Rap1b Regulates B Cell Development, Homing, and T Cell-Dependent Humoral Immunity

This information is current as Haiyan Chu, Aradhana Awasthi, Gilbert C. White II, of September 27, 2021. Magdalena Chrzanowska-Wodnicka and Subramaniam Malarkannan J Immunol 2008; 181:3373-3383; ; doi: 10.4049/jimmunol.181.5.3373

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rap1b Regulates B Cell Development, Homing, and T Cell-Dependent Humoral Immunity1

Haiyan Chu,* Aradhana Awasthi,* Gilbert C. White II,† Magdalena Chrzanowska-Wodnicka,‡ and Subramaniam Malarkannan2*§

Rap1 is a small GTPase that belongs to Ras superfamily. This ubiquitously expressed GTPase is a key regulator of integrin functions. Rap1 exists in two isoforms: Rap1a and Rap1b. Although Rap1 has been extensively studied, its isoform-specific functions in B cells have not been elucidated. In this study, using knockout mice, we show that Rap1b is the dominant isoform in B cells. Lack of Rap1b significantly reduced the absolute number of B220؉IgM؊ pro/pre-B cells and B220؉IgM؉ immature B cells in bone marrow. In vitro culture of bone marrow-derived Rap1b؊/؊ pro/pre-B cells with IL-7 showed similar proliferation levels but reduced adhesion to stromal cell line compared with wild type. Rap1b؊/؊ mice displayed reduced splenic marginal zone ؊ ؊ (MZ) B cells, and increased newly forming B cells, whereas the number of follicular B cells was normal. Functionally, Rap1b / Downloaded from mice showed reduced T-dependent but normal T-independent humoral responses. B cells from Rap1b؊/؊ mice showed reduced migration to SDF-1, CXCL13 and in vivo homing to lymph nodes. MZ B cells showed reduced sphingosine-1-phosphate-induced migration and adhesion to ICAM-1. However, absence of Rap1b did not affect splenic B cell proliferation, BCR-mediated acti- vation of Erk1/2, p38 MAPKs, and AKT. Thus, Rap1b is crucial for early B cell development, MZ B cell homeostasis and T-dependent humoral immunity. The Journal of Immunology, 2008, 181: 3373–3383. http://www.jimmunol.org/ ␣ ␤ he small GTPase, Rap1, belongs to the Ras superfamily effector of Rap1-GTPase in immune cells and associates with L 2 and cycles between an inactive GDP-bound and an active integrin (9). RIAM contains Ras-associated and pleckstrin homol- T GTP-bound conformation (1, 2). This ubiquitously ex- ogy domains and interacts with profilin and Ena/VASP pressed molecular switch regulates cell proliferation, differentia- that regulate actin dynamics (10). Rap1 signaling is terminated by tion, and adhesion by distinct mechanisms through integrin acti- GTPase-activating proteins (GAPs), such as SPA-1 (signal-in- vation and MAPK cascades (3, 4). Multiple extracellular stimuli duced proliferation-associated 1) and RapGAPs, which can activate Rap1. This activation is mediated by various guanine convert active GTP-bound to inactive GDP-bound form (11, 12). 3 nucleotide exchange factors (GEFs), such as C3G, CalDAG-GEF, Integrins mediate cellular contacts to extracellular matrix or to their by guest on September 27, 2021 and EPAC (5–7). C3G is activated by receptor-associated protein counter receptors, thereby regulating cell motility, polarity, growth, tyrosine kinases (5). CalDAG-GEF responds to calcium and diac- and survival (13). Rap1 is expressed in multiple hematopoietic cells ylglycerol, downstream of phospholipase C signaling (8). EPAC is and one of its key functions is to regulate integrin-mediated cell ad- a cAMP-dependent GEF (7). Active Rap1-GTPase recruits multi- hesion (4). This amplifies a series of hematopoietic cellular processes ple effectors (e.g., RapL/Nore1B and RIAM) to execute their cel- including aggregation, migration, extravasation, and homing (4, 14, lular functions (9, 10). RapL, a Rap1-binding molecule, is a major 15). Mice lacking CalDAG-GEFI show impaired platelet adhesion and aggregation leading to impaired thrombus formation (16). Defi- ciency of C3G, one of the major Rap-specific GEFs, resulted in severe *Laboratory of Molecular Immunology, †Laboratory of Platelet, ‡Vascular Signaling, Blood Research Institute, and §Division of Neoplastic Diseases and Related Disor- embryonic lethality, partly due to aberrant cell adhesion and spreading ders, Department of Medicine, and Department of Microbiology and Molecular Ge- (17). Overexpression of Rap1, RapGEFs, GAPs, or their mutant pro- netics, Medical College of Wisconsin, Milwaukee, WI 53226 teins altered the activation of Rap1 and helped to uncover its signaling Received for publication November 15, 2007. Accepted for publication June 26, 2008. functions in different cell types (18–21). Rap1 can be activated through Ag or chemokine receptors (18, 22, 23). Active Rap1 plays an The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance important role in immunological synapse formation between T cells with 18 U.S.C. Section 1734 solely to indicate this fact. and APCs (22). 1 This work was supported in part by an American Cancer Society Scholar Grant Recent studies have well documented that interference in Rap1 RSG-02-172-LIB (to S.M.), by Roche Organ Transplantation Research Foundation Grant 111662730 (to S.M.), by National Institutes of Health Grants R01 functions can result in severe pathological conditions. In human, A1064826-01 (to S.M.), U19 AI062627-01 (to S.M.), NO1-HHSN26600500032C (to leukocyte adhesion deficiency syndrome III is associated with de- S.M.), and HL-45100 (to G.C.W.), and by American Heart Association Grant fective Rap1 activation and impaired stabilization of integrin SDG0235127N (to M.C.W.). H.C. is a recipient of Wisconsin Breast Cancer Show House Postdoctoral Fellowship. S.M. is a recipient of The American Society for bonds (24). Additionally, knockout mice for SPA-1 or RapL have Blood and Marrow Transplantation Young Investigator Award. provided critical insights into in vivo functions of Rap1-GTPase 2 Address correspondence and reprint requests to Dr. Subramaniam Malarkannan, (25, 26). SPA-1 is the major Rap1GAP in the hematopoietic pro- Laboratory of Molecular Immunology, Medical College of Wisconsin, 8701 Water- Ϫ/Ϫ town Plank Road, Milwaukee, WI 53226. E-mail address: [email protected] genitors in bone marrow (26). SPA-1 mice show an increase in the hematopoietic stem cells and eventually develop leukemia 3 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MZ, marginal zone; FO, follicular; TNP, trinitrophenol; (26). SPA-1 deficiency also results in aberrant BCR repertoire KLH, keyhole limpet hemocyanin; S1P, sphingosine-1-phosphate; SDF, stromal cell- and autoimmune diseases (27). RapL deficiency causes defec- derived factor; LN, lymph node; WT, wild type. tive chemokine-triggered lymphocyte adhesion and migration Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 (25). However, many of these RapGEFs, GAPs, and effectors www.jimmunol.org 3374 Rap1b IN B CELL DEVELOPMENT AND FUNCTION are promiscuous and can associate with multiple Rap1 GTPases; Immunohistochemistry staining therefore the direct evaluation of Rap1 functions is limited in the Spleens were embedded in Tissue-Tek (Sakura Finetek), frozen with dry described knockout mouse models. ice-isopropanol mixture, and kept at Ϫ80°C until sectioning. The 10-␮m Rap1 is closely related to the Rap2 family (Rap2a/2b/2c) and cryostat sections were generated, air-dried for1hatroom temperature, and shares 60% homology (1, 2, 28). Rap1 exists in two isoforms, fixed in acetone for 10 min. The sections were blocked with 0.1% BSA/ Rap1a and Rap1b, which are encoded by different . Rap1 PBS containing anti-CD16/CD32 mAb for 45 min, incubated for1hat room temperature in dark with the rat anti-mouse CD3␧-PE, anti-B220- isoforms share more than 95% amino acid homology and differ Cy5, MOMA-1-FITC, anti-mouse IgM-PE, or anti-mouse IgD-FITC only in eight amino acids with five located at their C termini. Due mAbs. Sections were mounted in aqueous mounting medium and visual- to the high homology of Rap1a and Rap1b, their functions have ized on inverted Nikon Eclipse TE200 fluorescence microscope. Images been studied undistinguished in some studies (22, 23, 29). How- are at a magnification of ϫ40. ever, the different subcellular localization and tissue expression Bone marrow transplantation patterns of these two isoforms have been described and indicate Bone marrow cells from tibias and femurs of wild-type (WT) and Rap1Ϫ/Ϫ their distinct functions (30–35). Studies have indicated the role of mice were prepared and resuspended in 2% FBS/RPMI 1640 (Invitrogen Rap1 isoforms in neuron, T cells, and platelets (19, 34, 35). Rap1a Life Technologies). A total of 5 ϫ 106 bone marrow cells (200 ␮l) were has been shown to positively regulate T cell functions in both injected i.v. to reconstitute 6- to 8-wk-old, sublethally irradiated (300 rad) Ϫ Ϫ Ϫ Ϫ transgenic and knockout mouse models (19, 36). Recently, we JAK3 / and lethally irradiated (1100 rad) Rap1b / mice 4 h after the have generated Rap1b gene knockout mice (34). Approximately irradiation. The recipient mice were sacrificed 8 wk later and the spleens were analyzed for reconstitution of B cells by flow cytometry. 40% of the Rap1bϪ/Ϫ fetuses die during embryogenesis. Surviving Ϫ Ϫ Rap1b / mice show bleeding disorder due to reduced platelet Rap1-GTPase pull-down assay and Western blotting Downloaded from Ϫ/Ϫ aggregation that is similar to the phenotype in CalDAG-GEFI B cells were purified from spleens and resuspended in HEPES buffer (10 mice (16, 34). Irrespective of these findings, the differential ex- mM HEPES/0.5% FCS/RPMI 1640). A total of 15 ϫ 106 B cells were used pression of Rap1 isoforms and their specific roles in B cell have for each lane. Cells were stimulated with 50 ␮g/ml goat anti-mouse IgM Ϫ/Ϫ Ј yet to be determined. In this study, using Rap1b mice, we show F(ab )2 or 100 ng/ml SDF-1 for 2 min at 37°C or left unstimulated. Stim- that Rap1b is the dominant Rap1 isoform in B cells. Rap1b is ulation was terminated by adding one volume of 2X ice-cold lysis buffer (100 mM Tris (pH 7.4), 150 mM NaCl, 2% Nonidet P-40, 1% deoxycholic crucial for early B cell development, marginal zone (MZ) B cell acid, 0.2% SDS, 2 mM sodium orthovanadate, with Phosphatase Inhibitor http://www.jimmunol.org/ maturation, and T-dependent humoral responses. Additionally, ab- Cocktail (PhosSTOP; Roche Diagnostics)) and Proteinase Inhibitor Cock- sence of Rap1b leads to the reduced B cell adhesion and chemo- tail (Sigma-Aldrich). Lysates were incubated for 30 min on ice and cen- ϫ ϫ taxis and in vivo homing. However, Rap1b is dispensable for trifuged at 15,000 g for 15 min at 4°C. An aliquot of cell lysates (1 106 cells) was taken out for evaluating the total Rap1 loading. The remain- BCR-mediated splenic B cell phosphorylation of Erk1/2, p38, ing cell lysates were incubated with glutathione-agarose beads that pre- and AKT. coupled with GST-RalGDS-Ras binding domain at 4°C (37). This assay is based on the fact that RalGDS binds with high affinity to Rap1-GTPase but not to Rap1-GDP (37). The beads were washed, and 30 ␮l of 1X Laemmli buffer was added and heated at 95°C to elute the pull-down proteins. The Materials and Methods active Rap1 was analyzed by an Ab that recognizes both Rap1a and Rap1b. by guest on September 27, 2021 Mice and reagents For Western blotting, cell lysate aliquot (corresponding to 1 ϫ 106 cells)

Ϫ/Ϫ ϩ/Ϫ or pull-down protein pellets were separated by SDS-PAGE, transferred to Rap1b mice were previously described (34). The Rap1b mice were polyvinylidene difluoride membrane, and probed with primary and sec- crossed with Rap1bϩ/Ϫ or Rap1bϪ/Ϫ mice to obtain Rap1bϩ/ϩ and Ϫ/Ϫ ondary Abs conjugated with HRP. The signal was detected by ECL (GE Rap1b mice. The following Abs and reagents were used in this study: Healthcare Life Sciences). recombinant proteins Rap1a-GST and Rap1b-GST (85–184 aa) (Novus Biologicals); rabbit anti-Rap1 polyclonal Ab, goat anti-rabbit mouse IgG IL-7-driven bone marrow culture and BrdU labeling (HϩL) HRP (Santa Cruz Biotechnology); rabbit anti-Rap1b mAb (36E1), rabbit anti-Erk1/2 polyclonal Ab, rabbit anti-phospho-Erk1/2 mAb Primary pre-B cell cultures were previously described (38). Briefly, eryth- (197G2), rabbit anti-p38 polyclonal Ab, rabbit anti-phospho-p38 mAb rocyte-depleted total bone marrow cells were cultured with 2 ng/ml re- (3D7), rabbit anti-AKT polyclonal Ab, rabbit anti-phospho-AKT (Ser473) combinant murine IL-7 (R&D Systems) at 2 ϫ 106/ml for 5–7 days. The mAb (193H12), and anti-GST mAb (Cell Signaling Technology); rabbit medium was replaced with fresh IL-7 every second day. For BrdU labeling, anti-␤-actin Ab (Abcam); rat anti-mouse B220 (RA3-6B2), rat anti-mouse on day 5, bone marrow cells treated with IL-7 were seeded into 24 wells IgM (R6-60.2), rat anti-mouse IgD (11-26c.2a), rat anti-mouse CD19 and cultured for another 3 days with IL-7. These cells were pulsed for 3 h (1D3), rat anti-mouse CD1d (1B1), rat anti-mouse CD21/CD35 (4E3), rat- with 10 ␮M BrdU at the end of day 3. Cells were stained with anti-B220 anti-mouse CD23 (B3B4), Armenian hamster anti-moue CD3␧ (145- and anti-IgM Abs, resuspended in cold PBS, fixed with ice-cold ethanol 2C11), rat anti-mouse CD16/CD32(93), rat anti-mouse CD43, rat anti- (95%), and incubated on ice for 30 min. Cells were fixed or permeabilized mouse CD25 and IL-4 (eBioscience); rat anti-mouse CD4 (H129.19), rat with 1% paraformaldehyde/0.01% Tween 20 at 4°C overnight. Cells were anti-mouse c-Kit, and anti-mouse CD8 (53-6.2) (BD Pharmingen); resuspended with 50 Kunitz DNaseI/ml in 0.15 M NaCl, 4.2 mM MgCl2 Ј MOMA-1-FITC (Serotec); goat anti-mouse IgM F(ab )2 (Jackson Immu- (pH 5) for 10 min at 37°C, washed, stained with 1/50 anti-BrdU-FITC (BD noResearch Laboratories); mouse anti-GAPDH LPS (Escherichia coli Biosciences) for 30 min, and analyzed. O111:B4; Sigma-Aldrich); CFSE (C-1157) and TAMRA (C-300) (Molec- 3 ular Probes); and stromal cell-derived factor (SDF)-1 and CXCL13 (R&D [ H]Thymidine incorporation assay Systems). Purified B cells were plated into 96-well plates in triplicates (2 ϫ 104 ␮ Ј cells/well) and stimulated with 10 g/ml goat anti-mouse IgM F(ab )2,10 ␮ Ј ␮ g/ml goat anti-mouse IgM F(ab )2 plus 10 ng/ml IL-4, or 1 g/ml LPS in Flow cytometry, cell separation, and cell sorting 10% FBS for 72 h. A total of 1 ␮Ci of [3H]thymidine (GE Healthcare Life Sciences) was added 16 h before harvest. Labeled DNA from cells was Single-cell suspensions were prepared by gently mincing the dissected or- collected on GSC filters (Whatman), and the radioactivity was measured in gans through 70-␮m cell strainers. T cells and resting B cells were purified a microplate scintillation counter. by anti-CD3-PE mAb/anti-PE Microbeads (BD Pharmingen) and B cell isolation kit (Miltenyi Biotec). Separated cells were stained with anti-CD19 Immunization and anti-CD3␧ mAbs, and their purity was Ͼ95% as confirmed by flow cytometry. Flow cytometry analysis was conducted in LSR-II and analyzed For the T-independent immune response, 6- to 8-wk-old mice were i.p. with FACSDiva software (BD Biosciences). For isolating newly forming, immunized with 100 ␮g of trinitrophenol (TNP)-Ficoll (Biosearch Tech- follicular (FO), and MZ B cell populations, single-cell suspensions from nologies) in PBS. A total of 50 ␮l of blood was collected from the retro- spleens were stained with anti-B220-Pacific Blue, anti-CD21-FITC, and orbital venous plexus with capillary tubes on days 0 and 7 after immuni- anti-CD23-PE-Cy7 mAbs and sorted using FACSAria (BD Biosciences). zation. For the T-dependent response, mice were i.p. injected with 100 ␮g The Journal of Immunology 3375

FIGURE 1. Rap1b expression and its role in B cell development. A, Ex- pression of total Rap1 and Rap1b in B and T cells from WT and Rap1bϪ/Ϫ spleens. Total Rap1 was detected with anti-Rap1 polyclonal Ab (upper) and Rap1b was detected with anti-Rap1b mAb (middle). Membranes were stripped and reprobed for actin as load- ing control (lower). B, Western blot of recombinant GST-Rap1a and GST- Rap1b (85–184 aa). A total of 100 ng of recombinant proteins were detected with anti-Rap1 polyclonal Ab and anti- GST. C and D, Early B cell develop- ment in the bone marrow is impaired in Ϫ/Ϫ Rap1b mice. Bone marrow cells Downloaded from were stained with anti-B220, anti-IgM, CD43, c-Kit, and CD25 mAbs. The ab- solute cell number was calculated as fol- lowing: cell number (ϫ106) ϭ cellular- ity ϫ (percentage of lymphatic cell) ϫ (percentage of lymphocyte subsets).

The B cell subset percentage and the ab- http://www.jimmunol.org/ solute cell number are presented. Data presented are the representative of n ϭ 3–5 mice. E, Peripheral blood was stained with anti-B220, IgM, and IgD. The percentage of each subpopulation was shown. Data presented are repre- sentative of n ϭ 5 mice. F, IL-7-driven bone marrow culture of WT and Rap1bϪ/Ϫ mice led to the similar purity by guest on September 27, 2021 of B220ϩIgMϪ pro/pre-B cells (top) for n ϭ 3 ϩ 3 mice and showed similar proliferation rate of BrdU incorporation (bottom) for n ϭ 3 ϩ 3 mice. G, Re- duced Rap1bϪ/Ϫ pro/pre-B cells to M2- 10B4 stromal cell line. IL-7-driven bone marrow-derived pro/pre-B cells from WT and Rap1bϪ/Ϫ mice were labeled with CFSE and TAMRA respectively, added to stromal cell line and incubated for 5 h. Adherent cells were analyzed by flow. A dye swap was performed, which showed the same result (data not shown).

of TNP-keyhole limpet hemocyanin (KLH) in 200 ␮l of aluminum hy- rectly added to the lower compartment. For sphingosine 1-phosphate droxide adjuvant and Imject Alum (Pierce) and boosted with 10 ␮gof (S1P)-induced migration assay, different concentrations of S1P (Biomol) in TNP-KLH without adjuvant on day 21. Sera were collected on days 0, 7, HEPES buffer were added to the lower compartments and the RBC-de- and 28. Sera were analyzed by ELISA for basal and TNP-specific Ig iso- pleted splenocytes from WT and Rap1bϪ/Ϫ were added to the upper cham- types using Clonotyping System (Southern Biotechnology Associates). bers. At 3 h later, the cells that migrated into the lower compartment were stained with anti-B220, anti-CD21, and anti-CD23 Abs. In vitro chemotaxis assay In vitro adhesion assay Chemotaxis assay was performed in triplicates in 5.0-␮m Transwell (5.0-␮m pore, 6.5 mm; Costar). The lower compartment contained 600 ␮l S1P-induced adhesion of B cells to ICAM-1 was described (39). Briefly, of 150 ng/ml SDF-1 or 500 ng/ml CXCL13 in HEPES buffer (RPMI 1640, Immulon 4 HBX 96-well flat-bottom plates (ISC BioExpress) were coated 10 mM HEPES, 0.5% FBS). Purified B cells were resuspended to 5 ϫ with 1% fat-free BSA/PBS, recombinant mouse ICAM-1/Fc 10 ␮g/ml 106/ml in the same buffer. The 100 ␮l of B cell suspension was added to (R&D Systems), triplicates, keep at 4°C overnight. Splenocytes were in- the upper chamber and allowed to migrate for 3 h. The number of viable cubated in 10% FBS/RPMI 1640 for 30 min at 37°C in a tissue culture flask cells that migrated into the lower compartment was determined by flow to eliminate adherent macrophages. Nonadherent floating cells were cytometry and expressed as a percentage of the input cells that were di- collected and RBCs were lysed. Cells were resensitized in RPMI 1640 3376 Rap1b IN B CELL DEVELOPMENT AND FUNCTION AC −/− WT Rap1b−/− WT Rap1b p=0.023 80 30 p=0.0002 FO 12

+ 47.5 45.6 40 15 6 ±4.0 ±9.1 CD23 % B220 Cells Cell # 0 0 0 NF MZ FO NF MZ CD21 B220 6 p=0.0009 MZ B WT Rap1b−/− 3 % Cells IgM

0 IgMHi CD21 Hi

IgD WT

(MZ) Downloaded from CD21 Rap1b-/-

6

IgMIgM p=0.0015 30 20 6 p=0.0488 3 MZ % Cells 15 10 3 IgM http://www.jimmunol.org/ % Cells 0 0 _ 0 0 _ Hi Hi IgM IgD++++IgM IgD IgM IgD IgM CD1d CD1d (MZ)

30 D WT Rap1b-/-

15 by guest on September 27, 2021 % Cells IgD 0 B220+ IgM− IgM+ IgD+ IgD+ IgM FIGURE 2. Reduced MZ B cells in the spleens of Rap1bϪ/Ϫ mice. A and B, Single-cell suspensions derived from spleens were stained with anti-B220, anti-IgM, and anti-IgD mAbs. Histograms of B220ϩ cells from the WT and Rap1bϪ/Ϫ mice are shown (A). The staining pattern of IgM and IgD of spleen cells were shown (B). The percentage of subsets is shown (bottom)(n ϭ 7 mice for both experiment). C, Splenocytes were stained with anti-B220, anti-CD21, and anti-CD23 mAbs. B220ϩ splenocytes were separated into CD21low/ϪCD23low/Ϫ newly forming (NF) B cells, CD23highCD21low/Ϫ FO B cells, and CD21highCD23low/Ϫ MZ B cells (upper). Splenocytes were also stained with anti-CD21 and anti-IgM (middle) or anti-CD1d and anti-IgM mAbs (lower). D, Single-cell suspensions derived from auxiliary and inguinal LNs were stained with anti-B220, anti-IgM, and anti-IgD mAbs. The percentage of B220ϩ, IgMϩIgDϩ, and IgMϪIgDϩ cells were shown (right). B–D, n ϭ 6–8 mice. containing 0.5% fatty acid-free BSA and 10 mM HEPES at 37°C for 1 h. TAMRA as bone marrow pro-B cells and resuspended in 2% FBS medium. The plates were gently washed with PBS twice and blocked with 1% fatty A mixture of 20 ϫ 106 CFSE- and TAMRA-labeled cells was i.v. injected acid-free BSA/PBS at 37°C for 1 h. A total of 3 ϫ 105 cells in 100 ␮lof into recipient WT mice. At 3 h later, single-cell preparations from spleen, buffer with or without 100 nM S1P and incubated at 37°C for 60 min peripheral lymph nodes (LNs) (inguinal and auxiliary), and mesenteric LN nonadherent cells wells were washed away and adherent cells were re- of recipient mice and a portion of labeled cells were stained with anti- leased by keeping the plates on ice for 20 min with RPMI 1640/5 mM B220-allophycocyanin or anti-CD3-PE-Cy7 mAbs. The homing efficiency EDTA. Collected cells were stained for B220, CD23, and CD21 markers. of Rap1bϪ/Ϫ/WT B and T cells were calculated compared with the input Adhesion was calculated as a percentage of input cells. ratio, which was normalized to 1. A dye swap was also performed. To quantify the adhesion of B cell progenitors from bone marrow, IL- 7-driven bone marrow cells were labeled on day 7 with 2 ␮M CFSE and Statistics 10 ␮M TAMRA for 10 min at room temperature, and the reaction was Data were presented as mean Ϯ SE, and statistical significance was cal- terminated by adding one volume of FBS. Cells were washed and resus- Ͻ pended in 10% FBS medium. A mixture of 0.5 ϫ 106 CFSE- and TAMRA- culated using the Student t test. A value of p 0.05 was considered labeled cells was added onto M2-10B4 confluent stromal cell layer (Amer- significant. ican Type Culture Collection) and incubated for 5 h. Unbound cells were washed away with warm plain medium and adherent cells were released Results with 5 mM EDTA/plain medium. Stromal cell layers were removed by Rap1b is the dominant isoform in B cells filtering before flow cytometry. Because Rap1 exists in two isoforms, we first analyzed the relative In vivo homing assay levels of Rap1a and Rap1b in B cells. Expression of total Rap1 RBC-depleted splenocytes from WT and Rap1bϪ/Ϫ were resuspended to protein was assayed using an affinity purified polyclonal IgG that 20 ϫ 106 cells/ml in PBS and labeled with 2 ␮M CFSE and 10 ␮M recognizes an identical epitope in the C terminus of both Rap1a The Journal of Immunology 3377

and Rap1b isoforms. Fig. 1A shows that the total Rap1 was readily WT Rap1b−/− detectable in the WT-derived B cells. However, only a residual A level of Rap1 protein was present in B cells from Rap1bϪ/Ϫ mice. With recombinant GST-Rap1A full-length protein and GST- Rap1B (85–184 aa), we show that anti-Rap1 has similar affinities H&E to both Rap1a and Rap1b proteins compared with anti-GST Ab (Fig. 1B). Therefore, the residual protein band in Rap1bϪ/Ϫ B cells represents Rap1a. Similar Rap1 expression pattern was also ob- served in T cells (Fig. 1A). This finding demonstrates that the B Rap1b is the major isoform in B and T cells. Rap1b-specific mAb confirmed the absence of Rap1b in B and T cells from Rap1bϪ/Ϫ CD3 mice (Fig. 1A). The dominant expression of Rap1b suggested its MOMA B220 potential role in B cell development and function. Lack of Rap1b impairs B cell development in the bone marrow As Rap1b is highly expressed in B cells, we analyzed its role in B C cell development. The total cellularity of bone marrow in Rap1bϪ/Ϫ mice was significantly less compared with that of WT IgM Ϫ Ϫ (WT: 45.4 Ϯ 8.8 ϫ 106 cells vs Rap1b / : 31.0 Ϯ 6.4 ϫ 106 cells; MOMA Downloaded from n ϭ 6ϩ6 mice; and p ϭ 0.014). The percentage of B220ϩ B cells were comparable between Rap1bϪ/Ϫ and WT mice. However, be- cause of reduced cellularity, the absolute number of B220ϩ B cells was significantly reduced in Rap1bϪ/Ϫ mice (Fig. 1C). Based on D B220 and IgM expression, B cells in the bone marrow can be IgM divided into different maturation stages: pro/pre-B cells IgD http://www.jimmunol.org/ (B220ϩIgMϪ), immature B cells (B220ϩIgMϩ), and recirculating high mature B cells (B220 ). The percentages of pro/pre-B cells were 100µΜ reduced in Rap1bϪ/Ϫ mice compared with WT. Likewise, the ab- solute number of both pro/pre-B cells and immature B cells were FIGURE 3. Reduced MZ compartment in the spleens of Rap1bϪ/Ϫ mice. Ϫ/Ϫ significantly reduced in Rap1b mice (Fig. 1C). In contrast, the A, H&E staining of cryosection of WT and Rap1bϪ/Ϫ spleens. B–D, Immu- high number of recirculating mature B cell B220 population was nostaining of spleen cryosections with anti-CD3-PE (red), MOMA-1-FITC Ϫ Ϫ comparable between Rap1b / and WT mice (Fig. 1C). B cell (green), and anti-B220-Cy5 (blue) mAbs (B); with anti-IgM-PE (red) and development in bone marrow can be further characterized through MOMA-1-FITC (green) mAbs (C); and with anti-IgM-PE (red) and anti-IgD- the expression of CD43 (leukosialin), CD117 (c-Kit), and CD25 FITC (green) mAbs (D). MZ areas are indicated (open arrowheads). A min- by guest on September 27, 2021 (IL-2R␣). Early pro-B cells are marked by the expression of imum of 20 white pulps was analyzed and data presented are representatives ␮ B220ϩCD43ϩ, which develop into B220ϩCD43Ϫ B cells. As pro/ of two mice in each group. Scale bar represents 100 M. pre-B cells mature, c-Kit expression decreases while CD25 level Ϫ Ϫ Ϯ / Ϯ ϩ Ϫ Ϫ Ϫ increases. The relative (WT: 2.6 0.7% and Rap1b : 2.8 B220 IgM B cells in WT and Rap1b / mice (Fig. 1F, top). In Ϯ ϫ 6 Ϫ Ϫ 0.5%) and the absolute (WT: 0.41 0.167 10 cells and addition, the Rap1b / pro/pre-B cells showed similar BrdU in- Ϫ/Ϫ Ϯ ϫ 6 ϩ ϩ Rap1b : 0.264 0.076 10 cells) B220 CD43 cell num- corporation as WT cells (Fig. 1F, bottom), thereby excluding a Ϫ/Ϫ bers did not differ significantly between Rap1b and WT bone defect in the proliferation as a probable cause for our observations. ϭ ϩ ϭ marrow (n 6 6 mice; p 0.0855) (Fig. 1D). However, However, consistent to the role of Rap1b in regulating integrin medium Ϫ Ϫ/Ϫ B220 CD43 B cells were much lower in Rap1b in both activation, we observed a significantly decreased adhesion of relative percentages (WT: 35.2 Ϯ 8.4% and Rap1bϪ/Ϫ: 25.9 Ϯ 10.3%) and absolute number (WT: 6.5 Ϯ 2.09 ϫ 106 cells and Rap1bϪ/Ϫ: 2.58 Ϯ 1.27 ϫ 106 cells, n ϭ 6 ϩ 6 mice; p ϭ 0.003), which is consistent with the reduced B220ϩIgMϪ populations (Fig. 1D, upper). The increased B220ϩc-kitϩ population in Rap1bϪ/Ϫ and with relatively decreased B220ϩCD25ϩ population (Fig. 1D, middle and lower) indicate a delay in B cell maturation in Rap1bϪ/Ϫ mice. In peripheral blood, there is an increased IgMϩIgDϪ B cell population in Rap1bϪ/Ϫ mice (Fig. 1E). Thus, the lack of Rap1b resulted in a developmental impairment of B cell progenitor maturing into pro/pre-B cell and immature B cell stages. FIGURE 4. MZ B cell defect in Rap1bϪ/Ϫ mice is cell autonomous. Bone marrow cells derived from Rap1bϪ/Ϫ and WT mice were i.v. trans- Adhesion but not proliferation of bone marrow-derived, ferred to sublethally irradiated JAK3Ϫ/Ϫ or lethally irradiated Rap1bϪ/Ϫ Ϫ Ϫ IL-7-driven pre-B cells is impaired in Rap1b / mice mice in three groups: WT to JAK3Ϫ/Ϫ (A), Rap1bϪ/Ϫ to JAK3Ϫ/Ϫ (B), WT Ϫ/Ϫ Ϫ Ϫ to Rap1b (C) mice. At 8 wk later, reconstituted cells from recipient To further gain the insight of B development in Rap1b / mice, spleens were stained with anti-B220, anti-CD21, and anti-CD23 mAbs. we conducted in vitro proliferation of pro/pre-B cells derived from Gated B220ϩ cells were separated into CD21low/ϪCD23low/Ϫ newly form- IL-7-driven bone marrow culture. The reduced pro/pre-B cells in ing (NF) B cells, CD23highCD21low/Ϫ FO B cells, and CD21highCD23low/Ϫ Ϫ/Ϫ the bone marrow of Rap1b mice may be due to either reduced MZ B cells. Number indicated is percentage of B cell subsets from one proliferation or adhesion to stromal cells. Bone marrow cells cul- representative mouse of each recipient group (n ϭ 4 mice for all ϩ tured with IL-7 contained similar purity of B220 and experiments). 3378 Rap1b IN B CELL DEVELOPMENT AND FUNCTION

−/− ABWT Rap1b 8 50 FO B 93.7 91.4

4 25

MZ B 98.0 95.4 H]Thymidine incorporation H]Thymidine

3 0 0 (CPM x1000) [ NoneIgM IgM+IL4 LPS CFSE WT WT Rap1b−/− C None IgM SDF-1 E 1 min NoneIgM SDF-1 None IgM SDF-1 Rap1-GTP exposure 0.4 0.94 1.0 P-Erk1/2 10 min Rap1-GTP exposure Erk1/2 0.97 1.18 1.0 Total Rap1 P-p38 1.06 1.03 1.0 p38

Actin Downloaded from

−/− D WT Rap1b F WT Rap1b−/− None IgM SDF-1 None IgM SDF-1 -’52 ’15’ -2’5’15’ Rap1-GTPase P-AKT-S473

Rap1 AKT http://www.jimmunol.org/ 1 23 456 FIGURE 5. Rap1b is dispensable for B cell proliferation, Erk1/2, p38, and AKT phosphorylation. A, Splenic B cells were stimulated with 10 ␮g/ml goat Ј ␮ Ј ␮ 3 anti-mouse IgM F(ab )2,10 g/ml goat anti-IgM F(ab )2 plus 10 ng/ml IL-4, 1 g/ml LPS, or left untreated for 72 h. [ H]Thymidine incorporation is shown as average of triplicate wells. B, Sorted spleen FO and MZ B cells were labeled with 1 ␮M CFSE and then stimulated with 10 ␮g/ml LPS. The cell division was analyzed by flow cytometry after 72 h. The percentage of cells that have divided is shown. C, Activation of Rap1 after BCR cross-linking and SDF-1 ␮ Ј stimulation in WT B cells. Purified splenic B cells were stimulated with 50 g/ml goat anti-mouse IgM F(ab )2 or 100 ng/ml SDF-1. An aliquot of total lysate was used to detect the total Rap1 expression as a loading control (lower). Remaining lysates were incubated with GST-RalGDS and active Rap1-GTPase was detected with anti-Rap1 polyclonal Ab (upper, short exposure; middle, longer exposure; lower, total Rap1 protein loading control). D, Ϫ/Ϫ Ϫ/Ϫ

Activation of Rap1 after BCR cross-linking and SDF-1 stimulation was diminished in Rap1b B cells. Purified splenic B cells from WT and Rap1b by guest on September 27, 2021 mice stimulated as in C and 10-min long exposure was shown in the residual Rap1a in Rap1bϪ/Ϫ B cells. E, Activation of Erk1/2 and p38 after BCR ␮ Ј cross-linking and SDF-1 stimulation. Purified splenic B cells were stimulated with 50 g/ml goat anti-IgM F(ab )2 or 100 ng/ml SDF-1 for 2 min. The active Erk1/2 and p38 were detected by anti-phospho-Erk or phospho-p38 mAbs. The total Erk1/2, p38, and actin were used as protein loading controls. Data in Ј C and D are representative of three independent experiments. F, Purified splenic B cells were stimulated with goat anti-IgM F(ab )2 polyclonal at indicated time points. The phosphorylated AKT (Ser473) and total AKT protein were detected with anti-phospho-AKT-S473 and anti-AKT Abs, respectively. Data shown are representative of two independent experiments.

Rap1bϪ/Ϫ pro/pre-B cells to the mouse stromal cell line M2-10B4, into newly forming B cells (CD21low/ϪCD23low/Ϫ), FO B cells reduced to 60% of WT (Fig. 1G). (CD23highCD21low/Ϫ), and MZ B cells (CD21highCD23low/Ϫ). The Cells from auxiliary and inguinal LNs were stained with anti- percentages of FO B cells were comparable between Rap1bϪ/Ϫ and B220, anti-IgM, and anti-IgD Abs. The percentage of B220ϩ, WT mice. However, the MZ B cells were significantly reduced IgMϩIgDϩ, and IgMϪIgDϩ cells was comparable between (ϳ55%), whereas the newly forming B cells were relatively increased Rap1bϪ/Ϫ and WT mice (Fig. 2D). Additionally, we examined the in Rap1bϪ/Ϫ mice (Fig. 2C, upper panels). MZ B cells can also be T cell development in Rap1bϪ/Ϫ mice. The cellularity of thymus, defined by their exclusive high-level expression of IgM, CD21, and spleen, and LN from Rap1bϪ/Ϫ mice was comparable to that of CD1d (40). Staining for these markers confirmed the reduction in WT (thymus, WT: 120.7 Ϯ 8.9 ϫ 106 cells; Rap1bϪ/Ϫ: 119.0 Ϯ IgMhighCD21high and IgMhighCD1dhigh B cell population (Fig. 2, D, 40.8 ϫ 106 cells, spleen, WT: 107.6 Ϯ 24.6 ϫ 106 cells; middle and lower panels). Rap1bϪ/Ϫ: 99.7 Ϯ 16.2 ϫ 106 cells; and LN: 4.8 Ϯ 1.1 ϫ 106 MZ B cells reside in a unique location around the follicles in the cells; Rap1bϪ/Ϫ: 4.8 Ϯ 2.4 ϫ 106 cells; n ϭ 6–8 mice). T cell spleen. To examine the lymphocyte organization, we performed development appears normal in thymus, spleen, and LN based on H&E staining of spleen cryosections. Rap1bϪ/Ϫ mice displayed CD4 and CD8 expression in the absence of Rap1b (data not normal spleen architecture of red and white pulps (Fig. 3A). Stain- shown). These results indicate Rap1b plays a role in B but not in ing with anti-CD3␧ and anti-B220 mAbs revealed a normal B and T cell development. T cell organization within the white pulps of the spleens (Fig. 3B). To view the MZ, we used MOMA-1 mAb, which stains the met- Rap1b regulates the homeostasis of MZ B cells allophillic macrophages. The location of metallophillic macro- Next, we analyzed the B cell maturation in the spleen. The percentage phages defines the boundary between the FO and MZ. The spleen B220ϩ B cells were comparable between Rap1bϪ/Ϫ and WT mice sections from the WT mice revealed a clearly defined band of (Fig. 2A). However, when we stained splenocytes with anti-IgM and B220ϩ MZ area outside of the MOMA-1ϩ metallophillic anti-IgD Abs, Rap1bϪ/Ϫ mice showed a reduction in IgMϩIgDlow/Ϫ macrophages. However, similar regions were greatly reduced and population (Fig. 2B). B220ϩ splenic B cells can be further separated less contiguous in Rap1bϪ/Ϫ mice (Fig. 3C). Staining splenocytes The Journal of Immunology 3379

WT +/+ A Rap1b−/− +/+ −/− +/+ −/− +/+ −/− +/+ −/− +/+ −/− +/+ −/− 2000 1000

100 Serum Ig [ µ g/ml] 10 IgM IgG1IgG3 IgG2a IgG2b IgA B C TI-IgM TI-IgG3 TD-IgM TD-IgG1 TD-IgG3 3 3 3 3 4 * * Preimmune 3 Downloaded from 2 2 2 2 2 day 7 1 1 1 1 1 day 28 0 0 0 0

Absorbance (450 nM) Absorbance 0 0 Absorbance (450 nM) Absorbance g p<0.03 400 800 400 800 400 800 400 800 400 800 * http://www.jimmunol.org/ 1600 3200 6400 1600 3200 6400 1600 3200 6400 1600 3200 6400 1600 3200 6400 12800 12800 25600 51200 12800 25600 102400 Serum Titration (1: X) Serum Titration (1: X) FIGURE 6. T-dependent but not T-independent humoral response is impaired in Rap1bϪ/Ϫ mice. A, Basal Ig levels (␮g/ml) of 6- to 8-wk-old mice were determined by ELISA (gray circle, WT; F, Rap1bϪ/Ϫ, n ϭ 7 mice). B, T-independent immune responses were intact in Rap1bϪ/Ϫ mice. Mice were i.p. injected with TNP-Ficoll, and sera were collected at days 0 and 7 after immunization. TNP-specific IgM and IgG3 isotype levels at days 0 and 7 were measured by ELISA. The values were presented as average of absorbance of serial dilution at 450 nm of each group. Preimmune (gray circle, WT; F, Rap1bϪ/Ϫ) and day 7 (u, WT; f, Rap1bϪ/Ϫ) mice are shown for n ϭ 4 mice. C, T-dependent immunization. Mice were i.p. injected with TNP-KLH and were boosted at day 21. TNP-specific Ab responses at day 0, 7, and 28 were determined by ELISA. Day 28 (u, WT; Œ, Rap1bϪ/Ϫ) mice are shown for n ϭ 4 mice. SD was excluded from the data because of the variability in a few individual sera. by guest on September 27, 2021 with anti-IgM and anti-IgD mAbs also demonstrated a reduction in tion in MZ B cells in Rap1bϪ/Ϫ mice could be due to defective cell IgMhighIgDϪ MZ B cells in the Rap1bϪ/Ϫ mice (Fig. 3D). The proliferation. We purified B cells from spleens and stimulated Ј Ј immunostaining results are consistent with the flow cytometry them with goat anti-IgM F(ab )2, goat anti-IgM F(ab )2 plus IL-4, data. The significant alteration in the newly forming B cell to MZ or LPS. [3H]Thymidine incorporation assay showed comparable B cell ratio indicates that the Rap1b plays a critical role in the proliferation of Rap1bϪ/Ϫ B cells as that of WT (Fig. 5A). To homeostasis of MZ B cells. further investigate the proliferation of B cell subsets, FO B cells and MZ B cells were sorted, CFSE-labeled, and stimulated with MZ B cell defect is cell autonomous Ϫ Ϫ LPS. Percentages of dividing cells between Rap1b / and WT Rap1b is expressed in multiple cell lineages (4). Therefore, the mice were compared and found to be equivalent (Fig. 5B). There- defects we observed in MZ B cells can be cell intrinsic or due to fore, it seems that Rap1b does not regulate splenic B cell prolif- an abnormality in the stromal microenvironment. To distinguish eration under current stimulation conditions. between these two possibilities, we performed bone marrow trans- plantation experiments. As expected, the transfer of WT bone mar- Ϫ/Ϫ Ϫ Ϫ Diminished Rap1-GTPase activation in the Rap1b B cells row cells into JAK3 / mice led to the reconstitution of newly forming, FO, and MZ B cells (Fig. 4A). However, similar recon- Earlier studies have shown that BCR engagement and chemokine stitutions using Rap1bϪ/Ϫ bone marrow cells into JAK3Ϫ/Ϫ mice stimulation lead to increased GTP-bound active Rap1 in B cells gave rise to normal FO B cells, but significantly reduced MZ B (18, 23, 42). However, the specific isoform activated is not known. cells ( p ϭ 0.007; n ϭ 4 mice) and slightly increased newly form- Therefore, we purified resting B cells from spleens and stimulated Ϫ/Ϫ Ј ing B cells, which mimicked the phenotype in Rap1b mice them with goat anti-IgM F(ab )2 and SDF-1. The active Rap1-GTP (Fig. 4B). Additionally, transfer of WT bone marrow cells to sub- was analyzed by a pull-down assay. In WT B cells, Rap1-GTP was lethally irradiated Rap1b1bϪ/Ϫ mice showed they were able to increased to more than 2-fold compared with the basal level (Fig. reconstitute the MZ population in the Rap1bϪ/Ϫ host (Fig. 4C). 5C, upper). Overexposure led to the diminished difference between Taken together, our results demonstrate that the B cell defects in control and activated samples (Fig. 5C, middle). As the Rap1a the spleen of Rap1bϪ/Ϫ mice were cell intrinsic, and the splenic level was extremely low compared with Rap1b in B cells and no environment in Rap1bϪ/Ϫ mice was able to support the develop- good commercial Rap1a specific Ab are available, we used anti- ment of WT B cells. Rap1 Ab that recognizes both the isoforms. The basal level of Rap1-GTP in Rap1bϪ/Ϫ resting B cells was hardly detectable even Rap1b is dispensable for splenic B cell proliferation after a prolonged exposure (Fig. 5D, lane 4), which led to the Earlier study indicated a role for Rap1 signaling in lymphocyte saturation of Rap1-GTP in WT sample. Upon stimulation, Rap1- proliferation (41, 42). Therefore, we hypothesized that the reduc- GTP was also increased in Rap1bϪ/Ϫ B cells. However, it was 3380 Rap1b IN B CELL DEVELOPMENT AND FUNCTION Downloaded from http://www.jimmunol.org/

FIGURE 7. In vitro chemotaxis, adhesion, and in vivo homing of Rap1bϪ/Ϫ B cells are reduced. A, A total of 150 ng/ml SDF-1- or 500 ng/ml CXCL13-induced migration of Rap1bϪ/Ϫ B cells is impaired. The migration rate is presented as a percentage of migrated cells compared with the input cells that were added directly the lower chamber. Data presented are representative of three independent experiments. B, Reduced migration of Rap1bϪ/Ϫ MZ and FO B cell to S1P. RBC-depleted splenocytes were added to Transwell with S1P gradient (0–1000 nM) and allowed to migrate for 3 h. The migrated by guest on September 27, 2021 cells were stained with anti-B220, CD21, and CD23 mAbs. The migration rate is presented as a percentage of migrated cells compared with the input cells that were added directly the lower chambers. Data presented are representative of three independent experiments. C, Reduced S1P-induced adhesion to ICAM-1 of Rap1bϪ/Ϫ B cells. The 96-well plates were coated with BSA or recombinant mouse ICAM-1 10 ␮g/ml as described in Materials and Methods and cells were incubated with or without 100 nM S1P and incubated at 37°C for 60 min. After washing to remove the nonadherent cells, adherent cells were stained for B220, CD23, and CD21. Adhesion was calculated as a percentage of input cells. D, Lack of Rap1b impairs the in vivo homing of B and T cells. RBC-depleted WT and Rap1bϪ/Ϫ splenocytes were labeled with CFSE and TAMRA, respectively, and injected into WT recipient mice. At 3 h later, cells from spleens, auxiliary or inguinal LN, and mesenteric LN of recipient mice were stained with anti-B220-allophycocyanin or anti-CD3-PE-Cy7. E, A representative recipient mouse for B cell homing is shown (right) for data from n ϭ 7 mice. The homing efficiency is shown as the ratio of homed Rap1bϪ/Ϫ to WT B or T cells after corrected to the ratio of injected Rap1bϪ/Ϫ to WT B or T cells (left) for data obtained from n ϭ 3 mice.

Ј much lower than that in WT cells (Fig. 5D, lanes 5 and 6). These goat anti-IgM F(ab )2 Ab and found similar levels of AKT phos- results indicate that although both Rap1a and Rap1b can be con- phorylation at its Ser473 residue in Rap1bϪ/Ϫ as that of WT. This verted to active GTP-bound forms through BCR or chemokine finding indicates that Rap1b-GTPase does not regulate AKT phos- receptor-mediated activation, the major isoform is Rap1b. phorylation during BCR signaling in splenic B cells (Fig. 5F).

Lack of Rap1b does not affect the phosphorylation of Erk1/2, Rap1b regulates T-dependent but not T-independent humoral p38, or AKT immunity Activation of Erk1/2, p38, and AKT are critical for lymphocytes The present study indicates that Rap1b is the dominant isoform in proliferation and survival (43, 44). Yet the role of Rap1b in the B cells and regulates B cell development. To test whether Rap1b activation of these molecules is not clear. Overexpression of Rap- plays a role in B cell-mediated immunity, we first analyzed the GAPII in B cells, which inhibited the activation of both Rap1 and basal Ig levels in the sera of Rap1bϪ/Ϫ mice. Fig. 6A shows that Rap2, did not affect Erk1/2 and p38 phosphorylation but led to the preimmune Ig IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels increased AKT activation (45). To test the role of Rap1b in these were comparable between WT and Rap1bϪ/Ϫ mice. Because MZ events, we examined the BCR or SDF-1-stimulated splenic B cells. B cells have been shown to play an important role in Fig. 5E shows that the levels of Erk1/2 and p38 phosphorylation T-independent Ab generation (46), we challenged the mice with were comparable between Rap1bϪ/Ϫ and WT. It is important to T-independent Ag TNP-Ficoll and collected sera to analyze the note that the BCR- or SDF-1-mediated activation resulted in only presence of Ag-specific Abs. Rap1bϪ/Ϫ mice were able to generate a minimal increase of Rap1-GTP in Rap1bϪ/Ϫ B cells (Fig. 5D). similar amounts of TNP-specific IgM and IgG3, the two major Ig Therefore, Rap1b does not seem to regulate the phosphorylation of isotypes generated during T-independent responses, as generated Erk1/2 and p38. Furthermore, we stimulated splenic B cells with by WT mice (Fig. 6B). Optimal Ab response in Rap1bϪ/Ϫ mice The Journal of Immunology 3381 indicates that the remaining MZ B cells were sufficient for T-in- generated (25, 26, 36). Deletion of SPA-1, a principle Rap1GAP in dependent Ab generation. Next, we examined the T-dependent im- lineage negative (LinϪ) bone marrow cells, resulted in an accu- mune responses by i.p. injection of TNP-KLH. In the primary re- mulation of Rap1-GTP and lead to an increase of the hematopoi- sponse, the generation of TNP-specific IgM, IgG1, and IgG3 was etic progenitors (27). Expression of constitutively active Rap1, comparable between Rap1bϪ/Ϫ and WT mice (Fig. 6C). However, RapE63, resulted in the LinϪ bone marrow cell expansion, after Ag boost, the TNP-specific IgG1 was significantly lower in whereas SPA-1 led to the suppression (27). However, in the ab- the Rap1bϪ/Ϫ mice compared with that of WT (Fig. 6C). Although sence of Rap1b, bone marrow-derived B220ϩ B cell lineage not significant, the TNP-specific IgG3 were also lower in showed similar proliferation rates as that of WT. Interestingly, lack Rap1bϪ/Ϫ mice during the secondary responses (Fig. 6C). There- of Rap1b resulted in reduced adhesion to bone marrow stromal cell fore, we conclude that Rap1b is required for optimal T-dependent lines. The reduced bone marrow pro/pre-B cells in Rap1bϪ/Ϫ may but not T-independent humoral responses. be due to reduced adhesion rather than a decreased expansion of B cell lineage. In addition, although lack of Rap1b did not adversely Rap1b regulates B cell chemotaxis, adhesion, and in vivo affect the number of B220ϩCD43ϩ B progenitors, there was a homing delayed maturation as indicated by more c-kitϩ and fewer CD25ϩ Rap1 regulates cell adhesion and migration (30). To better under- cells in Rap1bϪ/Ϫ bone marrow. In peripheral blood, a higher per- stand the functional role of Rap1b in B cell migration, we first centage of B cells displayed IgMϩIgDϪ immature phenotype com- examined the chemokines SDF-1 and CXCL13 and lipid S1P-in- pared with that of WT. Reduced adhesion of pro/pre-B cell to duced chemotaxis in Transwell assays. Rap1bϪ/Ϫ splenic B cells stromal cells could also cause a corresponding decrease of B pro- Ϫ Ϫ displayed significantly reduced migration toward chemokine gra- genitors in the bone marrow of Rap1b / mice. Taken together, Downloaded from dient (Fig. 7A). S1P is important for lymphocyte migration (47, these results indicate a possible differential regulation of LinϪ and 48). Similar to SDF-1 and CXCL13, S1P has been shown to ac- Linϩ bone marrow cells by Rap1b. The reduced bone marrow tivate Rap1 and integrin activation is required for the correct lo- pro/pre-B cells in Rap1bϪ/Ϫ may be due to reduced adhesion calization of MZ B cells (39, 49). Consistent with published data rather than an augmented expansion of B cell lineage. (50), MZ B cells showed robust response to S1P and showed Spleen has a unique architecture composed of stromal and he- Ϫ/Ϫ higher migration toward S1P. In contrast, Rap1b B cells matopoietic cells that are vital for B cell maturation and function. http://www.jimmunol.org/ showed dramatically reduced migration compared with WT (Fig. A strong reduction of MZ B cells in Rap1bϪ/Ϫ mice may be due ␣ ␤ Ϫ/Ϫ 7B). MZ B cells have higher L and 2 integrin expression and to the deficiency in B or splenic stromal cells. Rap1b mice show higher adhesion to ICAM-1 compared with FO B cells (39). showed normal organization of spleen and the defect of MZ B cells Similarly, S1P-induced adhesion to ICAM-1 was also reduced in was recapitulated in our bone marrow transplantation experiment. Rap1bϪ/Ϫ B cells (Fig. 7C). To further investigate these defects, Together, these indicate the MZ B defect is cell intrinsic. A MZ B we performed an in vivo homing assay. Rap1bϪ/Ϫ and WT-de- cell defect is also observed in RapLϪ/Ϫ mice validating the role of rived splenocytes were separately labeled with TAMRA and Rap1-RapL signaling in MZ B cell development (25). However, CFSE-DA, respectively, premixed and i.v. injected into the recip- the MZ B cells in RapLϪ/Ϫ mice are nearly absent, which is a more ient mice. To provide a Rap1b-sufficient microenvironment, we severe phenotype compared with Rap1bϪ/Ϫ mice. The multiple by guest on September 27, 2021 used the WT mice as the recipients. Three hours later, cells from upstream RapL activators, Rap1 and Rap2 isoforms, could account spleen, auxiliary/inguinal and mesenteric LN were stained with for the phenotypic differences in these two mouse models (51). anti-B220 and anti-CD3␧ mAbs, and the ratio of Rap1bϪ/Ϫ to Expression of Rap2 in B cells has been previously established (18). WT-derived lymphocytes were quantified (Fig. 7D). The homing Immature B cells emigrate from the bone marrow and home to the of B and T cells from Rap1bϪ/Ϫ and WT donors into the recipient secondary lymphoidal organs such as spleen (40). Once migrated spleens were comparable. However, the ability of Rap1bϪ/Ϫ B into the spleen, newly formed B cells have the potential to differ- cells to migrate into the auxiliary or inguinal LN was significantly entiate into either FO or MZ B cells (40). Reduced adhesion of reduced (ϳ60%). Similarly, homing of Rap1bϪ/Ϫ B cells into pro/pre-B cell to stromal cells may lead to an augmented egress of mesenteric LN was also affected (Fig. 7E, left). We also observed immature B cells from bone marrow, which may account for the reduced ability of Rap1bϪ/Ϫ T cells to migrate into the auxiliary or normal FO B cells in Rap1bϪ/Ϫ mice. Rap1 signaling seems not inguinal LN (Fig. 7E, right). Together, these results demonstrate essential for egressed immature B cells to differentiate into FO B that Rap1b plays a critical role in chemokine-mediated migration cells in Rap1bϪ/Ϫ spleen. This is partly supported by bone marrow and in the in vivo homing of B cells. progenitors transfected with constitutively active RapE63 that showed similar differential potential as vector-transfected control Discussion cells (26). An increase in the newly forming B cells in Rap1bϪ/Ϫ In this study, we demonstrate that Rap1b is the dominant isoform mice could indicate the specific role of Rap1 in the maturation for in B cells. Using gene knockout mice, we show that Rap1b is MZ but not FO B cells. The role of integrin in the retention of MZ essential for pro/pre-B cell development in the bone marrow and B cells has been demonstrated (39). MZ B cells have higher ex- ␣ ␤ ␣ ␤ for the homeostasis of MZ B cells in the spleen. Furthermore, pression level of L 2 and 4 1 integrins than FO B cells. As S1P Rap1b is required for optimal T-dependent humoral immunity, induced activation of Rap1 (49), the dramatically reduced migra- chemokine- and S1P-stimulated migration, S1P-induced adhesion tion to S1P and S1P-induced adhesion to ICAM-1 of Rap1bϪ/Ϫ to ICAM-1, and in vivo homing of B cells. Rap1b is also important splenic MZ B cells may contribute to the defect in MZ B cells. In for B cell progenitor adhesion to stromal cell lines. Rap1b is not contrast, in the preleukemic SPAϪ/Ϫ mice, the development of FO required for splenic B cell proliferation and BCR-mediated acti- and MZ B cells is normal (27). This discrepancy between vation of Erk1/2, p38, or AKT. Rap1bϪ/Ϫ and SPAϪ/Ϫ mice is due to the differential expression of One of the key findings in this study is the role of Rap1b in B these two proteins in bone marrow and spleen B cells. SPA-1 cell development in the bone marrow. A reduction of pro/pre-B expression level is considerably higher in LinϪ than Linϩ cells, cells and immature B cells indicates that the requirement of Rap1b which suggests Rap1 signaling may regulate lineage development cannot be compensated by Rap1a or Rap2 isoforms. Several gene differentially at different stages. The reduced MZ B cells in knockout mice with altered Rap1 signaling have been recently Rap1Ϫ/Ϫ mice is not due to defective cell proliferation as we did 3382 Rap1b IN B CELL DEVELOPMENT AND FUNCTION not observe a role of Rap1b in LPS- or BCR-induced proliferation ical synapses that contain LFA-1, ICAM-1, CD40, and other mol- of total spleen B cells or sorted FO and MZ B cell, which is ecules (62). Rap1 regulates T cell polarization and thereby the supported by the normal proliferation of SPA-1Ϫ/Ϫ splenic B cells interaction with APCs (22). The dominant negative form of Rap1, (27). Thus, the role of Rap1 signaling in cell proliferation seems to Rap1N17, has been shown to block TCR-dependent increase in be cell type-dependent as it has been shown to positively regulate adhesion to ICAM-1. Furthermore, Rap1aϪ/Ϫ T cells failed to the proliferation of hematopoietic (26) and peritoneal B1 cells properly polarize LFA-1 upon CD3 stimulation (36). Thus, lack of (27). The relatively normal development of T cells in the absence Rap1b could impair the efficiency of T cell-APC interactions lead- of Rap1b could be due to a compensatory effect from a different ing to a reduction in the secondary T-dependent Ab generation. member of the Rap family such as Rap2. Our present observations were supported by the findings in the SPA- Cell proliferation, differentiation and survival can be regulated 1Ϫ/Ϫ mice (27). T cells from SPA-1Ϫ/Ϫ mice had selective impair- by Erk1/2, p38, and AKT-mediated signaling pathways (52). Ras ment in memory T cell responses against a T-dependent Ag with is one of the major activator of Erk1/2 and p38 (53). Rap1-GTPase normal primary Ab response. Lack of SPA-1 also resulted in an aug- has been shown to negatively regulate Ras-mediated Erk and p38 mented levels anti-dsDNA Abs from B1a B cells leading to lupus-like activation in some studies; however, it is controversial (1, 45, 54, autoimmune disease (27). Therefore, a regulated Rap1-signaling is 55). Our present study shows that phosphorylation of Erk1/2 and critical for optimal immune responses. Of note, during publication of p38 was not changed after BCR and SDF-1 stimulation in our results, Chen et al. published similar results (63). Rap1bϪ/Ϫ B cells, although Rap1-GTP was nearly absent. This However, the precise role of Rap1b in B cell development and finding is consistent with earlier studies in which expression of memory T-dependent humoral response are still not complete elu- Downloaded from constitutively active V12Rap1a in thymocytes, activation of Rap1 cidated. Induced deletion of Rap1b in B lineage could address this by cAMP in NIH3T3 cells, or overexpression of RapGAPII in B question more specifically. A conditional ablation of Rap1b will cells exhibited an unaltered Erk1/2 phosphorylation (19, 45, 56). help to dissect the distinct role of Rap1b in B cells, T cells, and Regulation of Erk1/2 activation by Rap1 has been shown to be cell dendritic cells, the major cell types that involve in T-dependent context-dependent. In SPA-1Ϫ/Ϫ mice, Rap1 signaling has been immunity. Rap1a and Rap1b have different C-terminal sequences shown to regulate Erk phosphorylation positively in LinϪ bone leading to distinct posttranslational modifications, which may con-

marrow cells and negatively in anergic T cells (26, 57). Earlier tribute to isoform-specific function (31, 32, 34). The incomplete http://www.jimmunol.org/ studies have also that Rap1 signaling AKT phosphorylation (41, loss of MZ B cells and intact T-independent immunity in 45). In contrast, the similar BCR-mediated activation of AKT Rap1bϪ/Ϫ mice may indicate the compensatory role of Rap1a or (Ser473) phosphorylation was observed in Rap1bϪ/Ϫ B cells as that Rap2 proteins. Continued research using Rap1a or Rap2-deficient in WT. This discrepancy may be due to the dual GAP activity of mouse models will greatly help us to understand the function of RapGAPII directed to both Rap1 and Rap2. It is most likely that Rap protein family. the Rap2 but not Rap1 that regulates the AKT phosphorylation as Rap2 has its unique effectors (58–60). Acknowledgments Cell migration is controlled by multiple coordinated processes We thank Jillian Dargatz for helping with mouse care, Barbara including chemoattraction, adhesion, and transmigration through A. Fleming for cryosectioning, and Asanga Samarakoon for technical support. by guest on September 27, 2021 the blood vessels. Chemokine-directed migration of B cells is es- sential for B cell development, differentiation, and Ab generation Disclosures Ϫ Ϫ (61). The defect of Rap1b / B cells in vitro chemotaxis assay and The authors have no financial conflict of interest. in vivo short-term homing to LN is consistent with the established role of Rap1b in cell migration. Interestingly, the LN cellularity of References Ϫ Ϫ Rap1b / mice was normal. The defect in the short-term homing 1. Kitayama, H., Y. Sugimoto, T. Matsuzaki, Y. Ikawa, and M. Noda. 1989. A assay may reflect only a slower cell migration. During the long Ras-related gene with transformation suppressor activity. 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