Somatically Diversified and Proliferating Transitional B Cells: Implications for Peripheral B Cell Homeostasis This information is current as Venkata A. Yeramilli and Katherine L. Knight of September 29, 2021. J Immunol published online 27 April 2011 http://www.jimmunol.org/content/early/2011/04/27/jimmun ol.1003897 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/04/27/jimmunol.100389 Material 7.DC1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 27, 2011, doi:10.4049/jimmunol.1003897 The Journal of Immunology Somatically Diversified and Proliferating Transitional B Cells: Implications for Peripheral B Cell Homeostasis Venkata A. Yeramilli and Katherine L. Knight The peripheral B cell compartment in mice and humans is maintained by continuous production of transitional B cells in the bone marrow. In other species, however, including rabbits, B lymphopoiesis in the bone marrow abates early in life, and it is unclear how the peripheral B cell compartment is maintained. We identified transitional B cells in rabbits and classified them into T1 (CD24high CD21low) and T2 (CD24highCD21+) B cell subsets. By neutralizing B cell-activating factor in vivo, we found an arrest in peripheral B cell development at the T1 B cell stage. Surprisingly, T1 B cells were present in GALT, blood, and spleen of adult rabbits, long after B lymphopoiesis was arrested. T1 B cells were distinct from their counterparts in other species because they are proliferating and the Ig genes are somatically diversified. We designate these newly described cells as T1d B cells and propose a model in which they develop in GALT, self renew, continuously differentiate into mature B cells, and thereby maintain peripheral B cell homeo- Downloaded from stasis in adults in the absence of B lymphopoiesis. The Journal of Immunology, 2011, 186: 000–000. uring B cell development, immature B cells in the bone cells as T1-like and T2-like based on the differential expression marrow (BM), designated transitional B cells, exit the of CD24 and CD38 (9) or IgD and CD38 (10). Recently, Suryani BM and migrate to the spleen, where they develop into et al. (11), using CD21 as a marker, identified two transitional D low high mature B cells (1, 2). Transitional B cells are identified by several B cell subsets (CD21 and CD21 ) in peripheral blood and http://www.jimmunol.org/ cell surface markers expressed on newly formed B cells in the demonstrated that the CD21low subset is the precursor to the BM. One such marker, CD24, is expressed at high levels on both CD21high B cells. human and murine transitional B cells and is downregulated on Transitional B cells mark a crucial link between immature BM mature B cells (3, 4). Using CD24 and AA4 (C1qR), several B cells and mature peripheral B cells. Whereas many studies of subsets of transitional B cells have been identified in mice. Loder peripheral B cell development have been performed in mice and et al. (2) classified the CD24high transitional B cells into two humans, essentially no such studies are available in rabbits or other stages, as follows: transitional type 1 (T1) and type 2 (T2) based mammals that use GALT for B cell expansion and somatic di- on the differential expression of CD21, CD23, and IgD. Using versification of Ig genes (12–17). The mechanism by which B cells AA4, a type I transmembrane protein, Allman et al. (5) identified undergo proliferative expansion in GALT is not known. Further- by guest on September 29, 2021 three populations of transitional B cells, as follows: AA4+CD232 more, in rabbits, and most likely in other species, B lymphopoiesis IgMhigh (T1), AA4+CD23+IgMhigh (T2), and AA4+CD232IgMlow in primary lymphoid organs abates early in life (18, 19), and it is (transitional type 3). Adoptive transfer experiments revealed that unclear how the peripheral B cell compartment is maintained in T1 cells give rise to T2 and mature B cells (2), and Schiemann the absence of ongoing B lymphopoiesis. Weill and Reynaud (14) et al. (6) demonstrated that this maturation is dependent on B cell- proposed that the GALT-derived B cells in these species might activating factor (BAFF). T1 B cells are found in the BM, blood, serve as transitional-like B cells. and spleen, whereas T2 B cells are restricted to spleen (2). Tran- In this study, we used several cross-reactive Abs to identify sitional B cell subsets exhibit distinct functional characteristics. transitional B cells (T1 and T2) in rabbit. Using anti-CD24 and For example, T2 B cells proliferate upon BCR cross-linking, anti-CD21 mAb, we identified transitional B cell subsets in blood, whereas T1 B cells die (7). Transitional B cells in humans are spleen, and GALT of adult rabbits, long after the arrest of B largely described as a single subset that is CD10+ (8) or CD24++ lymphopoiesis in the BM. Using soluble decoy receptors that CD38++ (4), although some investigators have classified these inhibited cell–cell and cell–cytokine interactions, we identified several signals required for the proliferative expansion of B cells in GALT. We describe a model for peripheral B cell development Department of Microbiology and Immunology, Loyola University Chicago, May- and maintenance in which proliferating and somatically diversi- wood, IL 60153 fied transitional B cells in adults develop in GALT, continuously Received for publication November 30, 2010. Accepted for publication April 5, 2011. differentiate into mature B cells, and thereby maintain peripheral This work was supported by National Institutes of Health Grants AI050260 and B cell homeostasis. AI068390 (to K.L.K.). Address correspondence and reprint requests to Dr. Katherine L. Knight, Department of Microbiology and Immunology, Loyola University Chicago, 2160 South First Materials and Methods Avenue, Maywood, IL 60153. E-mail address: [email protected] Rabbits and reagents The online version of this article contains supplemental material. Rabbits were from the colony maintained by K. L. Knight at Loyola Abbreviations used in this article: Ad, adenoviral/adenovirus; Apx, appendix; BAFF, University Chicago. Adult rabbits used in this study ranged from 4 mo to 2 y B cell-activating factor; BGG, bovine g globulin; BM, bone marrow; CR2, comple- of age. All studies were reviewed and approved by the Institutional Animal ment receptor 2; CVF, cobra venom factor; GC, germinal center; MLN, mesenteric lymph node; PB, peripheral blood; PP, Peyer’s patch; rBAFF, recombinant BAFF; SR, Care and Use Committee of Loyola University Chicago (Maywood, IL). sacculus rotundus; T1, transitional type 1; T2, transitional type 2; TACI, transmem- We tested commercially available Abs for cross-reactivity to rabbit B brane activator calcium modulator and cyclophilin ligand interactor; Tg, transgenic. lineage cells. A list of Abs that cross-reacted and were used in this study is shown in Table I. Rabbit-specific Abs and secondary reagents were as Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 follows: anti-IgM (clone 367; BD Biosciences, San Jose, CA), anti-IgA www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003897 2 DIVERSIFIED AND PROLIFERATING TRANSITIONAL B CELLS Table I. Cross-reactive Abs used for flow cytometry and immunohistology Ab Specificity Clone Vendor CD10 Human CB-CALLA eBiosciences, San Diego, CA CD20 Human B9E9 Santa Cruz Biotechnology, Santa Cruz, CA; Immunotech, Marseille Cedex, France CD21 Human BL13 Immunotech CD23 Human 9P25 Immunotech CD24 Mouse M1/169 eBiosciences; BD Biosciences, San Jose, CA CD38 Human IB6 Miltenyi Biotec, Auburn, CA; also provided by Dr. Malavasi, University of Turin, Turin, Italy CD62L Human LAM-1 Provided by Dr. Tedder, Duke University, Durham, NC CD90 Human 5E10 BD Biosciences Ki-67 Human B56 BD Biosciences (clone 102; BD Biosciences), anti-rabbit L chain (KLK stock), anti-MHC one a3/a3 homozygous rabbit (1.8 y of age) were amplified by nested PCR. class II (clone 2C4; BD Biosciences), FITC anti-rabbit C3 (Southern First-round PCR was performed using the following pan VH primers: Biotechnology Associates, Birmingham, AL), Dylight 649 goat Fab anti- forward, 59-CTCTGGCACAGGAGCTC-39 and reverse, 59-AGTTGAG- mouse IgG, and streptavidin PE/allophycocyanin (Jackson ImmunoResearch TAGGAAGAGAGA-39. Aliquots (2 ml) of first-round PCR products were Laboratories, West Grove, PA). Rabbit recombinant BAFF (rBAFF) (20) used as template for the second-round PCR using primers, as follows: was biotinylated using NHS-LC biotin (Pierce Biochemicals, Rockford, IL). forward, 59-CACTCACCATGGAGACT-39 and reverse, 59-GAGTTGG- Downloaded from Cobra venom factor (CVF) was obtained from Calbiochem (San Diego, CAAGGACTCAC-39. Products from the second-round PCR were directly CA). sequenced, and the frequency of VH mutations was determined by com- paring sequences to germline V genes. Recombinant adenovirus H Immunization and ELISA Adenoviral (Ad) constructs expressing transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI)-Ig (extracellular portion To determine the efficacy of CTLA4-Ig in vivo, rabbits neonatally injected of human TACI fused to human Fcg) and mouse Fcg (as control) were with Ad-CTLA4-Ig were reinjected with Ad-CTLA4-Ig at 2 and 5 wk of http://www.jimmunol.org/ provided by T.