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An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-β and Retinoic Acid

An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-β and... Aims: In the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-b. Methods and Results: To study the influence of RA and TGF-b on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-b and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-b, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-b was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, ‘‘innate’’ B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - a b and 4 7 -/- CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1 recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients. Conclusion: Present study demonstrates the differential and synergistic effect of RA and TGF-b on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity. Citation: Roy B, Brennecke A-M, Agarwal S, Krey M, Du¨ ber S, et al. (2013) An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-b and Retinoic Acid. PLoS ONE 8(12): e82121. doi:10.1371/journal.pone.0082121 Editor: Simon Fillatreau, DRFZ, Germany Received May 31, 2013; Accepted October 21, 2013; Published December 6, 2013 Copyright:  2013 Roy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the German Research Council (DFG), the Ministry for Education and Research (BMBF) and the Helmholtz Association via the HZI Graduate School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Bishnudeo.Roy@helmholtz-hzi.de secreting cells (SCs) and their homing to gut is promoted by Introduction intestinal DCs and appears to be dependent on RA.[13] IgA is the most abundant class of antibodies present in Consistently, mice deficient in RA precursor vitamin A showed mammalian mucosal tissues. It forms a first-line of defense against reduced numbers of IgA producing cells in the small intestine even invasion by inhaled or ingested pathogens and plays an important though the IgA levels in the serum remained unchanged.[13] The role in the maintenance of immune homeostasis. Besides mucosal interplay between TGF-b and RA is still controversially discussed. tissues, IgA is also found at significant concentrations in the serum It has been demonstrated that TGF-b inhibits RA induced IgA of many species, where it mediates the elimination of pathogens CSR.[13] However, another study using splenic cells showed that that have breached the mucosa.[1] a combination of RA and TGF-b with additional factors (LPS, Class switch recombination (CSR) to IgA is orchestrated by APRIL, and IL5) acts synergistically to induce IgA switching in various cytokines and other factors.[2–4] Amongst them, TGF-b vitro.[16] and retinoic acid (RA) are most prominent.[2,5] A special role of In addition to TGF-b and RA, cytokines, like IL2, IL4, IL5, IL6 TGF-b in IgA CSR is most evident from the observation that mice and IL10, are also thought to induce CSR and promote IgA deficient for TGF-b or lacking TGF-b receptor II expression on B production either via TGF-b induction within B cells or by cells exhibit reduced levels of IgA.[6,7] In gut, TGF-b is produced enhancing the post-switch maturation.[4] Furthermore, cytokines by B cells (autocrine factor) [8,9], T cells [10] dendritic cells (DCs) of the tumor necrosis factor (TNF) ligand family called B cell [11], and stromal cells.[12] Some of the T cells that produce TGF- activating factor (BAFF/BLyS) and a proliferation inducing ligand + + b are claimed to be Foxp3 CD4 regulatory T cells.[2] Besides (APRIL) produced by gut DCs are also considered to be involved TGF-b, vitamin A metabolite RA is also a highly potent inducer of in IgA switching.[17,18] IgA CSR.[5] RA is produced by gut associated DCs and In mice, at least three mature B cell subpopulations – follicular macrophages.[13–15] In accordance, the generation of IgA (FO), marginal zone (MZ) and B1 B cells constitute the humoral PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA immune response, where B1 cells can be subdivided further into RT-PCR B1a and B1b cells.[19,20] FO B cells also known as B2 cells are Total RNA from sorted/cultured cells was prepared with TM present in the B cell follicles of secondary lymphoid organs and TriFast FL Reagent (peQLab) following manufacturer’s proto- respond to antigens T cell dependently. On the other hand, MZ B col. DNase (Qiagen) treated RNA was reverse transcribed using TM cells residing in the splenic marginal zone and B1 cells oligo-d(T) (Thermo Scientific) and RevertAid reverse 12–18 predominantly located in the peritoneal cavity (PEC) or pleural transcriptase (Fermentas). PCR amplification of cDNA was TM cavity, are believed to respond to antigens without T cell help and performed using HotstarTaq DNA polymerase (Qiagen). and play an important role in the early stages of pathogenic the following primers: Igm/a heavy chain variable (VH) region, for invasion.[21] Such B cells respond differentially to various stimuli VHcons 59-GAGGTGCAGCTGCAGGAGTCTGG-39 rev Cm2 and need multiple triggers to induce IgA class switching.[16, 59-CATTTGGGAAGGACTGA-39 or Ca259-GAGCTGGTG- 22–24] Thus, under minimal stimulatory conditions GGAGTGTCAGTG-39. (BLys+LPS+TGF-b) they switch to IgA preferentially in compar- ison to B2 cells from PEC or spleen.[24] Sequencing of Ig VH chain and sequence analysis The contribution of B1 cell subsets to IgA production in vivo is RT-PCR products of IgVH chain transcripts from 4 days not known. In this regard, we could recently show that most of the cultured cells were cloned using TOPO TA CloningH kit IgA producing cells in the PEC of unmanipulated mice belonged (Invitrogen) following manufacturer’s protocol. Plasmids were to B1b cell population.[25] However, differential switching of B1a isolated using QIAprep Spin Mini Prep Kit (Qiagen). Sequencing and B1b cells to IgA under stimulatory culture conditions has not was done using M13 Reverse primers by the department of been comparatively studied. In addition, the combination of TGF- Genome Analysis of Helmholtz Centre for Infection Research, b and RA that was supposed to have synergistic effects on naı ¨ve Braunschweig. IgA and IgM VH sequences derived from sorted splenic B cells to switch to IgA [16] was never tested on various PEC and splenic B cells were submitted to GenBank (accession peritoneal and splenic B1 cell subsets. no. KF207924 – KF208284). Thus, in the present work, we stimulated peritoneal B1a, B1b and B2 cells with LPS, BLys and TGF-b in vitro. Switching to IgA was Cell culture and ELISPOT assay severely enhanced in peritoneal B1b cells in comparison to their B1a Purified peritoneal and splenic B cell populations, resus- and B2 counterparts. Switching to IgA was further enhanced when pended in IMDM at a density of approximately 1610 cells/ml TGF-b was combined with RA and IL5. In vitro switched B1 cells were stimulated with, LPS (25 mg/ml; Escherichia coli, Sigma), also showed the presence of frequent nucleotide exchanges in their Recombinant Mouse BLys (100 ng/ml; R&D SYSTEMS), functionally rearranged VH gene segments. This indicates that Recombinant Human TGF-b1 (2 ng/ml; R&D SYSTEMS), besides IgA CSR, somatic hypermutation had also taken place. IL4 (1:400; home made), IL5 (1:400; home made), and all These findings could be extended in part also to splenic B1a and trans-Retinoic Acid (100 nM; Sigma). ELISPOT: single-cell MZ B cells. Altogether, these findings demonstrate differential suspensions at serial dilutions of 1:5 were plated on plates switching of B cell subpopulations to IgA in response to various coated with anti-mouse IgA (Pharmingen) or anti-mouse IgM stimuli in vitro. This suggests alternative mechanisms regulating the (Pharmingen). Cells were incubated overnight at 37uCwith5% IgA CSR of different B cell subsets in vivo. CO and 95% humidity. After washing with PBS+0.01% Tween 20, biotinylated anti-mouse IgA (eBioscience) or anti- Materials and Methods mouse IgM (SeroTec) was added. Bound antibodies were developed with Streptavidin-horseradish peroxidase using AEC Ethic Statement (3-amino-9-ethyl-carbazole; Sigma) in DMF (N,N-Dimethylfor- All experiments were performed in accordance with the mamide; Sigma) diluted in 0.1 M acetate solution added with German Law on Care and Use of Laboratory Animals and were H O as substrate. Spot development was stopped by washing 2 2 approved by the local authority (LAVES) under permission plates with water and spots marking antibody secreting cells number 33.9-42502-04-11/0390. (ASCs) were counted after drying. Mice ELISA Normal BALB/c mice, 7–10 weeks old, used for the experi- -/- Ig concentrations in serum or intestinal lavage were ments were purchased from Janvier. Rag1 mice on BALB/c measured by ELISA. In brief, 96 well plates (MaxiSorb TM background were obtained from our animal facility (Helmholtz Immunoplates, Nunc) were coated over night with anti-mouse Institute for Infection Research; Braunschweig, Germany). IgA (Pharmingen) or anti-mouse IgM (Pharmingen) or goat- anti-mouse IgG (Sigma) antibodies at 4 C and blocked for 1 h Cell preparation, FACS analysis and cell sorting with 3% BSA in 0.05% Tween 20. Appropriately diluted To prepare PEC cells, peritoneum was flushed two times with culture supernatant/sera/intestinal lavage from each mouse was 8 ml of normal cell culture medium (IMDM), collected washout added to the wells and incubated for 2 h at RT. Bound IgA/ was centrifuged and the pellet was resuspended in appropriate IgM/IgG was detected with biotinylated rat anti-mouse IgA amount of cell culture medium. Monoclonal antibodies against (eBioscience) or rat-anti-mouse IgM (SeroTec) or PO conjugat- mouse CD19, CD5, CD43, Mac-1, IgA, IgM, CD23, CD21, ed goat-anti-mouse IgG (Jackson ImmunoResearch) antibodies. CCR9 and a b conjugated with FITC, PE, APC, PE-Cy7, or 4 7 Biotinylated antibodies were revealed by using horseradish Biotin were obtained from Pharmingen or eBioscience or were peroxidase (HRP) conjugated streptavidin (BD). Bound HRP home made. Biotinylated antibodies were revealed by Streptavi- activity was visualized using o-Phenylendiamin (OPD) as din-PerCPCy5.5 (eBioscience). Flow cytometry was done using substrate and the results were read using an ELISA-reader LSR II (BD) and data were analyzed using FACS DIVA software. (BioRad 3550- UV microplate reader). Cell sorting was performed using FACSAriaH (Becton Dickinson). Reanalysis showed that cells were .95% pure PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA In vivo adoptive transfer Sort purified PEC B1a and B1b cells cultured under different stimulatory conditions for 3 days were collected, washed, -/- resuspended in PBS and injected i.p into Rag1 hosts. Statistics Paired two-tailed Student’s t-test was applied to determine the statistical significance (p value). p#0.05 was considered significant. *p,0.05; **p,0.01; ***p,0.001. Results Peritoneal cavity B1b cells switch preferentially to IgA in presence of TGF-b TGF-b has been known to promote switching of B cells to IgA in general.[2,26,27] Under the influence of this cytokine, combined with LPS and Blys, differential propensity of switching to IgA was observed among PEC and splenic B cell subtypes.[24] Interestingly, under such conditions, PEC B1 and splenic MZ B cells switched in vitro to IgA more prominently in comparison to PEC or splenic B2 cells. However, in this study, B1 cells were not differentiated into B1a and B1b cells and thus their differential capability to switch to IgA was not established.[24] Our previous in vivo finding had suggested differences between PEC B1a and B1b cells in that respect.[25] This prompted us to test their preferences to switch to IgA under in vitro stimulatory conditions. For this purpose, PEC B cells were FACS purified into – B1a, B1b and B2 cells (Figure S1). B cells expressing surface IgA were sorted out to exclude expansion of such cells in vitro. After 4 days of stimulation in the presence of different combinations of cytokines, IgA and IgM ELISPOT assays were applied to assess the number of IgA and IgM SCs. Under different stimulatory conditions, the combinations of LPS+Blys+TGF-b and LPS+Blys+TGF- b+IL4+IL5 led to significant switching amongst cultured B cells (Figure 1). In comparison to B2 cells, both B1 cell subpopulations showed higher numbers of IgA SCs (Figure 1). Quite interestingly, B1b cells produced constantly higher number of IgA SCs in Figure 1. TGF-b enhances IgA switching among PEC B1b cells in comparison to B1a cells (Figure 1). Presence of BLys was required vitro. Results of IgA and IgM ELISPOT assays performed in triplicates on for efficient IgA CSR as treatment with LPS+TGF-b alone led to sort purified PEC B cell subpopulations after 4 days of treatment with generation of very few IgA SCs (Figure 1). the indicated combination of stimulatory factors in culture. Surface IgA - hi + + + Parallel, ELISPOTs for IgM showed no apparent trend for the cells were excluded by sorting from B1a (IgA CD19 CD5 CD43 Mac-1 ), - hi - + + - lo - - B1b (IgA CD19 CD5 CD43 Mac-1 ) and B2 (IgA CD19 CD5 CD43 Mac- number of IgM SCs amongst three PEC B cell populations under 1 ) B cell subpopulations. Data show the results of one of three different stimulatory conditions (Figure 1). However, a decrease in independent experiments. Bars represent mean 6 SD. the number of IgM SCs was noted for conditions under which an doi:10.1371/journal.pone.0082121.g001 increase in the numbers of IgA SCs had been observed (Figure 1). observed under the condition (LPS+Blys+TGF-b+IL5+RA) in Pronounced switching of B1 cells to IgA when TGF-b is which, very high number of IgA ASCs was observed (Figure 2A). combined with RA This finding was further confirmed by ELISA that was done to RA has been shown to promote the switching of naı ¨ve splenic B determine the amount of IgA in the culture supernatants of the cells to IgA.[16] The differential effect of this vitamin A metabolite cultured PEC B cells (Figure 2B). Consistently, PEC B1 cells also on various PEC and splenic B cell subpopulations has not been showed the expression of Iga germ line transcripts after combined studied so far. Therefore, to examine the influence of RA on PEC treatment with TGF-b and RA (Figure S2). When tested for the and splenic B cell subpopulations, sorted peritoneal and splenic B reactivity against gut bacteria, secretory IgA present in the culture cells were subjected in vitro to different combinations of stimulatory supernatant of PEC B cells showed their binding towards these factors including RA. After 4 days of activation, the number of IgA commensals (Figure S3). This is consistent with our previous and IgM SCs were measured by ELISPOT assay. Consistent with finding that showed the binding of B1 cell derived Igs to gut earlier findings, considerably high numbers of IgA SCs were bacteria [28]. Furthermore, analysis of secretory IgG in the culture observed in the PEC B cell cultures supplemented with RA plus supernatants showed that the amount of IgG decreased to a TGF-b, although these reagents alone in combination with other minimum under conditions that favored the switching to IgA factors induced comparatively low numbers of IgA SCs (Figure 2B). Interestingly, under other conditions, the amount of (Figure 2A). Interestingly, under these circumstances, again PEC secretory IgG was found to be higher in B1b cell culture B1a and B2 cells had significantly less number of IgA ASCs than B1b cells. No differences in the numbers of IgM ASCs were PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 2. TGF-b in combination with RA synergistically induces IgA switching by PEC B cells. (A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified PEC B cell subpopulations after 4 days of treatment with the indicated combination of stimulatory factors in + - hi + + + - hi - + + culture. Surface IgA cells were excluded by sorting from B1a (IgA CD19 CD5 CD43 Mac-1 ), B1b (IgA CD19 CD5 CD43 Mac-1 ) and B2 - lo - - - (IgA CD19 CD5 CD43 Mac-1 ) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g002 supernatants in comparison to the other two PEC B cell In addition, these B cell types showed an uneven increase in their populations (Figure 2B). number under various culture conditions (Table 1). This might Among splenic B cell subpopulations (FACS sorted according to account for the slight differences between ELISPOT and ELISA the plan showed in Figure S1), mainly B1a and MZ B cells data, later being only qualitative under these circumstances. underwent pronounced switching to IgA compared to splenic B2 cells when treated with TGF-b plus RA (Figure 3A, B). Addition of In vitro differentiated B1 cells also exhibit somatic IL5 to the combination of RA and TGF-b did not increase the hypermutations number of IgA ASCs (Figure 3A). Presence of secretory IgA in the The enzyme AID responsible for CSR also mediates somatic supernatants of cultured B cells as determined by ELISA was hypermutation (SHM). Consistent with that, we had detected the consistent with the ELISPOT results (Figure 3A, B). IgG expression of AID among B cell populations cultured under production seemed to take place without the addition of TGF-b stimulatory conditions (data not shown). Thus, quite expectedly, or retinoic acid. However, TGF-b or RA appeared respectively to analysis of IgA VH sequences from PEC B1 cell and splenic B1a suppress or enhance the production of IgG in case of splenic B1a cell cultures supplemented with TGF-b or a combination of TGF- cells, (Figure 3B). b and RA exhibited the presence of frequent mutations Cell counts of cultured cells indicated strongly reduced (Figure 4A). Mutation frequencies observed under IgA CSR proliferation of all PEC and splenic B cell types in presence of inducing culture conditions were higher compared to unstimulated TGF-b and RA wherever IL5 was not added (Table 1). This is B cells (Figure 4A). Interestingly, frequent nucleotide exchanges consistent with the recent finding showing strong inhibition of B were also detected amongst IgM VH sequences derived from in cell proliferation in the cultures added with RA and TGF- b [29]. vitro stimulated B1 cells, (Figure 4A). Almost all of the VH PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 3. TGF-b in combination with RA synergistically induce IgA switching in splenic B1a and MZ B cells. (A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified splenic B cell subpopulations after 4 days of treatment with the indicated combination of + - + + - lo/- - + - stimulatory factors in culture. Surface IgA cells were excluded by sorting from B1a (IgA CD19 CD5 CD23 CD21 ), MZ (IgA CD19 CD5 C- lo/- hi - + - hi int D23 CD21 ) and B2 (IgA CD19 CD5 CD23 CD21 ) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g003 sequences were found to be functional and devoid of stop codons (Figure 5A, B). Without RA, no influence of TGF-b or IL5 on the or frame shift mutations in the coding VH region (Table 2). expression of such molecules was observed (Figure 5A, B). Analysis of replacement (R) vs. silent (S) mutation revealed a high However, addition of IL5 to RA with or without TGF-b appeared R/S ratio in the VH regions (Figure 4B). to enhance the expression of CCR9 on the treated B cell surface (Figure 5A, B). Notably, considerable percentages of B cells expressed either a4b7 or CCR9 on their surface though double Retinoic acid enhances the expression of gut homing expressers were found to be present as well (Figure 5A, B). Under molecules by B1 cells these conditions, PEC B1a and B1b cells showed higher Besides the induction of IgA switching, retinoic acid is known to percentages of B cells expressing these two molecules in induce the expression of gut homing surface receptors (a4b7 and comparison to PEC B2 cells (Figure 5A). Among splenic B cells, CCR9) on target B and T cells.[5,14] CCR9 is a chemokine B1a and MZ cells were also higher for such markers in comparison receptor for CCL25 which is secreted by gut epithelial cells and to B2 cells (Figure 5B). Thus, expression of these gut homing a4b7 is an integrin that binds to the mucosal adressin molecules seemed to be consistent with the switching tendency of MadCAM.[30,31] Expression of these two molecules was expect- B cell subpopulations to IgA as observed above as well as when ed to be induced after in vitro treatment of peritoneal and splenic B checked for the expression of surface IgA on PEC and splenic B cell subpopulations with TGF-b and RA as IgA producing plasma cell subpopulations (cultured under various stimulatory conditions) blasts are supposed to home to intestine. Consistent with earlier by flow cytometry (Figure 2, Figure 3 and Figure 5C, D). findings, treatment with RA led to high surface expression of a4b7 and/or CCR9 on some of the treated PEC and splenic B cells PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Table 1. Increase in cell numbers after 4 days of culture under various conditions. Fold increase in no. of cells after 4 days of culture PEC Spleen Culture condition B1a B1b B2 B1a MZ B2 LPS+BLys 5.9061.59 7.2060.42 3.1060.65 6.5461.31 6.8863.75 3.1261.70 LPS+Blys+TGF-b 2.9360.39 2.3361.73 1.6060.56 1.8160.71 1.9360.98 0.7960.40 LPS+Blys+RA 6.2361.59 3.5560.62 1.5560.70 2.9462.03 5.0762.56 0.9660.42 LPS+Blys+IL5 12.8465.79 8.3461.54 3.6761.64 4.8862.62 6.9163.25 2.1660.84 LPS+Blys+TGF-b+RA 1.2760.49 0.6360.24 0.3260.27 0.6660.56 0.4860.26 0.2560.15 LPS+Blys+TGF-b+IL5 6.0661.38 5.4961.09 1.8360.38 2.5061.41 3.7261.29 1.0460.34 LPS+Blys+RA+IL5 15.8465.53 16.5465.92 2.4460.18 4.4560.98 4.9461.06 0.9960.55 LPS+Blys+TGF-b+RA+IL5 7.5461.55 3.9261.43 0.4960.16 4.7062.87 1.0560.48 0.4660.32 1 - Sorted IgA PEC and splenic B cells were cultured under different conditions for 4 days and the fold increase in cell number was calculated by dividing the cell count after 4 days by the number of cells at the beginning of the cell culture. The values represent mean 6 SD calculated from three independent culture experiments. doi:10.1371/journal.pone.0082121.t001 The expression of gut homing molecules - a4b7 and CCR9 combination of TGF-b, LPS and BLys.[24] However, in this study after TGF-b and RA treatment should render these cells capable no attempt was made to distinguish between two B1 cell subtypes. of migrating to the gut. Therefore, the migratory capacity of such Our previous finding that among PEC B cells, almost exclusively, B cells was tested by adoptive transfer experiment. PEC B1a and B1b cells had switched to IgA in vivo[25] led us to speculate that B1b cells treated in vitro with RA and TGF-b for three days were B1a and B1b cells might differ in their IgA switching potential -/- adoptively transferred into lymphopenic Rag1 recipients. Day under stimulatory culture conditions. When tested, this speculation was found to be true as TGF-b in combination with LPS and BLys three was chosen because high expression of Iga germ line transcripts could be detected after this period of treatment (Figure led to significantly higher switching of B1b cells to IgA in comparison to the other two PEC B cell subpopulations. Thus, S2). The recipients were analyzed 2 weeks after transfer for the presence of IgA and IgM ASCs in the spleen, and the peritoneal IgA SCs must have resulted from CSR in vitro after treatment with TGF-b as the possibility of expansion of preexisting IgA cells cavity as well as for the secretory IgA and IgM in the serum and among B1b cells had been already excluded by sorting out IgA intestinal lavage. Interestingly, presence of IgA SCs could be cells before culture. detected in the recipients of PEC B1 cells that were triggered with TGF-b or RA (Figure 6A and Figure S4A). However, only the TGF-b together with RA has recently been shown to induce IgA switching synergistically.[16] In the present study, we used this splenocytes of recipients of B1b cells treated with TGF-b+RA+IL5 contained considerably high numbers of IgA SCs (Figure 6A and knowledge to determine the propensity of different peritoneal and splenic B cell subtypes to switch to IgA in response to RA with or Figure S4A). Higher amounts of secretory IgA could be detected in without TGF-b. The combined effect of these two factors together the serum and gut lavage of the recipients of B1a or B1b cells with LPS, IL5 and BLys was much stronger than any other treated with TGF-b+RA+IL5 in comparison to any other recipient combination of stimulants. Again, the frequency of switching to groups (Figure 6B and Figure S4B). Again the recipients of B1b IgA among PEC B cells was significantly higher in B1b cells cells were higher in comparison to B1a cell recipients. Flow compared to other PEC B cell types. Interestingly, among splenic cytometric analysis of PEC cells from transfer recipients showed B cells, B1a and MZ B cells showed significantly higher IgA CSR the presence and survival of transferred cells (Figure S4C). than B2 cells. Low switching frequency observed among PEC and Together, these findings demonstrate a synergistic and differ- splenic B2 cells can be explained by the fact that they require ential effect of RA and TGF-b on PEC and splenic B cell additional stimuli for proliferation and differentiation. For subpopulations for IgA switching. Surprisingly, B1b cells prefer- example, B2 cells expressing higher surface IgD show stronger entially switch to IgA independent of T cell help. proliferative response in presence of anti-IgD-Dex which is further enhanced by addition of IL4.[23] The considerable high Discussion propensity of splenic B1a cell to switch to IgA is consistent with Peritoneal cavity B cell subpopulations differ from each other studies where CD5 expressing splenic B1 cells have been claimed with regard to origin, phenotype and function. PEC B1 cells to contribute to IgA production.[35] differentiate faster into plasma cells after stimulation with mitogen Besides induction of CSR, the vitamin A metabolite RA is also than B2 cells from the same compartment.[25,32] In vivo, the two known to induce or upregulate the expression of gut homing subtypes of B1 cells – B1a and B1b B cells have been shown to molecules - integrin a4b7 and the chemokine receptor CCR9.[5] react differently to pathogenic infections.[20,33] In murine Quite consistent with that, in our in vitro experimental set up, we infection models, B1a cells have been shown to respond abruptly observed an upregulated expression of these two gut homing after infection whereas B1b cells give rise to long lasting memory molecules after treatment with RA. Interestingly, the expression of immunity against certain pathogens.[20,33] Moreover, these two a4b7or CCR9 was higher under conditions that led to enhanced B1 cell subtypes also have been shown to differ in their ability to induction of IgA CSR. PEC and splenic B cell subsets prone to produce IgA.[25,34] Along these lines, recent in vitro studies have enhanced IgA CSR showed higher expression of these homing demonstrated that compared to peritoneal and splenic B2 cells, molecules on their surface. In agreement with the upregulation of PEC B1 cells switch more preferentially to IgA in response to a gut homing molecules, when PEC B1 cells, treated with a PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 4. PEC and splenic B1 cell derived IgA VH sequences display high frequencies of nucleotide exchanges due to somatic hypermutation. Frequency of mutations observed in the IgM or IgA VH region sequences derived from PEC and splenic B1 cells cultured for 4 days with the indicated combinations of IgA CSR inducing factors. (A) Each colored sector represents a particular number of mutations found at a particular frequency. Total number of sequences is shown in the middle. (B) Number of replacement and silent mutations amongst IgM and IgA VH TM sequences derived from indicated B cell populations. Sequences were analyzed using SEQUENCHER Version 4.1 for Macintosh software (Gene Codes Corporation). Assignment of VH chain was done using VBASE2 database (http://www.vbase2.org/). Only functional sequences were included for the mutation analysis. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g004 combination of RA, TGF-b, LPS, BLys and IL5, were transferred sequences derived from the same B cell populations also showed -/- i.p. into Rag1 mice, IgA could be found in serum as well as in higher frequencies of mutations than unstimulated controls. gut lavage. This indicates gut migration of in vitro switched IgA SHM in vivo results in affinity maturation of a particular producing cells. BCR.[37] This is however accompanied by the generation of Isotype switching in B cells is mediated by the enzyme AID.[36] many nonfunctional BCRs due to the introduction of nonsense This enzyme is also responsible for nucleotide exchanges leading nucleotides by SHM. In accordance, one would expect to find to somatic hypermutation in the V regions of B cell receptors many non-functional VH sequences under in vitro conditions. This (BCRs) [36]. Quite expectedly, AID had been found to be was apparently not the case. The simplest explanation would be expressed in cultured B cells under stimulatory condition. This that B cells that had already undergone SHM, and started IgA raised the query whether B cells undergoing IgA CSR also CSR, are selectively expanded under our culture conditions. Such accumulate SHM under our experimental conditions. Interesting- cells might be qualitatively or quantitatively distinct from B cells ly, a considerable fraction of IgA VH sequences derived from that had not yet undergone such processes. Signaling molecules or RA+TGF-b treated PEC and splenic B1 cells showed nucleotide transcription factors might be up- or down-regulated in this state exchanges. Apparently, like CSR, SHM might also have been and the cells might preferentially respond to the stimuli in vitro. induced in vitro. Surprisingly, besides IgA VH sequences, IgM VH Alternatively, IgA CSR and SHM were induced in vitro. The low PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA 1 B1 cells are generally believed to react independent of CD4 T Table 2. Nonfunctional vs functional sequences. cell help.[39,40] Our findings are quite consistent with this notion. The fact that cytokines together with RA efficiently induced CSR and SHM in such cells without interaction with T cells or the Sequence Condition PEC B1a PEC B1b Spleen B1a involvement of CD40/CD40L suggests that B1 cells indeed can IgAVH LPS+Blys+TGF+IL5 1/27 1/22 1/27 undergo differentiation without T cell help. This is in contrast with IgAVH LPS+Blys+TGF+IL5+RA 1/26 2/22 1/27 the earlier report where signaling through BCR, CD40 and CD38 IgMVH LPS+Blys+TGF+IL5 2/26 0/29 3/24 were shown to be necessary for the in vitro induction of mutations presumably in B2 cells.[41] The factors required for such IgMVH LPS+Blys+TGF+IL5+RA 1/25 3/26 0/19 differentiation are probably provided in vivo by stroma or myeloid IgMVH Unstimulated 2/25 0/26 2/9 cells like intestinal DCs or macrophages. B cells themselves could Sequences having frame shift or stop codon in the VH coding region were be the source of some of such factors under in vivo conditions but considered to be nonfunctional. The numbers of nonfunctional vs. functional also T cells interacting with such B1 cells in a non-cognate fashion sequences are displayed below indicated cell population. might be involved. doi:10.1371/journal.pone.0082121.t002 In conclusion, our work shows that under stimulatory conditions in vitro, PEC B1b cells switch to IgA with a higher propensity in frequency of non-functional VH could be explained by an intrinsic comparison to PEC B1a or B2 cells. The differences among selection mechanism to generate functional BCRs. It is known that splenic B cell subpopulations with regard to induced switching to nonfunctional mRNAs are recognized and such cells might be IgA are also quite evident. The results of this study show eliminated.[38] Also B cells producing nonfunctional non- differential stimulatory requirements for different B cell subsets secretable VH or VL chains might be deleted by intrinsic and suggest the presence of distinct mechanisms regulating the mechanisms. This would imply a strong first round of selection induction of IgA CSR among them. for functional BCRs independent of antigen recognition. Figure 5. RA treatment leads to up-regulation of gut homing molecules on the surface of cultured PEC B cells. Expression of surface a4b7 and CCR9 on IgA PEC (A) or splenic (B) B cells sorted in fashion similar to previous experiments was checked by flow cytometry after four days of culture under various combinations of stimulatory conditions. Using the same cultures (as in A and B), expression of surface IgA and IgM on PEC (C) and splenic (D) B cells was checked by flow cytometry. Data show the results of one of two independent experiments. doi:10.1371/journal.pone.0082121.g005 PLOS ONE | www.plosone.org 8 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA cultured in IgA CSR inducing conditions for two or three days and checked for the expression of Iga germline, and RPS9 (house keeping gene) transcripts by RT-PCR. Results of semi-quantitative PCR, using three serial dilutions (UD – undiluted; 1:2 and 1:4) of respective cDNA as template have been displayed. Primers: RPS9, forward (for) 59- TTGACGCTAGACGAGAAGGAT-39 reverse (rev) 59-AATCCAGCTTCATCTTGCCCT -39;Iga germline transcript, for Ia259- CCAGGCATGGTTGAGATAGAGA- TAG -39 rev Ca259-GAGCTGGTGGGAGTGTCAGTG-39. PCR conditions were: 94uC for 20 s, annealing at various temperatures for 40 s, 72uC for 40 s; 33-38 cycles. (TIFF) Figure S3 IgA antibodies derived from TGF-b and RA treated PEC B cells displays reactivity towards gut bacteria. Binding of secretory IgA antibodies present in the culture supernatants of FACS purified IgA PEC B1a, B1b and B2 cells to gut bacteria was tested by flow cytometry. Sorted cells were cultured with indicated factors for 4 days before collecting the supernatant. After incubation of a mixture of gut bacteria isolated from the normal BALB/c mice (also the source of the PEC cells used for this experiment) with supernatant for 30 minutes, secretory IgA bound to bacteria was revealed by using FITC conjugated anti mouse IgA (BD) antibody. For isolating the gut bacteria, colonic content of the mouse was collected after flushing with PBS, effectively mixed by vortexing and centrifuged at 30 g for 30 minutes to remove the fecal material. Supernatant containing gut bacteria was collected and used for the experiment. Bacteria without addition of antibody (blank) or supernatant or added with isotype-matched control antibody was used as staining controls. (TIFF) Figure S4 Survival of cultured PEC B1 cells after in vivo transfer Figure 6. RA and TGF-b treated PEC B1 cells migrate to spleen -/- - into Rag1 recipients. Sorted IgA PEC B1a and B1b cells were and gut and produce serum and intestinal secretory IgA. IgA cultured under various IgA CSR inducing conditions for 3 days PEC B1a and B1b cells sorted in a fashion similar to previous and transferred intraperitoneally (12000 live cells/mouse) into experiments were cultured under various IgA CSR inducing conditions -/- for 3 days and transferred intraperitoneally (16000 live cells/mouse) into lymphopenic Rag1 recipients. (A) Unstimulated splenocytes and -/- lymphopenic Rag1 recipients. (A) Unstimulated splenocytes and 2 2 days LPS (25 mg/ml) stimulated PECs of the recipient mice were days LPS (25 mg/ml) stimulated PECs of the recipient mice were analyzed for the presence of IgA producing cells by ELISPOT 2 analyzed for the presence of IgA and IgM producing cells by ELISPOT 2 weeks after adoptive B cell transfer. (B) Levels of secretory Igs in weeks after adoptive B cell transfer. (B) Levels of secretory Igs in the the serum and gut lavage of recipient mice were determined by serum and gut lavage of recipient mice were determined by ELISA. Per ELISA. (C) Percentage (gated for live lymphocytes) and numbers group, 2-3 mice were used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay of CD19 B cells in the peritoneum of recipient mice were and it was done in triplicates. Data show the results of one of two determined by flow cytometry. PEC cells from individual recipient independent experiments. Bars represent mean 6 SD. mouse were analyzed by flow cytometry. Per group, 3–4 mice were doi:10.1371/journal.pone.0082121.g006 used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay and it was done in Supporting Information triplicates. Bars represent mean 6 SD. (TIF) Figure S1 Strategy for sorting (A) PEC and (B) splenic B cell subpopulations using FACS. The numbers above the respective Acknowledgments gates in the right most panels represent purity of the sorted cells in percentage. Cell sorting was performed using FACSAriaH (BD). The authors are grateful to Regina Lesch and Susanne zur Lage for Dead cells were excluded by applying appropriate gating strategy excellent technical help and Prof. Oliver Pabst for the critical reading of the in the side and forward scatter plot. Doublets from the lymphocyte manuscript. gated population were excluded by applying appropriate scatter gate. Reanalysis revealed that cells were .95% pure. Author Contributions (TIFF) Conceived and designed the experiments: BR AMB SA MK SD SW. Figure S2 Combined treatment with TGF-b and RA induces Performed the experiments: BR AMB SA MK SD SW. Analyzed the data: the expression of Iga germiline transcript. IgA PEC and splenic B BR AMB SA MK SD SW. Contributed reagents/materials/analysis tools: cells sorted in a fashion similar to previous experiments, were BR SW. Wrote the paper: BR AMB SA MK SD SW. PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA References 1. Woof JM, Kerr MA (2004) IgA function—variations on a theme. Immunology 22. Oliver AM, Martin F, Kearney JF (1999) IgMhighCD21high lymphocytes 113: 175 177. enriched in the splenic marginal zone generate effector cells more rapidly than 2. Stavnezer J, Kang J (2009) The surprising discovery that TGF beta specifically the bulk of follicular B cells. J Immunol 162: 7198–7207. induces the IgA class switch. J Immunol 182: 5–7. 23. Snapper CM, Yamada H, Smoot D, Sneed R, Lees A, et al. (1993) Comparative 3. Cerutti A, Rescigno M (2008) The biology of intestinal immunoglobulin A in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine responses. Immunity 28: 740–750. marginal zone and follicular B cells. J Immunol 150: 2737–2745. 4. Macpherson AJ, Geuking MB, McCoy KD (2012) Homeland security: IgA 24. Kaminski DA, Stavnezer J (2006) Enhanced IgA class switching in marginal immunity at the frontiers of the body. Trends Immunol 33: 160–167. zone and B1 B cells relative to follicular/B2 B cells. J Immunol 177: 6025–6029. 5. Mora JR, von Andrian UH (2009) Role of retinoic acid in the imprinting of gut- 25. Roy B, Shukla S, Lyszkiewicz M, Krey M, Viegas N, et al. (2009) Somatic homing IgA-secreting cells. Semin Immunol 21: 28–35. hypermutation in peritoneal B1b cells. Mol Immunol 46: 1613–1619. 6. van Ginkel FW, Wahl SM, Kearney JF, Kweon MN, Fujihashi K, et al. (1999) 26. Coffman RL, Lebman DA, Shrader B (1989) Transforming growth factor beta Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 specifically enhances IgA production by lipopolysaccharide-stimulated murine B knockout mice. J Immunol 163: 1951–1957. lymphocytes. J Exp Med 170: 1039–1044. 7. Cazac BB, Roes J (2000) TGF-beta receptor controls B cell responsiveness and 27. Sonoda E, Matsumoto R, Hitoshi Y, Ishii T, Sugimoto M, et al. (1989) induction of IgA in vivo. Immunity 13: 443–451. Transforming growth factor beta induces IgA production and acts additively 8. Snapper CM, Waegell W, Beernink H, Dasch JR (1993) Transforming growth with interleukin 5 for IgA production. J Exp Med 170: 1415–1420. factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated 28. Roy B, Agarwal S, Brennecke AM, Krey M, Pabst O, et al. (2013) B-1-cell murine B cells in vitro. J Immunol 151: 4625–4636. subpopulations contribute differently to gut immunity. Eur J Immunol 43: 2023– 9. Zan H, Cerutti A, Dramitinos P, Schaffer A, Casali P (1998) CD40 engagement 2032. 10.1002/eji.201243070 [doi]. triggers switching to IgA1 and IgA2 in human B cells through induction of 29. Seo GY, Jang YS, Kim HA, Lee MR, Park MH, et al. (2013) Retinoic acid, endogenous TGF-beta: evidence for TGF-beta but not IL-10-dependent direct S acting as a highly specific IgA isotype switch factor, cooperates with TGF-beta1 mu—.S alpha and sequential S mu—.S gamma, S gamma—.S alpha DNA to enhance the overall IgA response. J Leukoc Biol 94: 325–335. jlb.0313128 recombination. J Immunol 161: 5217–5225. [pii];10.1189/jlb.0313128 [doi]. 10. Chen ML, Yan BS, Bando Y, Kuchroo VK, Weiner HL (2008) Latency- 30. Bowman EP, Kuklin NA, Youngman KR, Lazarus NH, Kunkel EJ, et al. (2002) associated peptide identifies a novel CD4+CD25+ regulatory T cell subset with The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA TGFbeta-mediated function and enhanced suppression of experimental antibody-secreting cells. J Exp Med 195: 269–275. autoimmune encephalomyelitis. J Immunol 180: 7327–7337. 31. Farstad IN, Halstensen TS, Lazarovits AI, Norstein J, Fausa O, et al. (1995) 11. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, et al. Human intestinal B-cell blasts and plasma cells express the mucosal homing (2007) A functionally specialized population of mucosal CD103+ DCs induces receptor integrin alpha 4 beta 7. Scand J Immunol 42: 662–672. Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent 32. Tumang JR, Frances R, Yeo SG, Rothstein TL (2005) Spontaneously Ig- mechanism. J Exp Med 204: 1757–1764. secreting B-1 cells violate the accepted paradigm for expression of differenti- 12. Fagarasan S (2008) Evolution, development, mechanism and function of IgA in ation-associated transcription factors. J Immunol 174: 3173–3177. the gut. Curr Opin Immunol 20: 170–177. 33. Alugupalli KR, Leong JM, Woodland RT, Muramatsu M, Honjo T, et al. (2004) 13. Mora JR, Iwata M, Eksteen B, Song SY, Junt T, et al. (2006) Generation of gut- B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity homing IgA-secreting B cells by intestinal dendritic cells. Science 314: 1157– 21: 379–390. 1160. 34. de Waard R, Dammers PM, Tung JW, Kantor AB, Wilshire JA, et al. (1998) 14. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, et al. (2004) Retinoic Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 acid imprints gut-homing specificity on T cells. Immunity 21: 527–538. cells. Dev Immunol 6: 81–87. 15. Denning TL, Wang YC, Patel SR, Williams IR, Pulendran B (2007) Lamina 35. Rosado MM, Aranburu A, Capolunghi F, Giorda E, Cascioli S, et al. (2009) propria macrophages and dendritic cells differentially induce regulatory and From the fetal liver to spleen and gut: the highway to natural antibody. Mucosal interleukin 17-producing T cell responses. Nat Immunol 8: 1086–1094. Immunol 2: 351–361. 16. Watanabe K, Sugai M, Nambu Y, Osato M, Hayashi T, et al. (2010) 36. Longerich S, Basu U, Alt F, Storb U (2006) AID in somatic hypermutation and Requirement for Runx proteins in IgA class switching acting downstream of class switch recombination. Curr Opin Immunol 18: 164–174. TGF-beta 1 and retinoic acid signaling. J Immunol 184: 2785–2792. 37. Di Noia JM, Neuberger MS (2007) Molecular mechanisms of antibody somatic 17. Litinskiy MB, Nardelli B, Hilbert DM, He B, Schaffer A, et al. (2002) DCs hypermutation. Annu Rev Biochem 76:1–22: 1–22. induce CD40-independent immunoglobulin class switching through BLyS and 38. Honorine R, Mosrin-Huaman C, Hervouet-Coste N, Libri D, Rahmouni AR APRIL. Nat Immunol 3: 822–829. (2011) Nuclear mRNA quality control in yeast is mediated by Nrd1 co- 18. He B, Santamaria R, Xu W, Cols M, Chen K, et al. (2010) The transmembrane transcriptional recruitment, as revealed by the targeting of Rho-induced activator TACI triggers immunoglobulin class switching by activating B cells aberrant transcripts. Nucleic Acids Res 39: 2809–2820. through the adaptor MyD88. Nat Immunol 11: 836–845. 39. Martin F, Oliver AM, Kearney JF (2001) Marginal zone and B1 B cells unite in 19. Stall AM, Adams S, Herzenberg LA, Kantor AB (1992) Characteristics and the early response against T-independent blood-borne particulate antigens. development of the murine B-1b (Ly-1 B sister) cell population. Ann N Y Acad Immunity 14: 617–629. Sci 651: 33–43. 40. Hornquist CE, Ekman L, Grdic KD, Schon K, Lycke NY (1995) Paradoxical 20. Haas KM, Poe JC, Steeber DA, Tedder TF (2005) B-1a and B-1b cells exhibit IgA immunity in CD4-deficient mice. Lack of cholera toxin-specific protective distinct developmental requirements and have unique functional roles in innate immunity despite normal gut mucosal IgA differentiation. J Immunol 155: and adaptive immunity to S. pneumoniae. Immunity 23: 7–18. 2877–2887. 21. Martin F, Kearney JF (2000) B-cell subsets and the mature preimmune 41. Bergthorsdottir S, Gallagher A, Jainandunsing S, Cockayne D, Sutton J, et al. repertoire. Marginal zone and B1 B cells as part of a ‘‘natural immune (2001) Signals that initiate somatic hypermutation of B cells in vitro. J Immunol memory’’. Immunol Rev 175:70–9: 70–79. 166: 2228–2234. PLOS ONE | www.plosone.org 10 December 2013 | Volume 8 | Issue 12 | e82121 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-β and Retinoic Acid

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Pubmed Central
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© 2013 Roy et al
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1932-6203
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10.1371/journal.pone.0082121
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Abstract

Aims: In the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-b. Methods and Results: To study the influence of RA and TGF-b on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-b and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-b, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-b was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, ‘‘innate’’ B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - a b and 4 7 -/- CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1 recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients. Conclusion: Present study demonstrates the differential and synergistic effect of RA and TGF-b on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity. Citation: Roy B, Brennecke A-M, Agarwal S, Krey M, Du¨ ber S, et al. (2013) An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-b and Retinoic Acid. PLoS ONE 8(12): e82121. doi:10.1371/journal.pone.0082121 Editor: Simon Fillatreau, DRFZ, Germany Received May 31, 2013; Accepted October 21, 2013; Published December 6, 2013 Copyright:  2013 Roy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the German Research Council (DFG), the Ministry for Education and Research (BMBF) and the Helmholtz Association via the HZI Graduate School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Bishnudeo.Roy@helmholtz-hzi.de secreting cells (SCs) and their homing to gut is promoted by Introduction intestinal DCs and appears to be dependent on RA.[13] IgA is the most abundant class of antibodies present in Consistently, mice deficient in RA precursor vitamin A showed mammalian mucosal tissues. It forms a first-line of defense against reduced numbers of IgA producing cells in the small intestine even invasion by inhaled or ingested pathogens and plays an important though the IgA levels in the serum remained unchanged.[13] The role in the maintenance of immune homeostasis. Besides mucosal interplay between TGF-b and RA is still controversially discussed. tissues, IgA is also found at significant concentrations in the serum It has been demonstrated that TGF-b inhibits RA induced IgA of many species, where it mediates the elimination of pathogens CSR.[13] However, another study using splenic cells showed that that have breached the mucosa.[1] a combination of RA and TGF-b with additional factors (LPS, Class switch recombination (CSR) to IgA is orchestrated by APRIL, and IL5) acts synergistically to induce IgA switching in various cytokines and other factors.[2–4] Amongst them, TGF-b vitro.[16] and retinoic acid (RA) are most prominent.[2,5] A special role of In addition to TGF-b and RA, cytokines, like IL2, IL4, IL5, IL6 TGF-b in IgA CSR is most evident from the observation that mice and IL10, are also thought to induce CSR and promote IgA deficient for TGF-b or lacking TGF-b receptor II expression on B production either via TGF-b induction within B cells or by cells exhibit reduced levels of IgA.[6,7] In gut, TGF-b is produced enhancing the post-switch maturation.[4] Furthermore, cytokines by B cells (autocrine factor) [8,9], T cells [10] dendritic cells (DCs) of the tumor necrosis factor (TNF) ligand family called B cell [11], and stromal cells.[12] Some of the T cells that produce TGF- activating factor (BAFF/BLyS) and a proliferation inducing ligand + + b are claimed to be Foxp3 CD4 regulatory T cells.[2] Besides (APRIL) produced by gut DCs are also considered to be involved TGF-b, vitamin A metabolite RA is also a highly potent inducer of in IgA switching.[17,18] IgA CSR.[5] RA is produced by gut associated DCs and In mice, at least three mature B cell subpopulations – follicular macrophages.[13–15] In accordance, the generation of IgA (FO), marginal zone (MZ) and B1 B cells constitute the humoral PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA immune response, where B1 cells can be subdivided further into RT-PCR B1a and B1b cells.[19,20] FO B cells also known as B2 cells are Total RNA from sorted/cultured cells was prepared with TM present in the B cell follicles of secondary lymphoid organs and TriFast FL Reagent (peQLab) following manufacturer’s proto- respond to antigens T cell dependently. On the other hand, MZ B col. DNase (Qiagen) treated RNA was reverse transcribed using TM cells residing in the splenic marginal zone and B1 cells oligo-d(T) (Thermo Scientific) and RevertAid reverse 12–18 predominantly located in the peritoneal cavity (PEC) or pleural transcriptase (Fermentas). PCR amplification of cDNA was TM cavity, are believed to respond to antigens without T cell help and performed using HotstarTaq DNA polymerase (Qiagen). and play an important role in the early stages of pathogenic the following primers: Igm/a heavy chain variable (VH) region, for invasion.[21] Such B cells respond differentially to various stimuli VHcons 59-GAGGTGCAGCTGCAGGAGTCTGG-39 rev Cm2 and need multiple triggers to induce IgA class switching.[16, 59-CATTTGGGAAGGACTGA-39 or Ca259-GAGCTGGTG- 22–24] Thus, under minimal stimulatory conditions GGAGTGTCAGTG-39. (BLys+LPS+TGF-b) they switch to IgA preferentially in compar- ison to B2 cells from PEC or spleen.[24] Sequencing of Ig VH chain and sequence analysis The contribution of B1 cell subsets to IgA production in vivo is RT-PCR products of IgVH chain transcripts from 4 days not known. In this regard, we could recently show that most of the cultured cells were cloned using TOPO TA CloningH kit IgA producing cells in the PEC of unmanipulated mice belonged (Invitrogen) following manufacturer’s protocol. Plasmids were to B1b cell population.[25] However, differential switching of B1a isolated using QIAprep Spin Mini Prep Kit (Qiagen). Sequencing and B1b cells to IgA under stimulatory culture conditions has not was done using M13 Reverse primers by the department of been comparatively studied. In addition, the combination of TGF- Genome Analysis of Helmholtz Centre for Infection Research, b and RA that was supposed to have synergistic effects on naı ¨ve Braunschweig. IgA and IgM VH sequences derived from sorted splenic B cells to switch to IgA [16] was never tested on various PEC and splenic B cells were submitted to GenBank (accession peritoneal and splenic B1 cell subsets. no. KF207924 – KF208284). Thus, in the present work, we stimulated peritoneal B1a, B1b and B2 cells with LPS, BLys and TGF-b in vitro. Switching to IgA was Cell culture and ELISPOT assay severely enhanced in peritoneal B1b cells in comparison to their B1a Purified peritoneal and splenic B cell populations, resus- and B2 counterparts. Switching to IgA was further enhanced when pended in IMDM at a density of approximately 1610 cells/ml TGF-b was combined with RA and IL5. In vitro switched B1 cells were stimulated with, LPS (25 mg/ml; Escherichia coli, Sigma), also showed the presence of frequent nucleotide exchanges in their Recombinant Mouse BLys (100 ng/ml; R&D SYSTEMS), functionally rearranged VH gene segments. This indicates that Recombinant Human TGF-b1 (2 ng/ml; R&D SYSTEMS), besides IgA CSR, somatic hypermutation had also taken place. IL4 (1:400; home made), IL5 (1:400; home made), and all These findings could be extended in part also to splenic B1a and trans-Retinoic Acid (100 nM; Sigma). ELISPOT: single-cell MZ B cells. Altogether, these findings demonstrate differential suspensions at serial dilutions of 1:5 were plated on plates switching of B cell subpopulations to IgA in response to various coated with anti-mouse IgA (Pharmingen) or anti-mouse IgM stimuli in vitro. This suggests alternative mechanisms regulating the (Pharmingen). Cells were incubated overnight at 37uCwith5% IgA CSR of different B cell subsets in vivo. CO and 95% humidity. After washing with PBS+0.01% Tween 20, biotinylated anti-mouse IgA (eBioscience) or anti- Materials and Methods mouse IgM (SeroTec) was added. Bound antibodies were developed with Streptavidin-horseradish peroxidase using AEC Ethic Statement (3-amino-9-ethyl-carbazole; Sigma) in DMF (N,N-Dimethylfor- All experiments were performed in accordance with the mamide; Sigma) diluted in 0.1 M acetate solution added with German Law on Care and Use of Laboratory Animals and were H O as substrate. Spot development was stopped by washing 2 2 approved by the local authority (LAVES) under permission plates with water and spots marking antibody secreting cells number 33.9-42502-04-11/0390. (ASCs) were counted after drying. Mice ELISA Normal BALB/c mice, 7–10 weeks old, used for the experi- -/- Ig concentrations in serum or intestinal lavage were ments were purchased from Janvier. Rag1 mice on BALB/c measured by ELISA. In brief, 96 well plates (MaxiSorb TM background were obtained from our animal facility (Helmholtz Immunoplates, Nunc) were coated over night with anti-mouse Institute for Infection Research; Braunschweig, Germany). IgA (Pharmingen) or anti-mouse IgM (Pharmingen) or goat- anti-mouse IgG (Sigma) antibodies at 4 C and blocked for 1 h Cell preparation, FACS analysis and cell sorting with 3% BSA in 0.05% Tween 20. Appropriately diluted To prepare PEC cells, peritoneum was flushed two times with culture supernatant/sera/intestinal lavage from each mouse was 8 ml of normal cell culture medium (IMDM), collected washout added to the wells and incubated for 2 h at RT. Bound IgA/ was centrifuged and the pellet was resuspended in appropriate IgM/IgG was detected with biotinylated rat anti-mouse IgA amount of cell culture medium. Monoclonal antibodies against (eBioscience) or rat-anti-mouse IgM (SeroTec) or PO conjugat- mouse CD19, CD5, CD43, Mac-1, IgA, IgM, CD23, CD21, ed goat-anti-mouse IgG (Jackson ImmunoResearch) antibodies. CCR9 and a b conjugated with FITC, PE, APC, PE-Cy7, or 4 7 Biotinylated antibodies were revealed by using horseradish Biotin were obtained from Pharmingen or eBioscience or were peroxidase (HRP) conjugated streptavidin (BD). Bound HRP home made. Biotinylated antibodies were revealed by Streptavi- activity was visualized using o-Phenylendiamin (OPD) as din-PerCPCy5.5 (eBioscience). Flow cytometry was done using substrate and the results were read using an ELISA-reader LSR II (BD) and data were analyzed using FACS DIVA software. (BioRad 3550- UV microplate reader). Cell sorting was performed using FACSAriaH (Becton Dickinson). Reanalysis showed that cells were .95% pure PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA In vivo adoptive transfer Sort purified PEC B1a and B1b cells cultured under different stimulatory conditions for 3 days were collected, washed, -/- resuspended in PBS and injected i.p into Rag1 hosts. Statistics Paired two-tailed Student’s t-test was applied to determine the statistical significance (p value). p#0.05 was considered significant. *p,0.05; **p,0.01; ***p,0.001. Results Peritoneal cavity B1b cells switch preferentially to IgA in presence of TGF-b TGF-b has been known to promote switching of B cells to IgA in general.[2,26,27] Under the influence of this cytokine, combined with LPS and Blys, differential propensity of switching to IgA was observed among PEC and splenic B cell subtypes.[24] Interestingly, under such conditions, PEC B1 and splenic MZ B cells switched in vitro to IgA more prominently in comparison to PEC or splenic B2 cells. However, in this study, B1 cells were not differentiated into B1a and B1b cells and thus their differential capability to switch to IgA was not established.[24] Our previous in vivo finding had suggested differences between PEC B1a and B1b cells in that respect.[25] This prompted us to test their preferences to switch to IgA under in vitro stimulatory conditions. For this purpose, PEC B cells were FACS purified into – B1a, B1b and B2 cells (Figure S1). B cells expressing surface IgA were sorted out to exclude expansion of such cells in vitro. After 4 days of stimulation in the presence of different combinations of cytokines, IgA and IgM ELISPOT assays were applied to assess the number of IgA and IgM SCs. Under different stimulatory conditions, the combinations of LPS+Blys+TGF-b and LPS+Blys+TGF- b+IL4+IL5 led to significant switching amongst cultured B cells (Figure 1). In comparison to B2 cells, both B1 cell subpopulations showed higher numbers of IgA SCs (Figure 1). Quite interestingly, B1b cells produced constantly higher number of IgA SCs in Figure 1. TGF-b enhances IgA switching among PEC B1b cells in comparison to B1a cells (Figure 1). Presence of BLys was required vitro. Results of IgA and IgM ELISPOT assays performed in triplicates on for efficient IgA CSR as treatment with LPS+TGF-b alone led to sort purified PEC B cell subpopulations after 4 days of treatment with generation of very few IgA SCs (Figure 1). the indicated combination of stimulatory factors in culture. Surface IgA - hi + + + Parallel, ELISPOTs for IgM showed no apparent trend for the cells were excluded by sorting from B1a (IgA CD19 CD5 CD43 Mac-1 ), - hi - + + - lo - - B1b (IgA CD19 CD5 CD43 Mac-1 ) and B2 (IgA CD19 CD5 CD43 Mac- number of IgM SCs amongst three PEC B cell populations under 1 ) B cell subpopulations. Data show the results of one of three different stimulatory conditions (Figure 1). However, a decrease in independent experiments. Bars represent mean 6 SD. the number of IgM SCs was noted for conditions under which an doi:10.1371/journal.pone.0082121.g001 increase in the numbers of IgA SCs had been observed (Figure 1). observed under the condition (LPS+Blys+TGF-b+IL5+RA) in Pronounced switching of B1 cells to IgA when TGF-b is which, very high number of IgA ASCs was observed (Figure 2A). combined with RA This finding was further confirmed by ELISA that was done to RA has been shown to promote the switching of naı ¨ve splenic B determine the amount of IgA in the culture supernatants of the cells to IgA.[16] The differential effect of this vitamin A metabolite cultured PEC B cells (Figure 2B). Consistently, PEC B1 cells also on various PEC and splenic B cell subpopulations has not been showed the expression of Iga germ line transcripts after combined studied so far. Therefore, to examine the influence of RA on PEC treatment with TGF-b and RA (Figure S2). When tested for the and splenic B cell subpopulations, sorted peritoneal and splenic B reactivity against gut bacteria, secretory IgA present in the culture cells were subjected in vitro to different combinations of stimulatory supernatant of PEC B cells showed their binding towards these factors including RA. After 4 days of activation, the number of IgA commensals (Figure S3). This is consistent with our previous and IgM SCs were measured by ELISPOT assay. Consistent with finding that showed the binding of B1 cell derived Igs to gut earlier findings, considerably high numbers of IgA SCs were bacteria [28]. Furthermore, analysis of secretory IgG in the culture observed in the PEC B cell cultures supplemented with RA plus supernatants showed that the amount of IgG decreased to a TGF-b, although these reagents alone in combination with other minimum under conditions that favored the switching to IgA factors induced comparatively low numbers of IgA SCs (Figure 2B). Interestingly, under other conditions, the amount of (Figure 2A). Interestingly, under these circumstances, again PEC secretory IgG was found to be higher in B1b cell culture B1a and B2 cells had significantly less number of IgA ASCs than B1b cells. No differences in the numbers of IgM ASCs were PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 2. TGF-b in combination with RA synergistically induces IgA switching by PEC B cells. (A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified PEC B cell subpopulations after 4 days of treatment with the indicated combination of stimulatory factors in + - hi + + + - hi - + + culture. Surface IgA cells were excluded by sorting from B1a (IgA CD19 CD5 CD43 Mac-1 ), B1b (IgA CD19 CD5 CD43 Mac-1 ) and B2 - lo - - - (IgA CD19 CD5 CD43 Mac-1 ) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g002 supernatants in comparison to the other two PEC B cell In addition, these B cell types showed an uneven increase in their populations (Figure 2B). number under various culture conditions (Table 1). This might Among splenic B cell subpopulations (FACS sorted according to account for the slight differences between ELISPOT and ELISA the plan showed in Figure S1), mainly B1a and MZ B cells data, later being only qualitative under these circumstances. underwent pronounced switching to IgA compared to splenic B2 cells when treated with TGF-b plus RA (Figure 3A, B). Addition of In vitro differentiated B1 cells also exhibit somatic IL5 to the combination of RA and TGF-b did not increase the hypermutations number of IgA ASCs (Figure 3A). Presence of secretory IgA in the The enzyme AID responsible for CSR also mediates somatic supernatants of cultured B cells as determined by ELISA was hypermutation (SHM). Consistent with that, we had detected the consistent with the ELISPOT results (Figure 3A, B). IgG expression of AID among B cell populations cultured under production seemed to take place without the addition of TGF-b stimulatory conditions (data not shown). Thus, quite expectedly, or retinoic acid. However, TGF-b or RA appeared respectively to analysis of IgA VH sequences from PEC B1 cell and splenic B1a suppress or enhance the production of IgG in case of splenic B1a cell cultures supplemented with TGF-b or a combination of TGF- cells, (Figure 3B). b and RA exhibited the presence of frequent mutations Cell counts of cultured cells indicated strongly reduced (Figure 4A). Mutation frequencies observed under IgA CSR proliferation of all PEC and splenic B cell types in presence of inducing culture conditions were higher compared to unstimulated TGF-b and RA wherever IL5 was not added (Table 1). This is B cells (Figure 4A). Interestingly, frequent nucleotide exchanges consistent with the recent finding showing strong inhibition of B were also detected amongst IgM VH sequences derived from in cell proliferation in the cultures added with RA and TGF- b [29]. vitro stimulated B1 cells, (Figure 4A). Almost all of the VH PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 3. TGF-b in combination with RA synergistically induce IgA switching in splenic B1a and MZ B cells. (A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified splenic B cell subpopulations after 4 days of treatment with the indicated combination of + - + + - lo/- - + - stimulatory factors in culture. Surface IgA cells were excluded by sorting from B1a (IgA CD19 CD5 CD23 CD21 ), MZ (IgA CD19 CD5 C- lo/- hi - + - hi int D23 CD21 ) and B2 (IgA CD19 CD5 CD23 CD21 ) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g003 sequences were found to be functional and devoid of stop codons (Figure 5A, B). Without RA, no influence of TGF-b or IL5 on the or frame shift mutations in the coding VH region (Table 2). expression of such molecules was observed (Figure 5A, B). Analysis of replacement (R) vs. silent (S) mutation revealed a high However, addition of IL5 to RA with or without TGF-b appeared R/S ratio in the VH regions (Figure 4B). to enhance the expression of CCR9 on the treated B cell surface (Figure 5A, B). Notably, considerable percentages of B cells expressed either a4b7 or CCR9 on their surface though double Retinoic acid enhances the expression of gut homing expressers were found to be present as well (Figure 5A, B). Under molecules by B1 cells these conditions, PEC B1a and B1b cells showed higher Besides the induction of IgA switching, retinoic acid is known to percentages of B cells expressing these two molecules in induce the expression of gut homing surface receptors (a4b7 and comparison to PEC B2 cells (Figure 5A). Among splenic B cells, CCR9) on target B and T cells.[5,14] CCR9 is a chemokine B1a and MZ cells were also higher for such markers in comparison receptor for CCL25 which is secreted by gut epithelial cells and to B2 cells (Figure 5B). Thus, expression of these gut homing a4b7 is an integrin that binds to the mucosal adressin molecules seemed to be consistent with the switching tendency of MadCAM.[30,31] Expression of these two molecules was expect- B cell subpopulations to IgA as observed above as well as when ed to be induced after in vitro treatment of peritoneal and splenic B checked for the expression of surface IgA on PEC and splenic B cell subpopulations with TGF-b and RA as IgA producing plasma cell subpopulations (cultured under various stimulatory conditions) blasts are supposed to home to intestine. Consistent with earlier by flow cytometry (Figure 2, Figure 3 and Figure 5C, D). findings, treatment with RA led to high surface expression of a4b7 and/or CCR9 on some of the treated PEC and splenic B cells PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Table 1. Increase in cell numbers after 4 days of culture under various conditions. Fold increase in no. of cells after 4 days of culture PEC Spleen Culture condition B1a B1b B2 B1a MZ B2 LPS+BLys 5.9061.59 7.2060.42 3.1060.65 6.5461.31 6.8863.75 3.1261.70 LPS+Blys+TGF-b 2.9360.39 2.3361.73 1.6060.56 1.8160.71 1.9360.98 0.7960.40 LPS+Blys+RA 6.2361.59 3.5560.62 1.5560.70 2.9462.03 5.0762.56 0.9660.42 LPS+Blys+IL5 12.8465.79 8.3461.54 3.6761.64 4.8862.62 6.9163.25 2.1660.84 LPS+Blys+TGF-b+RA 1.2760.49 0.6360.24 0.3260.27 0.6660.56 0.4860.26 0.2560.15 LPS+Blys+TGF-b+IL5 6.0661.38 5.4961.09 1.8360.38 2.5061.41 3.7261.29 1.0460.34 LPS+Blys+RA+IL5 15.8465.53 16.5465.92 2.4460.18 4.4560.98 4.9461.06 0.9960.55 LPS+Blys+TGF-b+RA+IL5 7.5461.55 3.9261.43 0.4960.16 4.7062.87 1.0560.48 0.4660.32 1 - Sorted IgA PEC and splenic B cells were cultured under different conditions for 4 days and the fold increase in cell number was calculated by dividing the cell count after 4 days by the number of cells at the beginning of the cell culture. The values represent mean 6 SD calculated from three independent culture experiments. doi:10.1371/journal.pone.0082121.t001 The expression of gut homing molecules - a4b7 and CCR9 combination of TGF-b, LPS and BLys.[24] However, in this study after TGF-b and RA treatment should render these cells capable no attempt was made to distinguish between two B1 cell subtypes. of migrating to the gut. Therefore, the migratory capacity of such Our previous finding that among PEC B cells, almost exclusively, B cells was tested by adoptive transfer experiment. PEC B1a and B1b cells had switched to IgA in vivo[25] led us to speculate that B1b cells treated in vitro with RA and TGF-b for three days were B1a and B1b cells might differ in their IgA switching potential -/- adoptively transferred into lymphopenic Rag1 recipients. Day under stimulatory culture conditions. When tested, this speculation was found to be true as TGF-b in combination with LPS and BLys three was chosen because high expression of Iga germ line transcripts could be detected after this period of treatment (Figure led to significantly higher switching of B1b cells to IgA in comparison to the other two PEC B cell subpopulations. Thus, S2). The recipients were analyzed 2 weeks after transfer for the presence of IgA and IgM ASCs in the spleen, and the peritoneal IgA SCs must have resulted from CSR in vitro after treatment with TGF-b as the possibility of expansion of preexisting IgA cells cavity as well as for the secretory IgA and IgM in the serum and among B1b cells had been already excluded by sorting out IgA intestinal lavage. Interestingly, presence of IgA SCs could be cells before culture. detected in the recipients of PEC B1 cells that were triggered with TGF-b or RA (Figure 6A and Figure S4A). However, only the TGF-b together with RA has recently been shown to induce IgA switching synergistically.[16] In the present study, we used this splenocytes of recipients of B1b cells treated with TGF-b+RA+IL5 contained considerably high numbers of IgA SCs (Figure 6A and knowledge to determine the propensity of different peritoneal and splenic B cell subtypes to switch to IgA in response to RA with or Figure S4A). Higher amounts of secretory IgA could be detected in without TGF-b. The combined effect of these two factors together the serum and gut lavage of the recipients of B1a or B1b cells with LPS, IL5 and BLys was much stronger than any other treated with TGF-b+RA+IL5 in comparison to any other recipient combination of stimulants. Again, the frequency of switching to groups (Figure 6B and Figure S4B). Again the recipients of B1b IgA among PEC B cells was significantly higher in B1b cells cells were higher in comparison to B1a cell recipients. Flow compared to other PEC B cell types. Interestingly, among splenic cytometric analysis of PEC cells from transfer recipients showed B cells, B1a and MZ B cells showed significantly higher IgA CSR the presence and survival of transferred cells (Figure S4C). than B2 cells. Low switching frequency observed among PEC and Together, these findings demonstrate a synergistic and differ- splenic B2 cells can be explained by the fact that they require ential effect of RA and TGF-b on PEC and splenic B cell additional stimuli for proliferation and differentiation. For subpopulations for IgA switching. Surprisingly, B1b cells prefer- example, B2 cells expressing higher surface IgD show stronger entially switch to IgA independent of T cell help. proliferative response in presence of anti-IgD-Dex which is further enhanced by addition of IL4.[23] The considerable high Discussion propensity of splenic B1a cell to switch to IgA is consistent with Peritoneal cavity B cell subpopulations differ from each other studies where CD5 expressing splenic B1 cells have been claimed with regard to origin, phenotype and function. PEC B1 cells to contribute to IgA production.[35] differentiate faster into plasma cells after stimulation with mitogen Besides induction of CSR, the vitamin A metabolite RA is also than B2 cells from the same compartment.[25,32] In vivo, the two known to induce or upregulate the expression of gut homing subtypes of B1 cells – B1a and B1b B cells have been shown to molecules - integrin a4b7 and the chemokine receptor CCR9.[5] react differently to pathogenic infections.[20,33] In murine Quite consistent with that, in our in vitro experimental set up, we infection models, B1a cells have been shown to respond abruptly observed an upregulated expression of these two gut homing after infection whereas B1b cells give rise to long lasting memory molecules after treatment with RA. Interestingly, the expression of immunity against certain pathogens.[20,33] Moreover, these two a4b7or CCR9 was higher under conditions that led to enhanced B1 cell subtypes also have been shown to differ in their ability to induction of IgA CSR. PEC and splenic B cell subsets prone to produce IgA.[25,34] Along these lines, recent in vitro studies have enhanced IgA CSR showed higher expression of these homing demonstrated that compared to peritoneal and splenic B2 cells, molecules on their surface. In agreement with the upregulation of PEC B1 cells switch more preferentially to IgA in response to a gut homing molecules, when PEC B1 cells, treated with a PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA Figure 4. PEC and splenic B1 cell derived IgA VH sequences display high frequencies of nucleotide exchanges due to somatic hypermutation. Frequency of mutations observed in the IgM or IgA VH region sequences derived from PEC and splenic B1 cells cultured for 4 days with the indicated combinations of IgA CSR inducing factors. (A) Each colored sector represents a particular number of mutations found at a particular frequency. Total number of sequences is shown in the middle. (B) Number of replacement and silent mutations amongst IgM and IgA VH TM sequences derived from indicated B cell populations. Sequences were analyzed using SEQUENCHER Version 4.1 for Macintosh software (Gene Codes Corporation). Assignment of VH chain was done using VBASE2 database (http://www.vbase2.org/). Only functional sequences were included for the mutation analysis. Bars represent mean 6 SD. doi:10.1371/journal.pone.0082121.g004 combination of RA, TGF-b, LPS, BLys and IL5, were transferred sequences derived from the same B cell populations also showed -/- i.p. into Rag1 mice, IgA could be found in serum as well as in higher frequencies of mutations than unstimulated controls. gut lavage. This indicates gut migration of in vitro switched IgA SHM in vivo results in affinity maturation of a particular producing cells. BCR.[37] This is however accompanied by the generation of Isotype switching in B cells is mediated by the enzyme AID.[36] many nonfunctional BCRs due to the introduction of nonsense This enzyme is also responsible for nucleotide exchanges leading nucleotides by SHM. In accordance, one would expect to find to somatic hypermutation in the V regions of B cell receptors many non-functional VH sequences under in vitro conditions. This (BCRs) [36]. Quite expectedly, AID had been found to be was apparently not the case. The simplest explanation would be expressed in cultured B cells under stimulatory condition. This that B cells that had already undergone SHM, and started IgA raised the query whether B cells undergoing IgA CSR also CSR, are selectively expanded under our culture conditions. Such accumulate SHM under our experimental conditions. Interesting- cells might be qualitatively or quantitatively distinct from B cells ly, a considerable fraction of IgA VH sequences derived from that had not yet undergone such processes. Signaling molecules or RA+TGF-b treated PEC and splenic B1 cells showed nucleotide transcription factors might be up- or down-regulated in this state exchanges. Apparently, like CSR, SHM might also have been and the cells might preferentially respond to the stimuli in vitro. induced in vitro. Surprisingly, besides IgA VH sequences, IgM VH Alternatively, IgA CSR and SHM were induced in vitro. The low PLOS ONE | www.plosone.org 7 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA 1 B1 cells are generally believed to react independent of CD4 T Table 2. Nonfunctional vs functional sequences. cell help.[39,40] Our findings are quite consistent with this notion. The fact that cytokines together with RA efficiently induced CSR and SHM in such cells without interaction with T cells or the Sequence Condition PEC B1a PEC B1b Spleen B1a involvement of CD40/CD40L suggests that B1 cells indeed can IgAVH LPS+Blys+TGF+IL5 1/27 1/22 1/27 undergo differentiation without T cell help. This is in contrast with IgAVH LPS+Blys+TGF+IL5+RA 1/26 2/22 1/27 the earlier report where signaling through BCR, CD40 and CD38 IgMVH LPS+Blys+TGF+IL5 2/26 0/29 3/24 were shown to be necessary for the in vitro induction of mutations presumably in B2 cells.[41] The factors required for such IgMVH LPS+Blys+TGF+IL5+RA 1/25 3/26 0/19 differentiation are probably provided in vivo by stroma or myeloid IgMVH Unstimulated 2/25 0/26 2/9 cells like intestinal DCs or macrophages. B cells themselves could Sequences having frame shift or stop codon in the VH coding region were be the source of some of such factors under in vivo conditions but considered to be nonfunctional. The numbers of nonfunctional vs. functional also T cells interacting with such B1 cells in a non-cognate fashion sequences are displayed below indicated cell population. might be involved. doi:10.1371/journal.pone.0082121.t002 In conclusion, our work shows that under stimulatory conditions in vitro, PEC B1b cells switch to IgA with a higher propensity in frequency of non-functional VH could be explained by an intrinsic comparison to PEC B1a or B2 cells. The differences among selection mechanism to generate functional BCRs. It is known that splenic B cell subpopulations with regard to induced switching to nonfunctional mRNAs are recognized and such cells might be IgA are also quite evident. The results of this study show eliminated.[38] Also B cells producing nonfunctional non- differential stimulatory requirements for different B cell subsets secretable VH or VL chains might be deleted by intrinsic and suggest the presence of distinct mechanisms regulating the mechanisms. This would imply a strong first round of selection induction of IgA CSR among them. for functional BCRs independent of antigen recognition. Figure 5. RA treatment leads to up-regulation of gut homing molecules on the surface of cultured PEC B cells. Expression of surface a4b7 and CCR9 on IgA PEC (A) or splenic (B) B cells sorted in fashion similar to previous experiments was checked by flow cytometry after four days of culture under various combinations of stimulatory conditions. Using the same cultures (as in A and B), expression of surface IgA and IgM on PEC (C) and splenic (D) B cells was checked by flow cytometry. Data show the results of one of two independent experiments. doi:10.1371/journal.pone.0082121.g005 PLOS ONE | www.plosone.org 8 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA cultured in IgA CSR inducing conditions for two or three days and checked for the expression of Iga germline, and RPS9 (house keeping gene) transcripts by RT-PCR. Results of semi-quantitative PCR, using three serial dilutions (UD – undiluted; 1:2 and 1:4) of respective cDNA as template have been displayed. Primers: RPS9, forward (for) 59- TTGACGCTAGACGAGAAGGAT-39 reverse (rev) 59-AATCCAGCTTCATCTTGCCCT -39;Iga germline transcript, for Ia259- CCAGGCATGGTTGAGATAGAGA- TAG -39 rev Ca259-GAGCTGGTGGGAGTGTCAGTG-39. PCR conditions were: 94uC for 20 s, annealing at various temperatures for 40 s, 72uC for 40 s; 33-38 cycles. (TIFF) Figure S3 IgA antibodies derived from TGF-b and RA treated PEC B cells displays reactivity towards gut bacteria. Binding of secretory IgA antibodies present in the culture supernatants of FACS purified IgA PEC B1a, B1b and B2 cells to gut bacteria was tested by flow cytometry. Sorted cells were cultured with indicated factors for 4 days before collecting the supernatant. After incubation of a mixture of gut bacteria isolated from the normal BALB/c mice (also the source of the PEC cells used for this experiment) with supernatant for 30 minutes, secretory IgA bound to bacteria was revealed by using FITC conjugated anti mouse IgA (BD) antibody. For isolating the gut bacteria, colonic content of the mouse was collected after flushing with PBS, effectively mixed by vortexing and centrifuged at 30 g for 30 minutes to remove the fecal material. Supernatant containing gut bacteria was collected and used for the experiment. Bacteria without addition of antibody (blank) or supernatant or added with isotype-matched control antibody was used as staining controls. (TIFF) Figure S4 Survival of cultured PEC B1 cells after in vivo transfer Figure 6. RA and TGF-b treated PEC B1 cells migrate to spleen -/- - into Rag1 recipients. Sorted IgA PEC B1a and B1b cells were and gut and produce serum and intestinal secretory IgA. IgA cultured under various IgA CSR inducing conditions for 3 days PEC B1a and B1b cells sorted in a fashion similar to previous and transferred intraperitoneally (12000 live cells/mouse) into experiments were cultured under various IgA CSR inducing conditions -/- for 3 days and transferred intraperitoneally (16000 live cells/mouse) into lymphopenic Rag1 recipients. (A) Unstimulated splenocytes and -/- lymphopenic Rag1 recipients. (A) Unstimulated splenocytes and 2 2 days LPS (25 mg/ml) stimulated PECs of the recipient mice were days LPS (25 mg/ml) stimulated PECs of the recipient mice were analyzed for the presence of IgA producing cells by ELISPOT 2 analyzed for the presence of IgA and IgM producing cells by ELISPOT 2 weeks after adoptive B cell transfer. (B) Levels of secretory Igs in weeks after adoptive B cell transfer. (B) Levels of secretory Igs in the the serum and gut lavage of recipient mice were determined by serum and gut lavage of recipient mice were determined by ELISA. Per ELISA. (C) Percentage (gated for live lymphocytes) and numbers group, 2-3 mice were used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay of CD19 B cells in the peritoneum of recipient mice were and it was done in triplicates. Data show the results of one of two determined by flow cytometry. PEC cells from individual recipient independent experiments. Bars represent mean 6 SD. mouse were analyzed by flow cytometry. Per group, 3–4 mice were doi:10.1371/journal.pone.0082121.g006 used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay and it was done in Supporting Information triplicates. Bars represent mean 6 SD. (TIF) Figure S1 Strategy for sorting (A) PEC and (B) splenic B cell subpopulations using FACS. The numbers above the respective Acknowledgments gates in the right most panels represent purity of the sorted cells in percentage. Cell sorting was performed using FACSAriaH (BD). The authors are grateful to Regina Lesch and Susanne zur Lage for Dead cells were excluded by applying appropriate gating strategy excellent technical help and Prof. Oliver Pabst for the critical reading of the in the side and forward scatter plot. Doublets from the lymphocyte manuscript. gated population were excluded by applying appropriate scatter gate. Reanalysis revealed that cells were .95% pure. Author Contributions (TIFF) Conceived and designed the experiments: BR AMB SA MK SD SW. Figure S2 Combined treatment with TGF-b and RA induces Performed the experiments: BR AMB SA MK SD SW. Analyzed the data: the expression of Iga germiline transcript. IgA PEC and splenic B BR AMB SA MK SD SW. Contributed reagents/materials/analysis tools: cells sorted in a fashion similar to previous experiments, were BR SW. Wrote the paper: BR AMB SA MK SD SW. PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e82121 Influence of RA on Switching to IgA References 1. Woof JM, Kerr MA (2004) IgA function—variations on a theme. Immunology 22. Oliver AM, Martin F, Kearney JF (1999) IgMhighCD21high lymphocytes 113: 175 177. enriched in the splenic marginal zone generate effector cells more rapidly than 2. Stavnezer J, Kang J (2009) The surprising discovery that TGF beta specifically the bulk of follicular B cells. J Immunol 162: 7198–7207. induces the IgA class switch. J Immunol 182: 5–7. 23. Snapper CM, Yamada H, Smoot D, Sneed R, Lees A, et al. (1993) Comparative 3. Cerutti A, Rescigno M (2008) The biology of intestinal immunoglobulin A in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine responses. Immunity 28: 740–750. marginal zone and follicular B cells. J Immunol 150: 2737–2745. 4. Macpherson AJ, Geuking MB, McCoy KD (2012) Homeland security: IgA 24. Kaminski DA, Stavnezer J (2006) Enhanced IgA class switching in marginal immunity at the frontiers of the body. Trends Immunol 33: 160–167. zone and B1 B cells relative to follicular/B2 B cells. J Immunol 177: 6025–6029. 5. Mora JR, von Andrian UH (2009) Role of retinoic acid in the imprinting of gut- 25. Roy B, Shukla S, Lyszkiewicz M, Krey M, Viegas N, et al. (2009) Somatic homing IgA-secreting cells. Semin Immunol 21: 28–35. hypermutation in peritoneal B1b cells. Mol Immunol 46: 1613–1619. 6. van Ginkel FW, Wahl SM, Kearney JF, Kweon MN, Fujihashi K, et al. (1999) 26. Coffman RL, Lebman DA, Shrader B (1989) Transforming growth factor beta Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 specifically enhances IgA production by lipopolysaccharide-stimulated murine B knockout mice. J Immunol 163: 1951–1957. lymphocytes. J Exp Med 170: 1039–1044. 7. Cazac BB, Roes J (2000) TGF-beta receptor controls B cell responsiveness and 27. Sonoda E, Matsumoto R, Hitoshi Y, Ishii T, Sugimoto M, et al. (1989) induction of IgA in vivo. Immunity 13: 443–451. Transforming growth factor beta induces IgA production and acts additively 8. Snapper CM, Waegell W, Beernink H, Dasch JR (1993) Transforming growth with interleukin 5 for IgA production. J Exp Med 170: 1415–1420. factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated 28. Roy B, Agarwal S, Brennecke AM, Krey M, Pabst O, et al. (2013) B-1-cell murine B cells in vitro. J Immunol 151: 4625–4636. subpopulations contribute differently to gut immunity. Eur J Immunol 43: 2023– 9. Zan H, Cerutti A, Dramitinos P, Schaffer A, Casali P (1998) CD40 engagement 2032. 10.1002/eji.201243070 [doi]. triggers switching to IgA1 and IgA2 in human B cells through induction of 29. Seo GY, Jang YS, Kim HA, Lee MR, Park MH, et al. (2013) Retinoic acid, endogenous TGF-beta: evidence for TGF-beta but not IL-10-dependent direct S acting as a highly specific IgA isotype switch factor, cooperates with TGF-beta1 mu—.S alpha and sequential S mu—.S gamma, S gamma—.S alpha DNA to enhance the overall IgA response. J Leukoc Biol 94: 325–335. jlb.0313128 recombination. J Immunol 161: 5217–5225. [pii];10.1189/jlb.0313128 [doi]. 10. Chen ML, Yan BS, Bando Y, Kuchroo VK, Weiner HL (2008) Latency- 30. Bowman EP, Kuklin NA, Youngman KR, Lazarus NH, Kunkel EJ, et al. (2002) associated peptide identifies a novel CD4+CD25+ regulatory T cell subset with The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA TGFbeta-mediated function and enhanced suppression of experimental antibody-secreting cells. J Exp Med 195: 269–275. autoimmune encephalomyelitis. J Immunol 180: 7327–7337. 31. Farstad IN, Halstensen TS, Lazarovits AI, Norstein J, Fausa O, et al. (1995) 11. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, et al. Human intestinal B-cell blasts and plasma cells express the mucosal homing (2007) A functionally specialized population of mucosal CD103+ DCs induces receptor integrin alpha 4 beta 7. Scand J Immunol 42: 662–672. Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent 32. Tumang JR, Frances R, Yeo SG, Rothstein TL (2005) Spontaneously Ig- mechanism. J Exp Med 204: 1757–1764. secreting B-1 cells violate the accepted paradigm for expression of differenti- 12. Fagarasan S (2008) Evolution, development, mechanism and function of IgA in ation-associated transcription factors. J Immunol 174: 3173–3177. the gut. Curr Opin Immunol 20: 170–177. 33. Alugupalli KR, Leong JM, Woodland RT, Muramatsu M, Honjo T, et al. (2004) 13. Mora JR, Iwata M, Eksteen B, Song SY, Junt T, et al. (2006) Generation of gut- B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity homing IgA-secreting B cells by intestinal dendritic cells. Science 314: 1157– 21: 379–390. 1160. 34. de Waard R, Dammers PM, Tung JW, Kantor AB, Wilshire JA, et al. (1998) 14. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, et al. (2004) Retinoic Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 acid imprints gut-homing specificity on T cells. Immunity 21: 527–538. cells. Dev Immunol 6: 81–87. 15. Denning TL, Wang YC, Patel SR, Williams IR, Pulendran B (2007) Lamina 35. Rosado MM, Aranburu A, Capolunghi F, Giorda E, Cascioli S, et al. (2009) propria macrophages and dendritic cells differentially induce regulatory and From the fetal liver to spleen and gut: the highway to natural antibody. Mucosal interleukin 17-producing T cell responses. Nat Immunol 8: 1086–1094. Immunol 2: 351–361. 16. Watanabe K, Sugai M, Nambu Y, Osato M, Hayashi T, et al. (2010) 36. Longerich S, Basu U, Alt F, Storb U (2006) AID in somatic hypermutation and Requirement for Runx proteins in IgA class switching acting downstream of class switch recombination. Curr Opin Immunol 18: 164–174. TGF-beta 1 and retinoic acid signaling. J Immunol 184: 2785–2792. 37. Di Noia JM, Neuberger MS (2007) Molecular mechanisms of antibody somatic 17. Litinskiy MB, Nardelli B, Hilbert DM, He B, Schaffer A, et al. (2002) DCs hypermutation. Annu Rev Biochem 76:1–22: 1–22. induce CD40-independent immunoglobulin class switching through BLyS and 38. Honorine R, Mosrin-Huaman C, Hervouet-Coste N, Libri D, Rahmouni AR APRIL. Nat Immunol 3: 822–829. (2011) Nuclear mRNA quality control in yeast is mediated by Nrd1 co- 18. He B, Santamaria R, Xu W, Cols M, Chen K, et al. (2010) The transmembrane transcriptional recruitment, as revealed by the targeting of Rho-induced activator TACI triggers immunoglobulin class switching by activating B cells aberrant transcripts. Nucleic Acids Res 39: 2809–2820. through the adaptor MyD88. Nat Immunol 11: 836–845. 39. Martin F, Oliver AM, Kearney JF (2001) Marginal zone and B1 B cells unite in 19. Stall AM, Adams S, Herzenberg LA, Kantor AB (1992) Characteristics and the early response against T-independent blood-borne particulate antigens. development of the murine B-1b (Ly-1 B sister) cell population. Ann N Y Acad Immunity 14: 617–629. Sci 651: 33–43. 40. Hornquist CE, Ekman L, Grdic KD, Schon K, Lycke NY (1995) Paradoxical 20. Haas KM, Poe JC, Steeber DA, Tedder TF (2005) B-1a and B-1b cells exhibit IgA immunity in CD4-deficient mice. Lack of cholera toxin-specific protective distinct developmental requirements and have unique functional roles in innate immunity despite normal gut mucosal IgA differentiation. J Immunol 155: and adaptive immunity to S. pneumoniae. Immunity 23: 7–18. 2877–2887. 21. Martin F, Kearney JF (2000) B-cell subsets and the mature preimmune 41. Bergthorsdottir S, Gallagher A, Jainandunsing S, Cockayne D, Sutton J, et al. repertoire. Marginal zone and B1 B cells as part of a ‘‘natural immune (2001) Signals that initiate somatic hypermutation of B cells in vitro. J Immunol memory’’. Immunol Rev 175:70–9: 70–79. 166: 2228–2234. PLOS ONE | www.plosone.org 10 December 2013 | Volume 8 | Issue 12 | e82121

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Published: Dec 6, 2013

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