Selective targeting of B cells with agonistic anti-CD40 is an efficacious strategy for the generation of induced regulatory T2-like B cells and for the suppression of lupus in MRL/lpr mice (original) (raw)

. Author manuscript; available in PMC: 2014 Jul 5.

Published in final edited form as: J Immunol. 2009 Mar 15;182(6):3492–3502. doi: 10.4049/jimmunol.0803052

Abstract

We have previously reported that IL10+ regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the Transitional-2 immature (T2) B cell pool (T2Bregs). Therapeutic strategies facilitating their enrichment or enhancing their suppressive activity are highly attractive. Here we report that agonistic anti-CD40 specifically targets T2 B cells and enriches B regs upon short term in vitro culture. Whilst transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40-generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, induced the differentiation of IL-10+CD4+T cells and conveyed regulatory effect to CD4+T cells. In addition, in vivo administration of agonistic anti-CD40, currently on trial for the treatment of cancer, halted and reversed established lupus. Taken together our results suggest a novel cellular approach for the amelioration of experimental lupus.

Keywords: B cells, tolerance, IL-10, antibodies, autoimmunity

Introduction

Bregs are present in several murine models of chronic inflammation, including collagen induced arthritis (CIA), inflammatory bowel disease, and experimental autoimmune encephalomyelitis (EAE) (1-3). This regulatory function appears to be directly mediated by the production of IL-10 and by the ability of B cells to interact with pathogenic T cells to dampen harmful immune responses (4). Although the most widely used markers for B regs are the expression of IL-10 and CD19, combinations of other surface molecules including CD1d/CD21 (2), CD5/CD1d (5) or CD1d, CD21, CD23 and CD24 (transitional 2 B cells: (T2)) (6) have emerged as additional markers for the identification of this subset of B cells.

It has previously been shown that, in contrast to immunized wild type mice, chimeric mice lacking IL-10 or CD40 exclusively on their B cells fail to recover from EAE (3). In addition, transfer of B cells from TCRα−/− mice to B cell deficient TCRα−/− mice with established colitis, markedly decreased the number of pathogenic colonic CD4+ TCRα−ß+ T cells in recipient mice while B cells isolated from CD40KO mice failed to confer similar protection (7). We have also shown that stimulation of arthritogenic splenocytes with anti-CD40, stimulates B cells to produce IL-10 (1). Even though activation of B cells via CD40 appears to be a requisite for the production of IL-10, it remains to be established whether all B cells, or a discrete subset, are the direct targets of CD40-induced IL-10 production. Similarly to T regs, the low number of B regs (and the unknown phenotype) limits the possibilities of using them as cellular therapy. Thus, the discovery that a specific B cell subset is the target of anti-CD40 stimulation would lead to an optimization of Breg expansion, a key factor for cellular therapy.

Patients with systemic lupus erythematosus (SLE) and MRL_/lpr_ mice spontaneously develop a severe autoimmune disease characterized by hyper-gammaglobulinemia, immune-complex associated end organ disease of the kidney, and production of anti-dsDNA, anti-small nuclear ribonucleoprotein (snRNP) and rheumatoid factor specificity auto-antibodies (8, 9). Here we report an efficacious therapeutic strategy, which polyclonally enriches a defined set of B regs, contained within the T2 B cell subset, previously reported to control experimental arthritis (6). Upon adoptive transfer, these newly generated T2-like B regs reversed autoimmunity in MRL/lpr mice, suppressed Th1 responses and induced the differentiation of IL-10+CD4+T cells, able to convey suppressive effect to other T cells. This protective and immunosuppressive effect was abrogated by in vivo neutralization of IL-10. Finally, in vivo administration of anti-CD40 reversed nephritis and enhanced survival in mice with new onset disease by increasing the number of T2 B cells, which we show to be numerically impaired in diseased mice.

Materials and Methods

Mice

MRL/Mp-Tnfrsf6_lpr_ (MRL/lpr), C57/BL6 and MRL/Mp mice were purchased from Harlan (Oxon, UK) and housed in a specific pathogen-free animal facility at UCL. All animal studies were conducted in accordance with protocols approved by the Home Office (United Kingdom). hCD20-transgenic mice were previously made by using bacterial artificial chromosomes incorporating the hCD20 locus. To generate hCD20-transgenic MRL/lpr mice, we backcrossed the founder line to MRL/lpr mice for over 15 generations (10).

Antibodies

The treatment antibodies used were: rat IgG agonistic mAb reactive with CD40 (FGK45, provided by Prof D. Gray, Edinburgh, UK), rat IgG, blocking mAb reactive with mouse IL-10 receptor (1B1.2, purchased from ATCC), rat IgG1 anti-IL-10 (JES5-2A5 ATCC), anti-mouse IgM [F(ab’)2] (R6-60.2, BD Biosciences), AFRC-Mac-1 (isotype control) rat IgG anti-dog chlamydomonas cell wall glycoprotein (European Collection of Animal Cell culture, Salisbury, UK), anti-TGF-β antibodies (clones 1D11 was purchased from R&D systems). The mAbs were purified from culture supernatants by affinity chromatography, using a staphylococcal protein G column (Bioprocessing, Durham, UK) and filter sterilized. All antibodies used throughout the experiments were below the limit of detection for LPS levels using the Limulus Amebocyte lysate test (Bio Whittaker Inc.). The following anti-mouse antibodies were purchased from BD Biosciences, anti-CD19-Phycoerythrin(PE)-Cy7 (1D3), anti-CD21-Fluorescin isothiocyanate (FITC) and (7G6), anti-CD23-PE (B3B4), anti-IgM-Allophycocyanin (APC) (II/41), anti-CD24-biotin (M1/69), anti-CD4-FITC (H129.19), anti-CD25-PE (7D4), anti-CD1d-PE (1B1), anti-AA4-PE, anti-IL10-APC (JES5-16E3), anti-TNFα-APC (MP6-XT22), anti-IFNγ-APC (XMG1.2). Goat anti-IgG-FITC, anti-mouse IgG-Alkaline phosphatase (AP), IgG1-AP and IgG2a-AP from Southern Biotechnology Associates (USA). Purified anti-mouse CD3ε (145-2C11 (BD Biosciences) and F(ab’)2 Fragment goat-anti-mouse IgM (Jackson Imunoresearch) were used for in vitro assays.

Cell Culture

Murine cells were cultured in RPMI 1640 containing L-glutamine and NaHCO3 (R8758 Sigma-Aldrich, UK) supplemented with, 100 U/μg/ml penicillin/streptomycin (Life Technologies), and 10% FCS (BioWhittaker) in 96-well U-bottom plates (Nunc).

Murine lupus assessment

Survival was assessed in MRL/lpr mice that died of disease spontaneously and those sacrificed due to general debility. Urinary protein levels were assessed weekly semi quantitatively using reagent strips for urinalysis (Albustix; Bayer Corporation, Dublin, Ireland). Histopathology was evaluated on paraffin-embedded, formalin fixed tissue section by routine H&E staining. IgG immune deposits were determined by direct immunofluorescence on 5μM OCT-embedded frozen kidney sections, using FITC anti-mouse IgG. Sections were analysed with a Bio-Rad MRC 1024 confocal system (Bio-Rad Laboratories, Hertfordshire, UK) equipped with an argon and helium/neon laser for excitation at 522 and 585nm. Images were acquired with the LaserSharp 3.2 software and analysed using Confocal Assistant 4. Histopathological assessments were determined in a blinded fashion by one of us (A.K.C.) Kidneys were graded for glomerular inflammation, proliferation, crescent formation, and necrosis. Interstitial changes and vasculitis were also noted. Scores from 0 to 3 were assigned for each of these features and then added together to yield a final renal score. For example, glomerular inflammation was graded: 0, normal; 1, few inflammatory cells; 2, moderate inflammation; and 3, severe inflammation. Serum titers of IgG anti-dsDNA Abs were measured as previously described using alkaline phosphatase conjugated goat anti-mouse IgG (BD Biosciences). The assay were calibrated using pooled serum from 5 MRL/lpr mice, 19 week old, which was arbitrarily assigned a value of 1500U/ml at 1:100 dilution; all readings were related to that using appropriate 1/2 serial dilutions starting from 1/100. For analysis of anti-dsDNA IgG isotypes, anti mouse IgG1-AP and IgG2a-AP were used. Assessment of skin disease. All mice were free from skin disease at the start of experiments. Skin disease was scored as follows: 0 – no rash, 1 – beginning of rash but little or no hair loss, 2 – discrete hairless rash < ~10mm in length, 3 – extensive rash > ~20mm in length, 4 – extensive rash that includes the ears.

Flow Cytometric analyses and intracellular staining

All staining profiles were based on live-gated cells, as determined by 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, UK).

Mouse Cytokine Detection

Staining for flow cytometric analyses were performed on single cell suspensions in 96-well U-bottom plates. 5×105 cells were stimulated for 48h with combinations of agonistic anti-CD40 antibody (5μg/ml), anti-CD40 isotype control (5mg/ml), and anti-IgM [F(ab’)2] (10μg/ml), CpG (ODN 1826, at 25μg/ml), LPS (E. coli)(10μg/ml) for B cells, or 1μg/ml anti-CD3 for T cells/B-T co-cultures. GolgiStop™ (BD Biosciences), PMA (50ng/ml; Sigma-Aldrich, UK), and ionomycin (250ng/ml; Sigma-Aldrich, UK) were added for the last 6h of culture. To detect surface Ags, cells were washed with PBS/FCS/azide and then stained with combinations of CD19-PE-Cy7, CD4-FITC, CD21-FITC, CD21-Biotin, CD23-PE, CD24-Biotin. Cells were fixed and permeabilized by incubation in Cytofix™ (BD Biosciences) for 20 minutes at 4°C, followed by washing in 1 × Permwash™ (BD Biosciences) at 4°C. Permeabilized cells were incubated in 1 × Permwash™ with anti-mouse IFNγ-APC, TNFα-APC, IL-10-APC or appropriate APC-conjugated isotype controls (BD Biosciences). To demonstrate the specificity of staining, fixed/permeabilized cells were incubated with excess of unlabeled anti-IFNγ or anti-IL-10 (BD Biosciences) prior to incubation with APC anti-IFNγ or APC anti-IL 10 or APC anti-TNFα. The cells were acquired by FACS scan LSR (BD Biosciences) and analyzed using FlowJo v5.7.1 software (Tree Star, Inc. USA). Alternatively, supernatants were collected, before the addition of Golgi Plug™, and IL-10, IFNγ and TNFα levels were measured by ELISA (BD Biosciences), according to the manufacturer’s instructions.

Cell sorting and transfer experiments

For the purification of mouse B cells, single cell suspensions from spleens isolated from 8-10 week old MRL/lpr mice were incubated for 15 min at 4°C with 10 μl of anti-CD43 magnetic beads per 107 cells for negative selection or CD19+ magnetic beads for positive selection (Miltenyi Biotec Germany) per 90μl of cell suspension. The cells were washed twice to remove unbound beads re-suspended in 500μl of MACS buffer (PBS, 0.5% FCS, 2 mM EDTA) and purified using a magnetic activated cell-sorting (MACS) system. FACS staining for the B cell marker CD19 was used to control cell purity. This procedure normally yielded B cell preparations which were > 95% CD19+. Purified B cells were cultured in complete RPMI medium (11) (Sigma-Aldrich, UK) for 48h with combinations of agonistic anti-CD40 antibody (5μg/ml), anti-CD40/isotype control (5μg/ml), and anti-IgM [F(ab’)2] (10μg/ml). After 48h incubation B cells were stained with anti-mouse anti-CD19-Cy7, anti-CD21-FITC, anti-CD23-PE, anti-CD24-biotin, and DAPI and sorted by a MoFlo cell sorter (DakoCytomation) or a BD FACSAria (BD Biosciences). 5 × 105 cells of a given sorted subset were injected into the tail veins of 9-10 week old MRL/lpr, once a week for a total of three weeks. Alternatively T2 or MZ B cell subsets were firstly sorted by a MoFlo cell sorter (DakoCytomation) or a BD FACSAria (BD Biosciences) and then stimulated with anti-CD40 (5μg/ml) or with an isotype control (5μg/ml) for 48hr. For the purification of mouse CD4+CD25−T cells, splenic CD4+ T cells were isolated from 8-10 week old MRL/lpr mice by negative selection using a CD4 T cell Isolation Kit and passing through a MACS LS column (Miltenyi Biotec) as previously described (1). The purity was checked after purification and resulted to be 98% (data not shown).

In vivo treatment of MRL/lpr mice with anti-CD40

Nine to 10-week-old MRL/lpr mice were given a daily i.p. injection of ~ 300μg/day anti-CD40 (FGK-45) in PBS for two weeks. Following a two week break the treatment was repeated for a further two weeks. A control group of nine 10-week-old MRL/lpr mice received isotype control over the same time periods. Disease progression was monitored until the majority of control mice had succumbed to disease.

Proliferation assay

5×105 cells/well were incubated in triplicate in 96-well plates coated with 2.5μg/ml anti-mouse anti-CD3. After 72hr in culture, cells were pulsed with 1μCi of [3H]thymidine for the remaining 18hr of culture. Proliferation was measured using a liquid scintillation counter.

Statistical analysis

For the statistical analysis of the data, the Mann-Whitney U test and the Fisher Exact test were applied to analyze clinical results. Unpaired t-tests were applied on cytokine quantification experiments. p<0.05 was considered significantly different.

Results

Numerical and functional impairments of Bregs in MRL/lpr mice

The low frequency of IL-10 producing T2 B cell, represents the major obstacle to empowering this subset for the treatment of autoimmunity. The numbers of T2 and MZ B cells (gated as previously shown (6, 12)) were measured in the spleens of MRL/lpr mice at different ages. The results in fig. 1A show a significant reduction in the absolute numbers of T2 B cells with the progressing age but an increase in MZ B cells. The significant inverse correlation between proteinuria and the absolute numbers of T2 B cells further supports a relationship between disease progression and reduction of T2 B cell numbers (fig. 1B). MRL/Mp mice, which also develop lupus-like disease (although less rapidly than MRL/lpr mice), display an increased ratio of MZ to T2 B cells compared to non-prone C57BL/6 mice (fig. 1C and D). However, the numbers and percentages of MZ and T2 B cells did not change with age in C57BL/6 and MRL/Mp mice, suggesting that a decrease in T2 B cell numbers is a feature exclusive to MRL/lpr mice.

Fig. 1. T2 and MZ B cells subsets fail to confer protection to MRL/lpr mice.

Fig. 1

Splenic B cells were labelled with CD19, CD21, CD23, CD24. MZ, FO and T2 B cells were gated as previously described (6, 12). (A) Absolute numbers of MZ and T2 B cells in MRL/lpr mice at different age (B) Scatter plot showing a significant correlation between the proteinuria and the absolute number of T2 or MZ B cells at different age. Splenocytes were harvested from MRL/Mp or C57BL/6 mice at 9 and 24 weeks of age and stained with CD19, CD21, CD23, CD24. C) The percentage of T2 and MZ B cells at different ages. D) Absolute numbers of T2 and MZ B cells calculated as the numbers of cells/live events × the total number of cells in the spleen. 5 × 105 T2 or MZ B cells were transferred i.v. to 9-week old recipient MRL/lpr mice, once a week for three consecutive weeks (n=5/group). (E) Proteinuria was assessed at weekly intervals starting at 9 weeks of age. (F) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were sacrificed due to general debility. One group of mice was left untreated as control. The data shown are representative of three independent experiments.

We next assessed the suppressive capacity that T2 B cells isolated from MRL/lpr mice have in an adoptive transfer system. Purified T2 and MZ (as control) B cells were transferred to syngeneic mice. Proteinuria levels and the survival analysis showed no significant differences in mice treated with either B cell subset compared with the control group (fig.1 E,F and fig. S1A for example of purity post-sort). Therefore, these results suggest that T2 Bregs in MRL/lpr mice are functionally impaired.

Anti-CD40 stimulation induces IL-10 producing T2-like B cells

We addressed whether splenic B cells, isolated from MRL/lpr mice, cultured with agonistic anti-CD40 ±anti-IgM [F(ab’)2] (as a surrogate autoantigen) would expand IL-10 producing B cells. Supernatants collected from B cells stimulated with anti-CD40 contained significantly higher levels of IL-10 compared to supernatants from isotype and IgM treated B cells (fig. 2A). Simultaneous engagement of BCR and anti-CD40 lead to a significant reduction of IL-10 production compared to B cells stimulated with anti-CD40 alone. Further, intracellular staining revealed that the majority of the IL-10 producing B cells were contained within the T2 B cell subset (fig. 2B and fig. S1B). Further analysis of ex vivo CD40 stimulated T2 B cells demonstrated that they express high levels of CD1d, previously identified as a Breg marker (2) (5), and CD93, which in the spleen is expressed exclusively on immature B cells (fig. 2C). However, since stimulation with anti-CD40 is known to modulate the expression of CD23 on different B cells subset ((13) and our results supplemental data fig. S2A,B), hereafter T2 generated after stimulation with anti-CD40 will be referred as T2-like to distinguished them from un-stimulated immature T2 B cells.

Fig. 2. Anti-CD40 stimulation specifically expands IL10 producing T2-like B cells.

Fig. 2

Negatively purified splenic B cells from 9-10 week old MRL/lpr mice were cultured for 72h with: isotype controls (10μg/ml), anti-IgM [F(ab’)2] (10μg/ml)/isotype control (10μg/ml), anti-IgM [F(ab’)2] (10μg/ml)/anti-CD40 (5μg/ml) or anti-CD40 alone (5μg/ml)/isotype control (for anti-IgM [F(ab’)2]) (10μg/ml). (A) Supernatants were collected after 72h of culture and IL-10 was measured by ELISA. Data shown mean±se of triplicate wells, and are representative of three independent experiments each with a minimum of five mice. (B) Negatively purified splenic B cells were cultured for 48h with isotype control (5 μg/ml) or anti-CD40 (5 μg/ml). PMA/ionomycin and GolgiStop™ were added for the last 6h of culture. Cells were stained with CD19, CD21, CD23, and IL-10 mAbs. Data shown are representative of 12 mice. (C) The changes in the levels of expression of CD1d and CD93 (AA4) T2-like or mature FO-like and MZ-like B cell subsets, following 48h anti-CD40 stimulation of purified B cells. (D) T2 and MZ B cells were first sorted and then cultured with isotype control (5 μg/ml) or anti-CD40 (5 μg/ml) for 72h. IL-10 production in the supernatants was measured by ELISA. Data shown are representative of three independent experiments. (E) T2, MZ and FO B cells were first sorted and then cultured with isotype control (5 μg/ml), anti-CD40 (5 μg/ml), LPS (10μg/ml) or CpG (25μg/ml) for 48h. PMA/ionomycin and GolgiStop™ were added for the last 6h of culture. IL-10 producing B cells were measured by intracellular staining.

No differences in the IL-10 production were found if T2 B cells were sorted first and directly stimulated with anti-CD40, (as opposed to pre-stimulation of all B cells followed by sorting) (fig. 2D). Interestingly, CPG and LPS, which have been previously shown to up-regulates IL-10 producing B cells (14), do not have the same selective effect as anti-CD40 on purified T2, but induce the differentiation of equal frequencies of IL-10 producing MZ and T2 B cells (fig. 2E). Hence anti-CD40 stimulation induces T2-like B cells that make IL-10. Of interest, in contrast to BCR stimulation, anti-CD40 stimulation prevented the spontaneous differentiation of sorted T2 B cells into a “mature” B cells (fig. S3). Since anti-CD40 stimulation of purified B cells yielded higher numbers of viable T2 B cells (compared with anti-CD40 stimulation of purified T2-like B cells), we have used this protocol for the remainder of the experiments.

T2-like B cells suppress Th1 responses and convey suppressive capacity to CD4+CD25− T cells

To assess if T2-like B cells have acquired suppressive function, T2, MZ or FO-like B cells were cultured with anti-CD3 stimulated CD4+CD25− T cells. Only T2-like, but not FO or MZ-like, or isotype treated B cells, significantly inhibited the production of the IFNγ and TNFα by CD4+CD25−T cells (fig. 3A, B and fig. S4 for dose response). No IL-4 was detectable in any of the tested conditions (data not shown). The suppressive capacity of T2-like B cells was comparable to the inhibitory capacity exerted by Tregs (fig. 3C, D). In addition, only T2-like B cells induced the differentiation of IL-10 producing T cells (fig. 3E). The majority of the IL-10 producing T cells following co-culture with T2-like B cells were FoxP3− (around 90% of the IL-10+CD4+T cells are FoxP3− whilst around 10% are FoxP3+), and there was no overall increase in FoxP3 expression under the same conditions (fig. 3F,G). Despite their capacity to suppress the release of Th1-like cytokine no inhibition of T cells proliferation was detected (data not shown).

Fig. 3. In vitro, T2-like B cells suppress the production of inflammatory cytokines by CD4+CD25−T cells and induce the differentiation of suppressor T cells.

Fig. 3

Splenic B cells were incubated with anti-CD40 or an irrelevant isotype control for 48h. DAPI-T2, FO, MZ like B cell subsets, or isotype treated B cell subsets, were sorted according to the gates described in figS1. CD4+CD25−T cells were MACS sorted from age matched control. CD4+CD25−T cells were cultured alone or with T2-, FO-, MZ-like B cells (1:1) and stimulated with anti-CD3 (1μg/ml). A) CD4+TNFα+ T cells, B) CD4+IFNγ+ T cells were measured by intracellular staining.

CD4+CD25−T cells were cultured either alone, with T2-like B cells (1:1) and with both CD4+CD25+T cells:T2-like B cells (1:1:1) stimulated with anti-CD3. (C) CD4+TNFα+T cell, (D) CD4+IFNγ+T cell, or (E) CD4+IL-10+T cells. CD4+CD25−T cells were cultured either alone or with T2-like B cells (1:1) (F). Expression of IL-10 and FoxP3 on CD4+ gated T cells cultured alone (G) or with T2-like B cells. (H) MACS purified CD4+CD25-T cells were cultured either alone or with T2-like B cells and stimulated with anti-CD3 (1μg/ml). 72h later from the first round of stimulation, CD4+ T cells which have been in culture with either T2 B cells or were cultured alone were re-purified by negatively selection (MACS® kit) stained with CFSE (CFSE+CD4+), and cultured 1:1 (1×106/ml total cells) with freshly purified syngeneic CD4+CD25−T cells (CD4+) for an additional 72h with anti-CD3 (1μg/ml). TNFα produced by freshly isolated CD4+T cells alone (CD4+CTRL), or by CD4+CTRL cultured with CFSE+CD4+ T cells re-isolated from CD4:T2-likeB cells culture on the 1st round of stimulation, or by CFSE+CD4+CTRL cultured with CD4+T cells cultured alone on the first round, was measured by intracellular cytokine staining. All the data shown are representative of three independent experiments.

The generation of newly formed IL-10 producing T cells could be a mechanism by which T2-like B cells might control inflammation in vivo. Anti-CD3 stimulated CD4+CD25−T cells were cultured 1:1 with T2-like B cells for an initial 72h (1st stimulation). These CD4+T cells were then purified, stained with CFSE (CFSE+CD4+) and cultured 1:1 with freshly isolated syngeneic CD4+CD25−T cells (CD4+ CTRL) for an additional 2 days (2nd stimulation). The result in fig. 3H showed that only CFSE+CD4+ (CD4+T cells which have been in culture with T2-like B cells) have acquired suppressive function and inhibit the production of TNFα by “fresh” CTRL CD4+T cells in the second round of stimulation. Neither CD4+T cells derived from CD4+T: FO cell cultures, CD4+T:MZ cell cultures, nor T cells purified from isotype-control treated T2 B cells acquired suppressive activity (data not shown). The lack of expression of Foxp3, the capacity to produce IL-10 and the ability to suppress CD4+CD25−T cells are hallmarks of Tr1 cells (15), thus demonstrating that T2-like B cells suppress Th1 differentiation and induce the differentiation of Tr1 cells.

T2-like B cells control the progression of lupus, suppresses T cell proliferation and differentiation into Th1 cells

Our findings raised the possibility that ex vivo expansion with anti-CD40 might empower T2-like B cells with regulatory capacity and allow them to suppress disease. 5×105 T2, MZ and FO-like B cells were purified by flow cytometry (purity in fig. S1), and transferred to 9-10 week old MRL/lpr mice. Only transfer of T2-like B cells significantly decreased mortality of recipient mice. At 25 weeks of age, 100% of the control and over 70% of MZ-like and FO-like B cells treated mice had succumbed, while only 33% of T2-like B cell treated mice had died (fig. 4A). This decrease in mortality was mirrored by lower levels of proteinuria. At 3 months of age 71% of the PBS treated mice, or mice transferred with MZ-like B cells, had proteinuria levels higher than 300mg/dl compared to only 25% in the T2-like B cells treated group (fig. 4B). Histological examination of the kidneys from 23 week old mice receiving T2-like B cells revealed reduced levels of immunoglobulin deposition, minimal glomerular hypercellularity and interstitial infiltration and showed a relative preservation of structure. In contrast, kidneys from control or from mice treated with MZ or FO-like B cells demonstrated the classical severe histological picture that characterizes this strain of mice (fig. 4C,D,E). IgG antibody levels against dsDNA (fig. 4F) and against anti-Sm (data not shown) were also lower in T2-like treated mice. Interestingly, in the T2-like B cell treated group the isotype of anti-dsDNA switched from IgG2a to IgG1 suggesting a skew from a pathogenic Th1 response to a more favourable Th2 response (fig. 4F). A similar suppressive capacity was observed if sorted T2 B cells were stimulated directly with anti-CD40 and then transferred to syngeneic mice, whereas no suppression was observed by anti-CD40 stimulated MZ B cells (fig S5).

Fig. 4. T2-like B cells control the progression of lupus in MRL/lpr mice.

Fig. 4

Splenic B cells from 9-10 week old MRL/lpr mice were cultured for 48h with anti-CD40 (5μg/ml). DAPI− T2, MZ and FO-like B cells were FACS sorted and 5×105 cells of each subset (purity around or above 90%) were transferred once a week for 3 weeks to 9-10 week old MRL/lpr mice (n=6/group). Control mice received PBS injections. (A) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were sacrificed due to general debility. (B) Levels of proteinuria in recipient mice were measured once a week. (C) Glomerulonephritis in recipient mice was identified by H&E-staining of kidney sections and, (D) IgG deposition was identified by immunofluorescence with anti-mouse IgG. Kidneys in (C) and (D) were sectioned and stained at 23 weeks of age. (E) Composite renal disease. (F) Sera from 23 weeks old mice were assessed for total anti-dsDNA and anti-dsDNA IgG isotypes by ELISA. Splenocytes were isolated from MRL/lpr mice one week after receiving the final transfer of anti-CD40 stimulated B cell subsets. (G) Splenic cells were stimulated with anti-CD3 for 72 h. Proliferation was determined by 3[H]Tdr incorporation. Reduced proliferative response compared to control group, was also observed when CD4+T cells were purified from T2-like treated and re-stimulated in vitro with anti-CD3 (data not shown). IFNγ and IL-10 production in the supernatants was measured by ELISA. All the data shown are representative of three independent experiments.

The total numbers of CD4+, CD8+ T cells and double negative T cells were significantly reduced in mice given T2-like B cells compared with the other groups (fig. S6). Splenocytes from the T2-like B cell treated group also showed a weaker response to anti-CD3 stimulation than other groups. This defective responsiveness, together with a significant reduction in IFNγ production, was mirrored by an increase in IL-10 production (fig. 4G) by CD4+T cells (data not shown). Thus, transfer of T2-like B cells inhibits T cell proliferation and Th1 responses in favour of an anti-inflammatory IL-10 response.

The protective effect of T2-like B cell transfer is IL-10 dependent

To assess the requirement of IL-10 for the protective effect observed in vivo, T2-like B cells were transferred to syngeneic mice that were treated with isotype control, or with anti-IL10R/anti-IL10 mAbs (16). A group of mice was treated with anti-IL10R/anti-IL10 mAbs alone. 100% of mice treated with T2-like B cells were still alive at 20 weeks of age, compared with only 20% in the group treated with T2-like B cells and anti-IL-10R/anti-IL-10 or in the control group (fig.5 A,B). Whereas T2-like treated mice produced significantly less total IgG and IgG2a anti-dsDNA antibody (fig.5 C,D), neutralization of IL-10 restored the levels of total anti-dsDNA and the IgG2a antibodies to the same level as the control group. Interestingly, previous work in CIA model has shown that blockade of IL-10/IL-10r at an early stage of disease development does not affect disease course (17). Similarly in MRL/lpr mice the short period of blockade of IL-10, while sufficient to inhibit the immune-regulatory cascade initiated by B regs, did not exacerbate the disease.

Fig. 5. Neutralization of IL-10, but not TGFβ, abrogated protection conferred by transfer of T2-like B cells.

Fig. 5

MRL/lpr mice were treated once a week for 3 weeks with T2-like B cells. Mice were treated with anti-IL-10r/IL-10 and with matching irrelevant isotype controls for anti-IL-10R and anti-IL-10 as previously shown (control) (16). (A) Survival rate. (B) Levels of proteinuria was assessed at weekly intervals. (C, D) antibody levels. Sera from 20 week old mice were assessed for total anti-dsDNA IgG and anti-dsDNA IgG isotype by ELISA. The data shown are representative of two independent experiments. MRL/lpr mice were treated once a week for 3 weeks with T2-like B cells. Mice were treated i.p. with anti-TGF-β (2 mg/mouse) or with matching irrelevant isotype controls at day 1 of cell transfer and twice weekly for the duration of the experiment. (E) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were sacrificed due to general debility. (F) Levels of proteinuria in recipient mice were measured once a week. The data are representative of two independent experiments.

Unlike neutralization of IL-10, blocking TGFβ did not inhibit the suppressive capacity of T2-like B cells as shown by the unchanged levels proteinuria levels or mortality rate amongst the different groups (fig. 5E, F).

In vivo tracking of the developmental kinetics of T2-like B cells

To ascertain where T2-like Bregs exert their suppressive activity, anti-CD40 stimulated splenic hCD20Tg B cells (10) were transferred to wild type MRL/lpr mice. Analysis of the B cells subsets at day 1 post transfer showed that hCD20Tg B cells homed to the spleen and maintained a T2:MZ:FO B cell ratio similar to the endogenous ratios measured in the wild type mice (fig. 6A). Seven days after transfer, FO B cells had migrated from the spleen into the LN (fig. 6B). In agreement with the non-recirculating nature of “conventional” T2 and MZ B cells, hCD20+Tg T2 and MZ-like B cells were recovered prevalently from the spleen, suggesting that stimulation via CD40 does not alter their migratory capacity. No hCD20+TgB cells were found in the kidneys of the recipient mice (data not shown).

Fig. 6. Differentiation of transferred hCD20+T2-like B cells.

Fig. 6

Splenic B cells from 9-10 week old hCD20Tg MRL/lpr mice were cultured for 48h with isotype or anti-CD40. Approximately 2×107 hCD20Tg MRL/lpr B cells were transferred to age matched MRL/lpr mice. Host spleens and inguinal LN were harvested after transfer at the indicated time points, and re-stained with CD19, CD21, CD23, CD24 and anti-human CD20. Representative FACS plots of hCD20+ B cell subsets recovered from spleen and lymph node (A) 1 day post transfer (B) 7 days post transfer. 1-2 ×106 sorted hCD20Tg (C) T2 isotype control (D) or anti-CD40 treated T2-like B cells were transferred to 9-10 week old MRL/lpr mice. Recipient spleens were harvested 7 days after transfer, and re-stained with CD19, CD21, CD23, anti-humanCD20. Gates were pre-set on endogenous B cells subset. Representative FACS plots of one out of 4 independent transfers using a minimum of 5 wild type mice as recipients.

Our in vitro experiments suggest that CD40 engagement on T2 B cells alters their maturation. To assess T2-like maturation in vivo, we transferred sorted hCD20 T2-like or isotype treated T2 B cells to wild type mice. 7 days after transfer spleens and draining LN were harvested. Virtually all recovered hCD20+T2 B cells stimulated in vitro with isotype control had differentiated into FO or MZ B cells (fig. 6C). In contrast, only 40% of transferred T2-like B cells had differentiated into mature FO B cells with around 40% of the T2-like B cells retaining their original “immature-like” phenotype (fig. 6D). This was highly consistent over different experiments. Due to their very low frequency we have not been able to re-isolate undifferentiated transferred T2-like B cells and compare their suppressive function with those that have differentiate into mature B cells. Since the transfer of mature FO B cells failed to protect recipient mice from disease and, in vitro, FO B cells did not inhibit T cell inflammatory cytokine production (Fig. 3A,B) it is plausible to hypothesize that disease suppression is mediated by T2-like Bregs retaining their undifferentiated phenotype.

In vivo administration of anti-CD40 ameliorates lupus activity in MRL/lpr mice and increases T2 B cell numbers

Agonistic monoclonal antibodies to CD40 (CD40 mAbs) have a puzzling dual therapeutic effect in experimental animal models. CD40 mAbs induce tumor regression by potentiating anti-tumoral T-cell responses (18), yet we have previously shown that anti-CD40 exert immunosuppressive activity in chronic autoimmune inflammatory processes (19). Our results showing that the transfer of adaptive T2-like B reg protect MRL/lpr mice from disease suggested that T2 B reg cells could be more sensitive to agonistic CD40 stimulation in vivo than other B cells subset. MRL/lpr mice were treated for four weeks with 300μg/day anti-CD40 i.p., or with isotype control. Anti-CD40 treated mice survive significantly better than control treated mice (p = 0.0461) (fig. 7A), displayed significantly lower proteinuria than controls (p = 0.0145) (fig. 7B), suffered less severe skin disease (p < 0.05) (fig. 7C) and displayed a significantly reduced amount of anti-dsDNA IgG (fig. 7D). Importantly, analysis of B cells subset two weeks after the beginning of the treatment, showed a significant increase in T2 B cell numbers (fig. 7E,F). Identical suppressive results were also obtained using a different agonistic anti-CD40 (3/23) (data not shown), suggesting that the suppressive effect is not due to epitope-specificity of the FGK45 antibody. To ascertain the effect of anti-CD40 treatment on different cell populations in the spleens of treated mice compared to the control group, mice were treated for two weeks and splenocytes were collected at different time to establish the kinetic of cytokine production. The results in 7G show a significant increase in the frequency of CD19+IL-10+ cells over the treatment period compared to control treated group. In contrast, CD40+CD19− cells appeared to remain unaffected by the treatment and produced an equivalent amount of pro-inflammatory cytokines at each time point (fig. 7H). These results provide additional evidence that anti-CD40 mAb therapy targets T2 B regs and provides initial support for the notion that anti-CD40 treatment similarly to rituximab, could be used in treatment for autoimmune diseases.

Fig. 7. Therapeutic effect of agonistic anti-CD40 in MRL/lpr mice.

Fig. 7

A group of nine 10-week-old MRL/lpr mice were treated daily with 300μg/day anti-CD40 (FGK-45) for four weeks. A control group of nine 10-week-old MRL/lpr mice received isotype control injections (AFRC MAC-1) over the same time periods. Disease progression was measured by survival, proteinuria and skin disease. (A) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were scarified due to general debility. (B) Levels of proteinuria in recipient mice, were measured, once a week, by Albustix. Groups of mice were compared by ANOVA or Mann-Whitney U statistical test. All mice had detectable level of proteinuria at the start of treatment. C) Skin disease 10 weeks after the start of treatment (20 weeks of age). All mice had a skin disease score of zero at the start of treatment. Scoring system: 0 – no rash, 1 – beginning of rash but little or no hair loss, 2 – discrete hairless rash < ~ 10mm in length, 3 – extensive rash > ~15mm in length, 4 – extensive rash that includes the ears. The p values were calculated by 2 way ANOVA. The data shown are representative of two independent experiments. (D) Sera from 23 week old CD40 and isotype treated mice were assessed for total anti-dsDNA and anti-dsDNA IgG isotypes by ELISA. (E) Representative FACS plots showing an increase in T2 B cells after anti-CD40 treatment compared to isotype treated mice. (F) Bar chart showing the significant difference in the absolute B cell numbers between anti-CD40 and isotype treated mice. Mice were treated for two weeks with either 300μg/mouse/day of anti-CD40 or with an irrelevant isotype control. Splenocytes were isolated from treated mice at different time point, stimulated for 4 hrs with PMA, ionomycin and Golgi Stop. Frequencies of (G) IL-10+CD19+ B cells and (H) IFNγ+, TNFα+ and IL-10+ CD40+CD19− non-B cells were assessed by intracellular staining and flow cytometry.

Discussion

Adoptive cellular therapy for the control of autoimmune disorders has seen a surge of interest due to the feasibility of manipulating T regs in vitro (20). Even though it is now well established that B regs, like T regs, are directly involved in the maintenance of tolerance, no strategies for expanding them in vitro have been identified. In this study we demonstrate that in vitro B regs suppress Th1 differentiation, convey suppressive capacity to CD4+T cells and induces the differentiation of CD4+FoxP3−IL-10+ T cells. In vivo anti-CD40 generated T2-like B regs reverse lupus like disease and induce long-term tolerance via the inhibition of Th1 responses and T cell proliferation. It is interesting to observe that suppression of T cell proliferative responses was only observed after B reg transfer in vivo. These could be due to the initiation of a complex suppressive cascade reaction in vivo, which is unlikely to take place in vitro. The in vivo neutralization of IL-10 supports the hypothesis that IL-10 produced by the T2-like Bregs is responsible for the regulation of lupus-like disease in MRL/lpr mice. The role of Th17 in lupus, unlike in other autoimmune diseases, is not well understood (21). Since no IL-17 producing T cells were detected in the spleens of MRL/lpr mice, in the same experimental condition found to be optimal for T2-like B reg mediated Th1 suppression, it is at the moment difficult to conclusively surmise whether T2-like B regs suppress Th17 as well as Th1 differentiation. Our results are interesting in the context of the recent finding showing that the in vivo production of IL-10 induced by DC can lead to the differentiation of Tr1 (22). Our data together with those of others (17), suggest that Bregs should also be included in the “pool” of cells responsible for the differentiation of Tr1. It will be interesting in the future to assess whether chimeric mice lacking IL-10 exclusively on B cells, previously shown to lack Bregs (3), develop a “normal” pool of suppressive Tr1 cells. Unfortunately, the previously reported IL-10 deficient MRL/lpr mice known to develop an exacerbated disease compared to wild type mice (23), are no longer available. CD40-CD154 signalling is important for the differentiation of plasma cells and the production of auto-antibodies, however the same co-stimulatory pathway, depending on the density of CD154 expression, has also been implicated in preventing antibody production (24) and in inhibiting the development of chronic arthritis (19). Here we demonstrate that CD40 ligation halts the maturation of T2 B cells (fig. S3 and fig. 6) and induces the differentiation of IL-10+T2-like B cells. Our data (fig. S7) are also in agreement with previous studies showing that CD40 ligation rescues B cells and transitional B cells subset from apoptosis (25, 26) and prevents their further differentiation into mature FO B cells (27). From our data it appears that anti-CD40 specifically targets T2 B cells and induces the differentiation of IL-10 producing T2 Bregs, whereas anti-CD40/anti-IgM [F(ab’)2] stimulation induces the differentiation of TNFα and IFNγ producing mature B cells (data not shown), that may contribute to the pathogenesis of lupus disease. We speculate that the balance between antigen exposure (i.e BCR cross-linking) and the strength of CD40 engagement might not only influence B cell maturation (FO versus MZ differentiation for example) but could also affect the outcome of the immune response (activation versus regulation) (4).

Other B cell subsets have been associated with IL-10 production and immuno-regulation (14, 28, 29). Neonatal CD5+B cells, responsible for the lack of inflammatory responses in neonates, have been shown to secrete IL-10 upon TLR9 receptor stimulation and to control the priming capacity of neonatal dendritic cells. Moreover, adult CD5+CD1dhigh B cells have been shown to possess regulatory function in contact hypersensitivity (CHS) responses (5, 29). Gray et al have shown that MZ B cells produce higher levels of IL-10 than FO B cells, but made no comparison between T2 and MZ B cells and their capacities to produce IL-10 and suppressive capacity (14). We have analyzed the IL-10 production by purified B cells in response to CPG, LPS and anti-CD40 and show that whereas MZ produce more IL-10 in response to CPG than FO B cell, and equal amounts to T2-like B cells, the highest amount of IL-10 was observed when T2 B cells were stimulated with anti-CD40 (fig 2E). In addition, even though MZ B cells produce some IL-10 in response to anti-CD40 stimulation, given that in our model MZ-like B cells failed to confer protection we have excluded them as possible B reg candidates.

T2-like B cells retain all the archetypical characteristics of the T2 Bregs previously identified in the CIA model (6), including a CD23hiCD21hiCD24hiCD93CD1dhi phenotype (30) (31) (32) (33) (12). Nevertheless, markers like CD19, CD1d, CD21 and CD5, are consistently expressed by Bregs in different models (4) (5), suggesting the possible existence of a unique B reg lineage common to the different experimental models. It is feasible that the modulation of other markers expressed by Bregs (i.e CD5, CD23) may be up or down regulated according to environmental control (i.e cytokines or co-stimulatory signals provided by activated CD4+T cells). In this context, our data have shown that stimulation with anti-CD40 stimulation up-regulates the expression of CD23 on MZ B cells (fig. S2B). Therefore, Bregs generated after anti-CD40 stimulation, despite being phenotypically indistinguishable from “conventional” un-manipulated T2 B cells, might contain B cells originating from either MZ of FO B cells. Nevertheless, the data showing that the progression of spontaneous lupus-like disease in MRL/lpr mice correlates with a decrease in the absolute numbers and percentages of T2 and an increase in MZ B cell numbers, further support the existence of B regs within the “natural” T2 B cells in autoimmunity.

Taken together with the data showing that immature B cells are the repopulating subset after depleting anti-CD20 treatment both in RA patients (34, 35) and in the NOD mouse model (36), our observation that in vivo treatment with anti-CD40, increases IL-10 producing B cells, but has negligible effect on other CD40+cells, and expands T2 Bregs has important implications for clinical therapy. We could envisage a scenario where human immature B cells, also enriched of Bregs (P. Blair and C. Mauri unpublished results) could be isolated from rituximab treated SLE patients and expanded ex vivo with anti-CD40 and re-administered at the time of re-flaring as alternative to further B cell depletion. This could be coupled with lower amounts of immunosuppressant. These strategies could control the pathogenic response whilst re-establishing an enduring homeostatic balance.

Supplementary Material

01

Fig. S1. (A) B cells subsets purity. Splenic B cells were negatevely purified using anti-CD43 MACS Kit, according to the manufacturer’s instructions, from 9-10 week old MRL/lpr mice and cultured for 48h with anti-CD40 (5μg/ml). Cells were then stained with CD19, CD21,CD23,CD24 and DAPI and sorted by BD FACS ARIA (BD bioscience). Purity of B cell subset as indicated. (B) Splenocytes were cultured for 48h with isotype control (5μg/ml) or anti-CD40 (5 μg/ml). PMA/ionomycin and GolgiStop™ were added for the last 6h of culture. Cells were stained with CD19, CD21, CD23, and IL-10 mAbs. Data shown are representative of 10 mice.

Fig. S2. Anti-CD40 stimulation induces CD23 expression by purified B cell subsets. B cells were negatively purified from the spleens of 9-10 week old MRL/lpr mice and stained with CD19, CD21, CD23 and DAPI. DAPI− T2, MZ and FO B cells were purified by FACS. Purified B cells were cultured with isotype control or anti-CD40 (5μg/ml) for 48h. Cells were re-stained with CD19, CD21 and CD23. (A) Pre-sort B cells and post-sort purities. (B) Phenotypes of purified B cell subsets following isotype or anti-CD40 stimulation. Plots are representative of 4 experiments.

Fig. S3. Anti-CD40 stimulation prevents T2 B cells maturation into FO B cells. Purified CD19+CD21hiCD23hiCD24+ B cells were cultured with isotype control, anti-CD40 (5μg/ml) or anti-IgM F(ab’)2 (10μg/ml) for 96h. Cells were stained again with CD19, CD21, CD23 and CD24. Plots show purified T2 maturing into CD21intCD24− FO B cells following isotype or anti-IgM F(ab’)2 stimulation, but remaining T2 following anti-CD40 stimulation.

Fig. S4. CD4 + T cells:T2-like B cell dose response suppression. B cells were purified from the spleens of MRL/lpr mice by negative selection using the anti-CD43 MACS™ kit. Purified B cells were incubated with anti-CD40 (5μg/ml) or an irrelevant isotype control (5μg/ml) for 48h and then stained with CD19, CD21, CD23, CD24 and DAPI. T2-like B cells were sorted by MoFlo according to the gates in Fig S1. CD4+CD25− T cells were also purified from age matched MRL/lpr splenocytes by MACS™ kit. 5 × 105 CD4+CD25− T cells were cultured at different ratios with T2-like B cells. Cultures were stimulated for 48h with 1μg/ml anti-CD3. PMA, ionomycin and GolgiStop™ were added for the last 6h of culture. CD4+ TNFα+ and CD4+IFNγ+ T cells were measured by intracellular cytokine staining.

Fig. S5. Anti-CD40 stimulated T2 B cells control the progression of lupus in MRL/lpr mice. Splenic B cells from 9-10 week old MRL/lpr mice were stained with CD19, CD21, CD23, CD24 and DAPI. T2 and MZ B cells were purified by FACS sorting (purity around or above 90%) and were cultured for 48h with anti-CD40 (5μg/ml). 5×105T2 or MZ B cells were transferred once a week for 3 weeks to 9-10 week old MRL/lpr mice. Control mice received PBS injections. (A) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were sacrificed due to general debility. (B) Levels of proteinuria in recipient mice were measured once a week. (C) Sera from 23 weeks old mice were assessed for total anti-dsDNA (D) Composite renal disease.

Fig. S6. T2-like B cells inhibit T cell absolute numbers after adoptive transfer. B cells were purified from the spleens of MRL/lpr mice by negative selection using the anti-CD43 MACS™ kit. Purified B cells were incubated with anti-CD40 (5μg/ml) or an irrelevant isotype control (5μg/ml) for 48h and then stained with CD19, CD21, CD23, CD24 and DAPI. DAPI−T2-like, MZ-like and FO-like B cells were FACS sorted and 5 × 105 cells of each subset were transferred once a week for 3 weeks to 9-10 week old MRL/lpr mice (n=6/group). Control mice received PBS injections. Spleno cytes were isolated from MRL/lpr mice one week after receiving the final transfer of anti-CD40 stimulated B cell subsets. Absolute numbers of double negative (DN), CD4+ and CD8+ T cells in the spleens were assessed by flow cytometry and num bers are shown as mean ± SE.

Fig. S7. B cell subset viability following 48hr stimulation of MACS purified B cells. Splenic B cells were negatively purified using the anti-CD43 MACS® kit, according to the manufacturer’s instructions, from 9-10 week old MRL/lpr mice and cultured for 48h with isotype control (5μg/ml) or anti-CD40 (5μg/ml). B cells were then stained with CD19, CD21, CD23, CD24, DAPI. Cells were left unfixed. B cell subsets were gated based on their expression of CD21,CD23,CD24, and percentages of dead/live DAPI+/DAPI− T2-like, MZ-like, FO-like B cells were assessed by flow cytometry. (A) isotype control (B) anti-CD40.

Acknowledgments

The authors thank the staff of the Biological Service Facility for animal husbandry.

Footnotes

1

This work was supported by The Wellcome Trust (grant no. 068629 to C.M.), the UCLH charities CDRC (grant no. G140 to C.M.) and equipment ARC grant ID 17746. P.A.B is supported by the Oliver Bird Rheumatism Programme to D.A.I.. K.A.C. is supported by The Instituto Mexicano Del Seguro Social.

The authors declare no competing financial interests.

References

Associated Data

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Supplementary Materials

01

Fig. S1. (A) B cells subsets purity. Splenic B cells were negatevely purified using anti-CD43 MACS Kit, according to the manufacturer’s instructions, from 9-10 week old MRL/lpr mice and cultured for 48h with anti-CD40 (5μg/ml). Cells were then stained with CD19, CD21,CD23,CD24 and DAPI and sorted by BD FACS ARIA (BD bioscience). Purity of B cell subset as indicated. (B) Splenocytes were cultured for 48h with isotype control (5μg/ml) or anti-CD40 (5 μg/ml). PMA/ionomycin and GolgiStop™ were added for the last 6h of culture. Cells were stained with CD19, CD21, CD23, and IL-10 mAbs. Data shown are representative of 10 mice.

Fig. S2. Anti-CD40 stimulation induces CD23 expression by purified B cell subsets. B cells were negatively purified from the spleens of 9-10 week old MRL/lpr mice and stained with CD19, CD21, CD23 and DAPI. DAPI− T2, MZ and FO B cells were purified by FACS. Purified B cells were cultured with isotype control or anti-CD40 (5μg/ml) for 48h. Cells were re-stained with CD19, CD21 and CD23. (A) Pre-sort B cells and post-sort purities. (B) Phenotypes of purified B cell subsets following isotype or anti-CD40 stimulation. Plots are representative of 4 experiments.

Fig. S3. Anti-CD40 stimulation prevents T2 B cells maturation into FO B cells. Purified CD19+CD21hiCD23hiCD24+ B cells were cultured with isotype control, anti-CD40 (5μg/ml) or anti-IgM F(ab’)2 (10μg/ml) for 96h. Cells were stained again with CD19, CD21, CD23 and CD24. Plots show purified T2 maturing into CD21intCD24− FO B cells following isotype or anti-IgM F(ab’)2 stimulation, but remaining T2 following anti-CD40 stimulation.

Fig. S4. CD4 + T cells:T2-like B cell dose response suppression. B cells were purified from the spleens of MRL/lpr mice by negative selection using the anti-CD43 MACS™ kit. Purified B cells were incubated with anti-CD40 (5μg/ml) or an irrelevant isotype control (5μg/ml) for 48h and then stained with CD19, CD21, CD23, CD24 and DAPI. T2-like B cells were sorted by MoFlo according to the gates in Fig S1. CD4+CD25− T cells were also purified from age matched MRL/lpr splenocytes by MACS™ kit. 5 × 105 CD4+CD25− T cells were cultured at different ratios with T2-like B cells. Cultures were stimulated for 48h with 1μg/ml anti-CD3. PMA, ionomycin and GolgiStop™ were added for the last 6h of culture. CD4+ TNFα+ and CD4+IFNγ+ T cells were measured by intracellular cytokine staining.

Fig. S5. Anti-CD40 stimulated T2 B cells control the progression of lupus in MRL/lpr mice. Splenic B cells from 9-10 week old MRL/lpr mice were stained with CD19, CD21, CD23, CD24 and DAPI. T2 and MZ B cells were purified by FACS sorting (purity around or above 90%) and were cultured for 48h with anti-CD40 (5μg/ml). 5×105T2 or MZ B cells were transferred once a week for 3 weeks to 9-10 week old MRL/lpr mice. Control mice received PBS injections. (A) Survival of recipient mice was recorded weekly until mice died of disease spontaneously or were sacrificed due to general debility. (B) Levels of proteinuria in recipient mice were measured once a week. (C) Sera from 23 weeks old mice were assessed for total anti-dsDNA (D) Composite renal disease.

Fig. S6. T2-like B cells inhibit T cell absolute numbers after adoptive transfer. B cells were purified from the spleens of MRL/lpr mice by negative selection using the anti-CD43 MACS™ kit. Purified B cells were incubated with anti-CD40 (5μg/ml) or an irrelevant isotype control (5μg/ml) for 48h and then stained with CD19, CD21, CD23, CD24 and DAPI. DAPI−T2-like, MZ-like and FO-like B cells were FACS sorted and 5 × 105 cells of each subset were transferred once a week for 3 weeks to 9-10 week old MRL/lpr mice (n=6/group). Control mice received PBS injections. Spleno cytes were isolated from MRL/lpr mice one week after receiving the final transfer of anti-CD40 stimulated B cell subsets. Absolute numbers of double negative (DN), CD4+ and CD8+ T cells in the spleens were assessed by flow cytometry and num bers are shown as mean ± SE.

Fig. S7. B cell subset viability following 48hr stimulation of MACS purified B cells. Splenic B cells were negatively purified using the anti-CD43 MACS® kit, according to the manufacturer’s instructions, from 9-10 week old MRL/lpr mice and cultured for 48h with isotype control (5μg/ml) or anti-CD40 (5μg/ml). B cells were then stained with CD19, CD21, CD23, CD24, DAPI. Cells were left unfixed. B cell subsets were gated based on their expression of CD21,CD23,CD24, and percentages of dead/live DAPI+/DAPI− T2-like, MZ-like, FO-like B cells were assessed by flow cytometry. (A) isotype control (B) anti-CD40.