B cell-intrinsic expression of the HuR RNA-binding protein is required for the T cell-dependent immune response in vivo

Abstract

The HuR RNA binding protein post-transcriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted antibodies, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all antibody isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity antibodies after immunization with a T cell-dependent antigen. This failure of HuRΔ/Δ mice to mount a T-cell dependent antibody response contrasts with the ability of HuRΔ/Δ B cells to become “GC-like” in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of co-stimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T-cell dependent immune responses in vivo.

Introduction

The development and function of cells such as B lymphocytes require finely tuned and dynamic changes in gene expression. These changes are instigated by cell-intrinsic and cell-extrinsic factors and controlled by a combination of transcriptional, post-transcriptional, and post-translational mechanisms. Post-transcriptional mechanisms allow cells to rapidly alter protein expression by modulating the stability and/or translation of specific mRNAs, or by modifying mRNA processing events, such as alternative polyadenylation and splicing. RNA binding proteins (RBPs) are major post-transcriptional regulators of gene expression. RBPs bind their target sequences in 3′ untranslated regions (3′UTRs) or internal elements of mRNAs to positively or negatively regulate mRNA processing, stability, and/or translation in cellular context-dependent manners (1, 2). RBP function is regulated via changes in RBP abundance, subcellular localization, post-translational modification, and interactions with other mRNA-binding factors, permitting quick and specific changes in gene expression in response to developmental cues or other stimuli (1). Although RBPs are appreciated to play a role in lymphocyte biology and function, their role in regulating B cell development and function remains poorly understood (3).

B lymphocytes are comprised of two main populations, B-1 and B-2 cells, that develop through distinct programs that each link cell-intrinsic and cell-extrinsic signals with cellular survival, proliferation, and continued differentiation. The larger B-2 cell population arises in bone marrow (BM) from common lymphoid progenitors (CLPs) starting shortly after birth and extending throughout life (4, 5). The smaller B-1 population develops mainly from fetal liver precursors during fetal and early neonatal development, although these B-1 B cells may continue to develop at a low level in adult BM (4, 6). Developmental-stage specific assembly and expression of IgH and Igκ or Igλ light chain (IgL) genes results in expression of BCRs on immature B cells (5). Depending on their antigen specificity, these BCRs signal gene expression changes to induce apoptosis or mediate differentiation of immature transitional B cells (7, 8). Transitional B-2 cells migrate to the spleen and differentiate into mature naive quiescent marginal zone (MZ) or Follicular (Fo) B cells that express BCRs and traffic throughout lymphatic tissues (4, 7). In contrast, transitional B-1 cells migrate to serous cavities where they become mature B-1 cells that continually proliferate and secrete antibodies (4, 6).

B lymphocytes mediate protective humoral immunity through their ability to express cell surface BCRs and secrete antibodies that bind antigens. B-2 cells recognize and respond to antigens through T cell-dependent or -independent mechanisms (4, 9). During a T cell-dependent germinal center (GC) immune response, Fo B cells are activated by encounter with antigen, usually on the surface of a professional antigen-presenting cell, such as a dendritic cell (10). These B-2 cells induce their expression of surface molecules and cytokines that further activate antigen-primed CD4⁺ T cells and promote their differentiation into T follicular helper (Tfh) cells (11–13). Tfh cells in turn further activate B cells and direct them to become GC B cells (10, 12, 14–17). GC B cells rapidly proliferate, conduct IgH isotype switching and Ig somatic mutation, and alter gene expression to differentiate into short-lived high-affinity antibody-secreting plasma cells or long-lived memory cells (14). The GC reaction is critical for adaptive responses and affinity maturation of antibodies against an enormous number and variety of antigens, as well as for more rapid secondary responses against previously encountered antigens (14). B-1 cells predominantly recognize and respond to antigens through T cell-independent mechanisms that occur outside of GCs and largely do not involve antigen-driven IgH isotype switching or Ig mutation, although T-dependent responses by B1 B cells do occur (4, 6). B-1 cells express BCRs that bind common pathogen epitopes and also spontaneously secrete antibodies to protect against commensal and other opportunistic bacteria (4, 6). Throughout life, B-1 and B-2 cells function together to protect host organisms from universally encountered common foreign organisms and random unanticipated infections.

The development and function of B cells requires exquisite regulation of gene expression to coordinate cellular survival, proliferation, and differentiation. The ubiquitously expressed protein HuR (also called Elavl1) controls post-transcriptional expression of many genes that mediate these cellular processes (18–22). Mice with germline or postnatal global deletion of HuR are not viable (23, 24), while global HuR deletion in adult mice causes increased apoptosis and loss of immature but not mature B cells (24). Since global HuR deletion leads to death of mice within 14 days (24), the role of HuR in B cell function is not known. To determine the roles of HuR in B cell development and function, we established and analyzed mice with B lineage-specific deletion of HuR initiating in pro-B cells. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms or cellular processes. We discovered that HuR is required for normal numbers of splenic B-2 cells and peritoneal B-1 cells; however, HuR is not necessary for B cell development per se, enabling us to study roles of HuR in B cell function. Upon in vitro stimulation of splenic B cells, HuR is dispensable for B cell survival, isotype switching, and induction of GC B cell markers, and HuRΔ/Δ B cells exhibit only mild defects in proliferation and Ig secretion. In contrast, HuRΔ/Δ mice have dramatically low serum titers of all antibody isotypes and severely decreased GC B cells, GC structures, Tfh cells, and high-affinity antibodies after immunization with a T-cell dependent antigen. These data indicate HuR expression in B lineage cells is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of co-stimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. Since in vivo immune responses require activated B cells to undergo more nuanced and sophisticated processes than can be recapitulated in vitro, HuRΔ/Δ mice provide a model to better understand B cell-intrinsic contributions to T-cell dependent immune responses in vivo.

Materials and Methods

Mice

All mice were on a 129S1/SvImJ and C57BL6 mixed background, and housed, bred, and used under pathogen-free conditions at the Children’s Hospital of Philadelphia (CHOP). HuR^flox/flox mice were provided by Dr. Timothy Hla (24) and bred with Mb1-Cre mice (25), p53^flox/flox mice (26), or V_H147 (anti-GPI) IgH transgenic mice (27). For most breedings, Mb1-Cre males were bred to Cre-negative females since Mb1-Cre is expressed in the female germline (28). However, Mb1-Cre⁺HuR^flox/flox females were bred with HuR^flox/flox males to generate HuR^flox/− mice with one germline HuR-deleted allele. Animal husbandry and experiments were performed in accordance with national guidelines and regulations and were approved by the CHOP Institutional Animal Care and Use Committee.

Flow cytometry and cell sorting

Peritoneal lymphocytes were obtained by peritoneal lavage with PBS, or cells were isolated from spleen or bone marrow as previously indicated (28, 29). Equal numbers of cells were incubated with live dead viability dye (Life technologies) and then stained with antibodies against surface antigens in PBS with 3% FBS. Following washing, cells were either analyzed directly or treated with cytofix/cytoperm buffer (BD biosciences) and then stained with antibodies against intracellular antigens. The antibodies used are listed in Supplemental Table 1. Samples were run on a FACSCalibur or LSR Fortessa cytometer (BD Biosciences) and analyzed with Flowjo software (Treestar). Sorting was performed on a MoFlo Astrios (Beckman-Coulter). AnnexinV assays were performed according to manufacturer instructions (BD Biosciences) except that annexinV antibody was used at a 1:100 dilution. BrdU incorporation assays were performed by incubating cells in medium containing 10uM BrdU and staining as instructed (BD Biosciences).

ELISA assays

96-well polystyrene assay plate (Corning) medium binding were coated with goat-mouse Ig(H+L), NP4-BSA, or NP33-BSA (Biosearch Technologies). After blocking with 2% BSA, serum and unlabeled isotype standard dilutions were applied. NP standard kindly provided by Garnett H. Kelsoe. Detection of antibody isotype was achieved with appropriate goat anti-mouse conjugated antibody (see supplemental table 1). TMB substrate (OptiEIA, BD) was used to develop according to manufacturer’s instructions and 2M sulphuric acid to stop the reaction. Signal was read at 450nm on a Molecular Devices Emax.

In vitro stimulation

Splenic B cells were isolated using EasySep negative selection B cell isolation kits (Stem Cell Technologies) or follicular B cells were isolated by positive selection using biotinylated anti-mouse CD23 (B3B4, BD) in conjunction with streptavidin microbeads (Miltenyi Biotec) on an LS column (Miltenyi Biotec). Isolated cells were labeled with CFSE (Life Technologies) as described (30, 31). Equal numbers of cells were stimulated for indicated time periods with 25μg/ml LPS (0111:B4, Sigma) and 80ng/ul recombinant mouse IL-4 (R&D Systems), or 10μg/ml anti-mouse CD40 (HM40-3, Biolegend) and 10μg/ml F(ab′)2 fragment goat anti-mouse IgM (Jackson Immunoresearch) with or without 50ng/ml IL-21 (Shenandoah Biotechnolgoy). Where not specified, cells were stimulated in RPMI-1640 supplemented with 10% heat-inactivated FBS, antibiotics, 50μM β-mercaptoethanol, 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate, and non-essential amino acids.

Immunization

NP-OVA is the hapten 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to the ovalbumin carrier protein. NP-OVA (Biosearch Technologies) resuspended in PBS was added to a solution of 10% aluminum potassium sulfate and precipitated by dropwise addition of potassium hydroxide. All solutions were sterilized and precipitate was washed thoroughly with sterile PBS before injection of 50μg NP-OVA in alum into the peritoneal cavity of 8-week old HuRΔ/Δ or HuRf/f mice. Injected mice were euthanized and analyzed at 9 or 14 days post-immunization.

Western blotting

Cells were resuspended in a Tween-20 containing lysis buffer, and sonicated at intervals of 30 sec on 30 sec off for 5 min at 4°C. Cells were incubated for 5 min on ice then spun to remove insoluble material. 30μg lysate prepared under reducing conditions were loaded into each well of a NuPage 10% Bis-Tris gel (Life Technologies). Electrophoresed proteins were transferred to PVDF, and membranes were blocked with Odyssey blocking buffer (Li-Cor) and incubated with anti-HuR antibody (3A2, Santa Cruz) or anti-β-actin (polyclonal, Sigma) for 1h at RT or overnight at 4°C. After washing, blots were incubated with IRDye800 secondary antibodies (LiCor) for 1h at RT. Following washing, blots were scanned on an Odyssey infrared scanner (Li-Cor).

qPCR and qRT-PCR

Genomic DNA was isolated as described (32). Total RNA was isolated using Trizol reagent (Life technologies) and DNase treated according to manufacturer directions (Promega), primed with random nonamer (New England Biolabs), and reverse transcribed with M-MuLV (NEB). qPCR and qRT-PCR reactions were performed with SYBR green mastermix (Applied Biosystems) and run on an Applied Biosystems 7500 Fast machine. The primers for qPCR and qRT-PCR reactions are found in Supplemental table 2.

RNA-Immunoprecipitation

RNA-IPs for HuR were performed as previously described (33). Per IP, 100 μl protein G dynabeads (Life Technologies) were incubated with 15μg anti-HuR (3A2, Santa Cruz) or 15μg normal mouse IgG (Santa Cruz). B cells stimulated for 72h with LPS + IL-4 (6–9 × 10⁷ cells per IP pair) were lysed in polysome lysis buffer containing 0.5% NP-40 supplemented with protease inhibitor (Roche) and RNase inhibitor (NEB). Half of each sample was added to IgG or HuR-coated dynabeads and incubated with shaking for 2h at 4°C. After washing of beads, RNA was extracted using Trizol as for total RNA.

LC/MS/MS proteomics workflow

Cells were lysed in urea buffer (34), protein concentration was measured via micro BCA assay (Thermo), and peptides were prepared. 15×10^6 stimulated B cells at the start of stimulation yielded ~0.25mg of total protein. The proteins were digested in solution with trypsin (protein:trypsin=50:1) overnight and the tryptic peptides were desalted via SepPak (50mg SepPak, Waters). Tryptic digests were lyophilized and dissolved in solvent A (2% acetonitrile, 5mM ammonium formate, pH 10). 48ug of peptides were injected to H-Class HPLC (Waters) and were separated in C18 column at 300uL/min, 39min gradient (ZORBAX 300 Extend-C18, 2.1× 100mm, 3.5-Micron 300A, P.N. 761775-902). The separated peptides were collected in a 96 well plates for 39 fractions and 12 combined fractions were lyophilized for LCMS analysis. The peptides were dissolved in 0.1% TFA/water and analyzed by LC-MS/MS on a hybrid LTQ Orbitrap Elite mass spectrometer (Thermofisher Scientific) coupled with a nanoLC Ultra (Eksigent). Peptides were separated by reverse phase (RP)-HPLC on a nanocapillary column, 75 um ID × 15 cm Reprosil-pur 3um (Dr. Maisch, Germany) in a Nanoflex chip system (Eksigent). Mobile phase A consisted of 0.1% formic acid (Thermofisher Scientific) and mobile phase B of 0.1% formic acid/80% acetonitrile. Peptides were eluted into the mass spectrometer at 300 nl/min with each RP-LC run comprising a 90 minute gradient from 10 to 25 % B in 65 min, 25–40%B in 25 min, followed by column re-equilibration. The mass spectrometer was set to repetitively scan m/z from 300 to 1800 (R = 240,000 for LTQ-Orbitrap Elite) followed by data-dependent MS/MS scans on the twenty most abundant ions, with a minimum signal of 1500, dynamic exclusion with a repeat count of 1, repeat duration of 30s, exclusion size of 500 and duration of 60s, isolation width of 2.0, normalized collision energy of 33, and waveform injection and dynamic exclusion enabled. FTMS full scan AGC target value was 1e6, while MSn AGC was 1e4, respectively. FTMS full scan maximum fill time was 500 ms, while ion trap MSn fill time was 50 ms; microscans were set at one. FT preview mode; charge state screening, and monoisotopic precursor selection were all enabled with rejection of unassigned and 1+ charge states.

Analysis of proteomics data

Whole proteomes were analyzed together in MaxQuant version 1.5.1.2, using the Uniprot complete mouse reference proteome including isoforms (updated Apr 9, 2015) and common lab contaminants with a minimum peptide length of 7 amino acids and 1% false discovery rate; re-quantify and match between runs were turned off. Label-free quantification was used to identify MS counts for relative abundance.

Statistics

Except where otherwise indicated, p-values were generated by two-tailed unpaired Student’s t test using Prism (GraphPad Software). Error bars for all figures indicate the standard error and asterisks above data elements indicate a significant p value as follows: *p<0.01, **p<0.05, ***p<0.001.

Results

B lineage-specific deletion of HuR leads to decreased numbers of immature and mature B cells

To identify roles of the HuR RBP in B lymphocyte development and function, we generated and analyzed Mb1Cre:HuR^flox/flox (HuRΔ/Δ) mice with B lineage-specific deletion of the HuR gene initiating in pro-B cells. We performed cell counting and flow cytometry analyses for B cell developmental stage-specific markers on BM and spleen cells of 4 to 6 week-old HuRΔ/Δ mice and littermate HuR^flox/flox (HuRf/f or WT) mice. Compared to control mice, HuRΔ/Δ mice have 1.4-fold and 1.9-fold reduction in the numbers of B lymphocytes in their bone marrow and spleen, respectively (Fig. 1A). Although HuRΔ/Δ mice have higher than normal numbers of BM pro-B cells, they have reduced numbers of BM pre-B cells and BM B cells that represent immature B cells and recirculating mature B cells (Fig. 1B). We also evaluated cell surface expression of CD43, BP-1, and CD24 to divide BM B-2 cells into subpopulations referred to as “Hardy fractions” (35). While HuRΔ/Δ mice have normal numbers of cells in each of the pro-B cell subpopulations (Hardy fractions A, B, C, and C′), they have fewer cells in each of the pre-B cell and mature B cell subpopulations (D, E, and F) (Fig. 1C). The spleens of HuRΔ/Δ mice contain reduced numbers of T1 transitional, T2 transitional, MZ, and Fo B cells (Fig. 1D). HuRΔ/Δ mice have normal ratios of Igκ⁺ to Igλ⁺ B cells in their bone marrow and spleen (data not shown). We detected comparable substantial deletion of floxed HuR exons in pro-B, pre-B and mature B-2 cells of HuRΔ/Δ mice, and confirmed substantial loss of HuR protein in HuRΔ/Δ mature B cells (Supplemental Fig. 1A, 1B). Although Cre expression can negatively affect development independent of gene deletion (36, 37), we found no differences in numbers of mature B cells or B cells at each developmental stage in Mb1-Cre mice as compared to WT controls (Supplemental Fig. 1C). These data indicate that while HuR is not necessary for BM B-2 cell differentiation per se, HuR is required to generate normal numbers of B lineage cells at each developmental stage beyond the pro-B cell stage.

FIGURE 1.

B lineage-specific deletion of HuR leads to decreased numbers of immature and mature B cells. (A – D) Representative flow cytometry analysis and quantification of B lineage cell populations in the BM and spleens of 4–6 week old HuRΔ/Δ and HuRf/f mice. The population of live cells analyzed is depicted above each flow plot and the percentages of analyzed cells in each of the indicated gates are shown. Data are from three or more independent experiments conducted on a total of at least five HuRΔ/Δ mice and five control littermate HuRf/f mice. (A) B220⁺ cells in BM and spleen. (B) BM B220⁺CD43⁺ pro-B, B220⁺CD43⁻ pre-B, and B220⁺IgM⁺ cells. (C) BM Hardy fractions A (BP1⁻HSA⁻), B (BP1⁻HSA⁺), C (BP1⁺), C′ (BP1⁺HSA⁻), D (B220⁺IgM⁻), E (B220⁺IgM⁺), and F (B220-high IgM⁺). (D) Splenic IgM-high T1 (CD23⁻CD21⁻), T2 (CD23⁺CD21⁺), MZ (CD23⁻CD21⁺), and Fo (IgM-low CD21⁻) B cells.

Deletion of HuR impairs survival of B cells developing in the bone marrow

The decreased numbers of B-2 lineage cells at all developmental stages beyond the pro-B cell stage could reflect important roles of HuR in promoting IgH gene assembly, proliferation, and/or survival during B cell development. To investigate potential roles of HuR in these processes, we first generated and analyzed HuRΔ/Δ mice expressing the V_H147 IgH transgene (IgHTg) that blocks IgH gene assembly and enables formation of pre-BCRs to signal proliferation and survival in the absence of IgH gene recombination (27). The numbers of BM pre-B cells and B cells in the BM and spleens were all lower in 4 to 6 week-old IgHTg:HuRΔ/Δ mice relative to age-matched control IgHTg:HuRf/f mice (Fig. 2A). Since these differences were comparable to those between HuRΔ/Δ and HuRf/f mice (Fig. 1A), our data indicate that IgH gene assembly is grossly normal in HuRΔ/Δ mice. We next compared cell cycle profiles of pro-B cells and the most proliferating immature B cell subpopulation (fraction C) between HuRΔ/Δ and HuRf/f mice. By combining cell surface staining of B cell developmental markers with DNA content analysis, we detected similar percentages of pro-B cells and fraction C cells in S phase between HuRΔ/Δ and control HuRf/f mice (Fig. 2B). While these data suggest normal proliferation of immature HuRΔ/Δ B-2 cells, they cannot identify potential changes where cells spend proportionally more time in each cell cycle phase. Thus, we also quantified immature and mature splenic B-2 cell populations in HuRΔ/Δ and HuRf/f mice at 12 to 14 weeks of age when the mouse B cell compartment is replete (38), reasoning that the number of splenic B-2 cells in HuRΔ/Δ mice should reach the number of cells in WT mice at this age if HuRΔ/Δ B cells merely transit the cell cycle more slowly. We found that B cell numbers in the BM and spleens of these older HuRΔ/Δ mice are not restored to normal (Fig. 2C), indicating that the reduced number of splenic HuRΔ/Δ B cells is not caused by decreased proliferation of immature HuRΔ/Δ B-2 cells. Next, we assayed apoptosis of pro-B and pre-B cells in HuRΔ/Δ and HuRf/f mice by performing flow cytometry for B cell developmental stage-specific surface markers and the annexinV protein that is expressed on apoptotic and dead cells. We detected a near significant increase in the fraction of annexinV⁺ pre-B cells in HuRΔ/Δ mice as compared to HuRf/f mice (Fig. 2D), consistent with a role for HuR in promoting survival of immature B cells. Since HuR has been proposed to counter pro-apoptotic p53 signaling in HSCs and ELPs (24), we generated and analyzed Mb1Cre:p53^flox/floxHuR^flox/flox (p53Δ/Δ:HuRΔ/Δ) mice and control Mb1Cre:p53^flox/flox (p53Δ/Δ) mice. We detected no significant differences in the numbers of BM pro-B cells and pre-B cells between p53Δ/ΔHuRΔ/Δ and p53Δ/Δ mice, (Fig. 2E), indicating that HuR promotes survival of pre-B cells by countering p53-depedendent pro-apoptotic signals. Notably, deletion of p53 in HuRΔ/Δ B lineage cells did not restore to normal the numbers of mature splenic B-2 cells (Fig. 2E), despite inactivation of p53 rescuing immature B-2 cell numbers. Collectively, these data indicate that HuR supports B-2 lineage cells via developmental stage-specific mechanisms or cellular processes that antagonize p53-dependent elimination of immature but not mature B-2 cells.

FIGURE 2.

HuR protects immature B cells from p53-dependent elimination. (A – E) Representative flow cytometry analysis and quantification of B lineage cell populations, cell cycle distribution, or apoptosis in BM and spleens of the indicated mice. The population of live cells analyzed is depicted above each flow plot and the percentages of analyzed cells in each of the indicated gates are shown. Data are from three or more independent experiments conducted on a total of five or more mice of each genotype. (A) B220⁺CD43⁺ pro-B, B220⁺CD43⁻ pre-B, and B220⁺IgM⁺ B cells in BM or B220⁺IgM⁺ B cells in spleens of 4–6 week old IgHTg HuRΔ/Δ or IgHTg HuRf/f mice. (B) DNA content cell cycle profile of pro-B cells or Hardy fraction C cells (see Fig 1C) from 4–6 week old HuRΔ/Δ or HuRf/f mice. (C) Flow cytometry analysis of pro-B, pre-B, and B220⁺IgM⁺ B cells in BM or B220⁺IgM⁺ cells in spleens of 12–14 week old HuRΔ/Δ or HuRf/f mice. (D) AnnexinV staining of BM pro-B or pre-B cells of 4–6 week old HuRΔ/Δ or HuRf/f mice. (E) pro-B, pre-B, and B220⁺IgM⁺ B cells in BM or B220⁺IgM⁺ B cells in spleens of 4–6 week old p53Δ/Δ or p53Δ/Δ HuRf/f mice.

In vitro stimulated HuRΔ/Δ B cells exhibit a slight proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted antibodies

Despite the reduced numbers of immature and mature B cells in HuRΔ/Δ mice, we found that HuR is not necessary for the development of mature splenic B cells. Thus, we investigated roles of HuR in B cell function beginning with tractable in vitro approaches to monitor survival, proliferation, IgH isotype switching, and gene expression changes during B cell activation. We isolated total splenic B cells from HuRΔ/Δ and HuRf/f mice and incubated equal numbers of cells with LPS and IL-4, which mimics T cell-independent activation of B cells. Cell counting showed that HuRΔ/Δ cells expanded less than HuRf/f cells after 72 hours of stimulation (Fig. 3A). Although Cre expression can antagonize cell growth (39, 40), Mb1Cre⁺ B cells expand normally following addition of LPS and IL-4 (Supplemental Fig. 2A), suggesting that in vitro stimulated HuRΔ/Δ B cells exhibit increased apoptosis and/or reduced proliferation. To determine whether LPS- and IL-4-stimulated splenic HuRΔ/Δ B cells exhibit increased apoptosis, we labeled HuRΔ/Δ and HuRf/f cells with an amine-reactive viability dye to identify dead cells. We detected similar frequencies of dead cells in HuRΔ/Δ and HuRf/f B cell cultures after 72 hours stimulation and a lower frequency of dead HuRΔ/Δ cells after 96 hours stimulation (Fig. 3B). These data indicate that HuRΔ/Δ B cells do not exhibit increased apoptosis but instead survive better than HuRf/f B cells after in vitro stimulation with LPS and IL-4. To determine whether splenic HuRΔ/Δ B cells exhibit reduced proliferation, we labeled HuRΔ/Δ and HuRf/f B cells with the fluorescent dye CFSE, which is diluted 50% by each round of cell division, before 72 hours incubation with LPS and IL-4. We detected fewer cell divisions in HuRΔ/Δ cultures (Fig. 3C), indicating that LPS and IL-4 stimulated HuRΔ/Δ B cells exhibit reduced proliferation. As an independent means to monitor proliferation, we measured incorporation of BrdU into replicating DNA combined with DNA content staining to quantify cells in each cell cycle phase. We found that fewer HuRΔ/Δ cells entered S phase during the 45 minute BrdU pulse (Fig. 3D), providing further evidence that HuRΔ/Δ B cells stimulated by LPS and IL-4 exhibit reduced proliferation. To monitor IgH isotype switching, we measured surface expression of IgG1, since LPS and IL-4 promote IgH class switch recombination (CSR) predominantly to this isotype. After 72 hours of stimulation, HuRΔ/Δ cultures harbor IgG1⁺ B cells at the same frequency as HuRf/f cells (Fig. 3E). Consistent with normal signaling upstream of CSR, we detected normal levels of non-coding germline transcripts for IgG1 and IgE in stimulated HuRΔ/Δ cells (Supplemental Fig. 2B) (41). The data from our analyses of HuRΔ/Δ and HuRf/f B cells following in vitro stimulation with LPS and IL-4 indicates that HuR is necessary for optimal proliferation of B cells, yet dispensable for survival and isotype switching to IgG1.

FIGURE 3.

In vitro stimulated HuRΔ/Δ B cells exhibit a mild proliferation defect, enhanced survival, and normal IgH isotype switching. (A) Quantification of the fold expansion of splenic B cells from HuRΔ/Δ or HuRf/f mice cultured for 72 hours in LPS and IL-4. Data are from three independent experiments performed on a total of ten animals of each genotype. (B – E) Representative flow cytometry analysis and quantification of live cells (B), cellular divisions (C), cell cycle distribution (D), or switched Ig expression (E) following culture of HuRΔ/Δ or HuRf/f splenic B cells for 72 hours or where indicated 96 hours in LPS and IL-4. Data are from three or more independent experiments conducted on at least five mice of each genotype.

In addition to expressing cell surface BCRs, the ability to secrete antibodies is crucial for B cell function. Thus, we used ELISA assays to quantify IgM and IgG1 antibodies secreted into the supernatants of HuRΔ/Δ and HuRf/f cells stimulated in vitro with LPS and IL-4. Despite equivalent frequencies of IgM⁺ and IgG1⁺ B cells in HuRΔ/Δ and HuRf/f cultures, we detected modestly lower levels of IgM and IgG1 in supernatants from HuRΔ/Δ stimulations (Fig. 4A). Accounting for differences in cell numbers arising from impaired proliferation of HuRΔ/Δ B cells, these in vitro stimulated HuRΔ/Δ B cells secrete normal amounts of IgG1 but 50% less IgM than HuRf/f B cells (Fig. 4A). Membrane-bound and secreted IgH chains are generated from mRNAs that differ in 3′ translated sequences, with only membrane-bound mRNA forms encoding a transmembrane domain (Fig. 4B) (42, 43). Alternative polyadenylation controls the relative abundance of membrane-bound versus secreted transcripts (42, 43). Since HuR can regulate pre-mRNA processing (44), we investigated whether HuRΔ/Δ B cells normally generate mRNAs encoding secreted antibodies. We used distinct primer sets to amplify mRNAs encoding the membrane-bound or secreted isoforms of IgM after 72 hours of stimulation. We found that the levels of secreted IgM mRNAs were reduced while levels of membrane-bound IgM mRNAs were unchanged in HuRΔ/Δ cells as compared to HuRf/f cells (Fig. 4C), reflecting the relative amounts of secreted and membrane-bound IgM in the cultures. These data show HuR promotes generation of alternatively-polyadenylated IgH mRNAs and secretion of antibodies following in vitro stimulation by LPS and IL-4.

FIGURE 4.

HuR is required for antibody production and numbers of peritoneal B1 cells but dispensable for in vitro activation in response to anti-IgM and anti-CD40. (A) ELISA quantification of IgM or IgG1 secreted by HuRΔ/Δ or HuRf/f splenic B cells during a 72 hour culture in LPS and IL-4. Data are presented as raw values (left graph) or values normalized to the numbers of cells in each culture (right graph). Shown is a representative of three independent experiments. (B) Schematic of the final three exons of the μ constant region showing the genomic configuration and the mRNA forms generated by alternative polyadenylation. Arrows above the exons indicate primers used to detect secreted (sec) or membrane-bound (mem) IgM transcripts by qRT-PCR. (C) Quantification of IgM transcript variants in LPS and IL-4 stimulated HuRΔ/Δ or HuRf/f splenic B cells presented as their relative abundance to 18S mRNA. Data are from three independent experiments. (D – E) Representative flow cytometry analysis and quantification of live cells and cell divisions (D) or expression of GC and plasmablast markers (E) following culture of HuRf/f or HuRΔ/Δ splenic B cells for 60 hours without stimulation or with stimulation by anti-IgM, anti-CD40, with or without IL-21. Data are representative of two experiments performed on a total of four mice of each genotype. (F) ELISA quantification of serum Ig isotypes in non-immunized HuRΔ/Δ and HuRf/f mice. Data are from three or more independent experiments conducted on at least five 6–8 week old mice of each genotype. (G) Representative flow cytometry analysis and quantification of peritoneal B1 B cell subsets (CD11b⁺CD5⁺ B1a cells and CD11b⁺CD5⁻ B1b cells) following gating on live CD19⁺ lymphocytes. Bar graph shows numbers of total B1 cells from three experiments performed with 6–8 week old mice, 6 HuRf/f and 9 HuRΔ/Δ.

We next assayed in vitro activation of HuRΔ/Δ B cells by conditions that mimic a T cell-dependent B cell response. For this purpose, we isolated total splenic B cells from HuRΔ/Δ and HuRf/f mice and incubated equal numbers of cells with anti-IgM and anti-CD40 with or without IL-21. To assess whether splenic HuRΔ/Δ B cells also exhibit reduced proliferation when stimulated by these conditions, we labeled cells with CFSE prior to incubation for 60 hours with anti-IgM, anti-CD40, and/or IL-21. We observed an equivalent number of cell divisions in HuRΔ/Δ and HuRf/f cultures (Fig. 4D), indicating that HuRΔ/Δ B cells exhibit normal proliferation following in vitro stimulation under conditions that mimic T cell-dependent activation. We also noted that cell viability after stimulation was not decreased by HuR deletion (Fig 4D). To determine whether HuR affects the induction of GC markers on HuRΔ/Δ B cells in vitro, we measured expression of the BCL6, CD95, and TACI proteins in un-stimulated and stimulated cells. After stimulation for 60 hours, we found no significant difference in expression of each of these GC B cell markers between HuRΔ/Δ and HuRf/f cultures (Fig. 4E, Supplemental Figure 2F), revealing that HuRΔ/Δ B cells normally express GC markers following in vitro stimulation by conditions that mimic T cell-dependent activation. These data demonstrate that HuR expression in B-lineage cells is dispensable for the ability of naive B cells to become activated and “GC-like” in vitro.

HuRΔ/Δ mice exhibit paucities of GC B cells and GC Tfh cells during an in vivo T cell-dependent immune response

To determine whether HuR has a role in humoral immunity in vivo, we first analyzed standing serum antibody levels in non-immunized 8 to 10 week-old HuRΔ/Δ and WT mice. We observed that serum levels of all Ig isotypes were reduced in HuRΔ/Δ mice (Fig. 4F), indicating that HuR expression in B lineage cells is necessary for optimal antibody production in vivo. Since B-1 B cells produce the majority of standing IgM and IgA serum titers, we assessed the effects of HuR deletion on these B lineage cells. We detected 75% fewer peritoneal B-1 B cells in HuRΔ/Δ mice (Fig. 4G), revealing that HuR expression in B lineage cells is required to support normal numbers of B-1 cells. We also quantified GC cells, plasma cells (PC), and switched memory (Sw-mem) B cells, which arise from T cell-dependent B cell responses and together generate the majority of standing IgG and IgE serum titers. We detected reduced numbers of GC (50-fold lower) and PC (2-fold lower) cells, and a trending reduction in Sw-mem (2.5-fold lower) B cells in HuRΔ/Δ mice (Fig. 5A), indicating that expression of HuR in B lineage cells is required to support normal numbers of antibody secreting B-2 cells. Collectively, these data demonstrate that B-lineage intrinsic functions of HuR are necessary for normal numbers of antibody-secreting B-1 and B-2 cells and normal titers of standing antibodies of all isotypes.

FIGURE 5.

B cell-intrinsic HuR expression is required in vivo for generation of GC B cells and high-affinity antibodies. (A) Representative flow analysis and quantification of splenic GC, PC, and Sw-mem B cells from non-immunized mice. Flow plots are shown following gating on live IgD⁻dump⁻ lymphocytes. Data are from two independent experiments conducted on at least six 6–8 week-old mice of each genotype. (B – C) Quantification of B cell populations and NP-specific antibodies in HuRΔ/Δ and HuRf/f mice immunized with NP-OVA in alum. Data are from two independent experiments involving three and five mice of each genotype at 9 and 14 days after immunization, respectively. (B) Representative flow cytometry analysis and quantification of splenic naive, total GC cells, NP⁺ GC cells, and PCs. (C) ELISA quantification of low- and high-affinity anti-NP specific antibodies, indicated by NP25 and NP4, respectively. IgM anti-NP ELISAs were run without a standard, thus are displayed as a dilution series for each group. For IgM ELISAs, p-values are the result of two-way ANOVA. (D) Quantification of GC B cells from p53Δ/Δ and HuRΔ/Δ p53Δ/Δ mice at 14d after NP-OVA immunization. Representative plots show cells previously gated on live IgD− DUMP−, CD138− CD19+ lymphocytes. The experiment was performed three times with 5 mice per genotype in total.

We next evaluated the role of HuR in T cell-dependent humoral immunity in vivo by testing the ability of HuRΔ/Δ mice to mount a B cell response to immunization with a T cell-dependent antigen. For this purpose, we injected 8 week-old HuRΔ/Δ or WT mice with NP-OVA precipitated in alum. After 9 or 14 days, we quantified numbers of naive B cells, PCs, total GC B cells, and NP-specific GC B cells in immunized mice and non-immunized littermate controls. Since some antibodies generated from NP-OVA injection will recognize epitopes within OVA rather than NP, we measured total GCs as well as NP-specific GCs. The numbers of naive B cells in HuRΔ/Δ mice remained 50% lower than normal after immunization (Fig. 5B). WT mice mounted a robust humoral response, generating half a million or more total and NP-specific GC B cells by 9 and 14 days post-immunization (Fig. 5B). In contrast, HuRΔ/Δ mice were substantially impaired in their ability to mount a humoral immune response, generating only thousands of total and NP-specific GC B cells over the same period following immunization (Fig. 5B). Reflecting fewer GC B cells, the production of plasma cells was reduced 4.5-fold in HuRΔ/Δ mice relative to WT mice (Fig. 5B). We also performed ELISAs to quantify low- and high-affinity NP-specific antibodies in sera of HuRΔ/Δ and WT mice at 9 or 14 days post-immunization. We detected 6-fold reduced levels of low-affinity IgG NP-specific antibodies in HuRΔ/Δ mice at each time point assayed (Fig. 5C). Consistent with the requirement of GCs for robust affinity-maturation, we found a 20-fold increase in high-affinity IgG anti-NP antibodies in immunized WT mice between days 9 and 14, but only a 4-fold increase in HuRΔ/Δ mice over the same time (Fig. 5C). Further, HuRΔ/Δ mice had reduced levels of IgM anti-NP antibodies (Fig. 5C), consistent with the idea that low IgG antibody titers are not the result of a CSR defect in HuRΔ/Δ B cells. Notably, deletion of p53 in HuRΔ/Δ B lineage cells did not rescue GC numbers after immunization beyond those observed in mice with deletion of only p53 in B lineage cells (Fig. 5D). The lower than normal numbers of GC B cells after immunization of mice with deletion of only p53 in B lineage cells is consistent with the reported effect of p53 deletion on IgG2a switching (45). While we cannot rule out the possibility HuRΔ/Δ B cells undergo p53-independent death during an immune response, these in vivo data are consistent with our in vitro findings that HuR-deficiency does not impair viability of activated B cells.

HuR expression in B cells is critical for processes involving and proteins regulating B-T cell interactions

The compromised ability of HuRΔ/Δ mice to produce GC B cells and high-affinity antibodies in vivo after immunization contrasts with the normal ability of HuRΔ/Δ B cells to become “GC-like” and make antibodies in vitro. Since the formation of GC structures in lymphoid tissues are critical for differentiation of GC B cells and production of high-affinity antibodies in vivo (14, 46), we conducted histology to evaluate GCs in the spleens of HuRΔ/Δ and WT mice. As measured by H&E staining, gross splenic architecture was normal in HuRΔ/Δ mice before and after immunization with NP-OVA precipitated in alum (Fig 6A), consistent with the modest effects of HuR deletion on naïve B cell pools. As measured by H&E staining and immunohistochemical (IHC) staining for PNA, which marks GC B cells (47), GCs were detectable in the spleens of immunized WT mice, but not of immunized HuRΔ/Δ mice or un-immunized mice of either genotype (Fig 6A and data not shown). Since the light zone of the GC consists of rapidly dividing B cells (48), we also performed IHC staining for Ki67, which marks proliferating cells (49). Clusters of Ki67⁺ cells were detectable in the spleens of immunized WT mice, but not of immunized HuRΔ/Δ mice or un-immunized mice of either genotype (Fig 6A and data not shown). These data indicate HuR expression in B cells is required to support the GC structures needed for humoral immunity in vivo.

FIGURE 6.

HuR is critical for processes involving and proteins regulating B-T cell interactions. (A) Left two panels show representative H&E staining on spleens of un-immunized and immunized HuRf/f or HuRΔ/Δ mice 14 days after NP-OVA injection. Right two panels show representative immunohistochemical (IHC) staining for PNA or Ki67 in brown on spleens from mice 14 days after NPOVA injection. Staining was performed on spleens from two separate immunizations, with a total of two un-immunized mice and six immunized mice of each genotype. Scale bars are 400 μm (H&E) and 500 μm (IHC). Arrows indicate GC structures. (B) Representative flow analysis of TCRβ⁺CD4⁺CD62L⁻ lymphocytes from spleen of un-immunized mice or mice 14 days following NP-OVA injection. Percentages of PD-1⁺CXCR5⁺ total Tfh cells and PD-1^highCXCR5^high GC Tfh cells are shown on plots and quantified on the right. The quantification presented is from a single experiment of two experiments performed, each yielding similar results. (C) Flow cytometry to measure expression of co-stimulatory surface molecules on B cells after 48h stimulation with anti-IgM and anti-CD40. Representative histograms following gating on live cells is shown for significantly changed surface markers. Quantification of median fluorescence intensity (MFI) of HuRΔ/Δ cells is expressed as fold change over HuRf/f cells from the same experiment. Significant differences are the result of a one-sample t-test. The experiment was repeated at least twice for each marker shown, with a minimum of 5 animals per genotype. (D) RNA-IP qRT-PCR analysis performed on WT splenic B cells stimulated for 72h with anti-IgM and anti-CD40. Ct values for indicated transcripts were normalized to GAPDH for each IP, and HuR IP values are expressed as fold enrichment compared to IgG IP. The experiment was performed with 4 biological replicates over 2 experiments. No statistical tests were performed on these results, but typically RNA-IP enrichment over 2-fold (dotted line) is considered meaningful.

The formation of GCs requires activated B cells to interact with activated CD4⁺ T cells at the periphery of lymphoid follicles, promoting migration of both cell types into follicles where they differentiate into GC B cells and GC T follicular helper (Tfh) cells, respectively (11, 50). Since the failure of activated B cells to functionally interact with activated CD4⁺ T cells prevents GC Tfh formation and ablates the in vivo GC response (11, 51), we conducted flow cytometry to quantify total (CXCR5⁺PD-1⁺) and mature (GC) Tfh cells (CXCR5^highPD-1^high) in the spleens of immunized HuRΔ/Δ and WT mice. We found spleens of immunized HuRΔ/Δ mice had 1.6-fold and 17-fold lower numbers of total and GC Tfh cells, respectively, than spleens of WT mice (Fig. 6B). These data indicate HuR expression in B cells is required for the presence of GC Tfh cells after immunization of mice with a T-cell dependent antigen.

Our GC B and GC Tfh cell data indicate HuR expression in B cells is critical for T-cell dependent immune processes requiring B-T cell interactions within the follicular milieu. We sought to identify potential mechanisms by which HuR expression in B cells supports normal GC structures and populations of GC B and GC Tfh cells. Since we cannot detect HuRΔ/Δ GC B cells in vivo, we studied in vitro stimulated HuRΔ/Δ and control B cells to find differential expression of proteins that might account for the failure of HuRΔ/Δ mice to mount a normal GC response. Using unbiased label-free whole proteome quantification, we identified 6132 proteins in both cells, but limited our inspection to 4678 proteins for which we obtained at least 8 total mass spectra (MS) and had nonzero MS count values for 3 out of 4 biological replicates (Supplemental Table 2). We observed a greater number of proteins decreased in HuRΔ/Δ B cells than increased, consistent with the canonical role of HuR as promoting target gene expression (19, 22). HuRΔ/Δ B cells showed reduced expression of multiple proteins involved in chromatin modification and organization, suggesting roles of HuR-dependent chromatin regulation in B cell activation. HuRΔ/Δ B cells also had reduced expression of proteins involved in cytoskeletal remodeling, which could lead to impaired antigen presentation, cell migration, and/or cell-to-cell adhesion; however, our initial follow-up studies did not show a defect in any of these processes with HuRΔ/Δ B cells stimulated in vitro.

Since this proteomics approach lacks sensitivity to detect small magnitude changes in protein expression, we used a candidate-based approach to study expression of B cell factors known to regulate B-T cell interactions. Activated B cells interact with activated T cells via contacts between surface co-stimulatory receptors and ligands (15, 51, 52). Using flow cytometry to quantify surface expression of co-stimulatory molecules known to control B cell activation and/or B-T cell interactions, we found HuRΔ/Δ B cells showed reduced expression of the CD81, CD70, and CD86 proteins (Fig 6C) (29, 52–58). We then used RNA-immunoprecipitation to show HuR binds to CD81, CD70, and CD86 transcripts in B cells activated in vitro (Fig 6D), consistent with the notion that HuR directly regulates expression of these co-stimulatory molecules in activated B cells. Impaired expression of CD70, CD81, or CD86 has been observed on B cells of humans with Common Variable Immune Deficiency (CVID) (59–61). The subtle reduction in CD70 and CD86 expression on HuRΔ/Δ B cells mimics the modest lower levels of these molecules on B cells from CVID patients (60, 61). Although these clinical data from CVID patients suggests lower co-stimulatory factor expression may be sufficient to impair the GC response of HuRΔ/Δ mice, it is equally plausible that HuR controls expression of additional proteins, including those identified in our proteomics screen, that together with these co-stimulatory factors promote the GC response in vivo.

Discussion

Post-transcriptional regulation of gene expression contributes to the dynamic and precise regulation of intracellular processes and communication among cells. Here, we set out to determine roles of the ubiquitously expressed HuR RNA-binding protein in B cell development and function by analyzing mice with B lineage-specific HuR deletion. Deletion of HuR initiating in pro-B cells results in lower numbers of B cells at each developmental stage beyond the pro-B cell stage, including the GC and PC B cell populations that arise from Fo B cells that encounter and recognize antigen. Mature HuRΔ/Δ B cells display functional defects in vitro when stimulated under conditions that mimic a T cell-independent response but not by conditions that mimic a T cell-dependent GC immune response. In contrast with normal ability of HuRΔ/Δ B cells to become “GC-like” in vitro, HuRΔ/Δ B cells exhibit profound functional deficiencies in vivo, generating low levels of antibodies and failing to participate in a T cell-dependent humoral immune response. As we were submitting our work, another group published that a different strain of mice with B lineage-specific HuR deletion is defective in the GC reaction and antibody generation in response to T cell-dependent and -independent antigens (62). They concluded HuR loss results in defective mitochondrial metabolism that leads to large amounts of reactive oxygen species (ROS) and resultant death of activated B cells in vivo (62). Their conclusion was based on data showing in vitro stimulated HuR-deficient B cells exhibit increased ROS and death and impaired proliferation and isotype switching, which were all restored to normal upon addition of sodium pyruvate or other ROS scavengers to the culture media (62). In contrast, HuR-deficient B cells from our mouse strain exhibit normal apoptosis and isotype switching during in vitro stimulation regardless of whether the media has sodium pyruvate (Supplemental Fig. 2C, 2D). Moreover, the modest in vitro proliferation defect of our HuR-deficient B cells was equivalent in the presence or absence of sodium pyruvate (Supplemental Fig. 2C). While the reason for identical in vivo but different in vitro results between these two studies is unclear, possibilities include differences in experimental conditions and distinct roles for HuR on a 129SvEv versus a C57BL/6 background. Since our in vitro stimulated HuRΔ/Δ B cells survive, isotype switch, and induce expression of GC B cell markers normally under conditions that mimic a T-cell dependent response, altered B cell metabolism likely is not the major cause of the impaired GC reaction and T cell-dependent immune response of our HuRΔ/Δ mice. We cannot rule out that the ~50% lower than normal numbers of HuR-deficient mature B cells in either mouse strain might contribute to the more substantial reduction in GC B cells; however, we consider this possibility unlikely. Finally, although our data indicate HuR expression in B cells is critical for normal B and T cell interactions during a T-cell dependent immune response, they cannot distinguish among roles of HuR in promoting initiation of the GC B cell fate, maintaining survival of GC B cells, or directing functional communication between GC B cells and Tfh cells. Future analyses of both mouse strains lacking HuR in B cells will be needed to elucidate the B cell intrinsic mechanisms by which HuR is essential for supporting normal humoral immunity in vivo.

Our analyses of HuRΔ/Δ and HuRΔ/Δ:p53Δ/Δ mice indicate that HuR supports B lineage cells through developmental stage-specific mechanisms or cellular processes that antagonize p53-dependent elimination of immature but not mature B cells. A role for HuR in promoting survival of immature B-2 lineage cells had been suggested from analysis of adult mice with global HuR deletion (24). In contrast to our findings that B lineage-specific HuR deletion results in lower numbers of immature and mature B cells, T lineage-specific inactivation of HuR results in higher numbers of immature thymic T cells but decreased numbers of mature splenic T cells (63). These findings indicate that HuR has lineage-specific and developmental stage-specific roles in supporting normal numbers of B and T cells. The reduced numbers of naïve splenic HuRΔ/Δ B-2 cells in older mice and in the situation where p53 inactivation restores immature B-2 cell numbers suggest that HuR has B lineage-intrinsic functions in controlling homeostasis of naïve B-2 cells. In this context, the decreased numbers of peritoneal HuRΔ/Δ B-1 cells may reflect a role for HuR in promoting homeostasis of naïve B-1 cells. The BLyS family cytokine BAFF secreted from fibroblastic reticular cells and radiation-resistant stromal cells promotes B-2 cell homeostasis by signaling through BAFF-R, TACI, and BCMA receptors expressed on B cells (4, 64–66). We detected normal expression of TACI on stimulated and un-stimulated cells of both genotypes; however HuR might regulate expression of BAFF-R or BCMA, or their downstream signaling factors, such as the mTORC2 complex (67). While evidence suggests that the BLyS family cytokine APRIL secreted from peritoneal macrophages promotes B-1 cell homeostasis by signaling through heparin sulfate proteoglycans expressed on B cells (68), the cytokines and their receptors on B cells that regulate homeostasis of B-1 cells are less well understood than for B-2 cells. Identifying HuR mRNA targets in splenic B-2 and peritoneal B-1 cells may yield novel molecular insight into the molecular factors and mechanisms that govern B cell homeostasis.

Our analysis of HuRΔ/Δ mice demonstrates expression of HuR in B lineage cells is critical for normal antibody levels in non-immunized mice. The reduced serum titers of all antibody isotypes in HuRΔ/Δ mice are consistent with the reduced numbers of peritoneal B-1 cells and GC, PC, and Sw-mem B-2 cells. However, the impaired ability of HuRΔ/Δ B cells to produce mRNAs that encode the IgH chains of secreted antibodies suggests that decreased antibody secretion likely contributes to the low level of circulating antibodies. HuR may regulate alternative polyadenylation of IgH mRNAs by binding IgH pre-mRNAs and directly regulating pre-mRNA processing at the level of splicing or polyadenylation, which are in competition at the IgH locus (43). This role of HuR in mature B cells would be consistent with HuR regulating the splicing of many transcripts in other cells (22, 44). Alternatively, HuR might regulate the expression of an mRNA processing factor that acts on IgH mRNAs. We could not detect HuR bound to IgH pre-mRNAs in normal mature B-2 cells, while in HuRΔ/Δ B-2 cells we did not observe decreased expression of the ELL2, PTB, hnRNPF, or active XBP1 proteins (data not shown), which have been implicated in the switch between membrane and secreted forms of IgH mRNAs (42, 43, 69–72). Since the trans-acting factors and molecular mechanisms that regulate the switch from membrane to secreted forms of IgH mRNAs remain to be elucidated, the identification of mRNAs to which HuR binds in mature B cells could provide new insights into how post-transcriptional changes in gene expression promote antibody secretion.

Our analyses of HuRΔ/Δ B cells during in vitro stimulation and in vivo following immunization indicate B cell-intrinsic functions of HuR are required for GC structures, GC B cells, high-affinity antibodies, and GC Tfh cells each to be detectable at appreciable levels. Since HuRΔ/Δ mice lack HuR specifically in B lineage cells, any defects in Tfh cells are attributable to failure of B cells to appropriately coordinate gene expression programs during the immune response. In vivo GC responses require intimate co-dependent interactions between B and T cells (16, 17, 51). B cells traffic in and out of the follicle with defined kinetics in order to encounter antigen and subsequently engage antigen-primed CD4⁺ T cells (10, 12). Activated B cells must physically interact with antigen-primed CD4⁺ T cells to induce pre-Tfh cells to migrate into the follicle and differentiate into mature Tfh cells that provide survival and differentiation signals essential for generating GC B cells and high-affinity antibodies (11, 15, 50, 51). B cells drive contact with CD4⁺ T cells through B cell-surface MHC II molecules loaded with processed antigen fragments and through co-stimulatory proteins expressed on B cells (12, 51, 73). In addition to physical interactions with CD4⁺ T cells, B cells enhance Tfh differentiation by secreting IL6 and other cytokines (13, 74). We found that HuRΔ/Δ B cells exhibit normal expression of chemokine receptors that control B cell trafficking, MHCII proteins, and IL6 mRNA (Supplemental Figure 2E and not shown); however, we did not confirm chemokine signaling and antigen processing are normal in HuRΔ/Δ B cells. In contrast, we found that activated HuRΔ/Δ B cells had reduced expression of the CD81, CD70, and CD86 co-stimulatory molecules (29, 52–58). HuR binds to CD81, CD70, and CD86 transcripts in stimulated WT cells, consistent with the notion that HuR directly regulates expression of these molecules. Since CD86 binding to CD4⁺ T cells also activates B cell intracellular signals to promote antibody secretion (75), this reduced CD86 expression might impair humoral immunity through distinct mechanisms. Impaired expression of CD81, CD70, or CD86 has been observed on B cells of humans with Common Variable Immune Deficiency (CVID) (59–61). Notably, the subtle differences in CD86 and CD70 expression on HuRΔ/Δ B cells mimic the modest reduction of these co-stimulatory molecules on B cells from CVID patients (60, 61). Although to our knowledge, genetic mutations or polymorphisms of the HuR gene have not been associated with CVID, identifying HuR target mRNAs in naïve B-2 cells and antigen-activated GC B cells could lead to greater understanding and perhaps improved therapies for humans with impaired T cell-dependent immune responses.