FHL2 Is Essential for Spleen T Cell−Dependent B Cell Activation and Antibody Response

Talin Ebrahimian, France Dierick, David Simon, Maryam Heidari, Alexandre Orthwein, Koren K. Downloaded from Mann and Stephanie Lehoux

ImmunoHorizons 2020, 4 (5) 259-273 doi: https://doi.org/10.4049/immunohorizons.2000014 http://www.immunohorizons.org/content/4/5/259 http://www.immunohorizons.org/ This information is current as of September 27, 2021.

Supplementary http://www.immunohorizons.org/content/suppl/2020/05/20/4.5.259.DCSupp Material lemental References This article cites 70 articles, 30 of which you can access for free at: http://www.immunohorizons.org/content/4/5/259.full#ref-list-1 by guest on September 27, 2021 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts

ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE

Adaptive Immunity

FHL2 Is Essential for Spleen T Cell–Dependent B Cell Activation and Antibody Response

Talin Ebrahimian, France Dierick, David Simon, Maryam Heidari, Alexandre Orthwein, Koren K. Mann, and Stephanie Lehoux Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec H3T 1E2, Canada Downloaded from

ABSTRACT Four-and-a-half LIM domain 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal http://www.immunohorizons.org/ transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL22/2 mouse spleens displayed enlarged follicles with more B cells. When a T cell–dependent immune response was elicited using SRBC, FHL22/2 germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL22/2 mice, and circulating plasma cells were also reduced in FHL22/2. This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL22/2 B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell–deficient mMT mice transplanted with WT or FHL22/2 bone marrow, suggesting that the defects observed in FHL22/2 mice were not B cell intrinsic. However, spleen lysates from FHL22/2 mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to by guest on September 27, 2021 a T cell–dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2. ImmunoHorizons, 2020, 4: 259–273.

INTRODUCTION interacts with nuclear receptors, transcription factors, and transcription coregulatory , either enhancing or blocking Four-and-a-half LIM domain protein 2 (FHL2; also known as downstream expression. It binds to and alters the function SLIM3) is the best-studied member of the LIM-only subclass of of kinases, metalloproteinases, and adhesion molecules, altering the LIM protein superfamily. LIM proteins are defined by the protein activity and cellular localization (3–8). In turn, FHL2 presence of one or more LIM domains composed of a conserved modulates cellular processes such as cell survival, proliferation, cysteine-rich module that mediates protein–protein interactions migration, and signal transduction (2, 9, 10). The multiple actions (1). Multiple roles have been ascribed to FHL2, most of which can of FHL2 are described in detail in a recent review (2), and those be attributed to its interactions with specific proteins (2). FHL2 relevant to B cells are outlined in Supplemental Table I (11–24).

Received for publication March 16, 2020. Accepted for publication May 4, 2020. Address correspondence and reprint requests to: Dr. Talin Ebrahimian, Lady Davis Institute for Medical Research, 3755 Cote Ste-Catherine Road, Montreal, QC H3T 1E2, Canada. E-mail address: [email protected] ORCID: 0000-0003-3366-1123 (K.K.M.). This work was supported by a Canadian Institutes of Health Research (CIHR) project grant (to the S.L. laboratory); a CIHR project grant, a grant-in-aid from the Sir Mortimer B. Davis Foundation – Jewish General Hospital, and a transition grant from the Cole Foundation (to the A.O. laboratory); and a CIHR project grant (to the K.K.M. laboratory). A.O. is a Canada Research Chair (Tier 2) in genomic instability and hematological malignancies. Abbreviations used in this article: AID, activation-induced cytidine deaminase protein encoded by the Aicda gene; CSR, class-switch recombination; DZ, dark zone; FHL2, four-and-a-half LIM domain protein 2; Fo, follicular; FOXO1, forkhead box class O-1; GC, germinal center; LZ, light zone; Mz, marginal zone; PC, plasma cell; PNA, peanut agglutinin; S1P, sphingosine-1-phosphate; Tfh, T Fo helper cell. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2020 The Authors https://doi.org/10.4049/immunohorizons.2000014 259

ImmunoHorizons is published by The American Association of Immunologists, Inc. 260 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

Despite the fact that FHL2 regulates the susceptibility of mice Germany). Both strains were bred in-house, and all the experiments to infection and inflammation (25–28), its role in immune cell were performed on age-matched animals. To evaluate the role of function has hardly been studied. Overexpression of FHL2 in vivo FHL2 in T cell–dependent immune responses, mice were injected does not affect differentiation of the B cell lineages in the bone with 200 ml of 10% SRBC (equivalent to 1 3 108–5 3 108 SRBC; marrow (29), and FHL22/2 mice have a normal frequency of Innovative Research) or vehicle (saline), injected i.p. Animals myeloid cells, B cells, and T cells in the bone marrow, spleen, and were sacrificed at day 6 postinjection. Upon sacrifice, blood was (30). Nevertheless, there is evidence that thioglycollate- obtained by cardiac puncture and collected in EDTA-coated tubes induced B cell migration is enhanced in FHL2-deficient mice (31). (Sarstedt, Numbrecht,¨ Germany). Blood cell enumeration was FHL22/2 mice also show attenuated airway infiltration of B cells performed with a hematology analyzer (scil Vet abc). IgG1 and associated with decreased plasma IgE and IgG1 levels following IgM levels were measured in the plasma of vehicle or SRBC- OVA challenge (27). Hence, FHL2 may regulate B cell differen- injected mice using an ELISA kit (eBioscience, New York, NY) tiation or maturation in the secondary lymphoid organs. Notably, according to the manufacturer’sprotocol. FHL2 deletion alters the migration and cytokine release by dendritic cells and macrophages (26, 32, 33), two cell types that Bone marrow transplantation also reside within such organs and influence the trafficking and To evaluate the role of FHL2 specifically in B cells, we performed Downloaded from positioning of lymphocytes between light zones (LZ) and dark irradiation-transplantation experiments using B cell–deficient zones (DZ) of the germinal center (GC) (34, 35). mMT mice (The Jackson Laboratory, Bar Harbor, ME) as recipients. Beyond these effects of FHL2 on immune cells, several FHL2- mMT mice are deficient for IgM transmembrane tail exons, and bindingpartnersareknowntoinfluence B cell function and B cell development is blocked at the pro–B stage. Eight-week-old distribution within the spleen, further suggesting that FHL2 may mMT male mice were lethally irradiated with a single dose of http://www.immunohorizons.org/ be a leading regulator of these cells. For example, FHL2 interacts 10 Gy (0.19 Gy/min, 200 kV, 4 mA) total body irradiation (Andrex with forkhead box class O-1 (FOXO1) in the nucleus, resulting in Smart 225 Rontgen¨ source; YXLON International) immediately decreased transcriptional activity of FOXO1 and expression of before transplantation. Bone marrow was isolated by flushing the known FOXO target (36, 37). FOXO1 is important for the femurs and tibias of male WT or FHL22/2 mice with RPMI 1640 formation of the GC DZ (38) and is involved in somatic containing 10% FBS. Irradiated recipients received 2 3 107 bone hypermutation (17). Furthermore, FHL2 binds sphingosine kinase, marrow cells by retro-orbital sinus injection. After a 6-wk preventing activation of sphingosine-1-phosphate (S1P). S1P is recovery period, mice were injected with 10% SRBC or vehicle. involved both in sequestration of lymphocytes in secondary Mice were sacrificed 6 d thereafter. lymphoid organs (39) and in memory B cell migration from GCs (40). Migration is also influenced by the oxygen tension within Flow cytometry analysis by guest on September 27, 2021 the spleen, which itself regulates CXCR4 and CXCR5 signaling Splenocytes were prepared by gently mincing the spleen and through HIF-1ɑ (20). FHL2 directly interacts with and represses passing through a cell strainer (100-mm pores; BD Biosciences, transcriptional activity of HIF-1a (41). Finally, FHL2 acts as a San Jose, CA). Cells were centrifuged for 5 min at 400 3 g, transcriptional corepressor of the NR4A nuclear receptors, resuspended in 4% Fc block (MACS Miltenyi Biotec) and stained including Nur77 (42). Nur77 is recognized as a specific reporter with Abs targeting the surface markers B220, CD21, CD23, GL7, of the BCR signal strength (18), serving as a marker of B cell Ag CD138, IgG1, IgM, CD83, and CXCR4 (BioLegend, San Diego, CA, exposure (19). and eBioscience). Flow cytometry was performed on the BD LSR In this study, we set out to definetheroleofFHL2inBcell Fortessa (BD Biosciences). Fluorescence minus one control was maturation in the spleen. We used a knockout mouse model to used to determine fluorescence background and positively. Data demonstrate that the presence of FHL2 is essential to control analysis was performed using FlowJo software. Gating was first splenic B cell expansion, class-switch recombination (CSR), performed on forward versus side scatter to remove cell debris plasma cell (PC) differentiation, and Ab production in response and doublets before selection of live cells based on exclusion of a to a T cell–dependent Ag. Our data highlight the many ways by viability dye, 7-aminoactinomycin D (BioLegend), or Aqua (BD which FHL2 influences B cell and Ab response, possibly through Biosciences). B cells were selected based on their B220 marker regulation of cytokine production. expression. Among B cells, different subtypes were detected based on their differential expression of markers, CD212/intCD23+ for follicular (Fo) subsets, and CD21+CD232 for marginal zone (Mz) MATERIALS AND METHODS subsets. GC B cells were detected based on GL7 marker expression, and LZ and DZ cells were identified based on CD83 and CXCR4 Animal housing and immunization marker expression, respectively. Plasmablasts were defined as The McGill University Animal Care Committee approved the (B220hiCD138+)andPCsas(B2202CD138+). T Fo helper cells experimental protocol, and animals were handled in accordance (Tfh) cells were identified as PD1+CXCR5+ (among CD4+ Tcells). with institutional guidelines. Experiments were performed using Each cell population was considered positive or negative compared 8–10-wk-old male wild-type (WT; C57BL/6) or FHL22/2 mice with degree of fluorescence when stained with appropriate isotype (initially provided by Dr. Buttner,¨ University of Bonn, Bonn, control Ab.

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 261

Immunofluorescence as calibrator samples. The analyzed genes were expressed All immunofluorescence experiments were performed on OCT- relative to the murine Rps16 housekeeping gene (forward: embedded sections from mouse spleens. Naive, GC, LZ B cells, 59-ATCTCAAAGGCCCTGGTAGC-39, reverse: 59-ACAAAGGTAAA PCs, and total T cells and Tfh cells within spleens were identified CCCCGATCC-39). by immunofluorescence. Briefly, the sections were fixed with 4% paraformaldehyde and incubated with 5% BSA and 5% goat serum Western blotting (Sigma-Aldrich). The spleen sections were then incubated with Radioimmunoprecipitation assay buffer–soluble proteins (15–20 fluorescently labeled or unlabeled anti-B220 (eBioscience), anti- mg) from spleen or isolated B cells were separated by SDS-PAGE, GL7, anti-CD86, or anti-IgG1 (BioLegend) primary Abs. Sections transferred to nitrocellulose membranes, and probed with rabbit were rinsed and further incubated with fluorescently labeled anti-FHL2 Ab (1:1000; Abcam, Cambridge, MA) overnight at 4°C. secondary Abs (1:400; Invitrogen) as needed. For one set of Membranes were washed three times with TBST and incubated experiments, sections were incubated with rhodamine-coupled with HRP goat anti-rabbit Ab (1:1000) (Bio-Rad Laboratories, peanut agglutinin (PNA; Vector Laboratories, Burlingame, CA). Saint-Laurent, Canada). Membranes were revealed by chemilu- Sections were mounted with medium containing DAPI (Vector minescence with the ChemiDoc MP System molecular imager Laboratories). At least five sections per animal were stained. (Bio-Rad Laboratories) and quantified by densitometry using Downloaded from Negative controls were performed using sections incubated with Quantity One software. secondary Abs only. Cytokine Ab array Plasma Abs Proteins were extracted from spleens by Triton X-100 (1%). Splenic lysates were analyzed with a cytokine Ab array (R&D

Blood was obtained by cardiac puncture on EDTA. Plasma and http://www.immunohorizons.org/ cells were separated by centrifugation (5 min at 2000 3 g). Total Systems, Minneapolis, MN), according to the manufacturer’s plasma IgM and IgG1 levels were measured using ELISA instructions. Briefly, cytokine array membranes were blocked in kits according to the manufacturer instructions (Invitrogen, 2 ml of blocking buffer for 1 h and then incubated with 1 ml of the Carlsbad, CA). spleen samples and the Ab mixture overnight. The samples were then decanted from each container, and the membranes were B cell isolation washed three times with 20 ml of wash buffer before incubation Spleensingle-cell suspensions were preparedfromminced spleens for 30 min in 1:2000-diluted, HRP-conjugated streptavidin. The by dissociation through 70-mm filters using a syringe plunger. membraneswere exposed to a peroxidase substrate for1 min in the Bcell–enriched populations were separated by an autoMACS Pro dark before imaging with the ChemiDoc MP System molecular Separator (STEMCELL Technologies, Vancouver, BC) using a imager (Bio-Rad Laboratories) and quantified by densitometry by guest on September 27, 2021 Bcellpurification kit according to the manufacturer’s instructions. using Image Lab Software. B cells were cultured for 1–4 d in B cell medium (RPMI 1640, 2 mM GlutaMAX, 20 mM 2-ME, 1% penicillin/streptomycin, and 10% Statistical analysis FBS) in the presence of LPS (25 mg/ml) or anti-CD40 Ab (1 mg/ml) Statistical analyses were performed using GraphPad Prism and mouse IL-4 (IL-4; 10 ng/ml; PeproTech, Rocky Hill, NJ). software. Experimental groups were compared using a two- At days 1 and 4, B cells were collected, centrifuged for 5 min at tailed Student t test or Mann–Whitney U nonparametric test 400 3 g, resuspended in 4% Fc block (MACS Miltenyi Biotec), and (when n , 5). To assess comparisons between multiple groups, stained for IgG1, IgM, and IgD. B cells were counted using a Z2 cell data were analyzed using ANOVA after confirmation of normal counter (Beckman Coulter, Atlanta, GA). distribution using the Shapiro–Wilks test, followed by Newman– Keuls posttest. Data are presented as mean 6 SEM. A p value ,0.05 Analysis of splenic cell by real-time PCR was considered to be statistically significant. Total RNA from spleen was isolated using a Total RNA Mini Kit (Geneaid, New Taipei City, Taiwan). RNA was quantified on a spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scien- RESULTS tific, New York, NY). cDNA was synthesized from 1 mgRNAby using qScript cDNA SuperMix Kit (Quanta Biosciences, Beverly, FHL2-deficient mice display increased splenic follicles and CA). The expression level of activation-induced cytidine de- B cell subsets aminase protein encoded by the Aicda gene (AID) was analyzed We first verified that FHL2 was expressed in the spleen and by quantitative real-time PCR using 7500 Fast PCR (Applied particularly in spleen B cells. This was confirmed by Western Biosystems, New York, NY) under standard conditions of 60°C blot analysis of whole spleen and isolated B cells from WT and annealing temperature for 40 cycles. SYBR Green chemistry FHL22/2 mice, as well as spleen immunohistochemistry (Sup- (SYBR Green FastMix, Low ROX; Quanta Biosciences) was used plementalFig.1A).BecauseFHL2 was expressed in all the with specific primers (forward: 59-GCCACCTTCGCAACAAGTCT- regions of the spleen, we determined if its absence would trigger 39, reverse: 59-CCGGGCACAGTCATAGCAC-39; PrimerBank iden- a specific phenotype. Under basal conditions, FHL22/2 mice tifier 6753018a1). Results were analyzed using the DDCt method displayed significantly greater spleen/body weight ratio than

https://doi.org/10.4049/immunohorizons.2000014 262 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

WT (0.09 6 0.005 versus 0.06 6 0.001, respectively) and 50% class switch of IgM toward other Igs occurs. The DZ is where larger spleen follicles than WT, but equal follicle numbers (Fig. 1A). activated B cells undergo clonal expansion. Typically, these zones Flow cytometry of whole spleens was used to identify specificcell are identified by the surface expression of the CXCR4 chemokine subsets according to the gating strategy depicted in Supplemental (DZ) and CD83 (LZ) (45). To define whether increased Fig. 1B. Greater total B220+ B cell content (46.0 6 2.5 versus 31.0 6 GC size in FHL22/2 mice was associated with an abnormal GC 4.0%), including Fo (81.0 60.8 versus 66 62.5%) and Mz (6.6 60.7 architecture, we measured the expression of these markers by flow versus 3.8 6 1.0%) B cells, was quantified in FHL22/2 versus WT cytometry within GCs (B220+GL7+). Interestingly, a significant spleens. No differences in B2202 cells, including T cells, were 4.6-fold increase of DZ/LZ ratio was observed in SRBC- versus noted between genotypes (Fig. 1B). vehicle-injected FHL22/2, whereas no such increase was observed To address the potential role of FHL2 in splenic B cell responses in WT mice (Fig. 4A). This was confirmed with immunofluorescent to T-dependent Ag, WT and FHL22/2 mice were injected with 10% staining for CD86 and PNA and LZ and GC markers, respectively, SRBC, and the spleens were assessed 6 d postinjection. GC response showing a preferential expansion of the DZ in FHL22/2 mice is typically detected 4 d after immunization, peaks at day 6, and (Fig. 4B). Along with data above showing increased GC size, progressively diminishes over the ensuing 2 wk (43, 44). Both mouse these new data indicate altered DZ/LZ ratio in FHL2-deficient strains responded similarly to SRBC such that no further differences mice and further suggests that FHL2 regulates spleen DZ program Downloaded from were observed in terms of spleen/body weight ratio, spleen B cells, upon immunization with SRBC. and different B subsets (Fig. 2A). Total T cells were equivalent in SRBC-injected FHL22/2 versusWTmice(Fig.2B).Tovisualize FHL2-deficient mice display a defect in spleen and plasma spleen B and T cell distribution, immunofluorescence for B220 and SRBC-induced IgG1 and circulating PCs CD3 markers was performed. Data show a comparable increase in Upon full activation and CSR, B cells first become plasmablasts, http://www.immunohorizons.org/ B cell content by SRBC in both groups (WT, 3.5-fold; FHL22/2, expressing class-switched Abs, and then differentiate into Ab- 2-fold), with similar B and T cell localization (Fig. 2C). Supportive of secreting cells or PCs. PCs express high-affinity Abs against a given flow cytometry data, higher proportions of B cells were visualized in Ag, exit from the spleen, and secrete the Abs into the circulation. spleens from FHL22/2 mice than WT, but no difference was SRBC is a well-known Ag inducing IgG1 production by B cells (43). observed in CD3 T cell abundance (Fig. 2C). To examine whether the expanded DZ in FHL22/2 GCs affected We did not observe any differences between WT and FHL22/2 theimmuneresponsetoSRBC,wefirst measured B220high- mice in B cells in the bone marrow (Supplemental Fig. 2) or in CD138high spleen plasmablasts and B220lowCD138high spleen PC circulating B cells (see below), suggesting that FHL2 deficiency among live cells by flow cytometry. There was no difference in the mainly affects B cell subsets within the spleen rather than B cell abundance of both these populations between FHL22/2 and WT development. We also investigated the effects of FHL2 loss on lymph mice (Fig. 5A). However, the increase in SRBC-induced spleen by guest on September 27, 2021 nodes and in gut Peyer’s patches. In contrast to spleen, there was no B cells expressing IgG1, which averaged 2-fold in WT mice, was significant differences in lymph node size or B cell numbers or in the absent in FHL22/2 (Fig.5B).Immunofluorescence staining for IgG1/IgM immune phenotype of B cells in these other lymphoid IgG1 was similarly lower in the spleen of SRBC-injected FHL22/2 tissues, between FHL2 knockout and WT mice (data not shown). mice compared with WT. Correspondingly,SRBC induced a rise in plasma IgG1 in WT mice, but not in FHL22/2 mice, whereas IgM FHL2-deficient mice display enlarged GCs with a normal levels were similarly increased in both mouse strains (Fig. 5C). In induction in response to SRBC line with the impairedinductionof plasma IgG1, PCs were reduced ThefactthatFHL2influences spleen cell subsets and Fo zone by 32% (p , 0.05) in SRBC-immunized FHL22/2 versus WT mice, surface area prompted us to investigate if it also alters B cell with no change in total plasma naive B cells (Fig. 5D). These results distribution within the GCs. Among Fo cells (Supplemental Fig. 1B), suggest that decreased plasma IgG1 levels in FHL22/2 mice could the expression of GC B cell marker GL7 was equivalent in WT and be explained by decreased generation of PCs from the spleen. FHL22/2 mice at baseline, and it was increased to the same extent Because IgG1 expression and generation upon stimulation with (4-fold) in both strains 6 d after induction of the GC reaction by SRBC is the consequence of B cell CSR, we assessed the expression immunization with SRBC(Fig.3A).Immunofluorescence revealed of spleen Aicda which encodes the AID protein, the enzyme that thatthenumberofGCsperspleenwassimilarbetweenWTand initiates somatic hypermutation and CSR in activated B cells (46). FHL22/2 animals (Fig. 3B). However, the area of GL7 expressing TheriseinthemRNAexpressionofAicda was more modest in B cells, indicative of GC size, was 2-fold bigger (0.07 60.01 versus 0. spleens of SRBC-induced FHL22/2 compared with WT mice 03 6 0.01 mm2)inFHL22/2 than WT mice upon immunization. (3- versus 7-fold, respectively) (Fig. 5E). Hence, impaired IgG1 These results indicate that FHL2 regulates GC size without expression and production in FHL22/2 mice was likely due to a affecting GC B cell induction and number in response to SRBC. less-efficient activation of B cells. Because the presence of Tfh cells is essential for B cell FHL2-deficient mice display a defect in GC structure with activation within GCs (47, 48), the abnormal GC structure and an expanded DZ upon stimulation impaired immune response to SRBC in FHL22/2 mice might also Normal GCs are composed of two distinct zones. The LZ is be explained by the decrease or the absence of such T cells. typically where B cells interact with Th cells to get activated and Therefore, we compared spleens of immunized WT and FHL22/2

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 263

mice for total CD4+ T cells (Supplemental Fig. 1B), as well as T cells among the CD4+ subset coexpressing CXCR5 and PD-1, a combination of markers that is widely used in identifying Tfh (49). We did not find any significant difference in Tfh cell content or phenotype between FHL22/2 and WT mice (Fig. 5F), suggesting that FHL2 null mice do not present any defect in T cell activation. Overall, these results demonstrate that upon immunization with SRBC, the presence of FHL2 is an essential requirement in 1) the establishment of a normal GC structure, particularly for the regulation of the DZ phenotype, and 2) full B cell activation through regulation of AID leading to CSR, PC generation, and IgG1 production.

FHL2-deficient B cells display increased proliferation but no Downloaded from defect in CSR in vitro To better assess whether altered B cell function could account for the expanded DZ and reduced IgG1 in FHL22/2 mice, we verified two key aspects of the GC reaction in vitro: proliferation and CSR. Experiments were performed with isolated B cells from FHL22/2 http://www.immunohorizons.org/ or WT mice cultured for 4 d in the presence of LPS or an agonistic anti-CD40 Ab and IL-4 to mimic a T cell–independent or –dependent stimulation, respectively. Four days after stimulation with LPS, B cell numbers increased to the same extent (;70%) in WT and FHL22/2 cell fractions (Fig. 6A). However, FHL22/2 cell numbers displayed a greater increase (80%) when stimulated with anti-CD40 plus IL-4 than WT (;50%) (Fig. 6A), in line with our in vivo data showing DZ expansion. These results indicate that FHL2 controls B cell proliferation following a T cell–dependent type of stimulation. Concomitantly with B cell proliferation, we measured by guest on September 27, 2021 IgG1 and IgM/IgD among B220 cell fraction to assess CSR. CSR was equivalent between WT and FHL22/2 with both types of stimulations, LPS or anti-CD40 plus IL-4, showing reduced IgM/ IgD and increased IgG1 4 d after stimulation (Fig. 6B). Our data suggest that the defect in CSR in FHL2 deficient mice is not B cell intrinsic.

mMT chimeric mice display no intrinsic defect in FHL2-deficient B cells Our in vitro data prompted us to investigate whether impairment of CSR and IgG1 production in FHL22/2 mice might be due to an abnormal spleen environment rather than abnormal splenic B cells. Bone marrow transplantation experiments were performed

spleens, but larger follicles in FHL22/2.(B) Flow cytometry of mouse spleens indicates that the proportion of B cells (B220+) among live cells wasenhancedinFHL22/2 compared with WT, whereas non–Bcell 2 FIGURE 1. FHL2-deficient mice display increased splenic follicles and populations (B220 ) were equivalent in both genotypes. When measured B cell subsets. among the population of spleen B220+ B cells, the proportion of follicle 2 2 2 2 (A) Spleen/body weight ratio (both measured in grams) reveals an en- (CD21 /intCD23+) and Mz (CD21+CD23 ) B cells was greater in FHL2 / hanced relative spleen size in FHL2/2 mice. Immunofluorescence images than WT. T cells (CD4+) were present in equal proportions among live of spleens stained with Madcam-1 (green) reveal equal follicle numbers cells in both animal groups. Data are mean 6 SEM. Scale bar, 50 mm. per microscopic field (original magnification 35) in WT and FHL22/2 #p , 0.05, ##p , 0.01, ###p , 0.001 versus WT.

https://doi.org/10.4049/immunohorizons.2000014 264 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

using mMT as recipient mice (50). This model lacks any mature splenic B cells (Supplemental Fig. 3A). Transplant of lethally irradiated mMT mice with GFP-positive bone marrow cells indicated that 6 wk after reconstitution, ;95% of total spleen cells were from donor mice (GFP positive), including 98% of total B cells and 75% of total T cells (Supplemental Fig. 3B). Henceforth, lethally irradiated mMT-recipient mice were trans- planted with total bone marrow from WT or FHL22/2 mice, yielding mWT or mFHL22/2 animals. After 6 wk, the mice were injected with SRBC or vehicle (Fig. 7A). No difference was observed in spleen/body weights or B and T cell subsets between groups (Supplemental Fig. 3C). GC induction in response to SRBC was similar as well between mWT and mFHL22/2 chimeras. Importantly, the difference in GC size and DZ/LZ ratio observed between WT and FHL22/2 micewasnolongersignificant between Downloaded from mWT and mFHL22/2 animals (Fig. 7B). Furthermore, induction of spleen IgG1 determined by immunofluorescence, as well as splenic Aicda mRNA levels, were equally increased between both immunized groups (Fig. 7C). We did not observe a significant increase of plasma IgG1 levels with SRBC injection, http://www.immunohorizons.org/ and IgG1 levels were equal between mWT and mFHL22/2 mice, before and after SRBC injection (Fig. 7D). However, plasma IgM levels were elevated in mFHL22/2 compared with mWT mice, both before and after SRBC immunization (Fig. 7D). Overall, these results suggest that GC B cell hyperproliferation and the defect in IgG1 response to SRBC in FHL22/2 mice were not due to defective B cells but most likely to abnormal spleen microenvironment.

FHL2-deficient mice display reduced spleen CXCL12 and by guest on September 27, 2021 CXCL13 levels The proper activation of B cells in the spleen is directly related to their positioning and trafficking within GCs, and cell motility is orchestrated by the production of different cytokines. Conse- quently, we measured the main splenic cytokines known to regulate B cell function, using an unbiased array approach. The levels of three highly expressed splenic cytokines involved in B cell recruitment, CXCL5, soluble ICAM-1, and IL-16, were not different between groups (Fig. 8A). However, basal levels of CXCL13 and CXCL12 were significantly lower, by 16 and 35%,

proportions of B cells of B220+ among live cells, Fo B cells (CD212/intCD23+ among B220+), and Mz B cells (CD21+CD232 among B220+)inFHL22/2 are demonstrated by flow cytometry. (B) Proportions of T cells (CD4+ among live cells) remained indistinguishable between WT and FHL22/2. (C) Representative immunofluorescence images of spleen stained to reveal B cells (B220+, green), T cells (CD3+, red), and cell nuclei (DAPI, FIGURE 2. SRBC immunization has a similar effect on WT and blue). Quantification of relative spleen area covered by the different 2 2 FHL22/2 spleen B cell subsets. cell types shows that B cell zones expanded in both WT and FHL2 / WT and FHL22/2 mice were immunized i.p. with 10% SRBC or vehicle following SRBC, whereas T cell zones were not significantly altered. (Veh). Spleens were harvested at day 6 postinjection. (A) Spleen/body Data are mean 6 SEM. Scale bar, 50 mm. #p , 0.05, ##p , 0.01, weight ratio (both measured in grams) was elevated in FHL22/2 mice ###p , 0.001 versus WT-Veh or WT-SRBC; **p , 0.01 versus WT-Veh treatedwithVehorSRBC,comparedwithWT.Similarly,increased or FHL2-Veh.

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 265

A Veh SRBC 1% 5%

GC cells

** * WT WT +

1% 6% CD23 -/int % CD21 Downloaded from GL7 FHL2-/- FHL2-/- WT FHL2-/-

FSC http://www.immunohorizons.org/ B Red: B220, Green: GL7 (GC) WT FHL2-/- B cell area

T cell area SRBC T cell area GC

GC by guest on September 27, 2021 B cell area

GC number GC area +

dleifc ## ) 2 ipocsorcim/# within B220 within + area (mm GL7 WT FHL2-/- WT FHL2-/-

SRBC SRBC

FIGURE 3. FHL2-deficient mice display enlarged GCs with a normal cell number in response to SRBC. WT and FHL22/2 mice were immunized i.p. with 10% SRBC or vehicle (Veh). Spleens were harvested at day 6 postinjection. (A) Among Fo B cells (B220+ CD212/intCD23+), the number of cells that also harbored GC marker GL7 was enhanced following SRBC in both WT and FHL22/2,as assessed by flow cytometry. (B) Representative immunofluorescence images of WT and FHL22/2 spleens collected 6 d after SRBC injections, depicting B cells (B220+, red) and GCs (GL7+, green). Corresponding quantifications indicate that GC numbers per microscopic field (original magnification 35) did not vary between WT and FHL22/2 mice, but significant differences in GC size (B220+Gl7+/B220+ area ratio) were observed. Data are mean 6 SEM. Scale bar, 50 mm. *p , 0.05, **p , 0.01 versus WT-Veh or FHL2-Veh; ##p , 0.01 versus WT-SRBC.

https://doi.org/10.4049/immunohorizons.2000014 266 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

respectively, in FHL22/2 compared with WT mice. Nevertheless, the expression of CXCR5 and CXCR4, the respective recep- tors for CXCL13 and CXCL12, was equal in FHL22/2 and WT mice under basal conditions, whether measured as per- centage of positive cells (Fig. 8B) or as mean fluorescence intensity per cell (data not shown). These results suggest that the defect in GC structure observed in the absence of FHL2 could possibly be due to impaired induction of B cells by CXCL12 and CXCL13, leading to an improper positioning and activation within the GCs.

DISCUSSION

To our knowledge, our data demonstrate for the first time that Downloaded from FHL2 controls B cell proliferation, CSR, PC generation, and Ab production in response to a T cell–dependent Ag (Fig. 8C). Upon immunization with SRBC, FHL22/2 and WT mice displayed equivalent spleen GC induction and number, although the GCs were significantly enlarged in FHL22/2 mice. Nevertheless, http://www.immunohorizons.org/ SRBC-induced increases in spleen and plasma IgG1 were strongly reduced in FHL22/2 compared with WT mice, associated with reduced circulating PCs. This suggests a defect in immune response to SRBC in the absence of FHL2. Tight regulation of B and T cells in secondary lymphoid organs is crucial for a successful immune response to a specificAg. Dysregulation of immune cells could lead to inflammatory and autoimmune diseases or lymphoma development (51). Despite a growing body of literature on FHL2, there is very limited information on its role or effects in lymphocytes. FHL22/2 mice by guest on September 27, 2021 are reported to display enhanced B cell recruitment to the peri- toneal cavity in response to thioglycollate (10, 27) but reduced airway infiltration of B cells following OVA challenge (31). FHL2 is highly expressed in different regions of the spleen, including white and red pulp, and importantly in splenic B cells. This points to a potential important role for FHL2 in regulating immune cell activation. We first observed a significant spleen enlargement in FHL2-deficient mice, associated with bigger follicles and higher proportions of B cells compared with WT mice. However, there was no evidence that absence of FHL2 altered B cell maturation processes in the bone marrow (Supplemental Fig. 2) or circulating total B cells (Fig. 5D), suggesting a normal B cell progression to the spleen. Our data instead suggest that regulationofsplenic B cells by FHL2 occurs locally. In FHL22/2 mice, the percentages of Mz and Fo B cell FIGURE 4. FHL2-deficient mice display expanded GC DZ. subtypes under basal conditions were enhanced. We have WT and FHL22/2 mice were immunized i.p. with 10% SRBC or vehicle (Veh). previously shown that FHL2 knockdown increases early out- Spleens were harvested at day 6 postinjection. (A) Flow cytometry dot plots growth cell numbers (10), and absence of FHL2 is associated with depicting selection of B cells (B220+)withinGCs(GL7+), which are further an upregulation of S1P pathway in vascular and immune cells (10, identified as representative of DZ (CXCR4+CD832) and LZ (CXCR42CD83+). 52). S1P is a potent lysophospholipid that acts as a chemoattractant Only FHL22/2 mice displayed an enhanced proportion of DZ to LZ cells for a variety of immune cells (53–55), and it impacts on the egress of following SRBC. (B) Representative immunofluorescence images of WT and B and T cells from lymph nodes and the thymus (56). Furthermore, FHL22/2 spleens, showing GCs (PNA+, red), divided into LZ (CD86+,green), anS1Pgradientisessentialforpropertrafficking and positioning of and DZ (CD862). The arrows point to the follicles, LZ, and DZ within GCs. B cells in splenic Mz (57). Nevertheless, although more abundant 2 2 Data are mean 6 SEM.Scalebar,50mm. ***p , 0.001 versus FHL2-Veh. than in WT mice, B cells in FHL2 / animals were distributed

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 267 Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 5. FHL2-deficient mice display a defect in SRBC-induced IgG1 production and PC abundance in blood. WT and FHL22/2 mice were immunized i.p. with 10% SRBC or vehicle (Veh). Spleens were harvested at day 6 postinjection. (A) Representative flow cytometry dot plots and corresponding quantifications of spleen plasmablasts (PB; CD138+B220+) and PCs (CD138+B220lo) among live cells. No differences were noted between WT and FHL22/2 mouse spleens. (B) Representative flow cytometry dot plots with corresponding quantifications of spleen B220+IgG1+ cells (among live cells) reveal that SRBC enhanced B cell IgG1 only in WT mice. The data are corroborated in immunofluo- rescence images, showing enhanced staining for IgG1 (red) among B cells (B220+, green) in SRBC-injected WT mice compared with FHL22/2.(C) Plasma levels of IgG1 and IgM, measured by ELISA, demonstrate that IgG1 was only increased in WT mice following SRBC injection. In FHL22/2 mice, plasma IgM was increased instead following SRBC. (D) Flow cytometry quantifications of blood naive B cells (CD19+B220+, among live cells) and PCs (B220+CD138+, among live cells) in mice demonstrate an increase in circulating naive B cells following SRBC in WT mice only. SRBC did not alter PC numbers in WT but is associated with fewer PCs in FHL22/2 animals. (E) Quantitative PCR (qPCR) analysis of spleen lysates indicates that Aicda mRNA levels were only elevated by SRBC in WT mice, not FHL22/2.(F) No differences in quantities of spleen Tfh cells (PD1+CXCR5+, among CD4+ T cells) were noted between mice, irrespective of treatment. Data are mean 6 SEM. Scale bar, 50 mm. *p , 0.05, ***p , 0.001 versus WT-Veh or FHL2-Veh; #p , 0.05 versus WT-SRBC.

https://doi.org/10.4049/immunohorizons.2000014 268 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on September 27, 2021

FIGURE 6. FHL2-deficient B cell display increased proliferation but no defect in CSR in vitro. Splenic B cells were extracted from WT and FHL22/2 mice and cultured for 4 d in the presence of LPS (25 mg/ml), mimicking a T cell–independent stimulation, or agonistic anti-CD40 (1 mg/ml) plus IL-4 (10 ng/ml), mimicking a T cell–dependent stimulation. (A) B cell proliferation was assessed by flow cytometry evaluating total cell count at day 1 (d1) and d4 poststimulation. B cells of both genotypes responses identically to LPS, but FHL22/2 cells proliferated more when exposed to anti-CD40 plus IL-4. (B) IgG1 and IgM expressing B cells were quantified by flow cytometry. IgG1 increased equally in WT and FHL22/2 B cells stimulated by LPS, and FHL22/2 cells responded most strongly when exposed to anti-CD40 plus IL-4. Both cells types displayed an equivalent reductioninIgMwheninducedinvitro.Dataaremean6 SEM. ***p , 0.001, **p , 0.01, *p , 0.05 versus WT d1 or FHL2 d1; ###p , 0.001 versus WT d4. normally within the spleen follicles, showing expected proportions of the cells or cycling out from the DZ, rather than migration, was betweenMzandFo.Instead,theFHL22/2 GCs were expanded, impacted. This was particularly noticeable in mice immunized with an enhanced DZ/LZ ratio, suggesting that either proliferation with SRBC and paralleled a finding in vitro in which B cells from

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 269

FHL22/2 exposed to IL-4 plus anti-CD40 proliferated more than cells from WT. This points to two proteins that are active in B cells; FOXO1 has a predominant role in establishing the DZ (17), whereas Nur77 regulates B cell activation and survival (42). Because the transcriptional activities of these factors are repressed by FHL2 (36, 42), we cannot exclude their possible involvement in the disturbed GC structure observed in FHL22/2 mice. The global increase inB cellnumbernotwithstanding, the most strikingdifference between WT and FHL22/2 mice was the failure of the latter to mount an appropriate Ab response to Ag. In principle, upon full activation with a T cell–dependent Ag, B cells class switch their surface Abs before differentiating into Ab- secreting cells. The cytosine deaminase AID, which is expressed specifically on normal and transformed GC B cells, is responsible for the diversification of rearranged Ab genes in activated B cells. Downloaded from AID is absolutely required for CSR and somatic hypermutation (58, 59). Our results clearly show that in response to SRBC, mRNA levels of Aicda, which encodes the AID protein, were enhanced in WT, but not in FHL22/2, mouse spleens. Correspondingly, plasma IgM concentration remained higher in FHL22/2 mice than WT http://www.immunohorizons.org/ after SRBC, whereas plasma IgG1 levels rose only in WT animals. The decreased splenic and plasma IgG1 in SRBC-induced FHL22/2 mice could also be exacerbated by the reduced PC generation. In many ways, the FHL2 null mouse phenotype recapitulates the hyper-IgM syndrome, a primary immunodeficiency disorder that severely compromises the immune system, characterized by a defect in CSR, low to undetectable levels of IgGs, and increased IgM serum levels (60, 61). The most common form of hyper-IgM syndrome results from a defect or a deficiency in CD40L, affecting B cell interactions with T cells or dendritic cells (62). However, our by guest on September 27, 2021 bone-marrow transplantation data with mMT mice, showing high IgM levels in mFHL22/2 versus mWT mice irrespective of SRBC, suggest that this phenotype does not depend on B cell/T cell interaction. Moreover, in vitro CSR toward IgG1 was comparable

Flow cytometry quantifications of GC cells (GL7+)amongFoBcells (B220+, CD212/intCD23+) show no differences among groups, irre- spective of treatment. The proportion of DZ (CXCR4+CD682)toLZ (CXCR42CD68+) cells was only increased in mWT, but no differences were noted between mWT-SRBC and mFHL22/2SRBC. Representative immunofluorescence images of mWT and mFHL22/2 spleens collected 6 d after SRBC injections, depicting B cells (B220+, red) and GCs (GL7+, green), show no differences in GC size (C) Whether evaluated by immunofluorescence, staining for IgG1 (red) among B cells (B220+, green), or flow cytometry, IgG1 within the spleen was equivalent in mWT and mFHL22/2 mice.Similarly,quanti- tative PCR (qPCR) analysis of spleen lysates indicates similar Aicda 2 2 FIGURE 7. mMT chimeric mice display no intrinsic defect in FHL2- mRNA levels in mWT and mFHL2 / mice. (D)PlasmalevelsofIgG1 deficient B cells. and IgM, measured by ELISA, demonstrate that no changes in IgG1 (A) Recipient mMT mice were irradiated (10 Gy) and reconstituted were induced by SRBC, whereas plasma IgM was increased following 2 2 the same day with bone marrow from WT (mWT) or FHL22/2 mice SRBC in both mWT and mFHL2 / mice. Data are mean 6 SEM. Scale (mFHL22/2). Chimeric animals were immunized with 10% SRBC or vehicle bar, 50 mm. *p , 0.05 versus mWT-Veh; #p , 0.05, ###p , 0.001 2 2 (Veh) 6 wk later. Spleens were harvested at day 6 postimmunization. (B) versus mWT-Veh or mFHL2 / Veh.

https://doi.org/10.4049/immunohorizons.2000014 270 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

A Positive Positive sICAM-1 IL-16

1.4 WT FHL2-/- # ## CXCL13 1.2

WT 1 CCL5 Positive CXCL12 0.8

0.6

0.4

0.2 Mean pixel density WT) density (fold Mean pixel FHL2-/-

Negative 0 CXCL13 CXCL12 CCL5 SICAM-1sICAM1 IL-16

B Downloaded from 4% 89,7%

CXCR4+ cells CXCR5+ cells + +

2,5% 91,5% % B220 % B220 http://www.immunohorizons.org/

WT FHL2-/- CXCR5 WT FHL2-/- CXCR4

FSC FSC

C Spleen: WT mice Spleen: FHL2-/- mice Mz GC SRBC SRBC

IgG1 by guest on September 27, 2021 Fo Fo ProliferaƟon ProliferaƟon AID TĬ CSR

Naive B Short-lived PC 12 13 PC 12 13

IgM/IgD IgM/IgD MzB MzB IgM

FIGURE 8. FHL2-deficient mice display reduced spleen CXCL12 and CXCL13 levels. Cytokine/chemokine levels from WT and FHL22/2 spleen lysates were analyzed by protein array. (A) Representative images of the membrane spot signals corresponding to multiple cytokines is shown. The relative density of indicated spots was quantified (n =3).#p , 0.05, ##p , 0.01 versus WT. (B) Representative flow cytometry dot plots and corresponding quantifications of CXCR4- and CXCR5-expressing cells among spleen B cells (B220+). Data are mean 6 SEM. (C) Proposed model of splenic B cell activation in WT and FHL2-deficient mice upon immunization with SRBC. The Mz, follicle and GC are represented. During the activation process, B cells are segregated into two distinct zones within the GC, the LZ and DZ. Upon stimulation with a Tcell–dependent Ag (such as SRBC), proliferation occurs in the DZ, followed by AID induction and CSR in the LZ. The proper positioning and segregation between DZ/LZ within the GC are regulated by CXCL12 and CXCL13 production. Upon full activation, B cells differentiate into PCs that exit from the spleen and produce IgG1. FHL2 null mice display enlarged GCs and DZ possibly because of reduced CXCL12/CXCL13 levels and/or gradient and increased B cell proliferation associated with impaired AID induction and CSR, leading to decreased short-lived PCs and an absence of IgG1 production. between FHL22/2 and WT B cells, whether the cells were observation strengthens the hypothesis that B cell activation is stimulated with LPS (mimicking a T cell–independent response) defective in the absence of FHL2 in the spleen microenvironment. or anti-CD40 (mimicking a T cell–dependent response) (63). This Accordingly, we observed a reduction in basal CXCL13 and suggests that there was no intrinsic defect of FHL22/2 Bcell CXCL12 levels in spleens of FHL22/2 mice compared with WT. CD40R signaling or in B cell capacity to perform CSR. This CXCL12 and CXCL13 are two main cytokines that play a critical

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 271 role in B cell homing, trafficking, maturation, and differentiation 8. Wei, Y., C.-A. Renard, C. Labalette, Y. Wu, L. Lévy, C. Neuveut, within the spleen, influencing GC DZ and LZ segregation (64–67). X. Prieur, M. Flajolet, S. Prigent, and M.-A. Buendia. 2003. Identifi- In addition to the regulation of B cells mobility, CXCL12/CXCR4 cation of the LIM protein FHL2 as a coactivator of beta-catenin. J. Biol. Chem. 278: 5188–5194. interaction controls PC differentiation and maintenance (68). 2/2 9. Chan, K. K., S. K. Tsui, S. M. Lee, S. C. Luk, C. C. Liew, K. P. Fung, Decreased levels of CXCL12 and CXCL13 in FHL2 mice could M. M. Waye, and C. Y. Lee. 1998. Molecular cloning and character- possibly be responsible of an improper positioning and/or ization of FHL2, a novel LIM domain protein preferentially expressed trafficking of B cells, leading to the abnormal structure of GCs in human heart. Gene 210: 345–350. and impaired B cell activation. Furthermore, despite the fact that 10. Ebrahimian, T., O. Arfa, S. Simeone, C. A. Lemarié, S. Lehoux, and the reduction in chemokine levels was modest, our results do not S. Wassmann. 2014. Inhibition of four-and-a-half LIM domain protein-2 increases survival, migratory capacity, and paracrine function rule out a potential change in local CXCL12 and CXCL13 gradients of human early outgrowth cells through activation of the sphingosine that could further influence B cell migration. kinase-1 pathway: implications for endothelial regeneration. Circ. Finally, our data do not exclude an effect on other spleen cells, Res. 114: 114–123. including dendritic cells, whose migration and function are 11. Spaargaren, M., E. A. Beuling, M. L. Rurup, H. P. Meijer, M. D. Klok, affected by an absence of FHL2 (33, 69, 70). For example, absence S. Middendorp, R. W. Hendriks, and S. T. Pals. 2003. The B cell an- of FHL2 was associated with lower recruitment of B cells, T cells, tigen receptor controls integrin activity through Btk and PLCgamma2. Downloaded from J. Exp. Med. 198: 1539–1550. and dendritic cells in lungs of OVA-challenged mice, concomitant 12. Park, S.-Y., P. Wolfram, K. Canty, B. Harley, C. Nombela-Arrieta, with reduced IgE and IgG1 production (27). That study did not G. Pivarnik, J. Manis, H. E. Beggs, and L. E. Silberstein. 2013. Focal investigate the spleen, but it reinforces the notion that retention of adhesion kinase regulates the localization and retention of pro-B cells immune cells in the spleen, perhaps in conjunction with or in bone marrow microenvironments. J. Immunol. 190: 1094–1102. resulting from poor Ag presentation, may be a feature of FHL22/2 13. Shaheen, S., Z. Wan, Z. Li, A. Chau, X. Li, S. Zhang, Y. Liu, J. Yi, ff http://www.immunohorizons.org/ mice. Absence of FHL2 could impair dendritic cell interaction with Y. Zeng, J. Wang, et al. 2017. Substrate sti ness governs the initiation of B cell activation by the concerted signaling of PKCb and focal T cells, thus abating the immune response. The results in this study adhesion kinase. eLife 6: e23060. therefore provide some clues as to the role of FHL2 in the B cell 14. Yu, Q., W. J. Quinn III., T. Salay, J. E. Crowley, M. P. Cancro, and adaptive immune response. J. M. Sen. 2008. Role of beta-catenin in B cell development and function. J. Immunol. 181: 3777–3783. 15. Green, J. A., K. Suzuki, B. Cho, L. D. Willison, D. Palmer, DISCLOSURES C. D. C. Allen, T. H. Schmidt, Y. Xu, R. L. Proia, S. R. Coughlin, and

J. G. Cyster. 2011. The sphingosine 1-phosphate receptor S1P2 The authors have no financial conflicts of interest. maintains the homeostasis of germinal center B cells and promotes niche confinement. Nat. Immunol. 12: 672–680. 16. Sanjo, H., M. Hikida, Y. Aiba, Y. Mori, N. Hatano, M. Ogata, and by guest on September 27, 2021 REFERENCES T. Kurosaki. 2007. Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G. Mol. Cell. Biol. 27: 1236–1246. 1. Johannessen, M., S. Møller, T. Hansen, U. Moens, and M. Van Ghelue. 2006. The multifunctional roles of the four-and-a-half-LIM only 17. Inoue, T., R. Shinnakasu, W. Ise, C. Kawai, T. Egawa, and T. Kurosaki. protein FHL2. Cell. Mol. Life Sci. 63: 268–284. 2017. The Foxo1 controls germinal center B cell proliferation in response to T cell help. J. Exp. Med. 214: 1181–1198. 2. Tran, M. K., K. Kurakula, D. S. Koenis, and C. J. M. de Vries. 2016. fi Protein-protein interactions of the LIM-only protein FHL2 and 18. Ashouri, J. F., and A. Weiss. 2017. Endogenous Nur77 is a speci c functional implication of the interactions relevant in cardiovascular indicator of antigen receptor signaling in human T and B cells. – disease. Biochim. Biophys. Acta 1863: 219–228. J. Immunol. 198: 657 668. 3. Chen, D., W. Xu, E. Bales, C. Colmenares, M. Conacci-Sorrell, S. Ishii, 19. Zikherman, J., R. Parameswaran, and A. Weiss. 2012. Endogenous E.Stavnezer,J.Campisi,D.E.Fisher,A.Ben-Ze’ev, and antigen tunes the responsiveness of naive B cells but not T cells. – E. E. Medrano. 2003. SKI activates Wnt/beta-catenin signaling in Nature 489: 160 164. human melanoma. Cancer Res. 63: 6626–6634. 20. Hilchey, S. P., M. G. Palshikar, J. A. Emo, D. Li, J. Garigen, J. Wang, 4. Morlon, A., and P. Sassone-Corsi. 2003. The LIM-only protein FHL2 E. S. Mendelson, V. Cipolla, J. Thakar, and M. S. Zand. 2020. Cy- is a serum-inducible transcriptional coactivator of AP-1. Proc. Natl. closporine a directly affects human and mouse B cell migration in a Acad. Sci. USA 100: 3977–3982. vitro by disrupting a HIF-1 dependent, O2 sensing, molecular switch. 5. Muller,¨ J. M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, BMC Immunol. 21: 13. T. Breyer, C. Holubarsch, R. Buettner, and R. Schule.¨ 2000. FHL2, a 21. Heine, G., A. Dahten, K. Hilt, D. Ernst, M. Milovanovic, B. Hartmann, novel tissue-specific coactivator of the . EMBO and M. Worm. 2009. Liver X receptors control IgE expression in J. 19: 359–369. B cells. J. Immunol. 182: 5276–5282. 6. Purcell, N. H., D. Darwis, O. F. Bueno, J. M. Muller,¨ R. Schule,¨ and 22. Grotsch,¨ B., S. Brachs, C. Lang, J. Luther, A. Derer, U. Schlotzer-¨ J. D. Molkentin. 2004. Extracellular signal-regulated kinase 2 inter- Schrehardt, A. Bozec, S. Fillatreau, I. Berberich, E. Hobeika, et al. acts with and is negatively regulated by the LIM-only protein FHL2 2014. The AP-1 transcription factor Fra1 inhibits follicular B cell in cardiomyocytes. Mol. Cell. Biol. 24: 1081–1095. differentiation into plasma cells. [Published erratum appears in 2014 7. Samson, T., N. Smyth, S. Janetzky, O. Wendler, J. M. Muller,¨ J. Exp. Med. 211: 223.] J. Exp. Med. 211: 2199–2212. R. Schule,¨ H. von der Mark, K. von der Mark, and V. Wixler. 2004. 23. Chen, H.-C., J. C. Byrd, and N. Muthusamy. 2006. Differential role for The LIM-only proteins FHL2 and FHL3 interact with alpha- and cyclic AMP response element binding protein-1 in multiple stages of beta-subunits of the muscle alpha7beta1 integrin receptor. J. Biol. B cell development, differentiation, and survival. J. Immunol. 176: Chem. 279: 28641–28652. 2208–2218.

https://doi.org/10.4049/immunohorizons.2000014 272 FHL2 REGULATES SPLEEN B CELL ACTIVATION ImmunoHorizons

24. Akasaka, T., M. Muramatsu, N. Kadowaki, H. Ohno, K. Ishizaki, 41. Hubbi, M. E., D. M. Gilkes, J. H. Baek, and G. L. Semenza. 2012. Four- H. Yamabe, S. Fukuhara, and M. Okuma. 1996. mutation in B-cell and-a-half LIM domain proteins inhibit transactivation by hypoxia- lymphoid neoplasms with reference to oncogene rearrangements as- inducible factor 1. J. Biol. Chem. 287: 6139–6149. sociated with chromosomal translocations. Jpn. J. Cancer Res. 87: 42. Kurakula, K., E. van der Wal, D. Geerts, C. M. van Tiel, and C. J. M. de 930–937. Vries. 2011. FHL2 protein is a novel co-repressor of 25. Nordhoff, C., A. Hillesheim, B. M. Walter, E. Haasbach, O. Planz, Nur77. J. Biol. Chem. 286: 44336–44343. C. Ehrhardt, S. Ludwig, and V. Wixler. 2012. The adaptor protein 43. Wolniak, K. L., S. M. Shinall, and T. J. Waldschmidt. 2004. The FHL2 enhances the cellular innate immune response to influenza A germinal center response. Crit. Rev. Immunol. 24: 39–65. virus infection. Cell. Microbiol. 14: 1135–1147. 44. Shinall, S. M., M. Gonzalez-Fernandez, R. J. Noelle, and 26. Alnajar, A., C. Nordhoff, T. Schied, R. Chiquet-Ehrismann, K. Loser, T. J. Waldschmidt. 2000. Identification of murine germinal center T. Vogl, S. Ludwig, and V. Wixler. 2013. The LIM-only protein FHL2 B cell subsets defined by the expression of surface isotypes and dif- attenuates lung inflammation during bleomycin-induced fibrosis. ferentiation antigens. J. Immunol. 164: 5729–5738. PLoS One 8: e81356. 45. Victora, G. D., D. Dominguez-Sola, A. B. Holmes, S. Deroubaix, 27. Kurakula, K., M. Vos, A. Logiantara, J. J. T. H. Roelofs, M. A. Nieu- R. Dalla-Favera, and M. C. Nussenzweig. 2012. Identification of hu- wenhuis, G. H. Koppelman, D. S. Postma, C. A. Brandsma, D. D. Sin, man germinal center light and dark zone cells and their relationship Y. Bossé, et al. 2015. Deficiency of FHL2 attenuates airway in- to human B-cell lymphomas. [Published erratum appears in 2015 fl – ammation in mice and genetic variation associates with human Blood 126: 1262.] Blood 120: 2240 2248. Downloaded from bronchial hyper-responsiveness. Allergy 70: 1531–1544. 46. Crouch, E. E., Z. Li, M. Takizawa, S. Fichtner-Feigl, P. Gourzi, 28.Baranek,T.,E.Morello,A.Valayer,R.-F.Aimar,D.Bréa,C.Henry, C. Montano,~ L. Feigenbaum, P. Wilson, S. Janz, F. N. Papavasiliou, A.-G.Besnard,E.Dalloneau,A.Guillon,P.-F.Dequin,etal.2017. and R. Casellas. 2007. Regulation of AID expression in the immune FHL2 regulates natural killer cell development and activation dur- response. J. Exp. Med. 204: 1145–1156. ing Streptococcus pneumoniae infection. Front. Immunol. 8: 123. 47. Nurieva, R. I., Y. Chung, D. Hwang, X. O. Yang, H. S. Kang, L. Ma, 29. Qian, Z., L. Mao, A. A. Fernald, H. Yu, R. Luo, Y. Jiang, J. Anastasi, Y. H. Wang, S. S. Watowich, A. M. Jetten, Q. Tian, and C. Dong. 2008.

P. J. Valk, R. Delwel, and M. M. Le Beau. 2009. Enhanced expression Generation of T follicular helper cells is mediated by interleukin-21 http://www.immunohorizons.org/ of FHL2 leads to abnormal myelopoiesis in vivo. Leukemia 23: but independent of T helper 1, 2, or 17 cell lineages. Immunity 29: 1650–1657. 138–149. 30. Hou, Y., X. Wang, L. Li, R. Fan, J. Chen, T. Zhu, W. Li, Y. Jiang, 48. Vogelzang, A., H. M. McGuire, D. Yu, J. Sprent, C. R. Mackay, and N. Mittal, W. Wu, et al. 2015. FHL2 regulates hematopoietic stem cell C. King. 2008. A fundamental role for interleukin-21 in the generation functions under stress conditions. Leukemia 29: 615–624. of T follicular helper cells. Immunity 29: 127–137. 31. Kurakula, K., M. Vos, M. van Eijk, H. H. Smits, and C. J. M. de Vries. 49. Vinuesa, C. G., S. G. Tangye, B. Moser, and C. R. Mackay. 2005. Fol- 2015. LIM-only protein FHL2 regulates experimental pulmonary licular B helper T cells in antibody responses and autoimmunity. Nat. Schistosoma mansoni egg granuloma formation. Eur. J. Immunol. 45: Rev. Immunol. 5: 853–865. 3098–3106. 50. Kitamura, D., J. Roes, R. Kuhn,¨ and K. Rajewsky. 1991. A B cell- 32. Sawai, C. M., V. Sisirak, H. S. Ghosh, E. Z. Hou, M. Ceribelli, deficient mouse by targeted disruption of the membrane exon of the L. M. Staudt, and B. Reizis. 2013. Transcription factor Runx2 controls immunoglobulin mu chain gene. Nature 350: 423–426.

the development and migration of plasmacytoid dendritic cells. J. Exp. 51. Kuppers,¨ R. 2005. Mechanisms of B-cell lymphoma pathogenesis. Nat. by guest on September 27, 2021 Med. 210: 2151–2159. Rev. Cancer 5: 251–262. 33. Konig,¨ K., L. Diehl, U. Rommerscheidt-Fuss, C. Golletz, T. Quast, 52. Hayashi, H., H. Nakagami, Y. Takami, H. Koriyama, M. Mori, P. Kahl, W. Kolanus, P. Knolle, R. Buettner, and L. C. Heukamp. 2010. K. Tamai, J. Sun, K. Nagao, R. Morishita, and Y. Kaneda. 2009. FHL-2 Four-and-a-half LIM domain protein 2 is a novel regulator of suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activa- sphingosine 1-phosphate receptor 1 in CCL19-induced dendritic cell tion via interaction with sphingosine kinase-1. Arterioscler. Thromb. migration. J. Immunol. 185: 1466–1475. Vasc. Biol. 29: 909–914. 34. Kraal, G., and R. Mebius. 2006. New insights into the cell biology of 53. Ebrahimian, T., D. Simon, C. A. Lemarié, S. Simeone, M. Heidari, the marginal zone of the spleen. Int. Rev. Cytol. 250: 175–215. K. K. Mann, S. Wassmann, and S. Lehoux. 2015. Absence of four-and- 35. Pillai, S., and A. Cariappa. 2009. The follicular versus marginal zone a-half LIM domain protein 2 decreases atherosclerosis in ApoE-/- B lymphocyte cell fate decision. Nat. Rev. Immunol. 9: 767–777. mice. Arterioscler. Thromb. Vasc. Biol. 35: 1190–1197. 36. Yang, Y., H. Hou, E. M. Haller, S. V. Nicosia, and W. Bai. 2005. 54. Idzko, M., E. Panther, S. Corinti, A. Morelli, D. Ferrari, Y. Herouy, Suppression of FOXO1 activity by FHL2 through SIRT1-mediated S. Dichmann, M. Mockenhaupt, P. Gebicke-Haerter, F. Di Virgilio, deacetylation. EMBO J. 24: 1021–1032. et al. 2002. Sphingosine 1-phosphate induces chemotaxis of immature 37. Brun, J., O. Fromigué, F.-X. Dieudonné, C. Marty, J. Chen, J. Dahan, and modulates cytokine-release in mature human dendritic cells for Y. Wei, and P. J. Marie. 2013. The LIM-only protein FHL2 controls emergence of Th2 immune responses. FASEB J. 16: 625–627. mesenchymal cell osteogenic differentiation and bone formation 55. Graeler, M., and E. J. Goetzl. 2002. Activation-regulated expression through Wnt5a and Wnt10b. Bone 53: 6–12. and chemotactic function of sphingosine 1-phosphate receptors in 38. Dominguez-Sola, D., J. Kung, A. B. Holmes, V. A. Wells, T. Mo, mouse splenic T cells. FASEB J. 16: 1874–1878. K. Basso, and R. Dalla-Favera. 2015. The FOXO1 transcription factor 56. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, instructs the germinal center dark zone program. Immunity 43: V. Brinkmann, M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. 1064–1074. Lymphocyte egress from thymus and peripheral lymphoid organs is 39. Mandala, S., R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, dependent on S1P receptor 1. Nature 427: 355–360. J. Milligan, R. Thornton, G.-J. Shei, D. Card, C. Keohane, et al. 2002. 57. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, Alteration of lymphocyte trafficking by sphingosine-1-phosphate R. L. Proia, and J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 receptor agonists. Science 296: 346–349. promotes B cell localization in the splenic marginal zone. Nat. 40. Laidlaw, B. J., T. H. Schmidt, J. A. Green, C. D. C. Allen, T. Okada, and Immunol. 5: 713–720. J. G. Cyster. 2017. The Eph-related tyrosine kinase ligand Ephrin-B1 58. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, marks germinal center and memory precursor B cells. J. Exp. Med. N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 214: 639–649. 2000. Activation-induced cytidine deaminase (AID) deficiency causes

https://doi.org/10.4049/immunohorizons.2000014 ImmunoHorizons FHL2 REGULATES SPLEEN B CELL ACTIVATION 273

the autosomal recessive form of the hyper-IgM syndrome (HIGM2). 65. Nie, Y., J. Waite, F. Brewer, M.-J. Sunshine, D. R. Littman, and Y.-R. Cell 102: 565–575. Zou. 2004. The role of CXCR4 in maintaining peripheral B cell 59. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and compartments and humoral immunity. J. Exp. Med. 200: 1145–1156. T. Honjo. 2000. Class switch recombination and hypermutation re- 66. Okada, T., V. N. Ngo, E. H. Ekland, R. Forster,¨ M. Lipp, D. R. Littman, quire activation-induced cytidine deaminase (AID), a potential RNA and J. G. Cyster. 2002. Chemokine requirements for B cell entry to editing enzyme. Cell 102: 553–563. lymph nodes and Peyer’s patches. J. Exp. Med. 196: 65–75. 60. Durandy, A., T. Cantaert, S. Kracker, and E. Meffre. 2013. Potential 67. Victora, G. D., T. A. Schwickert, D. R. Fooksman, A. O. Kamphorst, roles of activation-induced cytidine deaminase in promotion or pre- M. Meyer-Hermann, M. L. Dustin, and M. C. Nussenzweig. 2010. vention of autoimmunity in humans. Autoimmunity 46: 148–156. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143: 592–605. 61. de la Morena, M. T. 2016. Clinical phenotypes of hyper-IgM syn- 68. Biajoux, V., J. Natt, C. Freitas, N. Alouche, A. Sacquin, P. Hemon, dromes. J. Allergy Clin. Immunol. Pract. 4: 1023–1036. F. Gaudin, N. Fazilleau, M. Espéli, and K. Balabanian. 2016. Efficient 62. Aghamohammadi, A., N. Parvaneh, N. Rezaei, K. Moazzami, S. Kashef, plasma cell differentiation and trafficking require Cxcr4 de- H. Abolhassani, A. Imanzadeh, J. Mohammadi, and L. Hammarstrom.¨ sensitization. Cell Rep. 17: 193–205. fi 2009. Clinical and laboratory ndings in hyper-IgM syndrome 69. Masemann, D., R. Leite Dantas, S. Sitnik, T. Schied, C. Nordhoff, with novel CD40L and AICDA mutations. J. Clin. Immunol. 29: S. Ludwig, and V. Wixler. 2018. The four-and-a-half LIM domain 769–776. fl fl

protein 2 supports in uenza A virus-induced lung in ammation by Downloaded from 63. Graham, J. P., K. M. Arcipowski, and G. A. Bishop. 2010. Differential restricting the host adaptive immune response. Am. J. Pathol. 188: B-lymphocyte regulation by CD40 and its viral mimic, latent mem- 1236–1245. brane protein 1. Immunol. Rev. 237: 226–248. 70. Zaal, A., B. Nota, K. S. Moore, M. Dieker, S. M. van Ham, and A. Ten 64. Allen, C. D. C., K. M. Ansel, C. Low, R. Lesley, H. Tamamura, N. Fujii, Brinke. 2017. TLR4 and C5aR crosstalk in dendritic cells induces a and J. G. Cyster. 2004. Germinal center dark and light zone organi- core regulatory network of RSK2, PI3Kb, SGK1, and FOXO tran- zation is mediated by CXCR4 and CXCR5. Nat. Immunol. 5: 943–952. scription factors. J. Leukoc. Biol. 102: 1035–1054. http://www.immunohorizons.org/ by guest on September 27, 2021

https://doi.org/10.4049/immunohorizons.2000014