The Journal of Immunology

Mice Lacking Endogenous IL-10–Producing Regulatory B Cells Develop Exacerbated Disease and Present with an Increased Frequency of Th1/Th17 but a Decrease in Regulatory T Cells

Natalie A. Carter,* Rita Vasconcellos,*,† Elizabeth C. Rosser,* Calogero Tulone,‡ Alba Mun˜oz-Suano,x Masahito Kamanaka,{,‖ Michael R. Ehrenstein,* Richard A. Flavell,{ and Claudia Mauri*

IL-10–producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10–producing B cells (IL-102/2B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-102/2 B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (mMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-102/2 mice increased Foxp3+ Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10+/+ B cells established longer contact times with arthritogenic CD4+CD252 T cells compared with IL-102/2 B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4+ T cells. Thus, IL-10–producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells. The Journal of Immunology, 2011, 186: 5569–5579.

nterleukin-10–producing B cells have been shown to be es- marginal zone precursor B cells (T2-MZP), which were identified sential for the restoration of tolerance in several autoimmune as the major producers of IL-10, prevents or ameliorates estab- I disorders and in the promotion of leukemia (1–4). These lished disease (6). Similarly, transfer of CD5+CD1dhi B cells cells can interfere with the development of several autoimmune (B10) controls the development of the contact hypersensitivity diseases, including experimental autoimmune encephalomyelitis response (3). In each instance, Bregs isolated from IL-10–deficient (5) and collagen-induced arthritis (6), by suppressing the devel- mice (IL-102/2) failed to suppress the development of autoim- opment of autoreactive Th1 responses (7). Similar mechanisms are mune diseases (5, 7, 9, 10). exploited by pathogens or in cancer to subvert host protective IL-10 mediates suppression of inflammation by a number of responses (8). Several regulatory B cell (Breg) subsets have now mechanisms, including downregulation of the production of pro- been identified, and most share the release of IL-10 as a common inflammatory cytokines, such as IFN-g and IL-17, and the expres- mechanism of action. In experimental arthritis, we have shown sion of MHC class II (11). In addition, it has been recently shown that the transfer of CD19+CD21hiCD23hiCD1dhi transitional 2 that the IL-10 produced by myeloid cells is crucial for the main- tenance of Foxp3 expression and regulatory T cell (Treg) func- *Division of Medicine, Centre for Rheumatology Research, University College Lon- tion and ultimately controls the severity of colitis (12). mMT mice don, London W1T 4JF, United Kingdom; †Departamento de Imunobiologia, Instituto display reduced numbers of Tregs (13) and develop exacerbated de Biologia, Universidade Federal Fluminese, Niteroi, Rio de Janeiro CEP 24020- Ag-induced arthritis (AIA) compared with wild-type (WT) mice 150, Brazil; ‡Division of Infection and Immunity, University College London, Lon- don W1T 4JF, United Kingdom; xMedical Research Council Laboratory of Molecular (A. Bosma, N.A. Carter, E.C. Rosser, and C. Mauri, unpublished Biology, Cambridge CB2 2HQ, United Kingdom; {Department of Immunobiology, observations). Altogether, this is suggestive that IL-10 produced Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520; and ‖Arai Hospital, Kuki, Saitama 346-0003, Japan by B cells may be important for maintaining the balance between Tregs and Th1/Th17 cells. Received for publication January 28, 2011. Accepted for publication March 7, 2011. Adoptive transfer of Bregs has been preferentially used as a This work was supported by Arthritis Research UK Programme Grant MP/17707 (to C.M.). method to dissect the function of these cells in vivo. Although this Address correspondence and reprint requests to Dr. Claudia Mauri, University Col- approach has provided immunological insights into the mecha- lege London, 46 Cleveland Street, London W1T 4JF, United Kingdom. E-mail ad- nisms of action of Bregs, the extent to which endogenous Bregs dress: [email protected] play a role in suppressing autoreactive responses in vivo remains The online version of this article contains supplemental material. completely unknown. Given the possible existence of several Breg Abbreviations used in this article: AIA, Ag-induced arthritis; Breg, regulatory B cell; 2 2 subsets, and to avoid the exclusion of any of these subsets, we have FO, follicular; IL-10 / , IL-10–deficient; LN, lymph node; mBSA, methylated BSA; MZ, marginal zone; RA, rheumatoid arthritis; T2-MZP, transitional 2 marginal zone addressed this question using mixed bone marrow chimeric mice precursor; Tr1, T regulatory 1; Treg, regulatory T cell; WT, wild-type; WTB, wild- with an IL-10 deficiency restricted to B cells. Chimeric mice type B. provide an invaluable tool for dissecting the effect of the lack of Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 specific genes in B cell populations during in vivo responses (14, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100284 5570 Bregs PRESERVE THE Treg POOL

15). In this study, we report a previously unappreciated liaison AIA experiments. The absence of B cells in the group reconstituted with between immunoregulatory and proinflammatory cells, showing 100% mMT bone marrow confirmed the total ablation of the host bone marrow by irradiation. In contrast, the three other groups showed numbers that IL-10 produced by B cells maintains Tregs over the in- + + 2/2 of CD19 B cells and CD4 T cells equivalent to numbers found in non- flammatory Th1 and Th17 pool. IL-10 B cell chimeric mice chimeric WT B6 mice. develop an exacerbated disease compared with WT mice and displayed significantly lower numbers of Foxp3+CD4+ T cells Dissection and digestion of the synovial membrane compared with WT B (WTB) cell control mice. In addition, these Synovial cells were isolated from the synovial membrane on day 5 post- CD4+ T cells displayed decreased levels of Foxp3 expression disease onset as described (17). Briefly, knee joints were removed, and the compared with the levels expressed in WTB cell control mice. The synovial membrane was excised under a dissecting microscope and reduced number of Tregs in IL-102/2 B cell mice was mirrored by digested with collagenase A (1 mg/ml; Sigma-Aldrich) and DNAse type IV (150 mg/ml Roche) for 45 min at 37˚C. Then cells were washed and an accumulation of Th1 and Th17 cells in the lymph nodes (LN) passed through a cell strainer before being analyzed by flow cytometry. draining the site of inflammation. We have also shown that only T2-MZP Bregs, and not marginal zone (MZ) or follicular (FO) Peritoneal macrophages enrichment B cell subsets, upregulated the frequency of Tregs after adoptive A total of 1.5 ml 4% thioglycolate fluid medium (Difco) was injected into transfer into B cell-deficient mice. In addition, we showed that the mice i.p. After 4 d, peritoneal lavage fluid was collected and cultured for 2 2 transfer of T2-MZP Bregs to IL-10 / mice increased the Treg 4 h. Cells adhering to the culture plate were used as the macrophage pool and reduced the severity of arthritis. In vitro analysis of the population (an aliquot was stained with CD11b and analyzed by flow cy- cross-talk between B and CD4+ T cells revealed that, upon Ag tometry). Macrophages were subsequently stimulated with purified Esch- +/+ erichia coli 0111:B4 LPS (1 mg/ml; Sigma-Aldrich) for 48 h. IL-10 in the stimulation, IL-10 B cells established contacts of longer dura- supernatant was measured by ELISA. tion with arthritogenic WT T cells than B cells isolated from IL- 102/2 mice. Of note, in the same culture conditions, upregulation Flow cytometric analysis of intracellular cytokine synthesis of Foxp3 on CD4+T cells, concomitantly to a downregulation of and ELISA lL-17, was observed. These data suggest that the increased contact Intracellular cytokine analysis was performed as previously described (2). time between IL-10–competent B cells and CD4+ T cells may be Splenocyte or LN cultures were suspended at 5 3 105 cells/ml in complete responsible for the differentiation of Tregs and the suppression of medium with PMA (50 ng/ml)/ionomycin (500 ng/ml) (Sigma-Aldrich) proinflammatory cytokines. In this study, we demonstrate a novel and GolgiPlug (BD Biosciences) for 5 h, unless otherwise stated. Cells were then stained with surface markers followed by permeabilization and function of Bregs in the primary induction of Tregs. incubation with intracellular Abs. The cells were acquired with an FACS- Calibur flow cytometer (BD Biosciences) and analyzed using FlowJo soft- ware (Tree Star). Supernatants from cell cultures were harvested and ana- Materials and Methods lyzed for cytokines using standard sandwich ELISA kit (R&D Systems) Abs and performed according to the manufacturer’s instructions. The following Abs were used: anti-CD3, anti-CD4, anti-CD19, anti-CD21, Treg suppression assay anti-CD23, anti-CD24, anti-Foxp3, anti–IL-10, anti–IFN-g and anti–IL-17, + 2 purchased either from BD Biosciences or eBioscience. Spleens or LN were removed postmortem, and Treg and CD4 CD25 T cells were isolated using the CD4+CD25+ Regulatory T Cell isolation kit Mice, induction, and assessment of arthritis for mouse (Miltenyi Biotec). Cells were cultured in round-bottom 96-well plates (Nunc) for 60 h with either complete medium alone or anti-CD3 (1 2 2 C57/B6, mMT/B6, and IL-10 / B6 mice were bred and maintained under mg/ml; BD Biosciences). Cultures were pulsed overnight with 1mCi [3H] specific pathogen-free conditions at the animal facility at University Col- thymidine, harvested, and counted in a scintillation counter (LKB Instru- lege London, London, U.K. Female C57/BL6 mice (8–12 wk old) were ments). Supernatants were also harvested for cytokine detection by ELISA immunized with 1 mg/ml methylated BSA (mBSA; Sigma-Aldrich) in prior to addition of thymidine. CFA. This was followed, 7 d later, by intra-articular injection of 10 ml20 mg/ml mBSA (disease onset). Knee swelling was measured using calipers Adoptive transfer of B cell subsets and calculated as the percentage increase in knee size as compared with 2 2 B cells isolated from the spleens of WT or IL-10 / mice in the remission day 0 (preinjection). The walking score was graded as follows: 4, no walking; 3, walking on three legs; 2, limping with the leg with in- phase of AIA were stained with CD19, CD23, CD21, and CD24. B cell flammation; 1, limping with the leg with inflamed knee only after pressure subsets were FACS sorted using gates drawn according to previous reports on the knee; and 0, normal walking (16). Affected joints were removed (6). Seven days prior to transfer/intra-articular injection, all recipients were immunized with mBSA in CFA. T2-MZP Breg, FO, and MZ B cells (2 3 postmortem, fixed in 10% (w/v) buffered formalin, and decalcified in 5% 2 2 6 / m EDTA. The joints were subsequently embedded in paraffin, sectioned, and 10 ) were transferred i.v. to syngeneic IL-10 C57/BL6 mice, MT stained with H&E. Normal = no damage; mild = minimal synovitis, car- C57/BL6 mice, or C57/B6 WT mice on the day of intra-articular injection. tilage loss, and bone erosion limited to discrete foci; moderate = synovitis The control group (no transfer) received a PBS injection. and erosion present, but normal joint architecture intact; and severe = Flow cytometric analysis of B–T cell conjugate formation extensive erosion and joint architecture disrupted. B and CD4+CD252 T cells were isolated from spleens of WT B6 and IL- Generation of chimeric mice 102/2 mice immunized with mBSA/CFA by negative selection. CD4+ m Chimeric mice were generated as previously described (14). Recipient mMt T cells were stained using Cell Tracker Green (0.5 M; Invitrogen) and B cells with Cell Tracker Blue (0.5 mM; Invitrogen). Stained B and CD4+ mice received 800 cGy gamma-irradiation via a caesium source. Five 3 hours following irradiation, recipients received 2 3 106 donor bone mar- T cells were combined at a ratio of 1:1 (total number of cells/well = 1 6 m row cells. Bone marrow preparations were depleted of T cells by negative 10 cells/ml) and cultured with or without 10 g/ml mBSA (Sigma- selection with an MACS magnetic column (Miltenyi Biotec). To generate Aldrich) and neutralizing anti–IL-10 (clone JES5–2A5 rat IgG1). Cells mice in which the absence of IL-10 was exclusively restricted to B cells, were incubated at 37˚C for up to 40 min. Cells were then fixed with chilled paraformaldehyde. Conjugates were defined as Green CD4+ T cells cos- mMT mice were reconstituted with mixture of bone marrow consisting of + 80% from mMT (no B cell differentiation) with 20% from IL-102/2 mice. taining with Blue-CD19 B cells. The cells were acquired with an LSRII Control mice received 80% from mMT and 20% bone marrow from WT flow cytometer (BD Biosciences) and analyzed using FlowJo software mice (to give a normal B cell compartment). Two additional control groups (Tree Star). were included: 100% of bone marrow from mMT into WT recipients Real-time imaging (control for the absence of B cells) or 80% WT and 20% IL-102/2 bone marrow into mMT recipients (this will assess the effect of 20% reduction in B and CD4+ T cells were isolated from mBSA-immunized WT B6 and IL- IL-10 production by non-B cell lymphocytes in the response observed). 102/2 mice using negative selection separation kits from Miltenyi Biotec. Chimeras were left to fully reconstitute over at least 8 wk before use in B cells were incubated at 37˚C for 1 h with 10 mg/ml mBSA and CD4+ The Journal of Immunology 5571

T cells with media alone. T cells were then stained using CFSE (5 mM; each cell type, to further confirm that the effect that we observed is Sigma-Aldrich) and B and T cells resuspended in RPMI 1640 clear (Life entirely due to the lack of IL-10–producing B cells, we have in- Technologies) for imaging. Cells were then plated out at 10,000 T cells and cluded an additional group of control mice. We reconstituted ir- 10,000 B cells per well with 10 mg/ml mBSA and imaged in real time over 2/2 20 min at 37˚C. Imaging was done using Nikon Eclipse TE 300 micro- radiated mMT recipients with 80% WT and 20% IL-10 bone scope, Plain Fluor ELWD 203/0.45 (Nikon) lens, and LaserSharp2000 marrow. This group of mice developed arthritis with the same acquisition software. The camera used was Laser System Radiance2100 severity as WTB cell chimeric mice and WT nonchimeric mice, (Bio-Rad). Analysis of cell interactions was done using Velocity (Perkin- which was significantly less severe than IL-102/2 B cell chimeric Elmer) software, and for each condition, all cells in the frame of view were numbered, and then 40 cells, picked using random number tables, were mice (data not shown). 2/2 blindly analyzed for cell–cell contacts. It has been previously shown that IL-10 mice develop ex- acerbated autoimmune diseases compared with WT mice (18, Statistical analysis 2 2 19). Similarly, IL-10 / mice develop a more severe arthritis as For the statistical analysis of the data, the Mann–Whitney U test and Fisher compared with WT mice (Fig. 1C–E; an enlarged representative exact test were applied to analyze clinical results. Unpaired t tests were area of inflammation is shown in Supplemental Fig. 2C,2D). It is p , 2 2 2 2 applied in all other experiments. A value 0.05 was considered sig- interesting to note that IL-10 / B cell and IL-10 / mice de- nificantly different. velop arthritis with equivalent severity, suggesting that B cells producing IL-10, in addition to other IL-10–producing cells (i.e., Results T cells) (11, 20), have an integral role in restraining the severity IL-10–competent B cells limit the severity and damage caused of the inflammatory responses (Fig. 1C–E; an enlarged region of by the inflammatory response interest is shown in Supplemental Fig. 2). First, we assessed the effect that the lack of endogenous IL-10– producing B cells has on the severity of inflammatory arthritis. IL-10 produced by B cells maintains Treg number and Foxp3 Bone marrow chimeric mice, in which B cells lack the capacity expression during inflammation to release IL-10, were generated as previously described (14) Next we investigated the effect that IL-10–producing B cells have (summarized in Supplemental Fig. 1A). Briefly, mMT mice were on the maintenance of Treg number and on the expression of reconstituted with either a mixture of bone marrow consisting of Foxp3 in CD4+T cells during the acute phase of arthritis. A de- 80% from mMT with 20% from IL-102/2 mice (IL-102/2 B cell) crease in the percentage and absolute number of CD4+Foxp3+ or with 80% from mMT and 20% bone marrow from WT mice Tregs was observed in the draining LNs of IL-102/2 B cell mice (WTB cell mice). One group received 100% of bone marrow from compared with the numbers measured in the LNs isolated from mMT (control for the absence of B cells). The results in Supple- control WTB cell mice (Fig. 2A). In addition, the levels of Foxp3 mental Fig. 1B confirm that B cells from IL-102/2 B cell mice do expression were significantly reduced in Tregs from IL-102/2 not produce IL-10 in response to CpG stimulation. In addition, B cell mice compared with Tregs from WTB cell mice (Fig. 2B). no significant differences were identified between the absolute Identical results were obtained if we measured the levels of Foxp3 numbers and the frequencies of repopulated CD4+ T cells, CD19+ on CD4+CD25+ T cells (data not shown). Despite the reduced B cells and different B cell subsets among the WT B cells and expression of Foxp3, CD4+CD25+ T cells isolated from IL-102/2 IL-102/2 B cell chimera compared with nonchimera WT mice B cell mice efficiently suppressed the proliferation of CD4+ (Supplemental Fig. 1C). T cell proliferative responses to anti-CD3 CD252 T cells isolated from WT mice (Fig. 2C). The numerical stimulation were equivalent in IL-102/2 B cell and WTB cell defect in Foxp3+ Tregs and reduced levels of Foxp3 expression chimeric mice (Supplemental Fig. 1D). The results in Supple- were confined to the Treg population isolated from the draining mental Fig. 1E show that peritoneal macrophages from IL-102/2 inguinal LN and were not found in Tregs isolated from non- B cell mice secreted similar amount of IL-10 to macrophages draining LN (data not shown), which is in agreement with the isolated from WTB cell chimeric mice and WT nonchimeric mice current concept of surveillance by Tregs in the draining LN (21, after stimulation with LPS in vitro. These results confirm that the 22). IL-10 production by the non-B cell compartment is quantitatively The reduced number of Tregs observed in the LNs of IL-102/2 equivalent in all groups. B cell mice could reflect their increased capacity to migrate and In the current study, we took advantage of the AIA model, as this accumulate in the target tissues. In general, IL-102/2 B cell mice model recapitulates both the DTH response and the development show a significantly increased accumulation of cells in the syno- of an autoimmune-like disease. AIA was induced in chimeric mice vium compared with control mice (Fig. 2D). The frequency of by immunization with mBSA/CFA, followed 1 wk later by intra- Foxp3+CD4+ T cells among synovial cells was significantly re- articular injection with mBSA. Unlike the experimental autoim- duced in the IL-102/2 B mice compared with control WTB cell mune encephalomyelitis model, in which the absence of IL-10– mice (Fig. 2E). The ratio between CD4+Foxp3+ Tregs and syno- producing B cells exclusively affected the recovery phase of dis- vial cells is reduced by more than half in IL-102/2 B cell mice ease (14), in AIA, the severity of arthritis was significantly ex- compared with WTB cell mice (Fig. 2F). However, equivalent acerbated in mice lacking IL-10–producing B cells (Fig. 1A; for absolute numbers of Tregs were measured in the two groups of clarity of representation, only days 3 and 7 are shown hereafter). chimeric mice (Fig. 2G). We could argue that the differences in Histological analysis revealed severe damage in 75% of the joints percentage observed in the joints is still of physiological relevance of IL-102/2 B cell mice as a result of the extensive expansion of as a dilution in the numbers of Tregs may reflect a reduced chance the synovial pannus, fibrin exudate, and massive infiltration of for Tregs to encounter, and hence suppress, effector cells. In con- mononuclear cells (Fig. 1B,1E; an enlarged representative area of trast to Tregs isolated from draining LNs, we did not observe any infiltration is shown in Supplemental Fig. 2A,2B). In contrast, differences in the levels of Foxp3 expression in synovial Tregs WTB cell mice developed mild to moderate arthritis, with only in the IL-102/2 B cell mice compared with WTB cell mice (Fig. 20% of the joints showing a severe loss of bone architecture (Fig. 2H). However, due to the low number of cells isolated from the 1B,1E). IL-102/2 B cell chimeric mice have populations of he- synovia of mice, we were unable to conduct a Treg suppression matopoietic cells that are IL-10–deficient besides B lymphocytes. assay and so are unable to comment on the functionality of Tregs Although these account for only 20% of the total population for from the synovia of IL-102/2 B cell mice. 5572 Bregs PRESERVE THE Treg POOL

FIGURE 1. Endogenous B cell-derived IL-10 constrains the severity of arthritis. AIA was induced in chimeric mice by immunization with mBSA/CFA, followed 1 wk later by intra-articular injection with mBSA. A, Bar charts represent walking score and knee swelling, calculated as a percentage increase relative to joint size prior to disease onset, of IL-102/2 B cell or WTB cell chimeric mice with AIA. Days 3 and 7 were chosen as representative days. One independent experiment, representative of three, is shown (n = 5). Data were compared by statistical analysis using the Fisher test. B, Representative histology of arthritic joints isolated from IL-102/2 B or WTB cell chimeric mice 7 d postdisease onset. Joints were processed as described in the Materials and Methods section (original magnification 310). The arrows indicate the areas where there has been an infiltration of cells into the joint. C, Bar charts represent walking score and knee swelling, calculated as a percentage increase relative to joint size prior to disease onset, for WT and IL-102/2 mice with AIA on day 3 postonset. One independent experiment, representative of three, is shown (n = 10). Data were compared by statistical analysis using the Fisher test. D, Representative histological analysis comparing arthritic knees of WT and IL-102/2 mice 7 d postdisease onset. The arrows indicate the areas where there has been an infiltration of cells into the joint. Original magnification 310. E, Bar chart shows the severity of damaged joints in WTB cell and IL-102/2 B cell chimeric mice as well as IL-102/2 and WT mice with AIA. Histological analysis was done 7 d postdisease onset, and five mice per group were examined. One experiment representative of three is shown.

IL-10–producing B cells suppress IL-17 and IFN-g production straining Th1/Th17 response and the severity of arthritis. Other at the site of inflammation IL-10–producing cells, including dendritic cells and myeloid cells, The involvement of the Th1 and Th17 subsets in the pathogenesis have been shown to contribute to the maintenance of healthy of arthritis is supported by accumulation of these cells and up- numbers of Tregs via the release of IL-10 (12). Next we set out to regulation of expression of their signature cytokines, IL-17 and understand and weight the sole contribution of IL-10–producing IFN-g, in the synovial fluid of individuals with rheumatoid ar- B cells in the maintenance/differentiation of Tregs in arthritis and thritis (RA) (23, 24). To address the effect that the lack of B cell- in the inhibition of Th1/Th17 response. T2-MZP Bregs cells, derived IL-10 has on the proinflammatory arm of the immune which we have previously shown to be the major producers of response, we next measured the frequencies of Th1 and Th17 in IL-10 in arthritis (6) (Supplemental Fig. 3A), were isolated from 2/2 WT mice in the remission phase of the disease and transferred draining LN of IL-10 B cell versus control WTB cell mice. We 2/2 found that both Th1 and Th17 responses were significantly in- i.v. to either syngeneic IL-10 mice or to WT mice on the 2/2 day of disease onset. The purity of the T2-MZP Bregs, after FACS creased in the draining LN of IL-10 B cell compared with 2/2 WTB cell mice (Fig. 3A,3B). This increase was mirrored by sorting, is shown in Supplemental Fig. 3B. A group of IL-10 a dramatic decrease in the frequency and in the number of CD4+ and WT mice were also left untreated and used as a control. IL-10+ T cells (Fig. 3C,3D). An overall increase in IFN-g and IL- Similar to our results in collagen-induced arthritis, transfer of T2- MZP Bregs protects WT mice from developing severe arthritis 17 production and a decrease of IL-10 released into the super- 2/2 natant was measured from the same LN culture upon stimulation (6) (Fig. 4A). Interestingly, despite IL-10 mice developing an with mBSA (Fig. 3E). exacerbated disease compared with WT mice (Figs. 1C–E,4A), transfer of T2-MZP Bregs conferred a modest, yet significant,

2/2 protection from AIA as shown by the reduction in knee swelling Transfer of T2-MZP Bregs into IL-10 hosts resets the compared with IL-102/2 mice treated with PBS (no cells trans- balance between Tregs and Th1/Th17 ferred) (Fig. 4A). Analysis of T cell differentiation revealed that Our data so far suggest that endogenous Bregs actively participate the protective effect was accompanied by increased percentages in the maintenance or differentiation of Foxp3+ Tregs and in con- of Foxp3+CD4+T cells and a suppression of Th1/Th17 cell dif- The Journal of Immunology 5573

FIGURE 2. Lack of B cell-derived IL-10 results in a decrease of Foxp3+ Tregs. AIA was induced in chimeric mice by immunization with mBSA/CFA, followed 1 wk later by intra-articular injection with mBSA. A, Representative dot plots showing the percentage of Foxp3+CD4+ Tregs in inguinal draining LN 5 d postdisease onset in WTB cell and IL-102/2 B cell chimeric mice. Bar chart shows mean 6 SEM (n = 5) of percentage and absolute numbers of Foxp3+CD4+ Tregs and is representative of three separate experiments. The p values were determined by unpaired t test. B, Fluorescence intensity, normalized to 100%, of Foxp3 on CD4+T cells. One of five representative histograms from one experiment is shown. Mean fluorescence intensity of Foxp3 gated on CD4+Foxp3+ (n = 5) is shown in the bar graph, representative of three independent experiments. The p values were determined by unpaired t test. C, CD4+CD25+ Tregs were isolated, respectively, from draining LN of arthritic IL-102/2 B or WTB cell chimeric mice and cocultured (1:1) with CD4+ CD252 T cells isolated from draining LN of arthritic WT mice for 72 h with anti-CD3 (1 mg/ml). [3H]thymidine was added 12 h before harvesting. Data shown are mean 6 SEM of triplicate wells and are representative of two independent experiments. D, Bar chart shows the live cells present in the synovia of IL-102/2 B cell and WTB cell chimeric, removed 5 d after disease onset, counted by trypan blue. One out of three independent experiments is shown. The p values were determined by unpaired t test. E, Representative dot plot showing the percentage of Foxp3+CD4+ T cells in the synovia. The synovial cells from five mice were pooled, and one independent experiment, representative of four, is shown. F, Bar chart showing the ratio of live synovial cells: synovial Tregs present in the IL-102/2 B cell and WTB cell chimeric 5 d after disease onset. Data shown are mean ratio 6 SEM from four independent experiments. For each experiment, the synovial cells from five mice were pooled for each group. The p values were determined by unpaired t test. G, Bar chart showing the absolute numbers of Foxp3+CD4+ T cells in the synovia. The synovial cells from five mice were pooled, and one independent experiment, representative of four, is shown. H, Representative histogram showing the fluorescence intensity, normalized to 100%, of Foxp3 in CD4+ T cells in the synovia of IL-102/2 B and WTB cell mice. The synovial cells from five mice were pooled, and one independent experiment, representative of four, is shown. ferentiation in LN draining the arthritic joint compared with the T cells levels in the IL-102/2 recipients (to WT levels); however, levels detected in untreated IL-102/2 mice (Fig. 4B,4C). In- it cannot further upregulate Foxp3+Tregs once they have reached terestingly, this increase in Foxp3+CD4+ T cells following T2- this threshold. MZP Breg transfer can only be seen in the IL-102/2 mice and Transfer of IL-10–deficient T2-MZP Bregs failed to suppress not WT recipients (Fig. 4B). This suggests that IL-10 produced by arthritis development in both WT and IL-102/2 mice (we have the T2-MZP Breg population is able to normalize Foxp3+CD4+ only included the results for WT for clarity of presentation in Fig. 5574 Bregs PRESERVE THE Treg POOL

FIGURE 3. Lack of B cell-derived IL-10 results in an increase in both Th1 and Th17 cell subsets. AIA was induced in chimeric mice by immunization with mBSA/CFA, followed 1 wk later by intra-articular injection with mBSA. Five days postdisease onset, draining LN cells were isolated from chimeric IL-102/2 B cell and WTB cell mice and cultured with PMA/ionomycin in the presence of brefeldin A for 5 h. A, Dot plots show representative staining for IL-17 and IFN-g in CD4+T cells. B, Bar charts show mean 6 SEM of percentage and absolute numbers of Th1 and Th17 cells, representative of three independent experiments (n = 5 for each group). The p values were determined by unpaired t test. C, Dot plots show the frequencies of IL-10+CD4+ T cells. D, Bar charts show mean 6 SEM of percentage and absolute numbers of CD4+IL-10+ T cells, representative of three independent experiments (n = 5 for each group). The p values were determined by unpaired t test. E, The same LN cells were also cultured for 48 h with mBSA (10 mg/ml), and the production of IFN-g, IL-17, and IL-10 was measured in the supernatants by ELISA. The p values were determined by unpaired t test.

4D). IL-102/2 T2-MZP Bregs also fail in upregulating Foxp3 Treg in arthritis (25). To address whether additional B cell subsets numbers and suppressing Th1/Th17 differentiation in both WT contribute to the induction of Treg in vivo, next we transferred FO, and IL-102/2 recipient mice, respectively (Fig. 4E and data not MZ, and T2-MZP Bregs isolated from AIA mice into arthritic shown), whereas T2-MZP Bregs from WT animals can suppress mMT mice. We used mMT mice as recipients in this set of Th1 and Th17 cells in these recipients. experiments to eliminate the potential participation of endogenous The aforementioned experiments suggest that T2-MZP Bregs B cells interacting with transferred IL-10+/+ B cells in the in- are responsible for the modulation of Treg numbers in vivo. How- duction of Tregs. The results in Fig. 4F report a significant de- ever, MZ B cells have also been previously shown to produce IL-10 crease in CD4+Foxp3+ Treg frequencies and absolute numbers in The Journal of Immunology 5575

FIGURE 4. Transfer of T2-MZP Bregs resets the balance between Tregs and Th1/Th17. T2-MZP cells (2 3 106), isolated from WT mice in AIA remission, were transferred i.v. to syngeneic IL-102/2 mice or to WT mice on the day of disease onset. Control group received a PBS injection. A, The bar chart shows the percentage increase in knee size on day 3 relative to the size of the same knee prior to disease onset. One independent experiment, representative of three, is shown (n = 5 for each group). All data were compared by statistical analysis using the Fisher test. B, Representative dot plots showing CD4+Foxp3+ T cells in draining LNs (isolated from different groups) and corresponding bar chart showing mean 6 SEM. One independent experiment, representative of three, is shown (n = 5 for each group). The p values were determined by unpaired t test. C, Bar chart shows mean 6 SEM of Th1 and Th17 frequencies in the draining LNs of IL-102/2 mice either with or without transfer of WT T2-MZP Bregs (2 3 106). One independent experiment, representative of three, is shown (n = 5 for each group). The p values were determined by unpaired t test. D, T2-MZP B cells (2 3 106), isolated from WT and IL-102/2 mice in the remission phase of arthritis, were transferred i.v. to syngeneic WT mice on the day of disease onset. Control group (no cell transfer) received a PBS injection. Chart shows the percentage increase in knee size on day 3 relative to the size of the same knee prior to disease onset. 5576 Bregs PRESERVE THE Treg POOL

FIGURE 5. IL-10 produced by B cells dictates the duration of contacts established with CD4+CD252 T cells. A, B and CD4+CD252 T cells were isolated from the spleens of WT or IL-102/2 mice with AIA (5 d postdisease onset) by negative selection. B cells were stained using Cell Tracker Blue, whereas CD4+ T cells were stained with Cell Tracker green. Total of 5 3 105 B cells were then cultured with 5 3 105 CD4+T cells with mBSA (10 mg/ml) and with or without neutralizing anti–IL-10 mAb (10 mg/ml). After 0 and 30 min, cells were fixed using 2% paraformaldehyde solution. The simultaneous staining for Cell Tracker Blue and Green defines the formation of conjugates. Representative dot plots (for 30 min time point) and a time course to show conjugate formation are shown. One independent experiment, representative of two, is shown (n = 5 for each group). The p values were determined by unpaired t test. B, Splenic B cells were isolated by negative selection from IL-102/2 or WT mice 5 d postdisease onset and were pulsed for 1 h with 10 mg/ml mBSA. Total of 1 3 104 pulsed B cells were cultured with 1 3 104 CFSE-stained CD4+CD252 T cells negatively isolated from LN of WT mice 5 d postdisease onset. T and B cells were cultured with mBSA (10 mg/ml) and imaged in real-time for 20 min at 37˚C. Imaging was done using Nikon Eclipse TE 300 microscope, Plain Fluor ELWD 203/0.45 (Nikon) lens, and LaserSharp2000 acquisition software. The camera used was Laser System Radiance2100 (Bio-Rad). Analysis of cell interactions was done using Velocity (Perkin-Elmer) software. Data show the mean contact time established between IL-102/2 B cell and WT CD4+CD252 T cells compared with IL-10+/+ B cell and WT CD4+CD252 T cells. For each condition, a minimum of 40 randomly assigned contacts between B and T cells were blindly analyzed, and one experiment representative of two is shown. The p values were determined by unpaired t test. C, Bar chart showing percentage of contacts within three different time categories, ,100 s, 100–799 s, and .800 s, established between IL-102 B cells with WT CD4+CD252 T cells and WT B cells with WTCD4+CD252 T cells. The p values were determined by unpaired t test.

B cell-deficient mice compared with WT mice in both draining IL-10–competent B cells establish longer contact time with 2 2 LN and the affected joints. Adoptive transfer of T2-MZP Bregs CD4+T cells than IL-10 / B cells alone, and not any of the other B cell subsets transferred, induced Several reports suggest that different modes of cell interaction are + an increase in the percentages and absolute numbers of CD4 pivotal in determining a tolerogenic versus activatory outcome (26, + Foxp3 Tregs as compared with the levels found in mMT mice in 27). IL-102/2 B cells and IL-10+/+B cells isolated from arthritic both draining LN and the affected joints (Fig. 4G, Supplemental mice were cultured with arthritogenic WT CD4+CD252 T cells Fig. 4A). The increase in Treg frequencies was also IL-10 de- (1:1) and stimulated with mBSA for up to 30 min. Upon Ag 2/2 pendent, as T2-MZP Bregs isolated from IL-10 mice fail to stimulation, IL-10+/+ B cells form a significantly higher number of upregulate CD4+Foxp3+T cells in mMT mice (Fig. 4H, Supple- conjugates with WT CD4+ T cells than IL-102/2 B cells (Fig. 5A). mental Fig. 4B for relative absolute numbers). Together, these In addition, to further confirm the role of IL-10 in this interaction results strongly suggest that the IL-10 produced by T2-MZP Bregs we neutralized IL-10 production in WT CD4+CD252 T cells and induces Tregs and is also responsible for the suppression of cy- IL-10+/+ B cells coculture and found that the numbers of con- tokines that drive inflammation. jugates were significantly reduced (Fig. 5A). Next, we addressed

One independent experiment, representative of three, is shown (n = 4 for each group). All data were compared by statistical analysis using the Fisher test. E, Bar chart shows mean percentage of Foxp3+CD4+ Tregs 6 SEM (n = 4) from inguinal draining LN 5 d postdisease onset and is representative of three separate experiments. In the same inguinal draining LN, Th1 and Th17 frequencies were assessed following 5 h culture with PMA/ionomycin and brefeldin A. One independent experiment, representative of three, is shown (n = 4 for each group). The p values were determined by unpaired t test. F, Bar chart shows mean percentage of Foxp3+CD4+ Tregs 6 SEM (n = 6) from inguinal draining LN of WT and mMT mice 5 d postdisease onset and is representative of three separate experiments. G, T2-MZP Breg, FO, and MZ B cells (2 3 106) were isolated from WT mice in the remission phase of AIA and were transferred i.v. to syngeneic mMT mice at the day of disease onset. A control group of arthritic mMT mice received a PBS injection. The percentage of Foxp3+CD4+ Tregs in the inguinal draining LN 5 d postdisease onset was calculated. The results display the mean 6 SEM percentage of increase/decrease of Foxp3+CD4+ Tregs in each group (recipient of different a B cell subset) compared with the percentage of Treg in control mMT group. H, T2-MZP B cells (2 3 106), isolated from WT or IL-102/2 mice in AIA remission were transferred i.v. to syngeneic mMT mice on the day of disease onset. Bar chart shows mean percentage of Foxp3+CD4+ Tregs 6 SEM (n = 4) from inguinal draining LN 5 d postdisease onset and is representative of two separate experiments. The Journal of Immunology 5577 whether the lack of IL-10 production by B cells altered the du- interaction was observed between B and T cells cocultured ration of interactions with arthritogenic WT CD4+T cells in re- without any stimulation (data not shown). sponse to mBSA stimulation. B cells were isolated from the 2/2 spleens of arthritic IL-10 or WT mice and cocultured with + + 2 2 IL-10–producing B cells convert effector CD4 T cell to arthritogenic WT CD4 CD25 T cells, also Foxp3 (purity of + + Foxp32 T cells are shown in Supplemental Fig. 4C). Contacts Foxp3 CD4 T cells between B and T cells were monitored for the first 20 min of Our data so far suggest that longer contact time between IL-10+/+ culture. Contacts were defined as two cells in close proximity with B cells and CD4+CD252 T cells might result in regulatory cell their membranes flattened against each other (Supplemental Fig. differentiation, whereas shorter interaction time, such as that estab- 4D,4E) as previously described (28, 29). The analysis of the lished between IL-102/2 B cells and CD4+CD252 T cells, results overall time that the cells spent in contact revealed a quantitative in the induction of inflammatory cell types. IL-102/2 B cells and difference between the two groups (Fig. 5B). We observed a IL-10+/+ B cells isolated from arthritic mice were cultured with higher percentage of long contacts (contacts exceeding 800 s) arthritogenic WT CD4+CD252 T cells and stimulated with mBSA. between IL-10–competent B cells and WT CD4+ T cells compared WT CD4+CD252 T cells were cultured alone with mBSA as with IL-102/2 B cells and WT CD4+ T cells, whereas an increased a control. Minute amounts of IL-17, but high secretion of IL-10, percentage of brief contacts was observed between IL-102/2 was measured when WT CD4+CD252 T cells were differentiated B cells and WT CD4+ T cells (,100 s) (Fig. 5C). Very minimal in the presence of WT IL-10+/+ B cells; in contrast, a dramatic

FIGURE 6. IL-10 produced by B cells influences Treg differentiation. A, As described in Fig. 5, spleens from WT and IL-102/2 mice with AIA were excised 5 d postdisease onset, and CD4+CD252 T cells and B cells were negatively purified. Total of 1.5 3 105 IL-102/2 and IL-10+/+ B cells were then cultured with WT CD4+CD252 T cells for 48 h with mBSA (10 mg/ml) (1:1). Total of 3 3 105 WT CD4+CD252 T cells, 3 3 105 WT B cells, and 3 3 105 IL-102/2 B cells were also cultured alone with mBSA (10 mg/ml) as controls. Supernatants were collected, and the production of IL-17 and IL-10 was measured by ELISA. Bar charts show mean 6 SEM, and one experiment, representative of three, is shown (n = 5 for each group). The p values were determined by unpaired t test. B, Foxp3 expression on CD4+ T cells was assessed by FACS, and representative plots are also shown. Bar chart shows mean 6 SEM, and one experiment, representative of three, is shown (n = 5 for each group). The p values were determined by unpaired t test. C, CD4+CD252 T cells were isolated by negative selection from IL-10 GFP reporter animals (TIGER mice) 5 d postdisease onset. Spleens from WT and IL-102/2 mice were excised 5 d postdisease onset, and B cells were negatively purified. Total of 1.5 3 105 IL-102/2 and IL-10+/+ B cells were then cultured with WT CD4+CD252 T cells for 48 h with mBSA (10 mg/ml) (1:1). Expression of IL-10 (GFP+ cells) and Foxp3 on CD4+ gated T cells was assessed by FACS. The plots are representative of three independent experiments and for each group n = 5. ND, not detectable. 5578 Bregs PRESERVE THE Treg POOL increase in IL-17 production was observed when WT CD4+ T cells ducing IL-10 induce expansion of CD4+ and CD8+ Tregs (36–38). were cultured with IL-102/2 B cells (Fig. 6A). It is possible that Altogether, our results support the concept that the immune sys- B cells isolated from IL-102/2 mice induce IL-17 production tem has implemented a series of sophisticated check points con- because they might be more proinflammatory than IL-10–pro- sisting of several cell types that independently or synergistically ducing B cells. However, B cells isolated from IL-102/2 mice and contribute to the maintenance of Treg homeostasis in vivo. The WT produce similar amounts of IFN-g and IL-1b upon Ag stim- results in Fig. 1 clearly show an exacerbated arthritis in mice ulation (data not shown), suggesting that it is the IL-10 that di- lacking IL-10–producing B cells, demonstrating that these check rectly or indirectly modulates the production of IL-17. points are still in place despite inflammation. This reduction in proinflammatory T cells was mirrored by an Our data in Fig. 2 showed that in the absence of endogenous IL- increase in the Foxp3+ T cell population (Fig. 6B). Interestingly, 10 released by B cells, the few remaining Tregs display a reduced only IL-10–competent B cells were capable of converting CD4+ expression of Foxp3 but retained their suppressive function in CD252 (Foxp32) T cells to Foxp3+CD4+ T cells in response to vitro. Recent evidence shows that IL-10 produced by myeloid mBSA stimulation (Fig. 6B, Supplemental Fig. 4C for CD4+ cells is crucial in the maintenance of the expression of Foxp3+ CD252Foxp32 purity). Moreover, by taking advantage of GFP– Tregs in mice with colitis. However, unlike in our model, Tregs in IL-10 reporter mice (30), which permit a higher resolution of IL- the colitis model have lost suppressive capacity (12). One possible 10–producing CD4+ T cells, we demonstrated that the IL-10+ explanation for these discordant results is that whereas in colitis CD4+ T cell population differentiating in the IL-10+/+ B cells model, the expression of Foxp3 is nearly abrogated, in IL-102/2 cultured with WT CD4+T cells is Foxp32 (Fig. 6C), confirming B cell mice, the expression of Foxp3 is reduced. Of note, Tregs our and others previous findings showing that Bregs are also im- isolated from peripheral blood of RA patients also display reduced portant in the induction of T regulatory 1 (Tr1) populations in vitro numbers of Tregs compared with healthy controls, but retained the (25, 31). capacity to suppress proliferation (39, 40). Our data are also in broad agreement with data from patients with active RA or juvenile arthritis showing that the frequencies of Discussion Tregs in the synovia exceed those present in peripheral blood (39, Results from multiple studies have demonstrated that Breg-derived 41). It also remains to be established whether Tregs isolated from IL-10 is important in the prevention of autoimmune disorders the joints of IL-102/2 B cell arthritic mice are less suppressive including arthritis or in the promotion of cancer (1, 2, 14, 31, 32). than those isolated from WT chimeric mice. It has been reported Adoptive transfer of in vitro-manipulated B cells has been shown that addition of proinflammatory cytokines, which are usually in- to recruit Tregs to the site of inflammation (33). Hence, the rel- creased in the joints of patients with active RA, to synovial Treg/ evance of immunoregulation mediated by endogenous B cell- Teffector cocultures impairs the suppressive capacity of Tregs derived IL-10 in vivo remains completely uninvestigated. Our (41). This may suggest that despite being numerically unchanged, results revealed the existence of a previously unappreciated reg- the Tregs differentiating in IL-102/2 B cell mice may have lost ulatory pathway that maintains the number of Tregs and down- their suppressive capacity due to the increased exposure to regulates the differentiation of Th1 and Th17 pathogenic cells proinflammatory cytokines (Figs. 3E,6A). Alternatively, as has in vivo. We have previously shown that transferred Bregs can been previously shown, effector T cells at the site of inflammation suppress, even if Tregs were depleted prior to transfer, which may be less susceptible to suppression (42). Unfortunately, due to suggested that Bregs work independently from thymically derived the very low number of cells obtained from the inflamed joints, it Tregs (6). In this study, we show that IL-102/2 B cells fail in is technically not possible to perform coculture assays to assess converting CD4+ T cells into Foxp3+ Tregs in vitro and lack of the suppressive capacity of Tregs from the synovia of these mice. B cell-derived IL-10 in vivo profoundly reduces Treg numbers. By We have recently translated our mouse work into humans and taking advantage of the IL-10 GFP reporter mice, we have shown demonstrated that CD19+CD24hiCD38hi B cells, previously de- that, at least in vitro, the Breg-induced IL-10–secreting Tr1 cells scribed as B cells at the immature-transitional stage of devel- are Foxp32. opment, comprised the highest percentage of IL-10–producing In addition, our data show that in vivo, the ability of IL-10– B cells in response to CD40 stimulation compared with other secreting B cells to induce Foxp3+ Tregs is confined to the T2- healthy peripheral blood B cells (43, 44). In addition, CD19+ MZP Breg subset. Unfortunately, the currently available tools did CD24hiCD38hi B cells inhibited the differentiation of IFN-g– and not allow us to unequivocally discern in vivo whether the IL-10– TNF-a–producing CD4+ T cells (44). The existence of B cells producing T cells are Foxp3 negative. However, it is plausible to with regulatory function in humans has also been recently con- propose that Bregs, via the release of IL-10, are important in the firmed in an independent study (45). The results presented in this maintenance of a peripherally induced suppressive CD4+T cell study, together with our recent findings in man, are particularly pool comprising both Foxp3+ and CD4+IL-10+ Tr1 cells and the interesting in view of the recent work showing that long-term renal downregulation of the Th1/Th17 proinflammatory response. Fur- transplant tolerance in humans is associated with an accumula- thermore, it will be integral for the understanding of Breg biology tion of CD19+CD24hiCD38hi B cells (also known as transitional to ascertain whether these cells mediate this suppression by pro- B cells) and a greater number of transitional B cells expressing cessing and presenting Ag in addition to the secretion of IL-10. IL-10. In addition, the same set of patients also showed an in- Our results could also have important implications in the cancer crease in FOXP3+ Tregs (46). Therefore, it is tempting to specu- field, in which it has been suggested that Bregs play a pathogenic late that the increase in FOXP3 observed in this set of patients role by suppressing T cell activation (4, 32). Because a high might be directly related to the increase in transitional IL-10– number of Tregs are observed in several types of cancer, including producing B cells. lymphoma (34), it is tempting to intimate that there is a positive In conclusion, our results show the importance of Bregs as correlation between the upregulation of Bregs and Tregs. arbiters of the inflammatory response that can evoke other regu- A direct correlation between the numbers of DC and Tregs and latory cells (including Tr1 and Foxp3+ Tregs) to act in unison for an inverse correlation with Th1 and Th17 proinflammatory cyto- the reduction of proinflammatory cells types and prevention of kines has been previously reported (35). In addition, DCs pro- tissue damage. The Journal of Immunology 5579

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