Endothelial Plasmalemma Vesicle–Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells

Endothelial Plasmalemma Vesicle−Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells

This information is current as Raul Elgueta, Dan Tse, Sophie J. Deharvengt, Marcus R. of September 26, 2021. Luciano, Catherine Carriere, Randolph J. Noelle and Radu V. Stan J Immunol 2016; 197:3970-3981; Prepublished online 14 October 2016;

doi: 10.4049/jimmunol.1501859 Downloaded from http://www.jimmunol.org/content/197/10/3970

Supplementary http://www.jimmunol.org/content/suppl/2016/10/13/jimmunol.150185 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 64 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/197/10/3970.full#ref-list-1

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Endothelial Plasmalemma Vesicle–Associated Protein Regulates the Homeostasis of Splenic Immature B Cells and B-1 B Cells

Raul Elgueta,*,† Dan Tse,‡,1 Sophie J. Deharvengt,‡ Marcus R. Luciano,‡ Catherine Carriere,x,{,1 Randolph J. Noelle,*,{ and Radu V. Stan*,‡,‖

Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap + lo results in a dramatic decrease of IgM IgD B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap Downloaded from demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM+IgDlo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM+IgDlo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM+ B cells in the spleen and peritoneal cavity. The Journal of Immunology, 2016, 197: 3970–3981. http://www.jimmunol.org/ he innate immune response is the host’s first and most favored as the first cells to sample Ags in the blood and gut. rapid response to infection with a pathogen, whereas the Moreover, MZ and B-1 B cells are well characterized as having a T adaptive immune response involves a complex process low activation threshold and their BCRs recognize a wide range of including activation, expansion, and differentiation of pathogen- microbial Ags (4). Both B cell subsets significantly contribute to specific B and T cells. The development of adaptive immunity levels of serum IgM and the production of natural Abs. Natural requires several days to weeks to generate a long-standing effector Abs in many cases can be specific to pathogen-encoded molecules and memory immune response (1, 2). A key transition from innate and be critical in the rapid neutralization of both viruses and to adaptive immunity is mediated by the marginal zone (MZ) B bacteria (5). and B-1 cells because they produce the first set of low-affinity Abs MZ B cells arise from bone marrow precursors through tran- by guest on September 26, 2021 against the pathogen (3). MZ B and B-1 cells are localized in the sitional B cells, which colonize the periarteriolar lymphoid sheath marginal sinus and peritoneal cavity, respectively, where they are (5). The differentiation of transitional B cells to MZ B cells is driven by a weak BCR activity through a dependent pathway Bruton’s tyrosine kinase (6–8). This and the interaction of NOTCH *Department of Microbiology and Immunology, Geisel School of Medicine at Dart- expressed on transitional B cells with the ligand, D-like 1, on † mouth, Lebanon, NH 03756; Department of Immune Regulation and Intervention, endothelial cells induce the differentiation to MZ B cells (9). The Medical Research Council Centre for Transplantation, King’s College London, Guy’s Hospital, London, SE1 9RT, United Kingdom; ‡Department of Pathology, Geisel homing of MZ B cells is dependent on circulating sphingosine-1- School of Medicine at Dartmouth, Lebanon, NH 03756; xDepartment of Medicine, { phosphate (S1P) binding to S1P1 and S1P3 receptors expressed in Geisel School of Medicine at Dartmouth, Lebanon, NH 03756; Norris Cotton Can- the endothelial cells of blood vessels of MZ (10, 11). After mi- cer Center, Geisel School of Medicine at Dartmouth and Dartmouth-Hitchcock Med- ical Center, Lebanon, NH 03756; and ‖Department of Biochemistry and Cell Biology, gration, MZ B cells are retained by the interaction of aLb2 and Geisel School of Medicine at Dartmouth, Lebanon, NH 03756 a4b1 with ICAM1 and VCAM1, respectively (12). 1Current address: ImmuNext, Lebanon, NH. In contrast, B-1 cells are competently produced before birth and ORCIDs: 0000-0002-7083-2430 (R.E.); 0000-0003-0766-2464 (S.J.D.); 0000-0003- throughout the first couple weeks after birth. The precursors for B-1 2969-1725 (R.V.S.). cells have been discovered in the splanchnopleural region, yolk sac Received for publication August 20, 2015. Accepted for publication September 18, and intraembryonic hemogenic endothelium, and fetal liver, but 2016. they are absent from adult bone marrow (13–16). B-1 cells con- This work was supported by National Institutes of Health Grants GM120592, stantly circulate to and from the peritoneal space across the CA175592, CA172983, CA023108, and S10OD010330. omentum in a process that involves CXCL13, which is likely Address correspondence and reprint requests to Dr. Radu V. Stan or Prof. Randolph J. Noelle, Department of Biochemistry and Cell Biology, Geisel School of Medicine at produced by macrophages (17). Collectively, these findings show Dartmouth, One Medical Center Drive, Lebanon, NH 03756 (R.V.S.) or Department that B cell progenitor migration is highly regulated by molecules of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, One expressed on endothelial cells. However, it is not known whether Medical Center Drive, Lebanon, NH 03756 (R.J.N.). E-mail addresses: radu.v. [email protected] (R.V.S.) or [email protected] (R.J.N.) molecules expressed on endothelial cells are involved in B cell The online version of this article contains supplemental material. differentiation and trafficking. Abbreviations used in this article: AF, Alexa Fluor; MZ, marginal zone; PLN, pe- Plasmalemma vesicle-associated protein (Plvap) is a vertebrate ripheral lymph node; Plvap, plasmalemma vesicle-associated protein; PP, Peyer’s gene (18, 19) whose product, Plvap, is a heparin-binding (20), patches; RT, room temperature; S1P, sphingosine-1-phosphate; T1, transitional 1; homodimeric, single-span type II membrane glycoprotein (21–23) T2, transitional 2; WT, wild type. critical for the formation of the stomatal diaphragms of caveolae, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 transendothelial channels, and vesiculo-vacuolar organelles, as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501859 The Journal of Immunology 3971 well as the diaphragms of fenestrae in both mice (24–27) and using Tie2/Tek-cretg/+ mice [JAX strain B6.Cg-Tg(Tek-cre)12Flv/J] (label PlvapECKO-Tie2, genotype PlvapL/L;Tek-cretg/+); 4) B cells in the embryo humans (28). Microscopic (19, 22) and genetic (26, 28, 29) lines KI/+ tm1(cre)Cgn of investigation led to the conclusion that Plvap is specifically using CD19-cre knock-in mice (JAX strain B6.Cg-Cd19 /J) (45) (label CD19CrexPlvapL/L, genotype PlvapL/L;CD19-creki/+); and 5) expressed in the endothelial cells of blood vessel capillaries and hematopoietic cells in the embryo using Vav1-cre+/2 transgenic mice (46), venules in select vascular beds and in the heart endocardium, and for deletion of Plvap in all the hematopoietic cell lineages, but not in is absent from lymphatic endothelial cells. This pattern of ex- endothelium (44, 46). For inducible deletion of Plvap in the endothelial tg/+ pression was fully supported by a large body of literature obtained cells of the adult mice, we used end-SCL-Cre-ERT transgenic mice to generate mice with the genotype PlvapL/L; end-SCL-CreERTtg/+(label with two endothelial-specific mAbs that bind Plvap, such as PlvapiECKO). Deletion of Plvap was achieved by dosing 4-wk-old mice MECA-32 (30, 31) in the mouse and PAL-E (32–34) in humans (both males and females) by gavage with seven doses of 4 mg of tamoxifen (reviewed in Refs. 35, 36). However, recently, Plvap expression spaced at 48 h. Experiments were carried out 2 wk after the last tamoxifen was also demonstrated in the sinus lymphatic endothelial cells of dose administration. Control animals for germline deletion were sex- and age-matched wild type (WT) or CMV-cretg/+ (labeled WT) and Plvap+/2 or peripheral lymph nodes (PLNs) while confirming its absence from 2 2 Plvap+/ ;CMV-cretg/+ (labeled Plvap+/ ) littermates. Control animals for peripheral lymphatics elsewhere (27) (http://immgen.org). At the tissue-specific deletions were sex- and age-matched WT or PlvapL/L lit- whole organism level, endothelial diaphragms formed by Plvap termates. (24, 37, 38) are critical for maintaining basal permeability of VEC-Plvap-HAtg/+ transgenic mice (26) that express Plvap-HA fusion fenestrated blood vessels, with their absence resulting in disrupted protein specifically in the endothelial cells under the control of cadherin 5 + (VE Cadherin) promoter were used to reconstitute Plvap in endothelial blood homeostasis and reduced survival (26, 28). In PLNs, Plvap cells in the context of the Plvap2/2 (label PV1ECRC) as described (26). The diaphragms in the sinus lymphatic endothelial cells control the same strategy was used to reconstitute Plvap in the context of PlvapiECKO iECRC L/L Ttg/+ entry of soluble Ags and lymphocytes in PLN parenchyma (27). (label Plvap , genotype Plvap ; end-SCL-CreER ; VEC-Plvap- Downloaded from tg/+ The expression of Plvap and diaphragm formation is increased in HA ). activated endothelial states associated with inflammation (39, 40) All animals were maintained in a pathogen-free facility at Dartmouth College. All procedures were approved by the local Institutional Animal and physiological and pathological angiogenesis (35, 41, 42) Care and Use Committee. where it has active roles. Plvap is required for cancer progression (43) and diapedesis of leukocytes into inflammation sites in vivo Abs, staining, and analysis by flow cytometry

(31). In vitro Plvap knockdown and Ab-mediated blockade ex- The following Abs and staining reagents were used: IgG1 (clone A85-1), http://www.jimmunol.org/ periments suggest that endothelial Plvap is important for the IgG2a/b (clone R2-40), CD138 (clone 281-2), IgM (clone 11–41), CD24 transcellular transmigration, but not for adhesion and rolling of (clone M1/69), CD4 (clone RM4-5), CD21/35 (clone 7E9), CD8 (clone 53- 6.7), CD25 (clone 3C7), CD62L (clone MEL-14), CD69 (clone H1.2F3), lymphoblasts, with no effect on neutrophils transmigration (31). CD44 (clone lM7), CD11c (clone HL3), CD80 (clone 16-10A1), CD86 Plvap is thought to control the transcellular migration of lymph- (clone GL1), CD11b (clone M1/70), c-Kit (CD117, clone 2B8), and borne lymphocytes into PLN parenchyma (27). Deletion of Plvap streptavidin-PerCP were from BD Pharmingen; Plvap (clone MECA-32) results in defective PLN morphogenesis with mild decreases in the was from Abcam; CD38 (clone 90), B220 (clone 6B2), CD23 (clone T cell compartment (both CD4 and CD8 T cells), hyperplastic B3B4), IgD (clone 11-26c), and FceRI (clone MAR-1) were from eBio- science; and peanut agglutinin was from Vector Laboratories. MECA-32 B cell follicles, and increases in both PLN B and T cell activation. mAb (anti-mouse Plvap rat IgG2a) secreting hybridoma was obtained from Intriguingly, Plvap deletion increases the entry of adoptively the Developmental Studies Hybridoma Bank (University of Iowa) and was by guest on September 26, 2021 transferred lymph-borne splenocytes (both B and T cells), whereas produced in serum-free conditions by BioXCell (Lebanon, NH). The Ab its ligation with MECA-32 Ab inhibits the recruitment of these was labeled with Alexa Fluor (AF) fluorochromes using the protein labeling kits for AF488 and AF647 (Invitrogen), as per manufacturer’s instructions. subsets (27). The mechanism of how Plvap mediates transendothelial Flow cytometry was performed on either a refurbished FACSCAN or a migration of immune cells is currently unclear. FACSCalibur running CellQuest software (BD), or a FACSCanto running In this study, we have examined whether Plvap plays a role in the FACSDiva software within the Norris Cotton Cancer Center DartLab Im- development and homeostasis of hematopoietic lineages, taking mune Monitoring Facility. The data analysis was performed using FlowJo advantage of recently created genetic models of Plvap gain and loss (Tree Star). of function and endothelial-specific reconstitution (26). Our studies Isolation of cells from spleen, PLNs, and Peyer’s patches show that deletion of Plvap results in an intense reduction of IgM+ To analyze B cells, T cells, and monocytes, we prepared single-cell sus- B cells in both spleen and peritoneal cavity. Tissue-specific de- pensions of lymphocytes from spleens by mechanical disruption in HBSS letion of Plvap demonstrates that the defect is B cell extrinsic, followed by passing the cells through a 70-mm cell strainer. In experiments because B cell and pan-hematopoietic Plvap deletion has no effect where dendritic cells were profiled, an enzymatic digestion step (45 min, on IgM+ B cell numbers. Endothelial-specific deletion of Plvap 37˚C) was included using DNAse (10 mg/ml; Roche) and Liberase (12.5 mg/ml; Roche) before mechanical disruption. Cells were collected by recapitulates full Plvap knockout, whereas endothelial-specific 3 + centrifugation (5 min, 500 g, 4˚C), and RBCs were lysed (2 min, 37˚C) reconstitution of Plvap rescues the IgM B cell phenotype. Taken using RBC Lysis buffer (BioLegend). Total number of cells and cell via- together, these results demonstrate that Plvap expression on endo- bility were determined using either a hemacytometer and trypan blue or a 5 6 thelial cells is key in the maintenance of IgM+ B cells into spleen Guava system (Millipore). Cells (10 –10 ) were stained with Ab cocktails, and peritoneal cavity. as noted. Analysis was performed after costaining with the mixture of Abs by flow cytometry. For isolation of peritoneal cells, the peritoneal cavity was flushed with 5 ml of warm (37˚C) PBS, 2% BSA, 2 mM EDTA, 0.02% Materials and Methods sodium azide, and 10 U/ml heparin. Conditional deletion of Plvap Analysis of Plvap expression in splenocytes of naive and Homozygous PlvaploxP (PlvapL/L) mice were generated by knock-in using LPS-treated mice homologous recombination in mice, as already described (26). PlvapL/L Female WT C57Bl6/J mice were treated with either 50 mgofLPS(E. coli 055: mice express Plvap at normal levels and have no overt phenotype (26). The PlvapL/L mice were bred to mice expressing the cre recombinase under the B5; Sigma) in PBS or an equal volume of PBS alone (control mice) i.p. 12 h control of different promoters to generate compound mice where Plvap was before spleens were harvested, enzymatically dissociated, as noted earlier, deleted in: 1) the germline (label Plvap2/2, genotype Plvap2/2;CMV-cretg/+) followed by Ab staining of splenocytes, and analyzed by flow cytometry. tg/+ (CMV-cre)1Cgn/J using CMV-cre transgenic mice (JAX strain BALB/c-Tg ); Immunofluorescence and laser confocal microscopy 2) endothelial and hematopoietic cells in the embryo using Ins-VEC- cretransgenicmice(44)(labelPlvapECKO-VEC, genotype PlvapL/L;Ins- Tissues were snap-frozen in optimal cutting temperature medium and VEC-cretg/+); 3) endothelial and hematopoietic cells in the embryo sectioned to 8 mm. Sections were collected on charged slides (Surgipath), 3972 ROLE OF Plvap IN IMMATURE B CELLS

fixed (220˚C, 10 min) with cold methanol, rinsed (3 3 2 min, room same reduction would also be found in peritoneal B-1 B cells. temperature [RT]) in PBS, encircled with hydrophobic barriers (PapPen), Profiling of the resident leukocytes in the peritoneum obtained by m blocked (30 min, RT) with 10% rat serum in PBS containing 10 g/ml peritoneal lavage demonstrated a drastic reduction in the total mouse Fc block, incubated (1 h, RT, in dark) with various fluorescently 2/2 labeled primary Ab cocktails in blocking buffer, rinsed (3 3 5 min, RT, in number of cells in the peritoneum of Plvap mice compared 2 the dark) again in PBS, stained (10 min, RT, in the dark) with 300 nM with WT or Plvap+/ littermates (Fig. 2A). The reduction in total DAPI (D1306; Life Technologies), and washed (33 5 min, RT) in PBS. viable leukocyte numbers in the peritoneal cavity was accompa- Labeled sections were mounted under #1.5 coverslips using a polymerizing nied by a low percentage and absolute number of IgD+ B cells and mounting medium (Fluoromount G; Southern Biotech). The Abs used were + low rat anti-mouse Plvap-AF568 (clone MECA-32), rat anti-mouse CD169- the absence of IgM IgD B cells (Fig. 2B–D). Taken together, FITC (clone MOMA-1; AbD Serotec), and rat anti-mouse/human B220- these results suggest that Plvap plays a role in the recruitment or AF647 (clone 6B2). Labeled sections were analyzed using a Zeiss retention of B cells into the peritoneal cavity and/or their survival. LSM510 Meta confocal microscope equipped with appropriate lasers (405, Because of the drastic reduction of cells in the peritoneum cavity 488, 532, 633 nm) and filters, all within the Norris Cotton Cancer Center 2/2 microscopy facility. The acquired images were processed for brightness of Plvap mice, we also analyzed the percentage of mast cells and contrast and analyzed using ImageJ (http://imagej.nih.gov/ij/), and the and monocytes in this compartment. Our results show that mast figures were mounted using Adobe Photoshop and Adobe Illustrator CS6. cell (cKit+ FceRI+ cells; Supplemental Fig. 2A) and monocyte + + Statistics (CD11b Gr-1 cells, data not shown) percentages were also reduced in Plvap2/2 mice compared with control mice, indicating Results are expressed as mean 6 SEM. A two-tailed Student t test with that Plvap is an important molecule in migration to or retention unequal variance was used to evaluate the statistical significance of the data. into the peritoneum. Downloaded from Plvap deletion within the hematopoietic compartment does not Results impact on the frequency of B cells in the spleen and peritoneum Deletion of Plvap in mice results in reduced numbers of splenic To understand the underlying mechanisms responsible for altered IgM+ B cells B cell frequencies in the spleen and peritoneum, we sought to Plvap is an endothelial protein that is involved in the diapedesis of determine whether these effects are B cell intrinsic or extrinsic. leukocytes at sites of inflammatory challenge and PLN sinuses. First, we inquired whether Plvap was expressed on hematopoietic http://www.jimmunol.org/ Diapedesis is a process that is central to the development and ho- cells, and second, we established the impact on B cell frequencies meostasis of hematopoietic lineages. To understand the role of Plvap of genetic B cell–specific and pan-hematopoietic deletion of in these processes, we used recently generated Plvap2/2 mice (26) to Plvap. characterize the function of this molecule on the numbers and subset LPS-activated or resting B cells or dendritic cells did not express 2/2 composition of leukocytes in the blood, spleen, PLNs, and Peyer’s Plvap, as detected with Ab staining and using Plvap splenocytes + + patches. Plvap2/2,Plvap+/2, and WT littermate control mice were as controls. Furthermore, CD4 and CD8 T cells were also profiled by flow cytometry to determine whether there are modifi- negative for Plvap expression (Fig. 3A–C). Examination of Plvap cations in terms of leukocyte subset numbers, frequency, or function. expression by confocal microscopy revealed that expression was No differences were found in cellular composition of peripheral limited to the splenic blood vessels in the MZ area (determined by by guest on September 26, 2021 + blood, LN, and Peyer’s patches with respect to percentages, number MOMA-1 macrophage localization) (Fig. 3D). Taken together, and viability of granulocytes, T and B lymphocytes, NK cells, and these data suggest that Plvap is not expressed within the hema- monocytes (Fig. 1A, Supplemental Fig. 1, and data not shown). In topoietic subsets studied, in agreement with recent data published the spleen, there was a drastic reduction in the percentage and ab- by the Immunological Genetic Consortium (http://immgen.org) solute number of IgM+ IgD2 B cells in Plvap2/2 mice (Fig. 1B, 1C), (49). whereas IgD+ B cells were not affected (Fig. 1B). Even though expression analysis conclusively established the In the spleen, several prominent B cell subsets are represented (47). lack of hematopoietic expression of Plvap, genetic deletion of To evaluate whether the reduction of this population is due to a Plvap in hematopoietic cells and subsets was used to confirm these L/L reduction of a specific subpopulation of splenic B cells, we stained findings. Plvap mice were interbred with CD19-Cre (45) or with a panel of Abs that identify those subpopulations (47). Neither Vav1-Cre mice (46) to obtain compound mice lacking Plvap in the the percentage of follicular (CD21/35int IgMint) or MZ B cells B cells (CD19Cre 3 PlvapL/L) or all hematopoietic cells (VavCre 3 (CD21/35+ IgM+) is affected in Plvap2/2 mice (Fig. 1D). Interest- PlvapL/L) (26), respectively. VavCre 3 PlvapL/L mice have .97% ingly, CD21/35lo IgM+ B cells are reduced in the Plvap2/2 mice Plvap deletion in the hematopoietic compartment (26). B cells compared with the WT or Plvap+/2 mice (Fig. 1D, 1E). Lastly, isolated from CD19Cre 3 PlvapL/L mice using magnetic separa- CD21/35lo IgM+ B cells can be subdivided in transitional 1 (T1) or tion showed .95% deletion of Plvap (data not shown). Immune transitional 2 (T1) B lymphocytes using the expression of CD23 and profiling of the peritoneal lavage showed that there was no effect HSA (Fig. 1F). Our results show that the proportion of T1 or T2 on B frequencies or phenotype in the spleen and peritoneum in B cells is not affected in the Plvap2/2 mice (Fig. 1F, 1G), indicating either CD19Cre 3 PlvapL/L (Fig. 4A–C) or Vav1Cre 3 PlvapL/L that both T1 and T2 B cells are reduced. Taken together, these results (Fig. 4D–F) mice. The combination of microscopy, flow cytom- show that among the splenic B cell populations, in Plvap-deficient etry, and genetic data clearly demonstrate that Plvap is not mice there is a selective reduction in transitional IgM+ B cells. expressed in the hematopoietic compartment and that the main- Similarly, no difference in the proportion of transitional B cells tenance of IgM+ B cells in both spleen and peritoneal cavity is due in Plvap2/2 mice (data not shown) was found when expression of to Plvap expression outside the hematopoietic compartment. CD93 was used for the analysis of transitional B cells (48). Thus, Plvap deletion on endothelial cells reduces IgM+ B cells in irrespective of the markers used to analyze transitional B cells, we spleen and peritoneum obtain the same results. + 2/2 Plvap is a molecule specifically expressed in endothelial cells of IgM B cells are reduced in the peritoneum of Plvap mice blood vessels (26, 29) and PLN sinus (27), suggesting that en- Based on the earlier observations showing a decrease of IgM+ dothelial cells might impact on the presence of IgM+ B cells in B cells in the spleen of Plvap2/2 mice, we hypothesized that the spleen and peritoneum. For this purpose, we made use of Tie2Cre The Journal of Immunology 3973 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. IgM+IgDlo B cells are reduced in the Plvap-deficient mice. Splenocytes from WT, heterozygous, or Plvap2/2 mice were analyzed for the proportion of different B cells subpopulation. (A) Representative histogram of B220 expression on splenocytes. The number in the corner represents the percentage of B220+ cells. (B) Representative contour plot of IgD and IgM expression in B220+ B cells. The number in the upper right corner represents the percentage of IgD+ B220+ cells, whereas the number in the bottom right corner shows the percentage of IgM+IgD2 B220+ B cells. (C) Quantification of percentage (top graph) and absolute number (bottom graph) of IgM+IgD2 B220+ B cells in spleen from WT, heterozygous, or Plvap2/2 mice. (D) Representative contour plot of CD21/35 and IgM expression on B220+ B cells. The number in each corner represents the percentage of different gates on which B cells were divided. (E) Quantification of CD21/35loIgM+ B cells in spleen from WT, heterozygous, or Plvap2/2 mice. (F) Contour plots of CD23 and HSA expression on CD21/35loIgM+ B cells. (G) Quantification of the percentage of T2 and T1 CD21/35loIgM+ B cells. n = 4 independent experiments with at least three Plvap2/2 mice per experiment. *p , 0.05. 3974 ROLE OF Plvap IN IMMATURE B CELLS

FIGURE 2. B cells are absent in the peritoneum of Plvap2/2 mice. (A) Quantiﬁcation of total number of cells in the peritoneum from WT or Plvap2/2 mice is shown in the graph. (B) A representative contour plot of

IgM and IgD expression on peritoneal B cells Downloaded from from WT, heterozygous, or Plvap2/2 mice is shown. The top and bottom numbers represent the percentage of IgD+/IgMlo and IgD2/IgM+ B cells, respectively. (C) Percentage and (D) absolute number of IgD+/IgMlo (left graphs) and IgM+/IgD2 (right graphs) B cells in peritoneum lavage from WT, heterozygous, or http://www.jimmunol.org/ Plvap2/2 mice. n = 4 independent experiments with at least three Plvap2/2 mice per experiment. *p , 0.05, **p , 0.01. by guest on September 26, 2021

3 PlvapL/L mice (26) where Plvap is efficiently (.95%) deleted Plvap under the control of the VE Cadherin promoter and 59 in the endothelial cells and the hematopoietic compartment at the intronic enhancer (50). We used VEC-Plvap-HA+/tg and Plvap+/2 embryonic stage. The results obtained in Tie2Cre 3 PlvapL/L mice to generate compound Plvap2/2; VEC-Plvap-HA+/tg (Plva- mice phenocopied those obtained in Plvap2/2 mice. There was a pECRC) mice, which display between 30 and 50% reconstitution of reduction in IgM+ IgD2 B cell percentage in the spleen from native endothelial Plvap levels (26). Tie2Cre 3 PlvapL/L compared with control littermates (Fig. 5A– As shown in Fig. 6, IgM+ IgDlow B cells are recovered to normal C). In addition, there also was a striking reduction in the per- levels in both spleen (Fig. 6A–C) and peritoneum (Fig. 6D–F) of centage of IgD+ and IgMpos IgD2 B cells in the peritoneal cavity PlvapECRC mice compared with Plvap2/2 mice. Moreover, IgD+ of Tie2Cre 3 PlvapL/L compared with control mice (Fig. 5D–F). B cells were also recovered in the peritoneum of PlvapECRC mice These data suggest that the expression of Plvap on endothelial (Fig. 6D–F). Together, all these results suggest that the expression cells regulates the numbers of IgM+ B cells in the spleen and of Plvap in endothelial cells is necessary to maintain normal levels peritoneal cavity. of B cells in the spleen and peritoneum compartment. Endothelial reconstitution of Plvap rescues the Plvap2/2 Plvap deletion in blood vessel endothelial cells does not affect phenotype and restores B cell frequencies in the spleen and the development of B cells in the bone marrow peritoneal cavity To determine whether Plvap deletion leads to a defect in B cell To demonstrate that Plvap2/2 phenotype is not due to a distortion development in bone marrow, we profiled B cells progenitors in of other genetic loci close to the Plvap locus, we transgenically the marrow of PlvapL/L; end-SCL-CreERTtg/+compound mice complemented the Plvap deficiency by the expression of Plvap- (labeled PlvapiECKO) and mice with endothelial reconstitution of HA in endothelial cells of Plvap2/2 mice (26). Previously, we Plvap in the context of the PlvapiECKO (labeled PlvapiECRC mice) have generated mouse lines (VEC-Plvap-HA+/tg) (26) expressing (see Materials and Methods). The promoter driving the expression The Journal of Immunology 3975 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Plvap expression on T cells, dendritic cells, and B cells. (A) Gate on CD4 and CD8 T cells (left panel), and Plvap expression on CD8 (middle panel) and CD4 T cells (right panel). (B) Plvap expression on dendritic cells (left panel, gated in CD11c) from mice immunized with (black line) or without (gray line) 50 mg of LPS 12 h before. As a control of activation of dendritic cells, CD80 expression is shown in the right panel (black line: immunized with LPS; gray line: nontreated). (C) Plvap expression on B cells from mice immunized with (right panel) or without (left panel) 50 mg of LPS 12 h before. B cells were gated on B220 and CD38 double positive. (D) Confocal micrographs of spleen frozen sections labeled with anti-Plvap (red), anti-B220 (blue), and CD169 (green). Original magnification 320. of CreERT fusion protein consists of SV40 virus minimal pro- marrow of PlvapiECKO mice as compared with controls or Plva- moter and the endothelial enhancer of the stem cell leukemia gene piECRC mice (Fig. 7E–G). These results together suggest that the (SCL) (51). This compound promoter confers activity in endo- defect observed in the splenic immature B cells and peritoneum thelial cells from select vascular beds, bone marrow included, as B cells are not due to a defect in the B cell development in the previously shown (51, 52). To minimize the effects on the peri- bone marrow. toneal cavity, we administered tamoxifen by gavage. As shown in Fig. 7A and 7B, IgM+ IgDlow B cells are reduced in Discussion both spleen (Fig. 7A, 7B) and peritoneum (Fig. 7C, 7D) of The development of effective immune responses is dependent on PlvapiECKO mice compared with control (tamoxifen-treated endothelial cell–mediated leukocyte migration into sites of in- PlvapL/L) mice, whereas endothelial reconstitution in PlvapiECRC flammation. To our knowledge, the results presented in this article mice reverses the effect. In addition, IgD+ B cells were also re- are the first to determine the critical role of Plvap in the mainte- duced in the peritoneum of PlvapiECKO mice (Fig. 7C, 7D), indi- nance of IgM+ B cells in spleen and peritoneum. We show that the cating that endothelial deletion in adult mice has a similar lack of Plvap results in a reduction of transitional splenic IgM+ phenotype as that obtained in Plvap2/2 mice with respect to B-1 B cells as well as B-1 B cells in the peritoneum. The tissue- and MZ B cells (Fig. 1). specific deletion of Plvap clearly demonstrates that the role of Using these models, we analyzed the proportion of immature Plvap is extrinsic to the B cell compartment, because B cell and B cells (B220+IgMhiIgD+), mature B cells (B220+IgMintIgD+), and pan-hematopoietic Plvap deletion has no effect on the IgM+ pre-B cells (B220+IgM+IgD+) from bone marrow (53, 54). How- B cells frequencies in either the spleen or the peritoneal cavity. In ever, no changes were detected in B cell subsets in the bone contrast, endothelial-specific deletion of Plvap recapitulates full 3976 ROLE OF Plvap IN IMMATURE B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Plvap expression on the hematopoietic compartment does not affect the localization of B cells in the spleen and peritoneum compartment. (A and D) A representative contour plot of IgM and IgD expression on spleen (top panel) and peritoneal (bottom panel) B cells from CD19Cre 3 PlvapL/L (A) or VavCre 3 PlvapL/L (D) with corresponding littermate controls is shown. The numbers at the top and bottom in each plot represent the percentage of IgD+/IgMlo and IgM+/IgD2 B cells, respectively. (B, C, E, and F) Quantiﬁcation of IgD+/IgMlo (right graph) and IgM+/IgD2 (left graph) B cells from spleen (B and E) and peritoneum lavage (C and F) from CD19Cre 3 PlvapL/L (B and C)orVavCre 3 PlvapL/L (E and F) mice. n = 3 independent experiments with at least three mice per experiment.

Plvap knockout phenotype, and endothelial-specific reconstitution marginal sinus of the spleen (55). Our observations confirm these of Plvap in the context of germline Plvap knockout rescues the findings by showing Plvap expression on vessels in the MZ of IgM+ B cell phenotype. Taken together, these results conclusively spleen (identified by colocalization of MOMA-1+ macrophages) demonstrate that Plvap expression in endothelial cells is key in as well as capillaries in the red pulp. Thus, Plvap is expressed at maintenance of IgM+ B cells in spleen and peritoneal cavity. The the site where it could regulate IgM+ B cell trafficking or retention reduced number of immature B cells in spleen and B-1 cells in the in this histological site. Although human Plvap expression has peritoneum of Plvap-deficient mice could also explain the previ- been reported in circulating human lymphocytes and monocytes ously reported marked reduction of IgM and IgA titers in Plvap- (i.e., PBMCs) by intracellular staining (31), we could not find deficient mice (26), further suggesting that Plvap may regulate the Plvap expression by Ab staining in a variety of circulating or abundance of B cells that produce natural Abs. parenchymal murine immune subsets. Our data are in accordance Previous reports have shown Plvap expression in spleen (19, with RNA sequencing data on Plvap expression, published by 55) (http://immgen.org), especially on the endothelial cells of the http://immgen.org (49, 56). In addition, we found that neither The Journal of Immunology 3977 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Localization of B-1 B cells in spleen and peritoneum is reduced in the lack of Plvap expression on endothelial cells. (A) A representative contour plot of IgM and IgD expression on spleen B cells from Tie2Cre 3 PlvapL/L (right panel) and littermate control mice (PlvapL/L, left panel) is shown. The number at the top and bottom in each plot represents the percentage of IgD+/IgMlo and IgM+/IgD2 B cells, respectively. (B) Percentage of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells in spleen from Tie2Cre 3 PlvapL/L and littermate control mice. (C) Absolute number of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells in spleen from Tie2Cre 3 PlvapL/L and littermate control mice. (D) A representative contour plot of IgM and IgD expression on peritoneal B cells from Tie2Cre 3 PlvapL/L (right panel) and littermate control mice (left panel) is shown. The number at the top and bottom in each plot represents the percentage of IgD+/IgMlo and IgM+/IgD2 B cells, respectively. (E) Percentage of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells from peritoneum lavage of Tie2Cre 3 PlvapL/L and littermate control mice. (F) Absolute number of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells from peritoneum lavage of Tie2Cre 3 PlvapL/L and littermate control mice. n = 3 independent experiments with at least three Plvap2/2 mice per experiment. *p , 0.05, **p , 0.01.

B cells nor dendritic cells express Plvap in basal or under in- MZ B cells. First, Plvap might hamper the generation of the B-1 flammatory (LPS stimulation) conditions. Furthermore, when we B cell hematopoietic progenitors in the vascular plexus of the abrogate Plvap expression on B cells or in the hematopoietic amniotic sac and hemogenic endothelium (16, 57, 58) where compartment, there is no effect on the abundance of IgM+ B cells Plvap is expressed very early (18, 59). Preliminary data show in the spleen or peritoneum. These findings suggest that if Plvap that the number of B-1 B cell progenitors in the amniotic sac has a B cell trafficking or retention function in the spleen or (M. Yoshimoto, M.C. Yoder, and R.V. Stan, unpublished obser- peritoneum, this role is not B cell or hematopoietic cell intrinsic. vations) at embryonic day 9 in the WT, PlvapL/L, and Plvap2/2 In contrast, when we used a mouse model where the lack of Plvap mice is similar. In addition, B cell precursors in the bone marrow was specific to endothelial cells, the defect in B cell migration or are also similar in the Plvap-deficient mice (Fig. 7), which makes retention was indistinguishable from that observed in the global Plvap involvement in B cell development unlikely. Plvap-deficient mice. Furthermore, the transgenic overexpression Second, Plvap controls B-1 and MZ B cell recruitment and/or of Plvap expression on endothelial cells in Plvap-deficient mice retention in the peritoneum and spleen, respectively. Integrins, rescued the phenotype. Together, these results demonstrate that chemokines, and other adhesion molecules have been shown to be Plvap expression in endothelial cells is key in the B cell trafficking involved in B cell migration (12, 60–62). Different types of ad- or retention in the spleen and peritoneum. hesion molecules regulate distinct events in lymphocyte extrava- Considering the data in the literature, there are several sation. We know from these studies that integrins play a role in mechanisms by which Plvap may reduce the number of B-1 and leukocyte tethering, whereas chemokines play a role in the rolling 3978 ROLE OF Plvap IN IMMATURE B CELLS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Localization of B-1 B cells in spleen and peritoneum is recovered with Plvap expression on endothelial cells. (A and D) A representative contour plot of IgM and IgD expression on spleen (A) and peritoneal (D) B cells from PlvapECRC (right panel), Plvap2/2 (middle panel), and WT (left panel) mice. The numbers at the top and bottom in each plot represent the percentage of IgD+/IgMlo and IgM+/IgD2 B cells, respectively. (B and E) Percentage of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells from spleen (B) and peritoneum lavage (E) of PlvapECRC (gray bar), Plvap2/2 (white bar), and WT (black bar) mice. (C and F) Absolute numbers of IgD+/IgMlo (left graph) and IgM+/IgD2 (right graph) B cells from spleen (C) and peritoneum lavage (F) of PlvapECRC (gray bar), Plvap2/2 (white bar), and WT (black bar) mice. n = 3 independent experiments with at least three mice per group. **p , 0.01. process (63). Immature B cells express aLb2 and a4b1, which addition, when we restore the expression of Plvap exclusively in retain B cells in the splenic marginal sinus, where they interact endothelial cells, we observed the rescue of B cells number with their ligand, ICAM1 and VCAM1 expressed in endothelial in steady-state, suggesting that Plvap expression in endothe- cells (12). In addition, an increased gradient of CXCL13 in the lial cells is the master regulator of immune cell migration to periarterial lymphocyte sheath induces the migration of B cells peritoneum. 2/2 from the MZ to the follicles (64). In contrast, S1P1 expressed on Third, Plvap vessels leak plasma components leading to B cells is able to induce the migration of B cells to the sinus of the hypoproteinemia and formation of ascites, a condition known to MZ and peritoneum (10, 11, 65). Conceivably, S1P1 expressed on reduce viability of resident peritoneal macrophages and other B cells could interact with Plvap, through vim, to permit the ex- immune cell subsets. Deletion of Plvap may induce conditions that travasation of B cell progenitors to the peritoneum and the splenic deplete the peritoneal progenitors of B-1 and MZ B cells. Ex- marginal sinus. However, additional studies are necessary to test periments involving Plvap deletion at adult stages should shed this hypothesis. light on which mechanism(s) are involved. Plvap may also be an integral mediator of inflammation-induced In conclusion, the expression of the glycoprotein Plvap on en- migration. It has been previously observed that the blocking of dothelial cells is a key regulator of B cells in the peritoneum, as well Plvap in vivo reduced the number of monocytes found in the as immature IgM+ B cells in the spleen. It also has a physiological peritoneum after the induction of peritonitis (31). Our results role in facilitating the production of natural IgM and IgA Abs by show that Plvap has a novel role in the progression of leukocyte peritoneal and splenic IgM+ B cells. Collectively, our findings migration to peritoneum, not just in inflammation but also in the point to a novel immunological significance for Plvap in innate steady-state. The global deletion or specific obliteration of humoral immunity. Future work will determine the precise mecha- Plvap in endothelial cells reduced the migration not just of nism by which Plvap regulates B cell subsets in the peritoneum and B cells but also others leukocytes by ∼80% in steady-state. In spleen. The Journal of Immunology 3979 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. Plvap does not affect the development of the B cells. Defect in the number of B cells is not due to a development issue. (A) Percentage of IgM+/IgD2 (left graph) and IgD+/IgMlo (right graph) B cells in spleen from PlvapL/L, PlvapiECKO, and PlvapiECRC mice. (B) Absolute number of IgM+/IgD2 (left graph) and IgD+/IgMlo (right graph) B cells in spleen from PlvapL/L,ERTCre 3 PlvapL/L, and ERTCre 3 PlvapECRC mice. (C) Percentage of IgM+/IgD2 (left graph) and IgD+/IgMlo (right graph) B cells from peritoneum lavage of PlvapL/L,PlvapiECKO,andPlvapiECRC mice. (D) Absolute number of IgM+/IgD2 (left graph) and IgD+/IgMlo (right graph) B cells from peritoneum lavage of PlvapL/L,PlvapiECKO,andPlvapiECRC mice. (E) A representative contour plot of immature (B220+IgMhiIgD+), mature B cells (B220+IgMintIgD+), and pre-B cell (B220+IgM+IgD+) from bone marrow of PlvapL/L,PlvapiECKO,andPlvapiECRC mice. The number in each plot represents the percentage of each subset. (F) Percentage and (G) absolute number of immature, mature, and pre-B cell from bone marrow of PlvapL/L,PlvapiECKO,andPlvapiECRC mice. n = 2 independent experiments with at least six mice per group per experiment. *p , 0.05, **p , 0.01. 3980 ROLE OF Plvap IN IMMATURE B CELLS

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