Ligation of Surface Ig by Gut-Derived Antigen Positively Selects Chicken Bursal and Peripheral B Cells This information is current as Dariush Davani, Zeev Pancer and Michael J. H. Ratcliffe of September 25, 2021. J Immunol 2014; 192:3218-3227; Prepublished online 24 February 2014; doi: 10.4049/jimmunol.1302395 http://www.jimmunol.org/content/192/7/3218 Downloaded from References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/192/7/3218.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 25, 2021 *average 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Ligation of Surface Ig by Gut-Derived Antigen Positively Selects Chicken Bursal and Peripheral B Cells Dariush Davani,* Zeev Pancer,† and Michael J. H. Ratcliffe* In many mammals and birds, B cell lymphopoiesis takes place in GALT, such as the avian bursa of Fabricius. Although BCR expression is sufficient for bursal colonization, the role of BCR ligation in the later stages of bursal B cell lymphopoiesis remains elusive. To address this directly, we introduced a surface Ig–related construct with defined Ag specificity containing the Ag-binding portion of a lamprey variable lymphocyte receptor specific for PE fused to a truncated chicken m-chain (VLRPETm) into developing chick embryos. VLRPETm expression supports bursal follicle colonization, clonal expansion, and Ig V gene diversifi- cation. VLRPETm-expressing B cells migrate to the periphery in the absence of the Ag starting from day 18 of embryogenesis. VLRPETm-expressing B cells declined rapidly in the bursa and periphery in the absence of Ag after hatch; however, intrabursal PE m+ injection of PE prolonged survival of VLR T bursal and peripheral B cells. Intrabursal introduction of Ag increased emi- Downloaded from gration of short-lived LT2+ B cells. Peripheral VLRPETm+ B cells were maintained following intrabursal PE application and contained both short-lived LT2+ and long-lived LT22 B cells. In the chicken bursa, the later stages of B cell development occur in the presence of gut-derived Ag; therefore, we conclude that Ag-mediated ligation of BCR in bursal B cells acts to positively select bursal B cells into both short-lived and long-lived peripheral B cell populations. The Journal of Immunology, 2014, 192: 3218–3227. lymphopoiesis in humans and rodents takes place in the It is becoming apparent that avian B cell development can be http://www.jimmunol.org/ fetal liver and, subsequently, in the bone marrow after divided into discrete stages. The early stages of chicken B cell de- B birth. However, in many mammals and birds B cell de- velopment include colonization of bursal mesenchyme by B cell velopment occurs in GALT, such as the appendix, Peyer’s patches, precursors, expression of a functional surface Ig (sIg) mole- and the bursa of Fabricius (1–7). Although GALT B cell lym- cule, and colonization of bursal follicles, with subsequent cell phopoiesis has been thought to be the dominant pathway in many proliferation and Ig diversification. Colonization of the bursal nonrodent and primate mammalian species (3–5, 7), this has be- mesenchyme with a single wave of B cell precursors takes place come less clear with a lack of demonstrable B cell development in during embryonic life in a germ-free environment and is inde- porcine ileal Peyer’s patches (8, 9). Moreover, the demonstration pendent of sIg expression (11). B cell precursors that have un- of B cell development in the gut of postweaning mice suggests dergone successful Ig gene rearrangement, leading to functional by guest on September 25, 2021 that bone marrow and GALT pathways of B cell development may BCR expression, migrate across the basement membrane and coexist (10). B cell development in both bone marrow and GALT expand to form the bursal buds from which follicles develop (12, generates a diverse pool of B cells; however, there are molecular 13). Strong evidence supports a model in which BCR expression and anatomical differences between these models of B cell lym- in the absence of ligation is necessary and sufficient to support phopoiesis. Foreign Ag is excluded from the bone marrow envi- transition through this developmental checkpoint (14–16). ronment, whereas B cell lymphopoiesis in GALT occurs in an The later stages of B cell development include redistribution to environment that is exposed to foreign Ag after birth. This begs form cortical and medullary B cell compartments within the bursal the question as to whether exogenous Ags play a role in the se- follicle, the establishment of bursal subpopulations distinguished lection of B cells during B lymphopoiesis. by differences in functional life span, and B cell migration to the periphery. These later stages coincide with the transportation of Ag through the bursal duct and into bursal follicles mediated by M cell–like follicle-associated epithelium (FAE) (17). Ectopic bursal *Sunnybrook Research Institute, Department of Immunology, University of Toronto, † grafts and bursae in which the bursal duct is ligated are colonized Toronto, Ontario M5S 1A8, Canada; and Department of Biochemistry and Molec- ular Biology, Institute of Marine and Environmental Technology, University of Mary- with B cell precursors during embryonic life but fail to continue land School of Medicine, Baltimore, MD 21202 development or diversification posthatch (18, 19). Although these Received for publication September 6, 2013. Accepted for publication January 28, results suggest the possibility that the presence of Ag in the me- 2014. dulla of bursal follicles can influence bursal B cell lymphopoiesis, This work was supported by the Canadian Institutes for Health Research (to M.J.H.R.). there is no direct evidence supporting such a model. D.D. was supported by a Canadian Institutes for Health Research strategic training grant in regenerative medicine. We demonstrated previously that B cell precursors expressing m Address correspondence and reprint requests to Dr. Michael J.H. Ratcliffe, Depart- a retrovirally transduced truncated sIgM (T ) that lacks Ag-binding ment of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON capacity supports the early stages of B cell development. Similarly, M5S 1A8, Canada. E-mail address: [email protected] a retrovirally transduced receptor in which the cytoplasmic domain Abbreviations used in this article: AID, activation-induced cytidine deaminase; CEF, of the chicken Iga is fused to the extracellular and transmembrane chicken embryo fibroblast; FAE, follicular-associated epithelium; FDC, follicular a dendritic cell; RCAS, replication competent avian leukosis virus with splice acceptor; domains of mouse CD8 also provides equivalent support for early RCAS(BP)B, RCAS (Bryan polymerase) subgroup B; sIg, surface Ig; Tm, truncated B cell development (20, 21). However, neither the Tm nor CD8a: PE sIgM; VLR, variable lymphocyte receptor; VLR , variable lymphocyte receptor Iga construct supports the later stages of B cell development, with with specificity for PE. cells expressing either construct being rapidly eliminated within the Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 first 10 d after hatch. Thus, BCR ligation by exposure to foreign www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302395 The Journal of Immunology 3219 ligands, as distinct from BCR expression, might be required for Calcium mobilization the later stages of B cell development (22). Changes in cytosolic calcium concentrations following receptor ligation To address this question, we generated chimeric receptors in were detected in INDO-1 (Molecular Probes, Eugene, OR)-loaded cells, as which the sequence of the diversity region of a lamprey variable described (26). Briefly, cells were washed with IMDM growth media and lymphocyte receptor (VLR) is fused to the chicken Tm.ThisVLR, loaded with 10 mM final concentration of INDO-1 at 37˚C for 45 min in 3 6 with specificity for PE (VLRPE),isexpressedonthecellsur- the dark, and 1 10 loaded cells were transferred to tubes. Samples were assayed on a BD LSR II and analyzed by BD FACSDiva software (BD face, and ligation of this receptor, when expressed in the chicken Biosciences). Calcium mobilization was assessed following exposure of B cell lymphoma DT40, induces calcium mobilization. Retroviral cells to anti-chicken H chain (HY18) or PE (26). transduction of this construct into developing B cell precursors Ag application supports the early stages of B cell development as effectively as does Tm, and, in the absence of cognate Ag, VLRPETm-expressing Ag, typically 100 ml at 1 mg/ml in PBS, was applied to the anal lips of B cells disappear rapidly after hatch.