The Tetraspanin CD53 Regulates Early B Cell Development by Promoting IL-7R Signaling This information is current as Zev J. Greenberg, Darlene A. Monlish, Rachel L. Bartnett, of September 23, 2021. Yihu Yang, Guomin Shen, Weikai Li, Jeffrey J. Bednarski and Laura G. Schuettpelz J Immunol published online 20 November 2019 http://www.jimmunol.org/content/early/2019/11/19/jimmun ol.1900539 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/11/20/jimmunol.190053 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published November 20, 2019, doi:10.4049/jimmunol.1900539 The Journal of Immunology The Tetraspanin CD53 Regulates Early B Cell Development by Promoting IL-7R Signaling Zev J. Greenberg,* Darlene A. Monlish,* Rachel L. Bartnett,* Yihu Yang,† Guomin Shen,‡ Weikai Li,† Jeffrey J. Bednarski,* and Laura G. Schuettpelz* The tetraspanin CD53 has been implicated in B cell development and function. CD53 is a transcriptional target of EBF1, a critical transcription factor for early B cell development. Further, human deficiency of CD53 results in recurrent infections and reduced serum Igs. Although prior studies have indicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. In this study, we show that CD53 expression, which is minimal on hematopoietic stem and progenitor cells, increases throughout bone marrow B cell maturation, and mice lacking CD53 have significantly decreased bone marrow, splenic, lymphatic, and peripheral B cells. Mixed bone marrow chimeras show that CD53 functions cell autonomously to promote 2 2 B lymphopoiesis. Cd53 / mice have reduced surface expression of IL-7Ra and diminished phosphatidylinositol 3 kinase and Downloaded from JAK/STAT signaling in prepro- and pro-B cells. Signaling through these pathways via IL-7R is essential for early B cell survival and transition from the pro-B to pre-B cell developmental stage. Indeed, we find increased apoptosis in developing B cells and an associated reduction in pre-B and immature B cell populations in the absence of CD53. Coimmunoprecipitation and proximity ligation studies demonstrate physical interaction between CD53 and IL-7R. Together, these data, to our knowledge, suggest a novel role for CD53 during IL-7 signaling to promote early B cell differentiation. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/ lymphopoiesis follows a series of well-defined, highly formation of a functional pre-BCR through the pro–B and pre–B regulated processes to confer broad immunity to foreign cell stages (8). Signaling through the pre-BCR on pre–B cells B pathogens and simultaneously prevent self-recognition (1, 2). initiates formation of the mature BCR, which drives the tran- Developing B cells depend on extracellular cues to facilitate mat- sition to IgM-expressing immature B cells. Immature B cells uration from the common lymphoid progenitor (CLP) to a mature then egress from the bone marrow, enter circulation, and migrate plasma cell (3, 4). B cell development begins in the bone marrow, to the spleen, where they form transitional B cells. Transitional but emigration from the marrow to the spleen is required for B cells continue to mature and differentiate into follicular (FO) complete differentiation. Commitment to B cell lineage during or marginal zone (MZ) B cells, which are both capable of forming transition from CLP to prepro–B cell requires IL-7 to induce immunocompetent B cells (9). by guest on September 23, 2021 expression of early B cell factor 1 (EBF1) (5). EBF1, along with In mice, IL-7 signaling is necessary for B cell development, E2A and PU.1, directs the expression of necessary B cell tran- specifically to promote progression from a pro–B cell to the pre–B cell scription factors, including Pax5, to specify and commit prepro–B developmental stage. IL-72/2 and IL-7R2/2 mice have a signifi- cells to the B lineage (6, 7). The next developmental hurdle is the cant impairment of early lymphocyte expansion and progression past the pro–B cell stage (10, 11). Ligation of IL-7 to its receptor activates phosphatidylinositol 3 kinase (PI3K) and Janus-associated *Department of Pediatrics, Washington University School of Medicine, St. Louis, kinase (JAK)–STAT signaling (12). Homeostatic IL-7 signaling MO 63110; †Department of Biochemistry and Molecular Biophysics, Washington promotes cell survival, proliferation, and differentiation through ‡ University School of Medicine, St. Louis, MO 63110; and College of Medicine, transcription of Ccnd1/3, Rag1/2, Pax5,andIl-7r, while also Henan University of Science and Technology, Luoyang, Henan 471003, People’s Republic of China repressing the expression of proapoptotic genes, Bax, Bad,and ORCIDs: 0000-0002-6234-7005 (Z.J.G.); 0000-0003-3374-9117 (D.A.M.); 0000- Bcl2l11 (BIM) (13–15). Lymphocytes are primed for IL-7 sig- 0003-3671-5641 (R.L.B.); 0000-0002-8711-1904 (W.L.); 0000-0001-6047- naling, as p-STAT5 translocation to the nucleus after IL-7R li- 3641 (L.G.S.). gation occurs within minutes. This primed state is achieved by the Received for publication May 9, 2019. Accepted for publication October 25, 2019. formation of microdomains in the plasma membrane surrounding This work was supported by funding from the Children’s Discovery Institute of the IL-7R, which organizes interacting proteins for near immediate Washington University and St. Louis Children’s Hospital (to L.G.S.), and the Training Program in Cellular and Molecular Biology (T32 GM007067-44) (to Z.J.G.). signal transduction (16). Tetraspanins are a family of transmembrane proteins important Address correspondence and reprint requests to Dr. Laura G. Schuettpelz, Depart- ment of Pediatrics, Washington University School of Medicine, 660 South Euclid for organization of the plasma membrane and regulation of cellular Avenue, Box 8116, St. Louis, MO 63110. E-mail address: [email protected] migration, adhesion, and activation (17, 18). These small hydro- The online version of this article contains supplemental material. phobic proteins, bearing four transmembrane domains, two short Abbreviations used in this article: BIM, Bcl2l11; CLP, common lymphoid progenitor; cytoplasmic tails, and two extracellular domains, are known to CyTOF, mass cytometry; EBF1, early B cell factor 1; Flt3-L, FMS-like tyrosine associate with other proteins in the membrane and cytosol, as well kinase three ligand; FO, follicular; HSC, hematopoietic stem cell; JAK, Janus-associated kinase; Lin2, lineage2; MPP, multipotent progenitor; MZ, marginal zone; PI3K, as other tetraspanins, to form specialized tetraspanin-enriched phosphatidylinositol 3 kinase; PLA, proximity ligation assay; qRT-PCR, quantitative microdomains (TEMs). Of the 33 identified tetraspanins, CD53 RT-PCR; TEM, tetraspanin-enriched microdomain; WT, wild-type. is one of four to be exclusively expressed on hematopoietic cells This article is distributed under The American Association of Immunologists, Inc., and is highly expressed on mature B cells (19, 20). A case of familial Reuse Terms and Conditions for Author Choice articles. CD53 deficiency was reported, with patients suffering recurrent Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 bacterial, viral, and fungal infections, as well as reduced serum www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900539 2 CD53 REGULATES B CELL DEVELOPMENT Ig levels, suggesting a role for CD53 in immune system function Cell sorting (21). CD53 is capable of organizing MHC class II on B cells Bone marrow cells were obtained by crushing long bones (femurs, tibiae, through TEMs into functional clusters on the cell surface, and humerii), pelvic bones, and vertebrae with a mortar and pestle in FACS suggesting that CD53 may interact with other surface proteins buffer supplemented with 2% BSA. Single-cell suspensions were filtered to modulate B cell activity (22). Stimulation of CD53 influ- using a 40-mm filter prior to staining with primary conjugated Abs + ences calcium influx, apoptosis, and proliferation in various (Supplemental Table I) on ice for 40 min. B220 cells were enriched prior to sorting by selection with B220-conjugated paramagnetic beads using the lymphocytes. A recent study showed that CD53 recruits pro- autoMACS Pro Separator (Miltenyi Biotec, Auburn, CA). Nonviable cells tein kinase C (PKC) to the plasma membrane to facilitate were excluded from analyses by DAPI staining. BCR-dependent PKC signaling (19, 23–25). Thus, multiple prior studies have suggested a role for CD53 in regulating B cells. IgM and IgG serum measurements However, the natural ligands for CD53 in B cells and the mech- Blood obtained by cardiac puncture was allowed to clot at room temper- anisms by which it influences B cell development and function are ature for 1 h, then centrifuged for 10 min at 6000 rpm. Collected serum was largely unknown. assessed for IgG and IgM levels using the Mouse Total IgG Uncoated ELISA Kit and the Mouse IgM Uncoated ELISA Kit (Invitrogen, Thermo In this study, we present the requirement of CD53 for normal Fisher Scientific, Santa Clara, CA) following the manufacturer’s bone marrow B cell development.