Ligation of Surface Ig by Gut-Derived 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

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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 © 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, 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 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 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 (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. However, following intra- neonatal chicks and repeated the following day. Uptake of Ag was visu- alized by inclusion of 10 ml india ink into 1 ml Ag solution, and dissected bursal application of Ag, Ag-specific B cells are maintained in the bursae were photographed after 10 min or 1 h. bursa, their emigration to the periphery is enhanced, and Ag-specific B cells are maintained for extended periods of time in the periphery. PCR amplification, gene-conversion analysis, and cloning of To our knowledge, this provides the first direct demonstration of V gene sequences Ag-mediated positive selection of specific B cells during their Bursal and splenic cells were stained, and aliquots of 1500 cells of defined development in a gut-associated lymphoid . phenotype were sorted directly into PCR tubes. Cells were incubated in m m

15 l10 g/ml proteinase K for 1 h at 50˚C, followed by heat inactivation Downloaded from at 85˚C for 20 min. VJL rearrangements were amplified using the primer Materials and Methods combinations described previously (14, 15). All amplification reactions Generation of replication competent avian leukosis virus with also contained the RAG59 and RAG39 combination of primers to amplify splice acceptor (Bryan polymerase) subgroup B–VLRPETm the single copy of chicken RAG2 sequence from genomic DNA as an internal standard. PCR products were electrophoresed, and bands were chimeric BCR quantitated by scanning densitometry (Bio-Rad). To generate a BCR with defined specificity, the diversity sequence of Gene-conversion events were assessed by restriction site analysis, as VLRPE (23) was fused to the chicken Tm sequence. The VLR diversity described, using the KpnI restriction enzyme (New England BioLabs) (27). http://www.jimmunol.org/ sequence was PCR amplified using Platinum Pfx polymerase (Invitrogen, VJL sequences were cloned into PCR2.1 (Invitrogen) and sequenced (TCAG, San Diego, CA) with the primer combination VLR59:59-ATTATTTGG- Toronto, ON, Canada). CCACGGCATGTCCCTCGCAGT-39 and VLR39:59-ATTATTCGGCCG- GGCATTTCGAGGGGCTAGTG-39 from the VLR-containing vector and Results cloned into Zero Blunt (Invitrogen). The cloned VLR was digested from Generation of RCAS(BP)B-VLRPETm–transduced chicken this shuttle vector using EaeI and EagI restriction sites embedded in the primers, and the VLR sequence ∼600 bp was gel purified and cloned into B cells the EagI site of replication competent avian leukosis virus with splice To generate sIg-related BCRs of defined specificity, we made use of m acceptor (Bryan polymerase) subgroup B [RCAS(BP)B]–T , between Ag-specific lamprey VLRPE isolated from a yeast surface display the chicken H chain leader sequence and Tm (24). by guest on September 25, 2021 library (23). The diversity region of the lamprey VLR was fused to PE m RCAS(BP)B-VLR T virus production and gene transfer the chicken Tm gene, using the chicken VH leader sequence. The in vivo and in vitro resulting constructs were cloned into the RCAS(BP)B productive The RCAS retroviral gene-transfer system was used to introduce VLRPETm retroviral vector (Fig. 1A) and transfected into CEFs. into chicken embryo fibroblasts and chicken embryos, as described As we observed in other studies, surface expression of chicken (15). Line 0 chicken embryo fibroblasts (CEFs) (Regional Poultry Research Tm does not require coexpression of Iga and Igb on CEFs and, Laboratories, East Lansing, MI), cultured in IMDM (Life Technologies), under these circumstances, does not signal when cross-linked (25); were transfected with RCAS(BP)B-VLRPETm plasmid by calcium chloride RCAS(BP)B-Tm–transfected CEFs expressed high levels of sur- precipitation, as described (15). Within7doftransfection,essentiallyall PE CEFs expressed the retrovirally containing chimeric receptors. face Tm. CEFs transfected with RCAS(BP)B-VLR Tm also ex- DT40 infection was performed as described previously (25). L chain– pressed this construct at similar levels on the CEF surface (Fig. 1B). deficient DT40 chicken bursal B lymphoma cells were infected with RCAS Staining with PE demonstrated that the RCAS(BP)B-VLRPETm PE m (BP)B-VLR T virus by coculturing serial dilutions of DT40 (starting at construct maintained the specificity of the original selected VLR 5 3 104 cells) with semiconfluent transfected CEFs in 6 wells of a 12-well plate for 48 h in IMDM with 2% chicken serum. Nonadherent DT40 cells (Fig. 1B). PE were harvested, expanded, and FACS sorted on a FACSAria (BD Bio- VLR Tm was expressed in the chicken B cell lymphoma DT40 sciences, Mississauga, ON, Canada). by coculture of transfected CEFs with DT40 cells (Fig. 1B). PE To generate chickens transduced with RCAS(BP)B-VLR Tm, day-3 VLRPETm-expressing DT40 cells were purified to homogeneity by incubated B line chick embryos (ISA North America A Hendrix Genetics, 3 6 . flow cytometric cell sorting and assessed for their ability to mo- Cambridge, ON, Canada) were inoculated with 1 10 CEFs ( 98% PE VLRPETm expressing) in 100 ml IMDM with 1% normal chicken serum. bilize calcium in response to VLR Tm ligation with soluble PE. As shown in Fig. 1C, cross-linking VLRPETm on VLRPETm-expressing Abs and flow cytometry DT40 cells resulted in levels of calcium mobilization equivalent to Surface expression of VLRPETm was detected using R-PE (Columbia those seen following receptor cross-linking with anti-IgM Abs (Fig. Biosciences, Columbia, MD) and/or anti-chicken m (HY18) Ab. 1C). Thus, when expressed on the B cell surface, VLR-Tm cross- Ex vivo B cells were stained with the anti-chicken pan B cell marker anti- a b m linking results in signaling consistent with association with Ig /Ig ChB6, anti-chicken (HY18), biotinylated or unlabeled anti-chicken Ig m m L chain, and R-PE (Columbia Biosciences), followed by FITC-conjugated (15). Consequently, although T and VLR-T can be expressed on anti-mouse Ig isotype Ab (Southern Biotechnology Associates, Birming- the cell surface in the absence of Iga/Igb,Tm and VLR-Tm will ham, AL) or Streptavidin-PerCP (BD Biosciences), as described (14, 15). associate with the Iga/Igb complex if available. Samples were analyzed on a FACSCalibur or sorted on a FACSAria (BD PE Biosciences). Expression of VLR Tm in the absence of ligation is sufficient For analysis of cellular DNA content, stained viable cells were sorted to support the early stages of B cell development using a FACSAria, pelleted, and resuspended in Vindelov’s solution, as PE described (15). DNA content of the resulting nuclei was analyzed on a Day-3 chicken embryos were inoculated with RCAS(BP)B-VLR FACSCalibur. Tm–transduced CEFs and analyzed neonatally for the presence of 3220 POSITIVE SELECTION IN BURSAL B LYMPHOPOIESIS

FIGURE 1. Functional expression of VLRPETm.(A) The diversity region of a VLRB specific for PE was fused to the chicken chm224 fragment (Tm) and cloned into the productive retroviral vector RCAS(BP)B. (B) CEFs were transduced with RCAS(BP)B-Tm (left panel) or RCAS(BP)B-VLRPETm (middle panel). Five days after transfection, CEFs were stained for the pres- ence of m and the capacity to bind PE. sIg2 DT40 were cocultured with RCAS(BP)B-VLRPETm–transduced CEFs and subsequently sorted for m expression and Downloaded from stained for the presence of m and the capacity to bind PE (right panel). Dot plots of 10,000 events gated on forward scatter and side scatter are shown. (C) sIg2 DT40 (upper left panel), sIg+ DT40 (lower left panel), and VLRPETm-expressing sIg2 DT40 were loaded with INDO-1 and stimulated with ligand, as indicated. http://www.jimmunol.org/ by guest on September 25, 2021

peripheral and bursal B cells expressing VLRPETm. VLRPETm- (Fig 2C). To ensure that digests were performed to completion, expressing B cells lacking endogenous IgM were detected in the the pUC18 plasmid containing a KpnI site was included in all PE bursa, as well as peripherally in the spleen and PBLs, of .30 reactions, as indicated. VJL sequences from bursal VLR Tm chicks analyzed in three independent experiments (Fig. 2A). Cell B cells were largely resistant to digestion with KpnI as were VJL cycle analysis showed that both sorted ex vivo bursal VLRPETm- sequences from bursal cells expressing endogenous sIg. Therefore, expressing B cells, as well as endogenous sIg-expressing (IgL+/ expression of VLRPETm supports the induction of gene conversion VLRPETm2) B cells, were proliferating at a high rate, which is as efficiently as does Tm or CD8a:Iga (14, 15). At days 18–19, characteristic of neonatal bursal B cells (Fig. 2B). Thus, surface most splenic B cells are prebursal, and the bursal microenviron- expression of a BCR, either sIg or VLRPETm, is sufficient and ment is required for the onset of gene conversion. Consistent with PE required for the early stages of bursal cell development. this, VJL sequences derived from splenic VLR Tm-expressing In addition, VLRPETm-expressing B cells showed a reduction in B cells were heavily digested with KpnI. This suggests that splenic endogenous L chain V to J rearrangement compared with B cells VLRPETm-expressing B cells have undergone less gene conversion expressing endogenous sIgM (data not shown). Semiquantitative than age-matched bursal B cells. This observation was confirmed by PCR of genomic DNA demonstrated that, in the absence of re- sequencing cloned VJL genes from the relevant B cell populations ceptor ligation, expression of the VLRPETm is sufficient to partially (data not shown). inhibit rearrangement of endogenous L chain genes in developing B cells. The levels of inhibition observed were similar to those seen Intrabursal exposure to Ag increases B cell migration to the in developing B cells expressing either the Tm receptor or the periphery CD8a:Iga receptor (14, 15). B cell migration from bursa to the periphery starts at about day 18 To assess gene conversion at the Ig L chain locus, we performed of embryonic life in chickens and continues posthatch until sexual restriction digests and sequence analysis of VJL sequences from maturity. Within the bursa after hatch, B cells redistribute and sorted VLRPETm-expressing B cells from the bursa and spleen of generate cortical and medullary populations separated by a cortico- PE RCAS-VLR Tm–transduced chicks. The germline VL1 sequence medullary junction that develops from the basement membrane. contains a KpnI restriction site that is partially lost upon gene This redistribution coincides with transportation of the contents of conversion during the diversification of the rearranged L chain the gut to the bursal medulla by FAE. Whether transport of Ag has allele (15, 27). Therefore, KpnI digests were performed on VJL any direct impact on the redistribution and migration of B cells to sequences amplified from sorted VLRPETm-expressing B cells the periphery remains unclear. The Journal of Immunology 3221 Downloaded from http://www.jimmunol.org/

FIGURE 2. VLRPETm expression supports the early stages of chicken B cell development. Day-3 chicken embryos were inoculated with RCAS(BP)B- VLRPETm–transfected CEFs. (A) Bursa, spleen, and PBLs of neonatal VLRPETm-transduced chicks were stained for the expression of ChB6, m H chain, Ig L chain, and PE binding. ChB6 flow cytometry graphs are gated on forward scatter, side scatter, and live cells and represent 50,000 events. The H chain/L by guest on September 25, 2021 chain dot plot is gated on ChB6+ cells. (B) B cells expressing VLRPETm and B cells expressing endogenous sIg were isolated by cell sorting and analyzed for their relative nuclear DNA content. Identification of cell cycle stages was assessed by FlowJo software. Graphs are representative of 10,000 cells. (C) PE + + PE pUC18- or PCR-amplified VJL of sorted VLR Tm or sIg B cells from neonatal chicks infected with VLR Tm-transduced CEFs were digested with KpnI and electrophoresed. The gene converted (GC) VJL is resistant to KpnI digestion. PCR was performed on pooled sorted bursal or splenic cells of three animals.

The contents of the cloaca are transported to the bursal duct by 1–2 d, which can be identified by the expression of a marker reverse peristalsis. Therefore, we applied Ag into the cloaca to mimic defined by the LT2 Ab (28). Subsequent to hatch, LT2 expression the physiological route of gut exposure to Ag. To visualize and is largely maintained among bursal B cells, although a clear confirm the effective transport of the injected material to the bursa population of LT2lo/2 cells emerges. Among peripheral B cells, lumen, the PE Ag was mixed with india ink. After 10 min, carbon the frequency of LT2+ emigrants increases with time, such that by particles were not detected in the cells of the FAE. However, 1 h after 1 mo of age there are clearly defined populations of peripheral application, carbon particles were detected in the follicular-associated LT2+ and LT22 cells. At this stage, although the LT2+ peripheral epithelial cells that map to the organization of B cell follicles (Fig. 3). cells have been shown to be short-lived bursal emigrants, the great To assess the consequences of BCR ligation on the fate of majority of LT22 peripheral cells are long-lived bursal emigrants. chicken bursal B cells, we introduced PE intrabursally into chicks Although intrabursal application of PE Ag did not substantially that had been transduced with RCAS(BP)B-VLRPETm. The pen- affect the overall frequency of peripheral VLRPETm–expressing etrance of the transduced VLRPETm was assessed on PBLs from PBL B cells, we used the LT2 Ag to specifically assess whether day-1 neonates. Chicks with similar VLRPETm-expressing B cell the intrabursal application of Ag affected the rate of emigration of populations were divided into two groups, and 100 ml of PE/india short-lived B cells from the bursa (Fig. 4A). In chicks exposed to ink solution was applied daily onto the anal lips of VLRPETm- intrabursal PE, we observed a higher frequency of LT2+ VLRPE transduced neonates for 2 d. Two days after PE application, the Tm-expressing B cells in the PBLs compared with control chicks. frequency of peripheral VLRPETm-expressing B cells was In contrast, in the same chicks, there was no difference in the assessed. Although there was a slight trend toward increased levels frequency of LT2+ B cells expressing endogenous sIg in the PBLs of VLRPETm-expressing B cells following PE exposure, the in- of the Ag-treated versus control chicks (Fig. 4A, 4B). Thus, in- crease was not significant (Fig. 3B). At a minimum, this result creased levels of VLRPETm+ LT2+ peripheral B cells were specific clearly indicates that intrabursal application of Ag does not me- for the Ag treatment. This is suggestive of Ag-driven emigration diate negative selection among peripheral B cells. of B cells from the bursa. It was shown that .90% of bursal emigrants to the periphery In the presence of intrabursally administered PE, the frequency of a 3-wk-old chickens are short-lived B cells, with a life span of of LT2+ PBL B cells was approximately equivalent when com- 3222 POSITIVE SELECTION IN BURSAL B LYMPHOPOIESIS

FIGURE 3. Intrabursal Ag treatment does not induce negative selection of peripheral B cells. (A) PE/india ink mixture was in- troduced into the bursal lumen. Uptake of the colloidal india ink by FAE was visual- ized as black staining on the follicular epi- thelium 10 min (Aiv)or1h(Ai, Aii, and Aiii) after application. Original magnifica- tions are: 33(Ai), 39(Aii), 330 (Aiii), 325 (Aiv). (B) Chick embryos were inoculated with VLRPETm-orTm-transduced CEFs, and PE/india ink was introduced into the bursal lumen of day-1–2 VLRPETm-transduced Downloaded from chicks. PBLs of day-3–5 chicks were as- sessed by flow cytometry for the frequency of VLRPETm2 or Tm-expressing ChB6+ B cells. Horizontal bars represent the mean of each group. http://www.jimmunol.org/

paring sIg2/VLRPETm+ B cells and sIg+/VLRPETm2 B cells from mine whether these VLRPETm+ B cells are recent emigrants the individual chicks (Fig. 4C, left panel). In contrast, in the ab- from the bursa or whether they are longer-lived cells whose sence of intrabursal PE, the proportion of LT2+ VLRPETm+ B cells presence is maintained in some way by exposure to Ag in the by guest on September 25, 2021 + + was significantly lower than the proportion of LT2 sIg B cells bursa. PE (Fig. 4C, right panel). This suggests that signals provided to VLR Among the peripheral B cells in the absence of intrabursal Ag, + Tm bursal cells in the presence of PE are functionally equivalent few, if any, VLRPETm-expressing cells were observed at day 21 to those provided within the normal bursal microenvironment to (Fig. 6A). Peripheral B cells at this stage include both LT2+ and B cells expressing endogenous sIg. LT22 populations, with the LT2+ population disappearing fol- Ag-dependent maintenance of B cells lowing bursectomy (Fig. 6A). In contrast, following Ag application PE m In the bursa, cells expressing either Tm or CD8a:Iga were main- in the bursa, VLR T -expressing B cells were maintained in tained for only ∼10dinRCAS-Tm– or RCAS-CD8a:Iga– the PBLs for extended periods of time (Fig. 6B, top row). These PE m + 2 transduced chicks, respectively (20, 21). Similarly, in the absence VLR T -expressing B cells also contained both LT2 and LT2 + PE of Ag, VLRPETm-expressing B cells in the bursa disappeared over cells, with the LT2 VLR Tm-expressing population disap- the first few weeks of life, such that by day 21, ,1% of VLRPETm- pearing rapidly within 7 d postbursectomy (Fig. 6A, middle row). expressing bursal cells remained (Fig. 5A). Thus, in the absence of Therefore, at least some peripheral VLRPETm+ B cells are recent cognate Ag, the VLRPETm construct behaves equivalently to those bursal emigrants whose emigration from the bursa is dependent constructs that lack Ag-binding capacity. However, in those chicks upon the maintenance of VLRPETm+ bursal B cells, which, in PE exposed to intrabursal PE, there was a clear indication that VLR turn, is driven by the presence of intrabursal Ag. Tm-expressing bursal cells were maintained for more extended Not all VLRPETm+ peripheral B cells expressed LT2 (Fig. 6), periods of time (Fig. 5A). and some VLRPETm+ PBL B cells in intrabursal PE-treated In the absence of intrabursal application of PE, the frequency VLRPETm-transduced chicks clearly fall into the long-lived emi- PE m of VLR T -expressing B cells declined in the periphery by ap- grant population, being maintained for $21 d following bursec- proximately one order of magnitude during the first week after tomy (Fig. 6B, bottom row). This was confirmed by assessing the hatch (Fig. 5B, left panel). This is consistent with the decline in frequency of VLRPETm+ splenic B cells 28 d after surgical bur- bursal B cells expressing VLRPETm in the absence of Ag. In sectomy in VLRPETm-transduced chickens that had been treated contrast, intrabursal application of Ag supported the continued maintenance of VLRPETm-expressing peripheral B cells (Fig. 5B, with PE intrabursally (Fig. 7). Despite the complete disappearance + right panel). Thus, the presence of Ag intrabursally clearly main- of splenic LT2 B cells, a significant population of PE-binding PE + tained the presence of VLRPETm-expressing B cells in the periphery B cells was maintained. Thus, some VLR Tm peripheral B cells 2 cells of those chicks sacrificed at day 7 (Fig. 5C). are long-lived LT2 emigrant B cells. Therefore, we conclude that Given that intrabursal application of Ag leads to maintained the presence of specific Ag in the bursa has the capacity to ligate VLRPETm+ B cells in the periphery (Fig. 5), we sought to deter- Ag-specific receptors on VLRPETm-expressing B cells, resulting in The Journal of Immunology 3223

FIGURE 4. BCR ligation increases mi- gration of LT2 Ag-expressing B cells from the bursa. Chick embryos were inoculated with VLRPETm-transduced CEFs, and PE/ india ink was applied to the bursal lumen of day-1–2 chicks. (A) Representative staining of PBLs assessed at days 3–5 for the ex- pression of ChB6, m chain, LT2 Ag, and PE binding. ChB6 graphs are gated on forward scatter, side scatter, and live cells and rep- resent 10,000 events. Contour plots are gated + B + on ChB6 cells. ( ) The frequency of LT2 Downloaded from cells within the VLRPETm+ B cell population (left panel) and sIg+ B cell population (right panel) in Ag-treated and control chicks. Each symbol represents an individual chick. The p values were calculated by Student t test. (C) Correlation of the frequency of + PE + + + LT2 VLR Tm and LT2 sIg PBL B cells http://www.jimmunol.org/ in the presence (left panel) or absence (right panel) of PE/india ink. Each symbol repre- sents an individual chick, and the statistics were performed by linear regression analy- sis. The p value indicates the significance of the slope. by guest on September 25, 2021

maintained viability within the bursa and sustained emigration to bursal follicles, undergo rapid proliferation, and undergo Ig di- the periphery (Fig. 8). versification by gene conversion. However, BCR expression in the absence of ligation is not Discussion sufficient to support the long-term maintenance of bursal B cells (20). In fact, in the absence of PE, VLRPETm+ B cells disappear Although B cell development in some mammalian species, such from the bursa within the first 10 d after hatch (Fig. 5). Disap- as human and mouse, occurs in an environment free of exog- m enous Ag, B cell development in other species occurs in GALT. pearance of B cells expressing the T chimeric receptor was shown This provokes the question as to whether the presence of Ag to be independent of competition with B cells expressing endoge- might impact B cell lymphopoiesis in the “so-called” GALT nous BCR (20). Moreover, in the chicken, all Ig gene rearrangement species (22). occurs during embryonic life, and the entire B cell repertoire is The current study addressed the consequences of gut-derived Ag derived from cells expressing sIg by embryonic day 17 (11, 33). PE m exposure on bursal B cell fate using the RCAS retroviral vector (29) Therefore, the disappearance of cells expressing VLR T cannot to introduce Ag-specific chimeric BCR into developing chicken simply be due to their replacement by newly developing B cells embryos. Because retroviral transduction is not 100%, this system undergoing Ig gene rearrangement. allows for direct comparisons to be made between cells expressing Ab-mediated cross-linking of chimeric CD8a:Iga BCR recep- the transduced BCR and cells expressing endogenous sIg (15, 24). tors in the bursa prolonged the survival of CD8a:Iga-expressing We demonstrated previously that BCR expression in the absence B cells (21), although the physiological relevance of Ab-mediated PE of ligation is sufficient to support B cell transition through the cross-linking of BCR can be questioned. However, the VLR Tm Ig-selection checkpoint, leading to productive bursal colonization construct allowed us to assess the role of Ag and specific BCR (14–16, 24, 30–32). Similarly, we show in this study that the ligation in chicken B cell development. Typically, BCR cross- VLRPETm receptor supported all early stages of B cell develop- linking on immature B cells has been considered to induce ment in the bursa, characteristic of B cells passing the Ig-selection tolerance (34, 35). Indeed, we provided evidence that exposure checkpoint. Thus, in the absence of Ag, VLRPETm+ B cells colonize of developing B cells in the chick embryo to a BCR ligand 3224 POSITIVE SELECTION IN BURSAL B LYMPHOPOIESIS

Developing B cells that encounter Ag presented by FDCs may additionally be exposed to costimulatory receptors expressed by the FDCs themselves. In contrast, developing prebursal B cells encountering i.v.-injected PE may receive signals through BCR in the absence of costimulatory signals provided by appropriate Ag presentation. Thus, gut-derived Ag, such as the PE adminis- tered in the current experiments, could induce positive selection of PE-specific B cells, whereas i.v.-injected Ag results in B cell deletion. It would be intriguing to examine the effect of specific Ag on chicken B cell development in embryos derived from IgG- deficient hens (37), in which intrabursally applied Ag would not be presented in the context of immune complexes. Normal sIg+ bursal B cells are maintained in the bursa until the involution of the organ at ∼6 mo of age. Bursal duct ligation (19, 38) and ectopic grafting of the bursa to sites in which there is no direct contact with the gut (18) result in the failure of continued bursal B cell development after hatch, suggesting that the signals supporting sIg+ bursal cells are gut derived. We show in this study

that maintenance of bursal B cells is a consequence of BCR li- Downloaded from gation, suggesting that such maintenance signals are not simply gut-derived mitogens acting through non-IgRs. The nature of the ligand for the BCR of normal sIg+ bursal cells remains unclear. Gut-derived bacterial superantigen positively selected rabbit B cells of a defined BCR allotype, independent

of receptor specificity (7, 39). Nonetheless, this superantigen http://www.jimmunol.org/ clearly signals through sIg. In our experiments, it is highly un- likely that a gut-derived superantigen would bind the VLR of the VLRPETm construct given that the lamprey VLR is not a member of the Ig supergene family. However, although we showed that Ag-mediated BCR ligation leads to the support of bursal B cells, we cannot distinguish between the presence of a B cell superantigen activating B cells through BCR, independent of their specificity, or the presence of a multiplicity of gut-derived Ags, each acting on Ag-specific B cells; indeed, these models are not mutually ex- by guest on September 25, 2021 clusive. After hatch, some bursal cells migrate back across the basement membrane to form the follicular cortex, whereas Ag taken up FIGURE 5. Intrabursal ligation of BCR by specific Ag maintains B cells through the FAE is retained in the follicular medulla (17, 32). PE in the bursa and periphery. Chick embryos were inoculated with VLR Bursal cells expressing either Tm or the chimeric CD8a:Iga BCR Tm-transduced CEFs, and PE/india ink was applied to the bursal lumen of A PE m + fail to migrate into the cortex (25), although there is some evi- day-1–2 chicks. ( ) The frequency of VLR T -expressing bursal ChB6 a a+ B cells was assessed by flow cytometry at days 7 and 21 following Ag dence that CD8 :Ig bursal cells migrate into the cortex when a application. (B) The frequency of VLRPETm-expressing PBL ChB6+ B exposed to anti-CD8 Abs intrabursally (21). It is tempting to PE + cells in control or intrabursal PE-treated VLRPETm-transduced chicks was suggest that, following intrabursal exposure to PE, VLR Tm assessed by flow cytometry at days 1 and 7. Lines connect the day-1 and -7 bursal cells are maintained and colonize the follicular cortex, al- results from individual chicks. (C) The relative rate of disappearance of though evidence supporting the latter is lacking. Migration of VLRPETm-expressing B cells in the PBLs of Ag-treated or control chicks VLRPETm-expressing B cells to the follicular cortex would place was assessed by dividing the frequency of VLRPETm-expressing PBLs at these cells into a physiologically distinct compartment within the PE day 7 by the frequency of VLR Tm-expressing PBLs at day 1. Each bursa from which gut-derived Ags are excluded (32). Thus, al- symbol represents an individual chick. Statistical analysis was performed though Ag-induced proliferation may be initiated in the follicular using unpaired t test. medulla, the transit of these cells into the Ag-free cortical envi- ronment may limit the proliferative burst. The bursal follicular cortex has been identified as a site of introduced i.v. induced a BCR-signaling–dependent deletion of Ig repertoire diversification by gene conversion, based on the B cells (36). Conversely, we show in this study that intrabursally high levels of activation-induced cytidine deaminase (AID) administered Ag maintains bursal B cells. Thus, we conclude that seen in cortical bursal cells (40). In the case of cells expressing the route of the Ag administration changes the outcome of BCR VLRPETm, diversification of the VLRPETm receptor by gene ligation. This could be due to the context in which the Ag is conversion could not occur because specificity is determined presented to the target B cell. The bursa contains sIgG-expressing by the retrovirally transduced construct. However, among nor- follicular dendritic cell (FDC)-like cells. The sIgG expressed on mal bursal cells, the high levels of cortical AID expression these cells is not autonomously produced but is acquired in the suggest that exposure to Ag in the follicular medulla might in- form of immune complexes (37) as a consequence of FcR ex- duce migration into the cortex, followed by continued prolifer- pression. Ag applied via the bursal lumen is taken up by the FAE ation and repertoire diversification by gene conversion. This and transported into the medulla of bursal follicles where it be- would generate a situation that, at first sight appears paradoxical comes associated with the surface of FDC-like cells (Fig. 8). but would represent an Ag-driven generation of a naive B cell The Journal of Immunology 3225 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Intrabursal Ag maintains peripheral LT2+ short-lived and LT22 long-lived postbursal cells. Chick embryos were inoculated with VLRPETm-transduced CEFs, and PE/india ink was applied to the bursal lumen of day-1–2 chicks, followed by surgical bursectomy on day 21 posthatch. VLRPETm+ PBL B cells in control (A)orPE-treated(B) chicks were assessed before and after bursectomy. VLRPETm+ B cells were assessed for LT2 expression by staining PBLs for m, PE, LT2, and ChB6. Data are representative of 50,000 cells gated on forward scatter and side scatter, and PE-binding graphs are gated on ChB6+ B cells. LT2 plots were gated on ChB6+ PE+ or ChB6+ PE2 cells, representative of three chicks analyzed. repertoire and preclude the possibility of the naive B cell rep- ligation clearly enhances the emigration of bursal cells to the ertoire becoming restricted to those specificities recognizing periphery. gut-derived Ags. This possibility is supported by the observation We showed previously that bursal emigrants fall into two phe- that SRBCs introduced into the normal neonatal chick bursa notypically and functionally distinct populations. Those B cells through the bursal lumen do not result in a detectable increase in expressing the LT2 marker are short-lived bursal emigrants and SRBC-binding bursal B cells (41). Indeed, not only does intra- are distinct from a longer-lived population of LT22 bursal emi- bursal Ag fail to result in increased numbers of Ag-binding bursal grants (28, 43). In the presence of PE, the proportion of LT2+ B cells, it also fails to induce Ag-specific Ab responses (42). VLRPETm+ cells that is found in the periphery is directly pro- Bursal B cells migrate to the periphery where they form the Ag- portional to the frequency of LT2+ sIg+ cells in the periphery responsive mature B cell population. This migration is initiated (Fig. 4C). In contrast, in the absence of PE, the proportion of LT2+ during the later stages of embryonic life, and we show in this study VLRPETm+ cells in the periphery is substantially lower than the that this can occur in the embryo independent of BCR ligation, proportion of LT2+ sIg+ peripheral B cells. Therefore, we can because VLRPETm-expressing B cells migrate to the periphery in conclude that the signals required to support the emigration of the absence of intrabursal PE. Nonetheless, intrabursal BCR short-lived LT2+ sIg+ cells from the bursa are functionally equiva- 3226 POSITIVE SELECTION IN BURSAL B LYMPHOPOIESIS

Ag-specific cells that subsequently would respond to peripheral challenge. Intrabursal exposure to PE generates a population of LT22 VLRPETm+ peripheral B cells that survive for extended periods following surgical bursectomy (Figs. 6, 7), suggesting that BCR ligation in the bursa can impact on the maintenance and/or emi- gration of longer-lived peripheral B cells. Therefore, it is possible that BCR ligation intrabursally induces a transition from short- lived to long-lived B cells. We demonstrated previously that, al- though short-lived LT2+ bursal emigrants are derived predomi- nantly from the follicular cortex, the longer-lived LT22 bursal emigrants are likely derived from the follicular medulla (43). We previously argued that because the follicular medulla is a site of gut-derived Ag, long-lived LT22 bursal emigrants may represent a population of B cells that have experienced Ag exposure. Therefore, the peripheral B cell compartment would contain a diverse population of naive B cells and an additional longer-lived population of LT22 cells enriched for specificities

present in the environment. Under these circumstances, BCR Downloaded from ligation in the medulla of bursal follicles could lead to both the generation of the diversified pool of naive B cells following redistribution to follicular cortex and subsequent emigration, as well as the generation of a longer-lived Ag-enriched peripheral population following emigration directly from the follicular

medulla. http://www.jimmunol.org/ 2 FIGURE 7. Maintenance of LT2 longer-lived splenic B cells in the Intrabursal exposure to PE additionally generates a population presence of specific Ag. Spleens of bursectomized chicks, inoculated of LT22 VLRPETm+ peripheral B cells that survives for extended PE m with VLR T -transduced CEFs, were assessed 28 d postbursectomy periods following surgical bursectomy (Figs. 6, 7). Thus, BCR for frequency of LT22 VLRPETm longer-lived B cells. Graphs are gated on ligation may induce a transition from short-lived to long-lived forward scatter, side scatter, and live cells and represent 50,000 events. PE + + B cell. Although short-lived LT2 bursal emigrants are derived Ag-binding graphs are gated on ChB6 B cells, and LT2 graphs are gated 2 on ChB6+, ChB6+ PE+, or ChB6+ PE2 B cells, as indicated. predominantly from the follicular cortex, longer-lived LT2 bur- sal emigrants are likely derived from the follicular medulla (43). Thus, because the follicular medulla is a site of gut-derived Ag, lent to the signals provided by PE to the LT2+ VLRPETm+ bursal long-lived LT22 bursal emigrants may represent a population of by guest on September 25, 2021 cells. Again, this is fully consistent with a role for BCR ligation in B cells that have experienced Ag exposure. support of the later stages of chicken B cell development, including In summary, the peripheral B cell compartment would contain emigration from the bursa. a diverse population of naive LT2+ B cells that have matured Our finding that intrabursal PE induces higher levels of LT2+ through the follicular cortex and an additional longer-lived pop- VLRPETm+ cells in the PBL within 3 d (Fig. 3) suggests that at ulation of medullary-derived LT22 cells enriched for specificities least one of the consequences of BCR ligation is a direct induc- present in the environment (Fig. 8). Under these circumstances, tion of bursal cells to emigrate to the periphery. This provides a BCR ligation in the medulla of bursal follicles could lead to both mechanistic explanation for experiments in which intrabursal ad- the generation of the diversified pool of naive B cells following ministration of Ag, while failing to induce an Ab response, enhanced their redistribution to the follicular cortex and subsequent emi- subsequent Ab responses to peripherally administered Ag (41). gration and the generation of a longer-lived Ag-enriched periph- Under these circumstances, intrabursal administration of Ag eral population following emigration directly from the follicular would enhance the emigration and peripheral maintenance of medulla.

FIGURE 8. A model of Ag-driven positive selection of B cells in the bursa. After hatch, gut-derived Ag (*) is transported across the FAE into the medulla of bursal follicles. Although some bursal cells (A) can emigrate in the absence of Ag binding, binding of Ag to B cells (B) can lead to their emigration from the bursa as long- lived cells. Alternatively, binding of Ag can lead to migration to the follicular cortex and the induction of proliferation and AID-induced repertoire diversification by gene conversion (C–F). Diversified B cells can emi- grate from the follicular cortex as short-lived cells in the absence of further Ag encounter (C and F). The Journal of Immunology 3227

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