Murine Glucocorticoid Receptors Orchestrate B Cell Migration Selectively between and Blood

This information is current as Derek W. Cain, Carl D. Bortner, David Diaz-Jimenez, Maria of September 27, 2021. G. Petrillo, Amanda Gruver-Yates and John A. Cidlowski J Immunol published online 22 June 2020 http://www.jimmunol.org/content/early/2020/06/19/jimmun ol.1901135 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 22, 2020, doi:10.4049/jimmunol.1901135 The Journal of Immunology

Murine Glucocorticoid Receptors Orchestrate B Cell Migration Selectively between Bone Marrow and Blood

Derek W. Cain,1 Carl D. Bortner, David Diaz-Jimenez, Maria G. Petrillo, Amanda Gruver-Yates, and John A. Cidlowski

Glucocorticoids promote CXCR4 expression by T cells, monocytes, macrophages, and eosinophils, but it is not known if gluco- corticoids regulate CXCR4 in B cells. Considering the important contributions of CXCR4 to B cell development and function, we investigated the glucocorticoid/CXCR4 axis in mice. We demonstrate that glucocorticoids upregulate CXCR4 mRNA and in mouse B cells. Using a novel strain of mice lacking glucocorticoid receptors (GRs) specifically in B cells, we show that reduced CXCR4 expression associated with GR deficiency results in impaired homing of mature B cells to bone marrow, whereas migration to other lymphoid tissues is independent of B cell GRs. The exchange of mature B cells between blood and bone

marrow is sensitive to small, physiologic changes in glucocorticoid activity, as evidenced by the lack of circadian rhythmicity in Downloaded from GR-deficient B cell counts normally associated with diurnal patterns of glucocorticoid secretion. B cellGRKO mice mounted normal humoral responses to immunizations with T-dependent and T-independent (Type 1) Ags, but Ab responses to a multivalent T-independent (Type 2) Ag were impaired, a surprise finding considering the immunosuppressive properties commonly attributed to glucocorticoids. We propose that endogenous glucocorticoids regulate a dynamic mode of B cell migration specialized for rapid exchange between bone marrow and blood, perhaps as a means to optimize humoral immunity during diurnal periods of activity.

The Journal of Immunology, 2020, 205: 000–000. http://www.jimmunol.org/

lucocorticoids are best-known for their immunosup- cues generated by tissues or act directly on to pressive properties, and synthetic glucocorticoids rep- modulate migration (10–13). Evidence for the latter includes ob- G resent a first-line of treatment for many inflammatory and servations that glucocorticoids upregulate CXCR4 expression by autoimmune diseases. Glucocorticoids bind intracellular gluco- eosinophils (14), monocytes (15), macrophages (16), T cells (17), corticoid receptors (GRs; encoded by Nr3c1) to repress proin- and hematopoietic progenitor cells (18). To our knowledge, it is flammatory transcription factors (1), activate anti-inflammatory not known if glucocorticoids regulate CXCR4 in B cells. CXCR4 (2–5), and induce apoptosis (6). However, the plays key roles in the retention of B cell precursors in bone cell type–specific contributions of GR signaling to multicellular marrow (19), migration of mature B cells from Peyer’s patches by guest on September 27, 2021 immune processes have been difficult to assess because of the (20), organization of germinal centers (21), and homing of plasma ubiquitous nature of GR expression. Recent studies have used cells to lymphoid tissues (22). If glucocorticoids regulate CXCR4 mice with genetic ablation of Gr in specific cell lineages to dissect in B cells, then these processes could be impacted by changes in direct glucocorticoid actions on T cells (7), macrophages (8), and glucocorticoid activity. We sought to determine if glucocorticoids dendritic cells (9) in vivo. regulate B cell expression of CXCR4 and, if so, to what extent Among their various effects, glucocorticoids alter leukocyte glucocorticoid-induced changes in CXCR4 expression affect trafficking. It is unclear if glucocorticoids regulate chemotactic B cell function.

Signal Transduction Laboratory, National Institute of Environmental Health Sci- Materials and Methods ences, National Institutes of Health, U.S. Department of Health and Human Services, Mice Research Triangle Park, NC 27709 1 Intact and adrenalectomized (ADX) C57BL/6 mice (Jackson Laboratories) Current address: Duke Human Vaccine Institute, Department of Medicine, Duke were housed in specific pathogen-free conditions under a 12 h /dark University, Durham, NC. cycle at the National Institute of Environmental Health Sciences. ADX ORCIDs: 0000-0002-5444-6628 (C.D.B.); 0000-0001-5999-9355 (M.G.P.). mice were provided saline drinking water. Mb1-Cre mice (23) were Received for publication September 18, 2019. Accepted for publication June 3, 2020. crossed with mice harboring Nr3c1 alleles containing loxp sites flanking exons 3 and 4 (24) to generate Mb1Cre/wtNr3c1fl/fl mice and Cre-negative This work was supported by the Intramural Research Program of the National Insti- littermates (Mb1wt/wtNr3c1fl/fl) on a C57BL/6 background. Mice were tutes of Health/National Institute of Environmental Health Sciences (to J.A.C.). provided with sterilized chow and water ad libitum. Male mice aged 8– D.W.C. designed, conducted, and evaluated experiments and wrote the manuscript. 12 wk were used in all studies unless otherwise stated. All experiments C.D.B., D.D.-J., M.G.P., and A.G.-Y. designed, conducted, and evaluated experi- were approved and performed according to the guidelines of the Animal ments. J.A.C. designed and evaluated experiments and wrote the manuscript. Care and Use Committee at the NIEHS. Address correspondence and reprint requests to Dr. John A. Cidlowski, National Institute of Environmental Health Sciences, F348 Rall Building, 111 TW Alexander Dexamethasone injections Drive, Research Triangle Park, NC 27709. E-mail address: [email protected] Dexamethasone (Dex) (Steraloids) was injected i.p. at 2 mg/kg; control The online version of this article contains supplemental material. mice received an injection of vehicle. Mice received one i.p. injection daily Abbreviations used in this article: ADX, adrenalectomized; Dex, dexamethasone; for 3 d and were sacrificed on day 4. GR, glucocorticoid receptor; KO, knockout; MIF, macrophage migration inhibitory factor; Q-PCR, quantitative PCR; viSNE, visualization of t-distributed stochastic Flow cytometry neighbor embedding. Bone marrow, blood, and spleen were prepared as single-cell suspensions Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 and subjected to RBC lysis with ACK buffer. Cells were stained with

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901135 2 GLUCOCORTICOID RECEPTORS REGULATE B CELL MIGRATION

fluorochrome-conjugated mAb for B220, IgM, IgD, CD138, CD43, CD3 or Adoptive transfer of B cells CD5, CXCR4, CD11c, and CD11b (eBioscience, BD Biosciences, and Splenic B cells were enriched by negative selection (Stem Cell Technol- BioLegend). The 7-aminoactinomycin D (7AAD) staining identified dead + cells. Data were acquired on an LSRII Flow Cytometer (BD Biosciences) ogies); the purity of B220 cells routinely exceeded 97%. Control B cells and analyzed with FlowJo software (FlowJo). were labeled with 10 or 100 nM CellTrace CFSE reagent (Thermo Fisher To detect intracellular GR, cells were stained for surface Ags and then Scientific) according to the manufacturer’s protocol. GR-deficient B cells fixed/permeabilized with eBioscience fixation/permeabilization reagents. were labeled with 100 nM CFSE. Recipient mice received a 100 mli.v. Cells were stained with anti-GR (clone D8H2; Cell Signaling Technology) injection of B cells (2–3 million cells) containing a 1:1 mixture of 10 nM or isotype-matched control mAb and then Alexa Fluor 647–conjugated anti- CFSE-labeled control B cells and 100 nM CFSE-labeled GR-deficient rabbit IgG (SouthernBiotech). visualization of t-distributed stochastic B cells, or a 1:1 mixture of 10 and 100 nM CFSE-labeled control B cells. neighbor embedding (viSNE) plots were generated in CytoBank using the After 18 h, recipient animals were sacrificed, and single-cell suspensions Barnes-Hut implementation of the t-SNE algorithm (25). from the blood, bone marrow, spleen, inguinal lymph nodes, and Peyer’s patches were analyzed by flow cytometry for the presence of CFSEdim Quantitative PCR and CFSEbright donor B cells. B cells were enriched by negative selection (EasySep Mouse B Cell Iso- Statistics lation Kit; StemCell Technologies). For Dex treatment studies, B cells were cultured in RPMI-1640, 10% charcoal-stripped FBS, 55 mM 2-ME, 10 mM Pair-wise comparisons were made with Wilcoxon Exact Tests. Comparisons HEPES, and penicillin-streptomycin containing 100 nM Dex or vehicle for transwell migration studies were made using Sign Tests for matched for 1, 3, and 6 h. Total RNA was isolated using Qiagen RNeasy kits. RNA pairs of wild-type and GR knockout (KO) cell count data for each assay (50–100 ng) was reverse transcribed and amplified with iScript One-Step condition. All statics were performed using JMP Pro version 15.0.0 (SAS). RT-PCR Kit (Bio-Rad Laboratories) using TaqMan primer/probe Sets Data sharing statement (Thermo Fisher Scientific). Real-time quantitative PCR (Q-PCR) was Downloaded from performed with a Bio-Rad CFX96 Sequence-Detection System. The For original data, please contact [email protected]. fluorescent signal from each transcript was normalized to the house- keeping Ppib using the 22DCt method. Results Transwell migration assay Glucocorticoids upregulate CXCR4 transcription in B cells Transwell plates (24 wells) with 5-mm pore polycarbonate membrane in- A link between GR signaling and Cxcr4 expression in B cells has serts were seeded with 105 splenic B cells. The lower chamber contained not, to our knowledge, been reported. We treated C57BL/6 splenic http://www.jimmunol.org/ medium alone, 100 ng/ml recombinant mouse CXCL12 (PeproTech), 200 ng/ml recombinant mouse macrophage migration inhibitory factor B cells with the synthetic glucocorticoid Dex or vehicle ex (MIF) (PeproTech or Abcam), 500 ng/ml recombinant mouse CXCL13 vivo, then evaluated transcription of the receptors (BioLegend), or 100 ng/ml recombinant mouse CCL19 (PeproTech). Cxcr4, Cxcr5,andCcr7, alongside the glucocorticoid target Plates were incubated at 37˚C incubator with 5% CO2 for 3 h. Cells in the gene Tsc22d3 (also known as Gilz). After 6 h of incubation, lower chamber were quantified by FACS, with a 30-s acquisition time for each sample. Gilz transcripts were significantly increased by Dex treatment, as expected (Fig. 1A). We also observed more Cxcr4 transcripts Immunizations in Dex-treated B cells compared with vehicle treatment The succinic anhydride ester of (4-hydroxy-3-nitrophenyl)-acetyl (NP) was (Fig.1A),whereasCxcr5 and Ccr7 transcripts were unchanged reacted with chicken g-globulin (CGG; Sigma-Aldrich) to generate (Fig. 1A). A time-course study revealed that B cells upregu- by guest on September 27, 2021 NP8-CGG. NP8-CGG was added to Alhydrogel 2% (Accurate Chemical). lated Cxcr4 transcription within 1 h of incubation with Dex Mice were injected i.p. with 10 mgofNP8-CGG per mouse. NP0.15-LPS (Fig. 1B). Dex-induced increases in CXCR4 surface protein (Biosearch Technologies) was diluted in PBS and injected i.p. at a dose were evident after 3 h of incubation (Fig. 1C). As previously of 10 mgNP0.15-LPS per mouse. NP49-AECEM-Ficoll (Biosearch Tech- nologies) was diluted in PBS and injected i.p. at a dose of 10 mg reported for myeloid and T cells (27), CXCR4 surface labeling NP49-AECEM-Ficoll per mouse. Mice were bled on days 0, 5, 8, 16, and gradually increased during the cultivation of both Dex-treated 24 after immunization, and serum was collected for ELISA. and control B cells; however, Dex treatment provided an ad- ELISA ditive effect (Fig. 1C). Notably, CXCR5 labeling diminished modestly with Dex treatment, suggestive of an inhibitory effect To measure IgM, IgG, and IgA in sera, wells of 384-well assay plates were coated with anti-Igk and anti-Igl (SouthernBiotech) in carbonate buffer. To of glucocorticoids on this , whereas CCR7 quantify NP-specific Ab, 384-well assay plates were coated overnight with labeling was unaffected (Fig. 1D). B cells were very sensitive NIP25-BSA. For quantification of anti-NP IgM, sera were subject to mild to Dex effects on CXCR4 expression, as increased CXCR4 reduction with 2-ME to dissociate pentameric IgM into monomers (26). labeling occurred at 1 nM Dex and saturated at 10 nM and Serial dilutions of serum were made in PBS containing 0.5% BSA. Anti- NP IgG and IgM mAb standards (clones H33Lg1 and B1-8, respectively) above (Fig. 1E). The opposing effects of Dex on CXCR4 and were run simultaneously as standards. HRP-conjugated anti-mouse IgM, CXCR5 expression suggested that glucocorticoids might alter IgG, or IgA (SouthernBiotech) was added to detect isotype-specific Ig. B cell–trafficking patterns. TMB substrate (BioLegend) was added to wells. 2 N H2SO4 was used to To investigate glucocorticoid effects on B cells in vivo, we stop the enzymatic reaction. OD450 readings were recorded with back- injected mice with Dex and enumerated B cell populations in ground subtraction at OD630. Serum IgE was quantified using a com- mercial ELISA (BioLegend). various lymphoid tissues. Mouse lymphocytes are sensitive to glucocorticoid-induced death, so it was not surprising that Dex- ELISpot treated mice had small spleens and generalized reductions in B For analysis of IgM-, IgG-, and IgA-secreting cells, 96-well High Protein and numbers in blood and tissues (Fig. 2A, 2B). All sub- Binding Plates (Millipore) were coated with anti-Igk and anti-Igl (South- populations of B cells analyzed, including IgM2IgD2–pro–/pre–, ernBiotech) in carbonate buffer. Bone marrow and spleen cell suspen- IgMlowIgD2–immature, IgMhiIgDlow–transitional, and IgMlowIgDhi– sions were added at 2.7, 0.9, 0.3, and 0.1 3 105 cells per well in duplicate. mature B cells, were dramatically reduced in the spleen and blood Cells were incubated at 37˚C in 5% CO2 atmosphere for 3 h. After re- moving cells, alkaline phosphatase–conjugated anti-mouse IgG or IgM, (Fig. 2A, 2B) (see Supplemental Fig. 1 for detailed B cell immu- or HRP-conjugated anti-mouse IgA were added to wells. SIGMAfast nophenotyping). However, the number of mature B cells (and T cells) BCIP/NBT reagent (Sigma-Aldrich) and AEC substrate (BD Biosci- in bone marrow was not changed by Dex treatment (Fig. 2B). Al- ences) were used to visualize alkaline phosphatase–conjugated and HRP- conjugated detection reagents, respectively. Frequencies of Ab-secreting though it is possible that the bone marrow selectively protects mature cells were calculated as the number of spots divided by the number of B cells from glucocorticoid-induced death, another explanation is that input cells. Dex upregulates CXCR4 expression by B cells, thereby promoting The Journal of Immunology 3 Downloaded from

FIGURE 1. Glucocorticoids enhance CXCR4 expression by murine B cells. (A) Splenic B cells from male C57BL/6 mice were cultured with vehicle

(gray bars) or 100 nM Dex (open bars) for 6 h, then analyzed by Q-PCR for Gilz, Cxcr4, Cxcr5, and Ccr7 transcription. Mean + SD number of gene http://www.jimmunol.org/ transcripts normalized to the housekeeping gene Ppib are shown. Data were pooled from three independent experiments (n = 8 biological replicates of each condition). (B) Cells were harvested after 1, 3, and 6 h of incubation with vehicle (gray) or 100 nM Dex (open) for Cxcr4 expression analysis by Q-PCR; the black bar shows Cxcr4 transcripts of freshly isolated B cells. Mean + SD numbers of Cxcr4 transcripts normalized to PPIB are shown. Data represent two independent experiments (n = 4 biological replicates of each condition). (C) The mean 6 SD median fluorescence intensities (MFIs) of anti-CXCR4 labeling of vehicle (filled, dotted line histogram)- versus Dex (open, solid line histogram)-treated B cells at different time points of incubation are shown. Data are pooled from two independent experiments (n = 4 biological replicates of each condition). (D) Mean 6 SD MFI of anti-CXCR5 and anti-CCR7 labeling of B cells after 6 h incubation with vehicle (gray) or 100 nM Dex (open). Data represent five independent experiments; n = 8–10 replicates per condition. (E) Anti-CXCR4 labeling (mean 6 SD MFI) of mouse B cells after 6 h of incubation with various Dex concentrations. Data are pooled from two independent experiments (n = 6 per condition). Experimental groups not connected by the same letter are significantly different (p , 0.01). *p , 0.05, **p , 0.01. by guest on September 27, 2021 their homing to or retention in bone marrow. In the second scenario, and spleens of control mice expressed GRs, anti-GR labeling was Dex-induced losses of mature B cells in bone marrow might be specifically reduced in B cells of B cellGRKO mice (Fig. 3A). In masked by Dex effects on B cell migration. B cellGRKO mice, GR was expressed by ,10% of bone marrow We next determined if ADX mice, which are deficient for en- pro–/pre–B cells and was undetectable in immature, transitional, dogenous glucocorticoids, exhibit alterations in B cell populations. and mature B cells (Fig. 3B). Moreover, B cells from B cellGRKO In bone marrow of ADX mice, the pro–/pre–B compartment was mice were protected from Dex-induced cell death in vitro, whereas enlarged, and immature and transitional B cell populations were T cells from B cellGRKO mice were fully sensitive (Fig. 3C), comparable to controls, but mature B cell and T cell numbers were confirming functional and specific deletion of GR in B cells. lower than intact animals (Fig. 2C). B and T cell numbers in the Decreased CXCR4 expression in GR-deficient B cells impacts blood, however, were supranormal in ADX mice, and the spleens chemotactic responses to CXCL12 contained higher numbers of B cell precursors but normal num- bers of mature B cells and T cells (Fig. 2C). These findings To determine if GR deficiency impacted Cxcr4 expression in GRKO suggested distinct effects of glucocorticoids at various stages of B cells, we isolated splenic B cells from B cell and control B cell development, yet it was unclear if changes represented mice for Q-PCR analysis. As expected, Gr and Gilz transcripts glucocorticoid effects on survival versus migration, or if gluco- were reduced in GR-deficient B cells, whereas transcription of the corticoids alter B cell populations intrinsically or extrinsically. mineralocorticoid receptor (Mr: encoded by Nr3c2) was unaltered (Fig. 4A). Cxcr4 transcripts in GR-deficient B cells were reduced Characterization of B cell–specific GRKO mice ∼50% compared with B cells from control mice (Fig. 4A), con- To determine direct versus indirect effects of glucocorticoids on sistent with a role for endogenous glucocorticoids in basal Cxcr4 B cells, we generated mice with B cell–specific GR deletion. expression. Cxcr5 transcripts were modestly, but not significantly, Mb1-Cre mice (23) were crossed with Nr3c1 floxed mice (24) to elevated in GR-deficient B cells (p = 0.054), whereas Ccr7 tran- generate B cellGRKO mice (Mb1Cre/wt Nr3c1fl/fl) and Cre-negative scripts were comparable in control and GRKO B cells (Fig. 4A). littermates (Mb1wt/wt Nr3c1fl/fl mice). B cellGRKO mice were born Ab labeling revealed a similar pattern of chemokine receptor at Mendelian ratios and exhibited normal weight, appearance, and expression; GR-deficient B cells had less CXCR4 on the cell behavior. Histological analyses revealed no evidence of general- surface than control B cells, whereas CXCR5 and CCR7 labeling ized pathology, and the lymphoid architecture in spleen and lymph were comparable between control and GR-deficient B cells nodes was normal (data not shown). (Fig. 4B). These results indicate that B cell CXCR4 is regu- We used flow cytometry to verify B cell–specific ablation of Gr lated in vivo by GR signaling at physiologic concentrations of in B cellGRKO mice. Whereas most cells in bone marrow, blood, glucocorticoids. 4 GLUCOCORTICOID RECEPTORS REGULATE B CELL MIGRATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Effects of glucocorticoid perturbation on B cells in vivo. Male C57BL/6 mice were injected i.p. with Dex (2 mg/kg) or vehicle once per day for 3 d, and then B cell populations in the bone marrow, blood, and spleen were enumerated by flow cytometry. (A) Representative dot plots from flow cytometric analyses of vehicle- and Dex-treated mice are shown. CD11b+CD11c+7AAD+ cells were excluded prior to gating on B220+CD32 cells (B cells) and B2202CD3+ (T cells). In bone marrow and spleen, B cells were further subdivided as pro–/pre–B cells (IgM2IgD2), immature B cells (IgMlowIgD2), transitional B cells (IgMhiIgDlow), and mature B cells (IgMlowIgDhi). In the spleen, the IgMhiIgDlow compartment comprises both transitional B cells and marginal zone B cells (T/MZ B cells). (B) The numbers (mean 6 SD) of B cells and T cells in the bone marrow, blood, and spleen in mice treated with vehicle (black) or Dex (open) are shown. Data were pooled from three independent experiments (n = 11 vehicle-treated mice and n = 11 Dex-treated mice). (C) The numbers (mean 6 SD) of B cells and T cells in the bone marrow, blood, and spleen of intact mice (black) and ADX mice (open) are shown. Data were pooled from three independent experiments (n = 14 intact mice and n = 12 ADX mice). *p , 0.05, **p , 0.01.

We analyzed CXCR4 expression by B cell populations in Next, we determined if GR deficiency impacts B cell migration the bone marrow, blood, and spleens of B cellGRKO and control toward the CXCR4 ligands CXCL12 (28, 29) and MIF (30). mice by flow cytometry. In both control and B cellGRKO mice, Splenic B cells from GR-deficient mice exhibited a modest but anti-CXCR4 labeling decreased with B cell maturation in bone reproducible reduction in migration toward CXCL12 in transwell marrow, but starting at the immature stage of differentiation, assays (p , 0.01) (Fig. 4D). Migration of control and GR- GR-deficient B cells exhibited less CXCR4 labeling than their deficient B cells toward CXCL12 was fully blocked by the control counterparts (Fig. 4C). Similarly, blood and splenic B cells CXCR4 antagonist AMD3100 (Fig. 4D), confirming that CXCL12 of B cellGRKO mice expressed lower levels of CXCR4 than in mediated B cell migration through CXCR4. However, we did not control mice (Fig. 4C), whereas T cells labeled comparably observe a chemotactic response of B cells, either control or GR (Fig. 4C). GR signaling plays an intrinsic role in regulating deficient, toward MIF (Fig. 4D) (recombinant MIF from two CXCR4 expression starting at the immature stage of B cell commercial sources at various concentrations were tested). In differentiation. contrast, GR-deficient B cells were fully competent to migrate The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Characterization of B cellGRKO mice. Mb1-Cre mice were crossed with Nr3c1fl/fl mice to generate animals lacking GR in B cells (B cellGRKO mice). (A) GR expression in bone marrow, blood, and spleen cells from control and B cellGRKO mice was determined via flow cytometry. After staining for surface Ags (B220, CD5, CD11b, and CD11c), cells were labeled intracellularly with anti-GR mAb. Multiparameter data were subjected to viSNE analysis based on B220, CD5, CD11b, and CD11c labeling, and discrete populations of B cells (B220+CD52CD11b2CD11c2), T cells (B2202CD5+CD11b2CD11c2), myeloid cells (B2202CD52CD11b+CD11c2), and dendritic cell (DCs) (B220+CD52CD11b2CD11c2) were identified in each tissue. GR labeling is depicted as a heat map on each viSNE plot. (B) GR expression by B cell precursors from bone marrow of control and B cellGRKO mice was investigated. B220+CD11c2CD11b2CD52 B cells were further subdivided into pro–/pre–B cells (IgM2IgD2), immature B cells (IgMlowIgD2), transitional B cells (IgMhiIgDlow), and mature B cells (IgMlowIgD+). Data are representative of three independent experiments (n = 3 mice of each ge- notype). (C) Susceptibility of GR-deficient B cells to Dex-induced cell death. Splenocytes from control (filled) and B cellGRKO (open) mice were cultured overnight with graded doses of Dex, and then viable B cells (B220+, left graph) and T cells (CD3+, right graph) were enumerated by flow cytometry. For each dose of Dex, the number of cells in each compartment was normalized to that in wells lacking Dex. The mean 6 SD relative number of cells from control (filled) and B cellGRKO (open) mice are shown. Data were pooled from three independent experiments, with three biological replicates per ex- periment. **p , 0.01. 6 GLUCOCORTICOID RECEPTORS REGULATE B CELL MIGRATION

toward CXCL13 and CCL19 (Fig. 4D), bound by CXCR5 and CCR7, respectively, that organize B cells in sec- ondary lymphoid tissues (31). GR deficiency does not impart a general defect in B cell migration but specifically impairs B cell responsiveness to CXCL12. Intrinsic GR deficiency alters B cell numbers in bone marrow and blood, but not in secondary lymphoid tissues To determine if GR deficiency affected B cell populations in tis- sues, we enumerated B cell populations in bone marrow, blood, and spleens of B cellGRKO mice and control littermates. B cellGRKO mice exhibited normal numbers of pro–/pre–, immature, and transitional B cells in bone marrow, but the number of mature B cells was reduced compared with controls (Fig. 5A), similar to ADX mice. B cellGRKO mouse blood contained 1.7-fold more B cells than controls (Fig. 5A), primarily because of excess mature B cells, although immature and transitional B cell numbers were also elevated (Fig. 5B). The spleens of GRKO Bcell mice were normal in size and in numbers of de- Downloaded from veloping and mature B cells (Fig. 5A). The lymph nodes of BcellGRKO were also normal size and contained similar num- bers of B cells as control mice (data not shown). Splenic T cell numbers were comparable across tissues in B cellGRKO and control littermates (Fig. 5A). B cellGRKO exhibited some of the

features observed in ADX mice, including decreased numbers http://www.jimmunol.org/ of mature B cells in bone marrow and increased numbers of B cells in blood, but B cell precursor numbers in spleen were not elevated in B cellGRKO mice as in ADX mice, suggesting that some aspects of the ADX phenotype reflect B cell–ex- trinsic effects of glucocorticoid deficiency. To investigate effects of GR deficiency on B cells in a non- lymphoid tissue, we lavaged the peritoneal cavities of control and B cellGRKO mice, where specialized B1a and B1b B cells are

common alongside canonical B2 B cells. As expected, B1a and by guest on September 27, 2021 B1b B cells from B cellGRKO mice lacked GR expression and, like B2 B cells, they exhibited significantly less surface CXCR4 than controls (Supplemental Fig. 2). However, we found no evidence that GR deficiency affected B1a, B1b, or B2 populations in the peritoneal cavity, as comparable numbers of each cell type were found in B cellGRKO mice and control mice (Supplemental Fig. 2). We expanded our studies of B cellGRKO animals to female mice and aged male mice. Female B cellGRKO mice (8–12 wk old) exhibited a similar B cell phenotype as males: decreased numbers of mature B cells in bone marrow, increased numbers of blood B cells, and normal counts of mature B cells in spleens (Supplemental Fig. 3). Notably, in female B cellGRKO mice, we observed a 1.7-fold increase in the number of immature B cells in the spleen compared with controls, a difference that was less apparent in male mice. In 12- to 15-mo-old male mice, mature B cell numbers in bone marrow approached that of control lit- FIGURE 4. Decreased CXCR4 expression in GR-deficient B cells. (A) termates (p . 0.05), but B cell numbers in blood were still ele- The mean + SD number of mRNA transcripts for Gr, Mr, Gilz, Cxcr4, vated (Fig. 5C). Splenic B cell numbers in aged B cellGRKO mice Cxcr5, and Ccr7 (normalized to Ppib) in control (gray bars) and GR-de- were similar to controls (Fig. 5C). The consistent phenotype of ficient (open) splenic B cells. Data represent five independent Q-PCR B cellGRKO mice across age and sex was an increase in blood experiments, with two mice of each genotype per experiment (n = 10); B cell numbers. except for Mr, where n =4.(B) The mean 6 SD median fluorescence intensities (MFIs) for APC anti-CXCR4, anti-CXCR5, and anti-CCR7 labeling of control (gray bars) and GR-deficient (open) splenic B cells. Data represent five independent flow cytometry experiments, with two mice of each genotype per experiment (n = 7–10). (C) The mean 6 SD assays toward CXCL12, CXCL12+AMD3100 (a CXCR4 antagonist), MIF, MFIs for anti-CXCR4 labeling of bone marrow (BM), blood, and spleen CXCL13, and CCL19. Each pair of connected points represents the mean cells from control and B cellGRKO mice, as defined in Fig. 2B. Data reflect number of control and GR-deficient B cells counted in the lower chamber of three independent experiments (n = 8 control mice and n = 6 B cellGRKO transwells from an independent study (n = 2–4 experimental replicates per mice). (D) Effect of GR deficiency on B cell migration in ex vivo transwell independent study). *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Redistribution of B cells from bone marrow to blood in B cellGRKO mice. B and T cell populations in male 8- to 12-wk-old control and B cellGRKO mice were enumerated by flow cytometry. (A) Representative dot plots from flow cytometric analyses of control (top row) and B cellGRKO (bottom row) mice are shown. CD11b+CD11c+7AAD+ cells were excluded prior to gating on B220+CD52 cells (B cells) and B2202CD5+ (T cells). In bone marrow and spleen, B cells were further subdivided as in Fig. 2B. The mean 6 SD numbers of B cell subsets and T cells in the bone marrow (femur and tibia), blood, and spleen of control mice (black) and B cellGRKO mice (open) are shown. Data were pooled from three independent experiments (n = 8 control mice and n = 6 B cellGRKO mice). (B) Blood B cells were subdivided by IgM and IgD expression, as in bone marrow and spleen. (C) The numbers (mean 6 SD) of cells in B cell subsets and T cells in bone marrow (femur and tibia), blood, and spleen were enumerated in aged (13–15 mo) male control (black) and B cellGRKO (open) mice. Data were pooled from two independent experiments (n = 7 control mice and n = 5 B cellGRKO mice). *p , 0.05, **p , 0.01.

Intrinsic GR signaling regulates diurnal exchange of B cells from morning to evening, despite a normal rise in serum corti- between blood and bone marrow costerone concentration (Fig. 6A). B cell GRs orchestrate circa- Diurnal patterns of glucocorticoid secretion drive circadian dian patterns of exchange between blood and tissues. rhythms of lymphocyte trafficking, with blood glucocorticoid To determine if GR deficiency affects B cell homing in a tissue- concentrations correlating inversely with blood lymphocyte counts specific manner, we employed competitive adoptive transfers. GRKO (17, 32). To determine if intrinsic GRs contribute to diurnal traf- Splenic B cells from control and B cell mice were differ- dim ficking patterns of B cells, we enumerated blood B cells in control entially labeled with CFSE to allow discrimination of CFSE GRKO bright dim and B cell mice at 9:00 AM and at 5:00 PM (Zeitgeber times and CFSE cells by flow cytometry. CFSE control B cells bright ZT2 and ZT10, respectively) when corticosterone concentrations and CFSE GR-deficient B cells were mixed at a 1:1 ratio and were low and high, respectively (Fig. 6A). In control mice, blood adoptively transferred into control mice. A second cohort of mice B cell counts were significantly higher in morning compared received a 1:1 mixture of B cells in which both CFSEdim and with evening (Fig. 6A), consistent with previous reports (32). CFSEbright cells were GR-sufficient. After 18 h, we determined the In B cellGRKO mice, however, circulating B cell counts were unchanged relative proportions of CFSEdim and CFSEbright cells in blood, 8 GLUCOCORTICOID RECEPTORS REGULATE B CELL MIGRATION

bone marrow, and various secondary lymphoid tissues of recipient mice. We calculated a Recovery Index as:

Recovery Index  À Á à Freq: of CFSEbright cells Freq: of CFSEdim þ CFSEbrightcells in tissue ¼ ½Freq: of CFSEbright cells in adoptive transfer

A Recovery Index .1 represented an enrichment of CFSEbright cells in tissue compared with the original cell mixture, whereas a value ,1 indicated a reduction in CFSEbright cells compared with the input. We observed a decreased Recovery Index of GR-deficient B cells in the bone marrow but not in the blood, spleen, lymph nodes, or Peyer’s patches (Fig. 6B, 6C). The Re- covery Index of GR-sufficient CFSEbright B cells, however, was comparable to the input mixture (Fig. 6B, 6C). We conclude that GR signaling in B cells enhances homing specifically to bone marrow. Humoral responses in B cellGRKO mice Downloaded from We hypothesized that GR deficiency in B cells would affect humoral immunity, either through dysregulated B cell migration or other modes of GR activity. However, serum concentrations of IgM, IgG, IgA, and IgE were comparable in control and BcellGRKO male mice (Fig. 7A), aged male mice (Fig. 7B), and female mice (Supplemental Fig. 3). We confirmed that GR was ablated in plasma cells of B cellGRKO mice (Supplemental Fig. 4) http://www.jimmunol.org/ but, consistent with serological data, the numbers of plasma cells/-blasts in the bone marrow and spleens of B cellGRKO mice were similar to those in littermate controls, as assayed by flow cytometry (Fig. 7C) and ELISpot (Fig. 7D). GR deficiency in the B cell lineage did not impact the basal generation ofIg-secreting cells. Lastly, we quantified Ab responses in B cellGRKO and control

mice to immunizations with various Ags. Following immunization by guest on September 27, 2021 with the T-dependent Ag NP-CGG, B cellGRKO mice generated Ag-specific IgM and IgG responses comparable to controls (Fig. 7E). Similarly, B cellGRKO and control mice mounted equivalent IgM and IgG responses to NP-LPS, a T-independent (Type 1) Ag (Fig. 7F). In response to the T-independent (Type 2) Ag NP-Ficoll, B cellGRKO mice mounted an NP-specific IgM re- sponse that was similar to control littermates, but generated sig- nificantly less NP-specific IgG than controls (Fig. 7G). The diminished IgG response of B cellGRKO mice to NP-Ficoll was surprising considering the immunosuppressive properties typically attributed to glucocorticoids, and indicates a positive role for GR signaling in certain aspects of humoral immunity.

Discussion In this study, we determined that glucocorticoids promote B cell expression of Cxcr4 (Fig. 1), and that alterations in GR signal- ing affect the distribution of B cells in blood and bone marrow (Figs. 2, 5). Using a B cell–specific GRKO mouse, we address FIGURE 6. GR deficiency in B cells disrupts trafficking between blood and bone marrow. (A) Effect of GR deficiency on B cell–circadian traf- ficking patterns. A cohort of male control and B cellGRKO mice were bled CFSEbright GR-deficient B cells (right column) were adoptively transferred 2 h after the start of the light cycle (9:00 h; Zeitgeber time ZT2) and into control recipient mice. Histograms show representative data of another cohort bled 2 h before the start of the dark cycle (17:00 h; ZT10). CFSEdim and CFSEbright B cells in the cell mixture used for adoptive Serum corticosterone concentrations were measured by ELISA; the left transfers (Input), and the proportions of CFSEdim and CFSEbright B cells in plot shows mean 6 SD corticosterone concentrations at each time point. recipient tissues 18 h after transfer (CFSE negative cells have been ex- The mean 6 SD number of blood B cells (B220+CD11b2CD11c2CD52) cluded). (C) A Recovery Index for control (closed) or GR-deficient (open) at each time point are shown at right. Data represent pooled data from B cells was calculated for each tissue of each recipient mouse. Each point three independent experiments, with n = 7–12 mice for each genotype at represents the recovery index from one recipient animal. Data reflect three each time point. (B) Effect of GR deficiency on B cell localization to independent experiments, with n = 6 recipients receiving control/control B different lymphoid tissues. A 1:1 mixture of CFSEdim control B cells and cell mixtures and n = 7 recipients receiving control/GRKO B cell mixtures. CFSEbright control B cells (left column) or CFSEdim control B cells and **p , 0.01, n.s., not significant. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 7. Humoral immunity in B cellGRKO mice. (A) Serum concentrations of IgM, IgG, IgA, and IgE from control (closed circles) and B cellGRKO (open circles) mouse are shown. Data reflect three independent ELISA experiments (n = 8 control mice and n = 7 B cellGRKO mice) (n = 7 control mice and n = 6 B cellGRKO mice for IgE). (B) Serum IgM, IgG, IgA, and IgE concentrations from aged (12- to 15-mo-old) naive male mice are shown. Data represent two independent ELISA experiments (n = 6 control mice and n = 4 B cellGRKO mice). (C) Representative dot plots of CD138hi FSCint plasma cells/-blasts in bone marrow (BM) and spleen from control (top row) and B cellGRKO (bottom row) mice are shown. The mean 6 SD number of plasma cells/-blasts are shown at right. Data were pooled from three independent experiments (n = 7 control mice and n = 6 B cellGRKO mice). (D) The mean 6 SD numbers of by guest on September 27, 2021 IgM-, IgG-, and IgA-secreting cells in BM (femur + tibia) and spleen are shown. Data were pooled from two independent ELISpot experiments( n =6 GRKO GRKO control [filled] and n = 6 B cell [open] mice). (E) T-dependent humoral responses to NP8-CGG/alum immunization of control and B cell mice. The mean 6 SD concentrations of NP-specific IgM and IgG from control (closed circles) and B cellGRKO (open circles) at each time point are shown. Data shown are results from one of two independent experiments (n = 4 for each genotype). (F) T-independent (Type I) humoral responses to NP-LPS by control and B cellGRKO mice. The mean 6 SD concentrations of NP-specific IgM and IgG from control (closed circles) and B cellGRKO (open circles) are shown (n = 7 for each genotype). (G) T-independent (Type II) humoral responses of control and B cellGRKO mice. The mean 6 SD concentrations of NP-specific IgM and IgG from control (closed circles) and B cellGRKO (open circles) on days 0, 5, 8, 16, and 24 are shown. Data were pooled from three independent experiments (n = 14 control mice and n = 9 B cellGRKO mice). **p , 0.01. long-standing questions regarding glucocorticoid effects susceptible to glucocorticoid-induced death regardless of tissue on lymphocyte migration and arrive at the following conclusions: location, but apoptotic losses in bone marrow are hidden by the 1) glucocorticoids act directly on B cells to promote homing glucocorticoid-induced influx of mature B cells from blood. specifically to bone marrow in association with increased CXCR4 Indeed, in the three mouse models we employed—a pharmaco- expression, and 2) GR-dependent regulation of B cell trafficking logic model (Dex treatment, Fig. 2A, 2B), a surgical model between blood and bone marrow occurs at physiologic concen- (adrenalectomy, Fig. 2C), and a genetic model (B cell–specific trations of glucocorticoids associated with diurnal patterns of GRKO mice, Fig. 5)—the common observation was that in- adrenal activity. creased glucocorticoid/GR activity supported mature B cell In humans and animals, glucocorticoid-induced changes in populations in bone marrow at the expense of blood, whereas immune cell numbers have typicallybeenattributedtowell- decreased glucocorticoid/GR activity had the reverse effect. We characterized lympholytic properties. Our study, however, posit that migration effects of glucocorticoids likely domi- highlights that glucocorticoid effects on B cell populations in vivo nate over survival effects in vivo, as increases in Cxcr4 tran- are complex and likely reflect regulation of both survival and scripts were evident at lower concentrations of Dex (1 nM, migration. This point is perhaps best illustrated in Dex-treated Fig. 1E) than death effects (10 nM, Fig. 3C). Moreover, our mice, in which B cell populations in blood and tissues were observations of altered B cell populations in bone marrow dramatically reduced, yet the number of mature B cells in bone and blood of B cellGRKO mice (Figs. 5, 6A), and defective mi- marrow remained stable, giving the appearance that this particular gration of GR-deficient B cells in adoptive transfer studies population was protected from glucocorticoid-induced death (Fig. 6B, 6C) occurred under physiologic conditions of adrenal (Fig. 2B). Based on our findings that glucocorticoids upregu- activity, not stress-induced or pharmacologic states in which late B cell CXCR4 and enhance homing to and/or retention in glucocorticoid-induced death is evident. We also suspect that survival bone marrow, we postulate that mature B cells are universally factors, such as BAFF, provide some protection for B cells from 10 GLUCOCORTICOID RECEPTORS REGULATE B CELL MIGRATION glucocorticoid-induced death in vivo, further separating the in vivo of serum autoantibody (36). Other than increased numbers of circu- concentrations at which glucocorticoids alter migration versus induce lating B cells, we found no evidence of generalized B lymphocytosis death. or pathology in B cellGRKO mice. GR-deficient B cells exhibited a Our finding that glucocorticoids regulate CXCR4 expression in 50% reduction in Gilz transcripts (Fig. 4A), indicating that residual, B cells to affect their migration to bone marrow explains the recent GR-independent Gilz expression prevents the pathology associated findings of Courties et al. (33), who observed in mice that ischemic with Gilz ablation. stroke provokes the same changes in B cell populations that we A fundamental tenet of immunology holds that recirculation observed with Dex treatment: decreased numbers of developing of B cells and T cells through lymphoid tissues promotes in- B cells but an abundance of mature B cells in bone marrow. Im- teractions between Ags and rare Ag-specific lymphocytes. Our portantly, this phenomenon occurred in association with a surge in adoptive transfer studies indicate that intrinsic GRs play a inflammatory cytokines and serum corticosterone, and was pre- minimal role in the recirculation of B cells through secondary vented by GR deletion in B cells. We suspect that glucocorticoid- lymphoid tissues, but may drive a distinct mode of B cell mi- induced changes in B cell migration may explain, at least in part, gration specifically through bone marrow (Fig. 6B, 6C). Be- the blood B lymphopenia associated with infection, inflammation, cause bone marrow is not subject to lymphatic drainage (37), and severe physical stress. we hypothesize that migration through bone marrow and sec- We observed similarities between B cellGRKO mice and B cell– ondary lymphoid tissues represent independent modes of specific CXCR4 KO mice, although the latter strain exhibits a recirculation, and that GR-mediated regulation of CXCR4 more dramatic phenotype. In B cell–CXCR4 KO mice, the bone provides a mechanism for rapid exchanges of B cells specif- marrow is nearly devoid of mature B cells, and substantial pop- ically between blood and bone marrow. Whereas B cell acti- Downloaded from ulations of B cell precursors occur in the blood and spleen, vation is normally associated with secondary lymphoid tissues, highlighting the role of CXCR4 in retaining both developing and recent studies suggest that bone marrow can support Ag-specific mature B cells in bone marrow (19). Similarly, in B cellGRKO humoral responses. Recirculating B cells localize to perivascular mice, the mature B cell population in bone marrow was reduced, spaces in bone marrow populated with T cells and dendritic cells and the numbers of immature and transitional B cells in blood (38), and mature B cells in bone marrow are capable of responding

were elevated (Fig. 5B), suggesting that glucocorticoid-dependent to blood-borne, multivalent Ags (39, 40). We speculate that the http://www.jimmunol.org/ regulation of CXCR4 supports the retention of precursors and shortage of mature B cells in the bone marrow of B cellGRKO mice mature B cells in bone marrow. Because CXCR4 expression is may explain the diminished IgG response to the multivalent Ag highest in early stages of B cell maturation [Fig. 4B and (19)], we NP-Ficoll (Fig. 7G); indeed, Ab responses to NP-Ficoll, but not the hypothesize that GR-independent expression of CXCR4 is sufficient T-dependent Ag NP-KLH, are also diminished in B cell–CXCR4 KO to retain most B cell precursors in bone marrow, but reduced mice, which lack mature B cells in bone marrow (19). Nonetheless, CXCR4 expression associated with GR deficiency in mature B cells glucocorticoids likely regulate other aspects of B cell biology, and impacts this population more severely, resulting in the redistribution determining the mechanism behind reduced humoral responses in of a substantial portion of this population to the blood. B cellGRKO mice warrants further investigation. Although glucocorticoids have been described to alter lymphocyte Based on findings that bone marrow contributes to humoral by guest on September 27, 2021 trafficking, it has been unclear if glucocorticoids act on tissues/ immunity (39), and that glucocorticoids regulate diurnal ex- endothelium to alter production of chemoattractants or if GR change of mature B cells between blood and bone marrow, we signaling in lymphocytes modifies responsiveness to migratory propose the following hypothesis. As an animal transitions from cues (10–13). Studies in humans showed that diurnal increases in resting to active periods of each day (morning for humans, circulating cortisol are accompanied by increased T cell expres- evening for rodents), adrenal production of glucocorticoids in- sion of CXCR4 and decreased T cell counts in blood, a correlation creases, promoting circulating B cells to migrate to bone mar- ablated by pharmaceutical antagonism of endogenous glucocor- row. During the active period of the day, in which the animal is ticoids (17). In mice, we similarly observed that blood B cell more likely to encounter pathogens and/or suffer injury in the counts correlated inversely with serum glucocorticoid concentra- process of foraging, avoiding predation, etc., GR-dependent tions throughout the day, and this relationship was uncoupled in maintenance of mature B cells in bone marrow ensures optimal B cellGRKO mice, which exhibited stable concentrations of B cells humoral defense against T-independent Ags. As glucocorticoid ac- in blood from morning to evening (Fig. 6A). We provide genetic tivity subsides with the transition to the resting period of the day, evidence that endogenous glucocorticoids act directly on B cells reduced CXCR4 expression results in release of mature B cells from to regulate diurnal traffic patterns. bone marrow pools into blood, allowing for migration into secondary Our study of B cellGRKO mice provides insight into the direct lymphoid tissues. versus indirect effects of glucocorticoids that were indiscernible in surgical and pharmaceutical models of glucocorticoid perturbation Acknowledgments (Fig. 2). Whereas ADX mice had increased numbers of B cell We thank the National Institute of Environmental Health Sciences Flow precursors in the spleen (Fig. 2C), the phenotype of B cellGRKO Cytometry Center for flow cytometry work. mice was subtler, with a B lymphocytosis that was primarily re- stricted to the blood (Fig. 5). In addition to glucocorticoids, the Disclosures adrenal glands produce mineralocorticoids and catecholamines, The authors have no financial conflicts of interest. and their absence in ADX mice may affect lymphocyte accumu- lation and/or turnover in peripheral lymphoid tissues. In contrast, ADX also disrupts the hypothalamic–pituitary–adrenal axis; dysreg- References ulated production of hypothalamic and/or pituitary hormones may 1. Go¨ttlicher, M., S. Heck, and P. Herrlich. 1998. Transcriptional cross-talk, the second mode of steroid action. J. Mol. Med. (Berl.) 76: 480–489. contribute to GR-independent features of the ADX phenotype (34). 2. Beaulieu, E., and E. F. Morand. 2011. Role of GILZ in immune regulation, gluco- Tsc22d3 (or Gilz), a well-characterized glucocorticoid target corticoid actions and rheumatoid arthritis. Nat. Rev. Rheumatol. 7: 340–348. 3. Scheinman, R. I., P. C. Cogswell, A. K. Lofquist, and A. S. Baldwin, Jr. 1995. gene, regulates B cell activation and survival (35, 36). GILZ KO Role of transcriptional activation of I kappa B alpha in mediation of immuno- mice exhibit B lymphocytosis (35) and increased concentrations suppression by glucocorticoids. Science 270: 283–286. The Journal of Immunology 11

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IgD

IgM

Transitional/ Spleen B cells Pro/Pre B Immature B Mature B Marginal Zone B

IgD

IgM

Supplemental Figure 1: B cell immunophenotyping by IgM/IgD expression. Viable CD11b- B220+ B cells were gated prior to IgM/IgD plots shown above. In each histogram, the gray line represents antibody labeling of all viable cells whereas the colored line represents labeling of the B cell subset defined by IgM and IgD expression.

Supplemental Figure 1 T cells A B1a B cells

B2 B1b

B B1a B1b B2 T cells

Control

B cellGRKO

GR

C D 600 2.0

500 Control

) 1.5 400 * 6

* B cellGRKO x10 300 * 1.0 200

No. cellsNo.( 0.5 APC CXCR4 APC (MFI) 100

0 0.0 B1a B B1b B B2 B T cells B1a B B1b B B2 B T cells cells cells cells cells cells cells

Supplemental Figure 2: Peritoneal B cells in control and B cellGRKO mice. (A) Gating strategy to identify T cells and B1a, B1b, and B2 B cells in peritoneal lavages. (B) GR expression by peritoneal T cells, B1a B cells, B1b B cells, and B2 B cells. Shaded histograms represent labeling with rabbit IgG isotype control and open histograms are anti-GR labeling. (C) The mean±SD MFI of APC anti-CXCR4 labeling of peritoneal B1a, B1b, and B2 B cells, as well as T cells from control (gray bars, N=5) and B cellGRKO (open bars, N=4) mice. (D) The mean±SD numbers of B1a B cells, B1b B cells, B2 B cells, and T cells in peritoneal lavages of control (gray bars, N=5) and B cellGRKO (open bars, N=4) mice. *, P<0.05.

Supplemental Figure 2 A Bone marrow Blood Spleen Spleen 7 8 1.8 50 Control ** 7 * )

6 6

) 1.5

)/ml 40 6 B cell-GRKO 6 6 5 5 1.2 4 30 4 0.9 3 3 20 0.6 2 2 (x10 No. cells

No. cells (x10 cells No. 10 No. cells (x10 No. cells 0.3 1 ** 1 0 0 0.0 0 Pro,Pre B Imm B Trans B Mature B B cells T cells Pro, Pre Imm B T/MZ B Mature B B

B Serum Immunoglobulin Serum IgE 1000 1000

100 100 Control

B cell GRKO

Ig (µg/ml) Ig 10 10 IgE (ng/ml)

1 1 IgM IgG IgA IgE

Supplemental Figure 3. Characterization of female B cellGRKO mice. (A) B and T cell populations in female 8-12 week-old control and B cellGRKO mice were enumerated by flow cytometry. B cell populations were defined through flow cytometric gating as shown in Fig. 5A. The mean±SD numbers of B cell subsets in the bone marrow (femur and tibia), blood, and spleen of control mice (black) and B cellGRKO mice (open) are shown. Data were pooled from three independent experiments; N=10 control mice and 8 B cellGRKO mice. *, P<0.05; **, P<0.01. (B) Concentrations of serum IgM, IgG, IgA (left panel), and IgE (right panel) in female 8-12 week-old control (filled) and B cellGRKO (open) mice are shown. N=10 control mice and 7 B cellGRKO mice. *, P<0.05; **, P<0.01.

Supplemental Figure 3 Control

B cellGRKO

Supplemental Figure 4. GR expression by plasma cells from control and B cellGRKO mice. Bone marrow cells from male 8-12 week-old control and B cellGRKO mice were analyzed for GR expression by flow cytometry. Plasma cells were gated as CD11b-CD11c-CD138hi cells. At right, the overlaid histograms show labeling with secondary detection reagent alone (gray) or anti-GR mAb (red). Data represent results of two independent experiments.

Supplemental Figure 4