mice (BatfΔZ/ΔZ) in which Batf protein is not produced. BatfΔZ/ΔZ mice contain normal numbers of B cells but show reduced numbers of peripheral CD4+ T cells. Analysis of CD4+ T helper (Th) cell subsets in BatfΔZ/ΔZ mice demonstrated that Batf is required for the development of functional Th type 17 (Th17), Th2, and follicular Th (Tfh) cells. In response to antigen immunization, germinal centers were absent in BatfΔZ/ΔZ mice and the maturation of Ig-secreting B cells was impaired. Although adoptive transfer experiments confirmed that this B cell phenotype can be driven by defects in the BatfΔZ/ΔZ CD4+ T cell compartment, stimulation of BatfΔZ/ΔZ B cells in vitro, or by a T cell–independent antigen in vivo, resulted in proliferation but not class-switch recombination. We conclude that loss of Batf disrupts multiple components of the lymphocyte communication network that are required for a robust immune response.

The development of the various lymphoid lineages is regulated by many transcription factors, including the dimerizing basic leucine zipper (bZIP) proteins collectively known as activator protein 1 (AP-1; Wagner and Eferl, 2005). The classical AP-1 transcription factor consists of a Jun:Fos heterodimer, although tissue-restricted bZIP proteins, including several of the Maf, Atf, and Batf proteins, provide alternative partner choices for Fos and/or Jun (Eferl and Wagner, 2003). Properties conferred on AP-1 by dimer composition and posttranslational modifications influence the DNA targets bound by AP-1 and, in some cases, convert what is normally a transcriptional activator into a transcriptional repressor (Eferl and Wagner, 2003; Hess et al., 2004; Amoutzias et al., 2006). It is not surprising, therefore, that AP-1 plays roles in cell growth, differentiation, and apoptosis (Hess et al., 2004) and that deregulated AP-1 activity is a feature of many pathologies, including cancer and neurological diseases (Eferl and Wagner, 2003; Raivich and Behrens, 2006).

Our laboratory studies Batf, an AP-1 protein which is expressed in immune cells and whose overall level of expression is regulated by developmental transitions (Li et al., 2001; Williams et al., 2001) and environmental cues (Senga et al., 2002; Johansen et al., 2003; Jung et al., 2004). Batf is the founding member of the Batf protein family (Batf, Batf2, and Batf3; Dorsey et al., 1995; Aronheim et al., 1997; Lim et al., 2006). All three Batf proteins compete with Fos for partnering with Jun and, in doing so, generate bZIP dimers that inhibit the transcription of AP-1 reporter genes (Echlin et al., 2000; Iacobelli et al., 2000; Su et al., 2008). Previous studies using a thymus-specific BATF transgene examined how constitutive AP-1 inhibition has an impact on the growth and development of T cells in vivo. Results showed that although the proliferative response of transgenic thymocytes was decreased in vitro, all T cell subsets, with the exception of NKT cells, were present in normal numbers in vivo (Williams et al., 2003; Zullo et al., 2007). The exquisite sensitivity of Vαi NKT cells to BATF overexpression provided the first evidence that downstream signaling through the invariant NKT cell receptor, which is largely responsible for the unique properties of these cells (Kronenberg and Engel, 2007), relies on the precise regulation of AP-1.

In this study, we report the immune system phenotype of mice (BatfΔZ/ΔZ) in which Batf protein expression has been eliminated. In these animals, the numbers of peripheral T cells, but not B cells, are affected. In agreement with a study published while this work was in progress (Schraml et al., 2009), we detect a significant decrease in CD4+ T cells and a dramatic reduction in Th17 cells. However, we also report that loss of Batf has a negative impact on Th2 cells, follicular Th (Tfh) cells, and the humoral immune response. Germinal centers (GCs) do not form in antigen-challenged BatfΔZ/ΔZ mice and B cells do not undergo productive Ig class-switch recombination (CSR), leading to dysgammaglobulinemia. These data identify essential roles for Batf in several Th cell lineages and in coordinating the transcriptional program required for the differentiation of peripheral B cells into antibody (Ab)-producing cells.

RESULTS AND DISCUSSION

Decreased numbers of peripheral CD4+ T cells in BatfΔZ/ΔZ mice

To examine the role of Batf in lymphocyte development, we first generated Batf knockin (Batf KI) mice in which exon 3, the ZIP coding region of Batf, is expressed with a C-terminal hemagglutinin antigen (HA) epitope tag (Fig. 1 A). This modified exon and the Pgk-neo cassette used for ES cell selection are flanked by loxP sites, permitting the excision of both elements using Cre recombinase. Batf KI mice were crossed to Cre-expressing mice (EIIa-Cre), producing heterozygous (Batf+/ΔZ) mice which were crossed to generate homozygous BatfΔZ/ΔZ mice and littermate Batf+/ΔZ and Batf+/+ mice for comparison (Fig. 1, A and B). BatfΔZ/ΔZ mice do not produce a functional Batf bZIP protein. Immunoblots using BatfΔZ/ΔZ splenocyte extracts and anti-HA antiserum failed to detect a protein (Fig. 1 C). As predicted, semi-quantitative PCR (qPCR) analysis of RNA isolated from BatfΔZ/ΔZ splenocytes using several primer sets detected transcripts representing exons 1 and 2 but no transcript specifying the Batf ZIP domain (Fig. S1, A and B).

Figure 1.
Profile of T and B cells in BatfΔZ/ΔZ mice. (A) Schematic of Batf KI and Batf exon 3–deleted (ΔZ) alleles. Batf exons 1–3 are numbered. Filled triangles indicate loxP sites. Arrows indicate genotyping primers. Numbered ...

Batf mRNA and protein are expressed in mouse B cells and in all major T cell subsets examined, with the exception of double-positive thymocytes (Williams et al., 2001) which, interestingly, lack all AP-1 activity (Rincón and Flavell, 1996). Mice expressing human BATF throughout T cell development in the thymus (p56lckHA-BATF) possess normal numbers of CD4+ and CD8+ T cells but are impaired in the development of Vαi NKT cells (Williams et al., 2003; Zullo et al., 2007). To determine if B or T cell development is altered by the absence of Batf, cells from the thymus, spleen, and Peyer’s patches (PPs) of Batf+/+ and BatfΔZ/ΔZ mice were analyzed by flow cytometry. No significant difference in thymic T cell populations was observed (Fig. S2 A). In the periphery, a trend toward a decreased number of T cells and an increase in B cell numbers was noted, yet statistical significance was established only for CD4+ T cells (Fig. 1 D). In agreement with a recent study (Schraml et al., 2009), we did not detect increases in any T cell subset in BatfΔZ/ΔZ mice, including Vαi NKT cells (unpublished data). This was unexpected based on the NKT cell–deficient phenotype of p56lckHA-BATF mice (Williams et al., 2001; Zullo et al., 2007) and on experimental evidence that BATF inhibits cell proliferation in several different contexts (Echlin et al., 2000; Williams et al., 2001; Senga et al., 2002; Thornton et al., 2006). Instead, this supports a model where the overexpression of an AP-1 inhibitor, such as Batf, can have a dramatic impact on cells, whereas the impact of deleting Batf might be masked by the compensatory actions of other AP-1 inhibitors (e.g., Batf3, JunD, FosB, and Atf3; Hess et al., 2004). Although a comparative profile of all AP-1 proteins expressed by various lymphocyte lineages has yet to be compiled, Batf and Batf3 are coexpressed in mouse Th1 cells, for example (Williams et al., 2001; Hildner et al., 2008). In this regard, transgenic mice in which either of these proteins is overexpressed during T cell development share phenotypes, including the NKT cell defect (unpublished data), whereas the absence of Batf or Batf3 has an impact on other cell types (Hildner et al., 2008; Schraml et al., 2009). Thus, it is the unique functions of Batf that will be revealed by a thorough analysis of BatfΔZ/ΔZ mice.

Th2 and Th17 cells require Batf

The decrease in peripheral CD4+ T cells associated with Batf deficiency prompted us to further investigate this phenotype. CD4+ T cells represent multiple T helper (Th) cell lineages (Zhou et al., 2009). To measure Th cell subsets in Batf+/+ and BatfΔZ/ΔZ mice, CD4+ T cells isolated directly from spleen and PP were analyzed by flow cytometry after a brief stimulation. IFN-γ and IL-4 are well characterized markers of the Th1 and Th2 lineages, respectively, and no statistically significant difference was noted for either cell type (Fig. 2 A). In contrast, a dramatic underrepresentation of CD4+ T cells expressing IL-17 (Th17 cells) was apparent in BatfΔZ/ΔZ mice (Fig. 2 A). A small but significant reduction in Foxp3+ CD4+ regulatory T (T reg) cells also was noted in BatfΔZ/ΔZ mice (Fig. S2 B).

Figure 2.
Th17 and Th2 differentiation is impaired in BatfΔZ/ΔZ mice. (A) Cells from spleens and PP of BatfΔZ/ΔZ and Batf+/+ mice were surface stained using anti-CD4 mAb. After stimulation with 50 ng/ml P + 1 µg/ml I with ...

To investigate if Batf deficiency affects the expression of genes that are markers for activated CD4+ Th cell subsets, RNA was prepared from CD4+ Batf+/+ and BatfΔZ/ΔZ splenocytes after stimulation with anti-CD3ε mAb for 48 h. qPCR was used to quantify transcripts unique to Th17 (IL-21, IL-23R, and IL-17), Th1 (T-bet), Th2 (Gata3 and IL-4), and T reg (Foxp3) cells. Results confirm the underrepresentation of Th17 cells, the normal levels of Th1 cells, and the modest reduction of T reg cells in BatfΔZ/ΔZ mice (Fig. 2 B). Interestingly, although no significant change in Th2 cells was detected by flow cytometry (Fig. 2 A), the low levels of IL-4 and Gata3 mRNA noted in Fig. 2 B suggest a role for Batf in Th2 responses. As confirmation that a difference in mRNA by this assay reflects a change in protein, ELISA was performed on media harvested from stimulated Batf+/+ and BatfΔZ/ΔZ splenocytes. Results showed that BatfΔZ/ΔZ cells secrete normal levels of IFN-γ, reduced levels of IL-4, and extremely low levels of IL-17 (unpublished data).

To compare the ability of Batf+/+ and BatfΔZ/ΔZ CD4+ T cells to respond to cues that polarize cells to distinct Th cell lineages, naive CD4+ T splenocytes, cultured under well defined Th1, Th2, Th17, and T reg cell conditions, were analyzed by flow cytometry. This general approach was used previously (Schraml et al. 2009) to demonstrate a role for Batf in Th17 differentiation. In agreement with those studies, we found that Batf+/+ and BatfΔZ/ΔZ cells were equally competent for Th1 differentiation (Fig. S2 C) and that the decreased levels of T reg cells noted in vivo did not reflect an inability of naive BatfΔZ/ΔZ T cells to differentiate to T reg cells in vitro (Fig. S2 D). Our results also confirmed that BatfΔZ/ΔZ cells cannot be directed toward the Th17 lineage under conditions where >40% of control Batf+/+ cells express IL-17 (Fig. S2 E). In contrast, attempts to assess Th2 polarization by flow cytometry produced inconsistent results for both Batf+/+ and BatfΔZ/ΔZ cells over several experiments, prompting us to rely on qPCR analysis of Th2 transcripts as an indicator of differentiation. qPCR with RNA from in vitro–polarized Th17 cultures was performed in parallel. As shown in Fig. 2 (C and D), when compared with control cells, BatfΔZ/ΔZ cells did not induce significant levels of either Th2- or Th17-specific transcripts. Although Schraml et al. (2009) did not describe a defect in polarized Th2 differentiation for their Batf-deficient cells, our results would indicate that there is, at minimum, a partial defect in the Th2 cell subset that contributes to a decreased level of IL-4 in BatfΔZ/ΔZ mice.

Impaired Ig production in BatfΔZ/ΔZ mice

IL-21 is required for the differentiation of Th17 cells and, in turn, is produced by Th17 cells (and other cells types) to stimulate IL-21–producing CD4+ Tfh cells and the B cell Ab response (King, 2009). Mice lacking IL-4 and the IL-21 receptor exhibit severe defects in Ab production (Ozaki et al., 2002). To test if the combined IL-4– and IL-21– deficient phenotype of BatfΔZ/ΔZ mice results in reduced Ig production, circulating IgM, IgG1, IgG2c, IgA, and IgE were quantified by ELISA. As shown in Fig. 3 A, when compared with Batf+/+ animals, BatfΔZ/ΔZ mice displayed a modest reduction in circulating IgM. Strikingly, the levels of all other Ig classes examined were barely detectable in BatfΔZ/ΔZ mice.

Figure 3.
Dysgammaglobulinemia in BatfΔZ/ΔZ mice. (A) ELISA was performed to measure the indicated Ig in sera of Batf+/+ and BatfΔZ/ΔZ mice. Shown are mean results from four mice per group (n = 4) assayed in duplicate. Error bars ...

To test if Ig production in BatfΔZ/ΔZ mice remains low in the presence of antigen challenge, Batf+/+ and BatfΔZ/ΔZ mice were injected with sheep RBC (sRBC) or mock injected with PBS. After 7 d, serum was isolated and circulating Ig quantified by ELISA. Again, although anti-sRBC IgM was induced in both Batf+/+ and BatfΔZ/ΔZ mice, induction of IgG by BatfΔZ/ΔZ mice was only 26% of the control (Fig. 3 B). Immunohistochemistry (IHC) confirmed the low levels of IgG1 and IgG2c in spleens of BatfΔZ/ΔZ mice (Fig. 3 C). The morphology of additional spleen sections from sRBC-challenged Batf+/+ and BatfΔZ/ΔZ mice was examined (Fig. 3 D). Although hematoxylin and eosin (H + E) and peanut agglutinin (PNA) staining demonstrated the presence of GCs in Batf+/+ mice, PNA+ GCs were conspicuously absent in BatfΔZ/ΔZ mice.

Reduced Tfh cell number and function in BatfΔZ/ΔZ mice

The production of high-affinity class-switched Ab relies on GC interactions between B cells and Tfh cells (King, 2009). Tfh cells are characterized by the expression of CXCR5 (CXC chemokine receptor 5), which directs the homing of Tfh cells to B cell follicles in the spleen and lymph nodes (Kim et al., 2001). There is strong evidence to suggest that IL-21 is critical for Tfh development and that the IL-21 produced by Tfh cells in GCs is essential for the B cell response (Nurieva et al., 2008; Vogelzang et al., 2008; King, 2009). To quantify Tfh cells in Batf+/+ and BatfΔZ/ΔZ mice, CD4+ T cells were stained with mAb specific for CXCR5 and CD62L and analyzed by flow cytometry (Fig. 4 A). Results show that in BatfΔZ/ΔZ mice, memory-type CD62LCXCR5+ Tfh cells are reduced by 70% in the spleen (Fig. 4 B, top) and by 90% in PP (Fig. 4 B, bottom). To examine if Tfh cells in BatfΔZ/ΔZ mice are functional, purified CD4+ T cells from PP were challenged in vitro to migrate to CXCL13, the CXCR5 ligand. Migrating cells were counted and expressed as a percentage of CD62LCXCR5+ cells in the initial suspensions. As shown in Fig. 4 C, ~25% of Batf+/+ Tfh cells were capable of chemotaxis, whereas <10% of BatfΔZ/ΔZ Tfh cells displayed this behavior. These results are further support for an essential role for Batf in CXCR5+ Tfh cells.

Figure 4.
Dysfunctional Tfh cells in BatfΔZ/ΔZ mice. (A) Cells from spleen and PP of BatfΔZ/ΔZ and Batf+/+ mice were stained with anti-CD4, anti-CD44, anti-CD62L, and anti-CXCR5 mAb (or isotype control) and Tfh cells detected by ...

Batf deletion has an impact on multiple CD4+ T cell lineages and, in doing so, generates an environment unfavorable to a robust Ab response. To demonstrate the T cell dependence of this phenotype, adoptive transfer was used to reconstitute T cell–deficient mice with CD4+ T cells purified from Batf+/+ or BatfΔZ/ΔZ mice. After transfer, the mice were challenged with sRBC and, 8 d later, Tfh cells were quantified and sera assayed for Ig. As predicted, when compared with mice reconstituted with Batf+/+ T cells, the spleens and lymph nodes of mice reconstituted with BatfΔZ/ΔZ T cells were not populated with CD62LCXCR5+ cells (Fig. 5, A and B) and sera from these animals contained less sRBC-induced IgM and IgG1 (Fig. 5 C).

Figure 5.
Limited response of BatfΔZ/ΔZ CD4+ T cells to T cell–dependent antigen. (A) CD4+ T cells from Batf+/+ or BatfΔZ/ΔZ mice were injected into T cell–deficient mice, which were immunized with 5 × 10 ...

BatfΔZ/ΔZ B cells do not express Aicda mRNA and do not undergo CSR

To this point, our data implicate defects associated with several CD4+ T cell subsets as the underlying cause of Ig deficiency in BatfΔZ/ΔZ mice. On the other hand, because Batf is expressed in mouse B cells (Williams et al., 2001) and functions as an inducible growth regulator in human B cells (Johansen et al., 2003), the loss of Batf could impact B cell function as well. To examine this possibility, resting B cells from spleens of Batf+/+ and BatfΔZ/ΔZ mice were cultured in control medium or in medium containing LPS, with or without added IL-4. Cells were analyzed for proliferation by BrdU staining after 40 h and for surface and secreted Ig after 4 d. Batf+/+ and BatfΔZ/ΔZ B cells proliferated similarly after exposure to LPS or LPS and IL-4 (Fig. 6 A). B cells of both genotypes also expressed surface and secreted IgM under all three growth conditions (Fig. 6 B). Strikingly, although control cells stimulated with LPS and IL-4 decreased IgM production and began producing IgG1 and IgE, BatfΔZ/ΔZ cells continued to produce high levels of IgM, indicating that Batf is required for efficient CSR.

Figure 6.
BatfΔZ/ΔZ B cells do not undergo CSR. (A) B cells purified from Batf+/+ and BatfΔZ/ΔZ mice were cultured in media, or media supplemented with 20 µg/ml LPS, with or without 20 ng/ml IL-4. After 24 h, DNA synthesis ...

The inability of BatfΔZ/ΔZ B cells to undergo CSR after stimulation was characterized further using qPCR to examine the expression of key genes known to participate in events critical for B cell maturation and CSR (Honjo et al., 2004; Fairfax et al., 2008). As a first indication that Batf was regulated as a part of this process, we observed that Batf mRNA is induced by LPS in Batf+/+ cells and was increased further by costimulation with IL-4 (Fig. 6 C). The absence of Batf did not dramatically affect the stimulation-induced down-regulation of Pax5 or Bcl-6 mRNA, nor did it prevent the up-regulation of Irf4, Prdm1, or Xbp1s mRNAs, although BatfΔZ/ΔZ B cells did appear to resist IL-4-induced modulation of this latter group of transcripts. Interestingly, expression of the Aicda gene encoding AID (activation-induced cytidine deaminase) was undetectable in stimulated BatfΔZ/ΔZ cells (Fig. 6 C). This finding is consistent with the lack of CSR and suggests that Batf participates in an essential molecular event downstream of B cell activation and upstream of Aicda expression, CSR, and somatic hypermutation (Fairfax et al., 2008; Park et al., 2009).

To confirm that this in vitro result reflects a B cell defect in vivo, Batf+/+ and BatfΔZ/ΔZ mice were injected with TNP-LPS. After 4 d, T cell–independent responses were assayed by ELISA and IHC. Although Batf+/+ animals induced TNP-LPS–specific IgG1 (Fig. 6 D) and their spleens displayed foci of both IgG1- and IgG2c-producing B cells (Fig. 6 E), BatfΔZ/ΔZ mice showed no T cell–independent antigen response by either assay.

The recent work of Schraml et al. (2009) clearly demonstrated a role for Batf in Th17 cell differentiation and cytokine gene regulation. Our studies have confirmed that role and have described additional roles for Batf in Tfh and Th2 cells that are required for the generation of a robust T cell–dependent antigen response in vivo. Moreover, our studies have revealed a role for Batf in the intrinsic responsiveness of B cells to T cell–independent stimulation in vitro and in vivo. Future studies, in which we exploit our conditional Batf ΔZ allele to disrupt Batf function in specific lymphocyte compartments or during key developmental transitions, will allow us to further dissect the molecular details of these intriguing Batf-dependent phenotypes.

MATERIALS AND METHODS

Generation of BatfKI/KI and BatfΔZ/ΔZ mice.

Batf primers with a 5′ loxP sequence and a 3′ HA epitope coding sequence (+ stop) were used to amplify a region of intron 2 plus exon 3 (− stop) of the Batf gene. This fragment was cloned into pBS KS and modified by insertion of the Batf 3′ UTR at SpeI and of a loxP-flanked Pgk-neomycin selection cassette at XbaI. The Batf KI sequence was excised using EcoRI and cloned into pBS KS ARMS containing 3.5 kbp of 5′ and 2.7 kbp of 3′ Batf genomic DNA. This plasmid, pBS KS CKO, was linearized and introduced into 129/SV mouse embryonic stem cells, and the correct targeting of drug-resistant clones was determined by PCR with forward (5′-GGACTAGTCATCTTGCCTT-3′) and reverse primers to detect endogenous (5′-GGAAGGCATGGGCACTCTATAC-3′) or recombined (5′-CGAGCATAGTGAGACGTGCTAC-3′) Batf. The Transgenic Mouse Core Facility of the Purdue University Center for Cancer Research produced germline chimeras which were crossed to C57BL/6 mice (Harlan). Batf+/KI mice were mated to produce BatfKI/KI mice which were crossed to EIIaCre mice (JAX). Batf+/ΔZ mice were backcrossed to C57BL/6 mice four to six times and were mated to generate BatfΔZ/ΔZ and littermate control Batf+/ΔZ and Batf+/+ mice that were used for experimentation at 7–12 wk of age. The genotyping primers for Batf ΔZ are forward, 5′-GCTTGTCTCTCACTAGTGAG-3′, and reverse, 5′-CTGTAGAGTGACTGGCTC-3′. All mice used in this study were maintained in a specific pathogen-free animal facility according to institutional guidelines. All animal protocols were reviewed and approved by the Purdue University Animal Care and Use Committee.

DNA blot hybridization.

20 µg DNA, isolated from tail tips by phenol/chloroform extraction, was digested with SpeI and resolved by 0.8% agarose gel electrophoresis. DNA was transferred to Zeta Probe membrane (Bio-Rad Laboratories), cross-linked, and probed using the Batf cDNA as previously described (Williams et al., 2001).

Immunoblot.

Protein was isolated from stimulated splenocytes using RIPA buffer supplemented with protease inhibitors. Immunoblots to detect Batf-HA and control of Hsp90 proteins were performed as previously described (Thornton et al. 2006; Zhu et al. 2007).

Flow cytometry.

Flow cytometry was performed using an FC500 (Beckman Coulter) or Canto II (BD) cytometer and data analyzed using FCSExpress3 (De Novo Software). Fc block prevented nonspecific Ab interactions. mAbs used are indicated in the figure legends. The identity and source of all Abs are listed in Table S2. Intracellular staining to detect CD4+ T cells reactive with IFN-γ, IL-4, and IL-17A mAbs was performed as previously described (Wang et al., 2009).

Cell culture.

Splenocytes were prepared and CD4+ T and resting B cells isolated using CD4 (L3T4) and CD43 magnetic bead separation, respectively (Miltenyi Biotec). Cells were cultured as previously described (Snapper et al., 1988; Williams et al., 2003). Stimulations and in vitro skewing conditions are detailed in figure legends. B cell proliferation was measured using a BrdU cell proliferation kit (Millipore).

Analysis of RNA.

Semi-qPCR was performed as previously described (Thornton et al., 2006) and qPCR was performed with SYBR green (Roche) and a real-time PCR system (7300; Applied Biosystems). Primers are listed in figure legends or Table S1. Results were normalized to Hprt or β-actin expression. ΔΔCt values were used to calculate relative expression of each mRNA.

ELISA.

Blood, collected by cardiac puncture, was allowed to clot at room temperature in a Microtainer Serum Separator tube (BD). Serum was isolated by centrifugation. ELISA for IgG2c and IgE was performed using OptEIA kits (BD). ELISA for IgG1, IgA, and IgM used mAb indicated in Table S2. Serum was diluted in Assay Diluent (BD) and applied to Ab-coated 96-well MaxiSorp plates (Nunc), and reactions were visualized with streptavidin-HRP and TMB substrate reagent (BD). ELISA plates and protocols for detecting sRBC-specific IgM and IgG were obtained from Life Diagnostics and IgG1 was obtained from Southern Biotech. ELISA to detect TNP-LPS–specific IgG1 used plates coated with 10 µg/ml TNP-BSA (Biosearch Technologies).

In vivo antigen stimulation and IHC.

For T cell–dependent response, mice were injected i.p. with 5 × 108 sRBC in 200 µl PBS or PBS alone (mock). On day 8, sera were isolated for ELISA and spleen tissue was processed for IHC as previously described (Zhu et al., 2007). For T cell–independent response, mice were injected with 30 µg TNP-LPS (Sigma-Aldrich) or an equal volume of PBS. On day 5, sera were isolated for ELISA and spleen tissue was processed for IHC. GCs were detected using biotinylated PNA (1:300) and visualized with RTU Vectastain, ABC reagent, and DAB peroxidase substrate (Vector Laboratories). For fluorescent detection, antigens were retrieved by boiling in citrate buffer (Vector Laboratories) and sections blocked using anti–mouse IgG and an avidin/biotin blocking kit (Vector Laboratories). B cells were detected using rat anti–mouse CD45R (B220; 1:200) and FITC rabbit anti–rat IgG (1:200). GC B cells were identified using biotinylated PNA (1:100) and Texas Red conjugated avidin (Vector Laboratories; 1:200). Nuclei were stained with DAPI (Santa Cruz Biotechnology, Inc.). Ig-producing cells were detected using anti-IgG1 (1:400) or anti-IgG2c (1:200), a biotin rabbit anti–goat (1:200) Ab, and a NovaRed alkaline phosphatase substrate kit (Vector Laboratories).

Adoptive transfer and immunization.

On day 0, 5 × 106 CD4+ T cells from Batf+/+ or BatfΔZ/ΔZ mice were injected i.v. into T cell–deficient mice (B6.129P2-Tcrbtm1Mom Tcrdtm1Mom/J; JAX) and animals immunized i.p. with 5 × 108 sRBC in 100 µl PBS. Mice were sacrificed on day 8 for the analysis of T cell subsets and Ig production.

Chemotaxis assay.

Chemotaxis assays were performed and analyzed as described previously (Lim et al., 2004). In brief, 5 × 105 lymphocytes from PP were added to the upper chamber of Transwell inserts (Corning) and allowed to migrate to media in a lower chamber containing 2.5 µg/ml rmCXCL13. After 3 h, cells were collected and analyzed by flow cytometry.

Online supplemental material.

Fig. S1 shows the expression of the mouse Batf gene in Batf+/+, BatfKI/KI, and BatfΔZ/ΔZ mice. Fig. S2 compares the thymic T cell profile, the T reg cell profile, and the in vitro T reg, Th1, and Th17 differentiation profiles of Batf+/+ and BatfΔZ/ΔZ mice. Table S1 contains sequences of the oligonucleotide primers used in this study. Table S2 provides information on the antibodies used in this study. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20091548/DC1.

Acknowledgments

The authors thank J.P. Robinson, K. Ragheb, C. Holdman, and the Purdue University Cytometry Laboratory for assistance with flow cytometry and S. Konieczny, J. Hallett, A. Kaufman, and the Transgenic Mouse Core Facility of the Purdue University Center for Cancer Research for the generation of Batf KI mice. Special thanks are extended to K. Williams and A. Zullo, whose Ph.D. thesis research provided a foundation for this work.

This study was supported by National Institutes of Health grants CA782464 and CA114381 (E. Taparowsky) and National Institutes of Health grant AI074745 (C.H. Kim). Predoctoral student support was provided by National Institutes of Health grant T32 GM08298 (K.L. Jordan-Williams and M.R. Logan) and by a Career Development Award from the Indiana CTSI (National Institutes of Health grant 5TL1 RR025759 to M.R. Logan).

The authors have no conflicting financial interests.

Footnotes

Abbreviations used:

Ab
antibody
AP-1
activator protein 1
bZIP
basic leucine zipper
CSR
class-switch recombination
GC
germinal center
HA
hemagglutinin antigen
IHC
immunohistochemistry
PNA
peanut agglutinin
PP
Peyer’s patch
qPCR
quantitative PCR
sRBC
sheep RBC
Tfh
follicular Th cell
T reg cell
regulatory T cell

References

  • Amoutzias G.D., Bornberg-Bauer E., Oliver S.G., Robertson D.L. 2006. Reduction/oxidation-phosphorylation control of DNA binding in the bZIP dimerization network. BMC Genomics. 7:107–117. doi: 10.1186/1471-2164-7-107. [PMC free article] [PubMed] [Cross Ref]
  • Aronheim A., Zandi E., Hennemann H., Elledge S.J., Karin M. 1997. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol. Cell. Biol. 17:3094–3102. [PMC free article] [PubMed]
  • Dorsey M.J., Tae H.J., Sollenberger K.G., Mascarenhas N.T., Johansen L.M., Taparowsky E.J. 1995. B-ATF: a novel human bZIP protein that associates with members of the AP-1 transcription factor family. Oncogene. 11:2255–2265. [PubMed]
  • Echlin D.R., Tae H.J., Mitin N., Taparowsky E.J. 2000. B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos. Oncogene. 19:1752–1763. doi: 10.1038/sj.onc.1203491. [PubMed] [Cross Ref]
  • Eferl R., Wagner E.F. 2003. AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer. 3:859–868. doi: 10.1038/nrc1209. [PubMed] [Cross Ref]
  • Fairfax K.A., Kallies A., Nutt S.L., Tarlinton D.M. 2008. Plasma cell development: from B-cell subsets to long-term survival niches. Semin. Immunol. 20:49–58. doi: 10.1016/j.smim.2007.12.002. [PubMed] [Cross Ref]
  • Hess J., Angel P., Schorpp-Kistner M. 2004. AP-1 subunits: quarrel and harmony among siblings. J. Cell Sci. 117:5965–5973. doi: 10.1242/jcs.01589. [PubMed] [Cross Ref]
  • Hildner K., Edelson B.T., Purtha W.E., Diamond M., Matsushita H., Kohyama M., Calderon B., Schraml B.U., Unanue E.R., Diamond M.S., et al. 2008. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 322:1097–1100. doi: 10.1126/science.1164206. [PMC free article] [PubMed] [Cross Ref]
  • Honjo T., Muramatsu M., Fagarasan S. 2004. AID: how does it aid antibody diversity? Immunity. 20:659–668. doi: 10.1016/j.immuni.2004.05.011. [PubMed] [Cross Ref]
  • Iacobelli M., Wachsman W., McGuire K.L. 2000. Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein. J. Immunol. 165:860–868. [PubMed]
  • Johansen L.M., Deppmann C.D., Erickson K.D., Coffin W.F., III, Thornton T.M., Humphrey S.E., Martin J.M., Taparowsky E.J. 2003. EBNA2 and activated Notch induce expression of BATF. J. Virol. 77:6029–6040. doi: 10.1128/JVI.77.10.6029-6040.2003. [PMC free article] [PubMed] [Cross Ref]
  • Jung M., Sabat R., Krätzschmar J., Seidel H., Wolk K., Schönbein C., Schütt S., Friedrich M., Döcke W.D., Asadullah K., et al. 2004. Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy. Eur. J. Immunol. 34:481–493. doi: 10.1002/eji.200324323. [PubMed] [Cross Ref]
  • Kim C.H., Rott L.S., Clark-Lewis I., Campbell D.J., Wu L., Butcher E.C. 2001. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center–localized subset of CXCR5+ T cells. J. Exp. Med. 193:1373–1381. doi: 10.1084/jem.193.12.1373. [PMC free article] [PubMed] [Cross Ref]
  • King C. 2009. New insights into the differentiation and function of T follicular helper cells. Nat. Rev. Immunol. 9:757–766. doi: 10.1038/nri2644. [PubMed] [Cross Ref]
  • Kronenberg M., Engel I. 2007. On the road: progress in finding the unique pathway of invariant NKT cell differentiation. Curr. Opin. Immunol. 19:186–193. doi: 10.1016/j.coi.2007.02.009. [PubMed] [Cross Ref]
  • Li J., Peet G.W., Balzarano D., Li X., Massa P., Barton R.W., Marcu K.B. 2001. Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition. J. Biol. Chem. 276:18579–18590. doi: 10.1074/jbc.M100846200. [PubMed] [Cross Ref]
  • Lim H.W., Hillsamer P., Kim C.H. 2004. Regulatory T cells can migrate to follicles upon T cell activation and suppress GC-Th cells and GC-Th cell-driven B cell responses. J. Clin. Invest. 114:1640–1649. [PMC free article] [PubMed]
  • Lim J., Hao T., Shaw C., Patel A.J., Szabó G., Rual J.F., Fisk C.J., Li N., Smolyar A., Hill D.E., et al. 2006. A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell. 125:801–814. doi: 10.1016/j.cell.2006.03.032. [PubMed] [Cross Ref]
  • Nurieva R.I., Chung Y., Hwang D., Yang X.O., Kang H.S., Ma L., Wang Y.H., Watowich S.S., Jetten A.M., Tian Q., Dong C. 2008. Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages. Immunity. 29:138–149. doi: 10.1016/j.immuni.2008.05.009. [PMC free article] [PubMed] [Cross Ref]
  • Ozaki K., Spolski R., Feng C.G., Qi C.F., Cheng J., Sher A., Morse H.C., III, Liu C., Schwartzberg P.L., Leonard W.J. 2002. A critical role for IL-21 in regulating immunoglobulin production. Science. 298:1630–1634. doi: 10.1126/science.1077002. [PubMed] [Cross Ref]
  • Park S.R., Zan H., Pal Z., Zhang J., Al-Qahtani A., Pone E.J., Xu Z., Mai T., Casali P. 2009. HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nat. Immunol. 10:540–550. doi: 10.1038/ni.1725. [PMC free article] [PubMed] [Cross Ref]
  • Raivich G., Behrens A. 2006. Role of the AP-1 transcription factor c-Jun in developing, adult and injured brain. Prog. Neurobiol. 78:347–363. doi: 10.1016/j.pneurobio.2006.03.006. [PubMed] [Cross Ref]
  • Rincón M., Flavell R.A. 1996. Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells. Mol. Cell. Biol. 16:1074–1084. [PMC free article] [PubMed]
  • Schraml B.U., Hildner K., Ise W., Lee W.L., Smith W.A., Solomon B., Sahota G., Sim J., Mukasa R., Cemerski S., et al. 2009. The AP-1 transcription factor Batf controls T(H)17 differentiation. Nature. 460:405–409. [PMC free article] [PubMed]
  • Senga T., Iwamoto T., Humphrey S.E., Yokota T., Taparowsky E.J., Hamaguchi M. 2002. Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells. Oncogene. 21:8186–8191. doi: 10.1038/sj.onc.1205918. [PubMed] [Cross Ref]
  • Snapper C.M., Finkelman F.D., Stefany D., Conrad D.H., Paul W.E. 1988. IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide. J. Immunol. 141:489–498. [PubMed]
  • Su Z.Z., Lee S.G., Emdad L., Lebdeva I.V., Gupta P., Valerie K., Sarkar D., Fisher P.B. 2008. Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN). Proc. Natl. Acad. Sci. USA. 105:20906–20911. doi: 10.1073/pnas.0807975106. [PubMed] [Cross Ref]
  • Thornton T.M., Zullo A.J., Williams K.L., Taparowsky E.J. 2006. Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro. Eur. J. Immunol. 36:160–169. doi: 10.1002/eji.200535215. [PubMed] [Cross Ref]
  • Vogelzang A., McGuire H.M., Yu D., Sprent J., Mackay C.R., King C. 2008. A fundamental role for interleukin-21 in the generation of T follicular helper cells. Immunity. 29:127–137. doi: 10.1016/j.immuni.2008.06.001. [PubMed] [Cross Ref]
  • Wagner E.F., Eferl R. 2005. Fos/AP-1 proteins in bone and the immune system. Immunol. Rev. 208:126–140. doi: 10.1111/j.0105-2896.2005.00332.x. [PubMed] [Cross Ref]
  • Wang C., Kang S.G., Lee J., Sun Z., Kim C.H. 2009. The roles of CCR6 in migration of Th17 cells and regulation of effector T-cell balance in the gut. Mucosal Immunol. 2:173–183. doi: 10.1038/mi.2008.84. [PMC free article] [PubMed] [Cross Ref]
  • Williams K.L., Nanda I., Lyons G.E., Kuo C.T., Schmid M., Leiden J.M., Kaplan M.H., Taparowsky E.J. 2001. Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus. Eur. J. Immunol. 31:1620–1627. doi: 10.1002/1521-4141(200105)31:5<1620::AID-IMMU1620>3.0.CO;2-3. [PubMed] [Cross Ref]
  • Williams K.L., Zullo A.J., Kaplan M.H., Brutkiewicz R.R., Deppmann C.D., Vinson C., Taparowsky E.J. 2003. BATF transgenic mice reveal a role for activator protein-1 in NKT cell development. J. Immunol. 170:2417–2426. [PubMed]
  • Zhou L., Chong M.M., Littman D.R. 2009. Plasticity of CD4+ T cell lineage differentiation. Immunity. 30:646–655. doi: 10.1016/j.immuni.2009.05.001. [PubMed] [Cross Ref]
  • Zhu L., Shi G., Schmidt C.M., Hruban R.H., Konieczny S.F. 2007. Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia. Am. J. Pathol. 171:263–273. doi: 10.2353/ajpath.2007.061176. [PubMed] [Cross Ref]
  • Zullo A.J., Benlagha K., Bendelac A., Taparowsky E.J. 2007. Sensitivity of NK1.1-negative NKT cells to transgenic BATF defines a role for activator protein-1 in the expansion and maturation of immature NKT cells in the thymus. J. Immunol. 178:58–66. [PubMed]

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