IL-10-GFP, TIM1^−/− C57BL/6 (see Figure S2Cii in Supplementary Material for assessment of genotype), TIM1^−/− BALB/c (see Figure S2Ci in Supplementary Material for assessment of genotype), C1q^−/−, complement receptor 2^−/− (CR2^−/−) (see Figure S2J in Supplementary Material for confirmation of phenotype), DO11.10 TcR Tg mice, and OTII-Ly5.1 TcR Tg mice (both OVA_323–339 peptide specific) were bred and maintained under specific pathogen free conditions in the Animal Facilities at the University of Edinburgh, UK. IL-10-GFP mice were kindly provided by Dr. Richard Flavell (Yale University, New Haven, CT, USA), TIM1^−/− mice by Prof. Andrew McKenzie (Cambridge, UK), C1q^−/− mice by Prof. M. Botto (Imperial) and CR2^−/− mice by Prof. Kevin Marchbank (Newcastle). Wild-type (WT) C57BL/6 and BALB/c mice were bred in house. Mice were used at 8–12 weeks of age and were sex and age matched. All experiments were covered by a Project License granted by the Home Office under the Animal (Scientific Procedures) Act 1986. Locally, this license was approved by the University of Edinburgh Ethical Review Committee.

For all staining, cells were stained in PBS with 2% FCS for 20 min at 4°C. BD Aria II was used for flow sorting and BD LSRII was used to collect data. For sorted cells, debris and dead cells were excluded using FSC-SSC. Doublets were excluded using both FSC and SSC singlet gating, then CD19^+ve B cells isolated. For Figures 1C and 2A, CD4 and CD3 stains were also included to exclude contaminating T cells. Antibodies used were specific for and labeled with CD21-FITC, CD3-PE TxR, CD4-PE, IgM-APC, GM-CSF-PE, IL-10-PE, IL-17-alexa fluor647, IFN-γ-FITC (BD Biosciences); CD11b-BV570, CD11b-PE Cy5, CD19-PE, CD19-BV605, CD1d-PerCP Cy5.5, CD21/35-APC, CD23-PE, CD23-alexa fluor 647, CD23-PE Cy7, CD3-FITC, CD3 PE/Dazzle 594, CD38-PE, CD4-PE, CD4-BV605, CD40-PE Cy7, CD43-FITC, CD43-APC, CD5-PE Cy5, CD80-PerCP Cy5.5, CD86-BV421, TIM1-PE, TNFα-BV605 (Biolegend); CD21/35-APC efluor780, CD24-APC efluor780, CD25-efluor450, CD9-APC, DO11.10 TCR-Biotin, IgD-efluor450, MHCII-PE Cy5, CD19-eFluor450, F4/80-APC, IFN-γ-PE Cy7, streptavidin-PE (eBioscience); IgM-TxR, IgM-alexa fluor647 (Southern Biotech); IgM-alexa488, cell tracker green (Molecular Probes), and CFSE (fluka). All analysis was performed using FlowJo Software.

Cells were stimulated for 4.5 h total with PMA [Sigma (20 ng/ml)] and Ionomycin [Sigma (1 μg/ml)]. After 1 h stimulation, Brefeldin A [Sigma (1 μg/ml)] was added for the remaining 3.5 h. Surface staining was performed before resuspending in fixation and permeabilization solution for 20 min (Cytofix/Cytoperm kit, BD Biosciences) followed by IC staining. All IC antibodies were used at 1:100 for 30 min in 1× Perm/Wash buffer (Cytofix/Cytoperm kit, BD Biosciences).

Thymi were removed from 4- to 6-week-old syngeneic mice, teased into single cell suspensions and cultured for 18 h in IMDM (supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 20 mM 2-mercaptoethanol, and 10% heat inactivated FCS). Cells prepared in this way give an average of 43% Annexin-V (AnV)^+ve/propidium iodide (PI)^−ve ACs and <5% AnV^+ve/PI^+ve secondary necrotic cells (14). Where ACs are needed for experiments involving C1q^−/− ACs were generated following culture in X-Vivo 15 media without serum.

Treatment with anti-CD95 (1 μg/ml) induced Jurkat cells to become apoptotic after 18 h in culture with serum free RPMI (supplemented with 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin). At this point 80% were AnV^+ve. Cells used in phagocytosis or interaction assays were labeled with cell tracker green (Molecular Probes) prior to induction of apoptosis.

Mouse splenocytes were “teased apart” to obtain single cell suspensions and depleted of red cells with red cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). B cells were isolated from spleen single cell suspensions or peritoneal lavages using positive selection with CD19⁺ microbeads as per manufacturer’s instructions (Miltenyi Biotech). Cells were then further sorted by FACS as per figure legends.

1 × 10⁵ FACS sorted B cells were cultured in complete IMDM along with 1 × 10⁶ apoptotic thymocytes and the relevant stimulation. After 72 h, supernatants were removed and cytokine levels checked by ELISA (All R&D Duoset ELISA kit except IL-35 Biolegend). Cells were stimulated with: TLR7/8 ligand R848 [InVivogen (0.1 μg/ml for B cell stimulation, 0.5 μg/ml for macrophage stimulation)], the mouse TLR9 ligand CpGB [ODN1826 Eurofins MWG Operon (1 μg/ml)], LPS [Sigma (2 μg/ml)], MOG35–55 [Cambridge Research Biochemicals (20 μg/ml unless otherwise stated)], or OVA323–339 [Cambridge Research Biochemials (2 μg/ml unless otherwise stated)].

IL-10-GFP mice were injected IV on D0, D2, and D5 with 20 × 10⁶ apoptotic thymocytes. On D7 mice were sacrificed and blood, peritoneal lavage and spleens harvested. Splenic CD19^+ve B cells were FACs sorted into IL-10-GFP^{+ve or −ve} fractions, phenotyped and cultured for 10 days in the presence of MegaAPRIL [Adipogen (200 ng/ml)], CpGB [ODN1826 Eurofins MWG Operon (1 μg/ml)], and IL-4 [R&D System (50 ng/ml)], after which culture supernatants were checked for IgM and IgG levels (Ready-SET-Go ELISA kit eBioScience).

Highly purified IL-10-GFP^+ve B1a cells, generated in vivo, were fused with SP2/0 cells to generate hybridomas. Cells were cultured with peritoneal macrophages for 7 days and supernatants screened (see Figures S3C,D in Supplementary Material) prior to subcloning. Colonies were selected on their ability to bind to ACs.

Apoptotic Jurkats were incubated with 20 μg/ml of IgM derived from the hybridoma supernatant followed by anti mouse IgM-alexa fluor 488 (1:400 dilution. Molecular probes). The plasma membrane was stained with Cell Mask Deep Red plasma membrane stain (1:1,000 in PBS) for 10 min at 37°C and following fixation with 3.7% formaldehyde the nucleus stained with DAPI. Cells, mounted in ProLong^® Gold Antifade Mountant were visualized on a Leica SP5 confocal microscope.

EIA/RIA (costar) 96-well plates were coated with 2 μg/ml (in 50 μl PBS) antigen overnight then blocked (PBS + 1% BSA) for 1.5 h. Antigens used were malondialdehyde-modified low-density lipoprotein (MDA-LDL), oxidized-LDL (Ox-LDL), citrullinated fibrinogen (Cambridge Biosciences); DNA, ssDNA, thyroglobulin, yeast RNA, alpha Actinin (Sigma); La, Ro, Smith, histone (Arotec Diagnostics); rheumatoid factor (Thermofisher Scientific), CWPS (Oxford Biosystems), ApoH (R&D Systems), PC-BSA (2B Scientific), and AnV (eBiosciences). Serum samples were initially diluted at 1:250. For in vitro-generated antibodies, supernatants were initially diluted 1:2. For hybridoma-generated antibodies, IgM was determined and supernatants were diluted to 0.5 μg/ml prior to serial dilutions. Secondary antibody (1:1,000 dilution) were either anti mouse IgM-HRP (Southern Biotech) or anti mouse IgG-HRP (Zymed). MRL/lpr mouse serum (diluted 1:250) was used as a positive control while IgM derived from IL-10^–ve clones that did not bind to ACs were used as negative controls.

Single cell suspensions of lymph node cells (from OVA Tg mice) were prepared and 5 × 10⁶ cells injected along with 20 × 10⁶ ACs (IV). For experiments with C1q^−/− mice, C1q^−/− thymocytes were cultured in serum free X-Vivo 15 media. Mice were immunised with 50 μl OVA323–339 peptide (1 mg/ml) emulsified in an equal volume of complete Freund’s adjuvant (CFA) sc into each hind leg. On D7 splenic single cell suspensions were generated prior to restimulation using 1 × 10⁶ splenic cells along with OVA323–339 peptide at 4, 2, 1, 0.5, and 0 μg/ml. After 72 h, cytokines were measured (R&D duoset ELISA).

Splenic cells were labeled with 1 μM CFSE before setting up in culture with 2 μg/ml OVA_323–339. Cells were harvested every 24 h and further stained with CD3 and Ly5.1 (for OTII). The mean florescence intensity (MFI) of CFSE for CD3^+ve Ly5.1^+ve cells was measured using flow cytometry.

Purified B cells were incubated with cell tracker green labeled AC-Jurkats in a ratio of 1:5. After 4 or 24 h, cells were stained for CD19 and CD43. Interaction was measured using flow cytometry by determining the percentage of B cells which were also positive for cell tracker green, indicating interaction between the B cell and AC.

Bone marrow-derived macrophages were isolated from the hind leg bones of C57BL/6 mice and cells cultured in compete IMDM + 10% L929-conditioned media (containing M-CSF) for 7 days in flat bottom plates.

1 × 10⁶ cell tracker green labeled AC-Jurkats were incubated with 20 μg/ml of hybridoma derived IgM and cocultured with BMDM for 30 m.

Peritoneal CD43^+veB1a or splenic follicular B (FOB) cells were activated with R848 (0.1 μg/ml) for 24 h and then cocultured with R848 (0.5 μg/ml) activated BMDM for 18 h. 20 μg/ml anti-IL-10 or isotype control antibody (Biolegend) was added for length of culture. Supernatants were assessed for released cytokines by ELISA (R&D duoset) and cells were stained for IC cytokines.

Experimental autoimmune encephalomyelitis was induced as described previously (15). On D3/4 mice were either given 1 × 10⁵ CD43^+ve B1 cells or splenic FOB, that had been previously activated with R848 (0.1 μg/ml) for 48 h. Following sacrifice, harvested organs were weighed and single cell suspensions from the spleen and LN generated. Spinal cords were collagenase/DNase digested before running over 70/30% Percoll gradient to obtain leukocytes. Re-stimulation assays were set up using 1 × 10⁶ splenic or LN cells or 1 × 10⁵ spinal cord cells along with MOG35–55 peptide at 20, 10, 5, 2.5, and 0 μg/ml. After 72 h, cytokines were measured (R&D duoset ELISA) and IC staining performed.

RNA was extracted from 10⁵ purified cells [TRI^® Reagent (Ambion)] followed by reverse transcription [High Capacity cDNA reverse transcription kit (Applied Biosystems)]. Bhlhe41 cDNA levels were quantitated by Taqman^® Gene Expression Assay predesigned primers (Mm00470512_m1) with intra-sample expression normalized to Eukaryotic 18S rRNA Endogenous control (FAM™/MGB probe) and run on an Applied Biosystems 7900HT Fast-Real Time System using SDS software (v2.4). Data were analyzed using the comparative CT method (Δ/ΔCT), where fold differences in gene expression between FOB and other B cell populations (ΔCT) were normalized to CT values of the 18S rRNA reference gene.

Experimental repeats are given under each experiment and ranged from 3 to 12. Data are expressed as mean and SEM. Statistical significance between the groups was assessed by GraphPad Prism Version 7.0 using the appropriate analysis as stated in the figure legends. P-values: *0.01–0.05, **0.001–0.01, and ***<0.001.

To identify naturally occurring populations of ACBregs, we sorted splenic B cells into transitional 2 marginal zone precursor B cells (T2-MZP-CD21^hiCD24^hiCD23^hi), FOB cells (CD21^loCD24^loCD23^+ve), and B cells found within the marginal zone (CD21^hiCD24^hiCD23^lo) (Figure 1A, i; Figure S1A in Supplementary Material). Subsequent stimulation of these B cell subsets with the TLR7/8 ligand R848 or the TLR9 ligand CpGB in the presence of ACs significantly augmented the level of IL-10 secretion from CD21^hiCD24^hiCD23^lo splenic B cells (Figure 1A, ii–iii, population A3), but barely at all from either T2-MZP B (Figure 1A, ii–iii, population A4) or FOB cells (Figure 1A, ii–iii, population A1). To further define the surface markers expressed by ACBregs, highly pure populations of splenic B cells were also sorted according to their expression of CD21, IgM, and IgD (Figure 1B, i; Figure S1B in Supplementary Material). ACs induced the highest secretion of IL-10 (but not IL-35), from activated CD21^hiIgM^hiIgD^lo B cells (Figure 1B, ii–iii, population C4).

To identify in vivo-derived ACBregs, apoptotic thymocytes were administered intravenously to mice and splenic IL-10^+ve and IL-10^−ve B cells harvested 7 days later (Figure S1C in Supplementary Material for sort strategy and purity). At this point the percentage of IL-10^+ve B cells had doubled (Figure 1C, i). Further analysis of the surface markers indicated that approximately 80% of the splenic IL-10^+ve B cells were B1a cells (CD19^hiCD43^+veCD5^+veCD23^lowCD38^+veCD25^+veCD1d^+veIgD^lowIgM^hi) (Figure 1C, ii; red line and Figure S1Ciii–iv in Supplementary Material). In line with possible in vivo activation (see Figure S1Cv in Supplementary Material), CD21 expression was reduced following AC infusion (30). Compared to IL-10^−ve B cells, ACBregs also expressed more CD86, Tim1, CD9, CD80, and CD40 (Figure 1C, iii).

To assess the differential responses of activated splenic B cells to ACs, highly purified splenic MZB (CD19^+veCD43^−veIgD^lowIgM^hi), B1a (CD19^+veCD43^+veIgD^lowIgM^hi), or FOB (CD19^+veCD43^−veIgD^+veIgM^lo) B cells were cocultured with OVA peptide, OVA-specific T cells, with and without ACs. Cultures containing B1a cells generated significantly more IL-10 than MZB (Figure 1D). However, MZB were also able to respond to ACs and secreted significantly more IL-10 than those cultures containing FOBs. This indicates that while splenic B1a B cells make up a major component of ACBregs, activated splenic CD43^−ve MZB cells can also respond to ACs by secreting IL-10.

Similar results were seen in the peritoneal cavity (PerC). Following an injection of ACs the percentage of IL-10^+ve B cells in the PerC had increased by 50% at day 7 when compared to naive mice (Figure 1E, i). Approximately 96% of the IL-10^+ve B cells were CD43^+ve and had the markers of PerC B1a B cells (Figure 1E, ii; red line and Figure S1Di–iii in Supplementary Material). Again, the expression of CD86, Tim1, CD9, CD80, MHCII, and CD40 were also increased compared to IL-10^−ve B cells (Figure 1E, iii). As expected, the CD5 staining on the B1a B cells was lower than that found on T cells (Figure S1Ei in Supplementary Material). However, prior treatment with ACs did not alter the percentage of cells that expressed CD43 or CD5 (Figure S1Eii–iii in Supplementary Material). In keeping with a recent report (31), CD43^+ve splenic and peritoneal B1a B cells preferentially expressed the transcription factor Bhlhe41, when compared to splenic FOB or marginal zone B cells and peritoneal CD43^−ve B cells (Figure 1F). In vitro, the percentage of IL-10^+ve B1a B cells is augmented by apoptotic thymocytes, but this requires a second signal such as TLR7/8 activation (Figure 1G).

We next asked if B1a B cells, isolated following an in vivo inflammatory immune response, could still induce antigen-specific T cells to secrete IL-10. Ovalbumin peptide (OVA_323–339)-specific D011.10 T cells were transferred into mice that had been given an intravenous injection of ACs at the time of OVA_323–339 peptide (emulsified in CFA) immunization. A week later, PerC B cells were sorted into CD19^hiCD43^+ve and CD19^+veCD43^−ve subsets (Figure S2A in Supplementary Material). Splenic CD19^+ve B cells were harvested and sorted into naive CD19^+veIgD^hi B cells and CD19^hiCD43^+ve splenic B1a cells (Figure S2B in Supplementary Material). These B cell subsets were used as antigen-presenting cells (APCs) to stimulate naive OVA_323–339-specific T cells and IL-10 production was assessed after 72 h (Figures 2A,B, i,iii). IC staining confirmed that the highest percentage of IL-10 producing cells was still seen among the splenic and PerC CD43^+ve B1a B cells. These same B1a B cells also induced a significantly higher percentage of CD4^+ve T cells to produce IL-10 (Figures 2A,B, ii). Hence splenic or PerC ACBregs were able to induce antigen-specific T cells to secrete IL-10, even following a proinflammatory in vivo stimulus.

T cell Ig and mucin domain (Tim1) has been reported to identify ACBregs and the loss of this molecule has been associated with impaired IL-10 production and the promotion of inflammatory T cell responses (32, 33). To ask if splenic B cells required Tim1 expression to induce IL-10 secretion following interaction with ACs, IgM^hiIgD^loCD21^hi splenic B cells from WT and Tim1-deficient (Tim1^−/−) mice were stimulated with the TLR7/8 ligand R848 or used as APCs to stimulate naive OVA_323–339-specific T cells in vitro. We did not detect any difference in the ability of TLR or T cell activated Tim1^−/− B cells to respond to ACs by secreting IL-10 (Figure 2C, i–ii) [on both the BALB/c or C57BL/6 background (see Figure S2Di–iv in Supplementary Material)]. Nor was there a difference in regulatory cell surface markers or their ability to interact with ACs (Figures S2E,F in Supplementary Material). Similar results were seen when splenic B cells were stimulated with the TLR9 ligand CpGB or the TLR4 ligand LPS (Figure S2Gi–ii in Supplementary Material). When ACs were given in vivo at the time of OVA_323–339/CFA immunization (as described for Figure 2A) no differences in IL-10 secretion (Figure S2H in Supplementary Material) or T cell proliferation upon re-stimulation (Figure S2I in Supplementary Material) was seen; leading us to conclude that Tim1 is not required for innate-like regulatory B cells to induce regulation.

Mammalian DNA has been shown to bind to CR2/CD21, which is most highly expressed by MZB and PerC B1 B cells (34, 35). ACs express DNA containing chromatin complexes on their cell surface and altered expression of CR2 is associated with SLE in mice (36). In addition, complement proteins, including C1q, bind to late ACs and increase their uptake by phagocytes (21, 37, 38); the loss of which is believed to contribute to the increased risk of SLE (20). Hence complement may also play an essential role in regulatory B cell function. To test this, we assessed the cytokine responses of WT or CR2^−/− splenic B cells, activated with TLR7/8 or TLR9 and cocultured with ACs. To avoid bias that may occur with B cell surface markers from these knockout mice, we assessed CD19⁺IgD^loIgM^hi B cells. In contrast to TIM1^−/− B cells, TLR stimulated CD19⁺IgD^loIgM^hi CR2^−/− B cells secreted significantly more IL-10, both with or without added ACs (Figure 2D; Figure S2K in Supplementary Material). Similar results were seen following the in vivo immunization with OVA_323–339 peptide in CFA, OVA_323–339-specific T cells and an infusion of ACs. Re-stimulation of splenocytes from these immunized mice a week later also generated significantly more IL-10 than WT controls (Figure 2E). The increase in IL-10 secretion may have resulted from the significantly higher percentage of splenic CD43^+ve B1a cells found among the CD19⁺IgD^loIgM^hi B cells (Figure 2F, i). The percentage of peritoneal B1 cells was also significantly increased (Figure 2F, ii). When equivalent numbers of splenic (CD19⁺IgD^loIgM^hiCD43⁺) or PerC (CD19^hiCD43⁺) B1 cells were isolated from CR2^−/− mice or WT controls and activated with TLR7/8 (R848) and ACs, similar amounts of IL-10 were generated (Figure 2G). Comparable results were seen with splenic CD21^hiIgD^loIgM^hi B cells from C1q^−/− mice (Figures S2L–N in Supplementary Material). This indicates that while ACBreg function is not affected by the absence of TIM1, it is augmented in both C1q- and CR2-deficient mice, likely as a result of the higher percentage of splenic and peritoneal B1a cells, able to respond to ACs.

Regulatory B cell function depends on the activation of endosomally located TLRs (15). Peritoneal B1 B cells have been reported to internalize beads and bacteria (39), but we could not detect whole ACs or apoptotic bodies within them (data not shown). However, in comparison to follicular cells, peritoneal B1a B cells made prolonged contact with ACs at both 4 and 24 h following coculture; with significantly more B1a cells still firmly bound to ACs (Figure 3A, i–ii; Figure S3A in Supplementary Material). These stable interactions may allow the recognition of AC expressed neoantigens via the B cell receptor (40), which we and others have previously reported to be required for regulatory B cell function (14, 41, 42).

Apoptotic cells express a range of self-antigens on their cell surface, including DNA, the proteins Ro and La, and the phospholipid phosphatidylserine, all of which are potent autoantigens in SLE and Sjogren’s syndrome (17, 19). Following PCD membrane LDLs also undergo peroxidation, resulting in the formation of neoepitopes including MDA-LDL, Ox-LDL, and phosphorylcholine (PC) (22). In contrast to the PerC, the spleen is a major site of antibody production by B1a B cells (28) and following an infusion of ACs, a rise in the titer of IgM (but not IgG) with specificities typical of NAbs was observed in the serum (Figure 3B) (22). To ask whether IL-10^+ve splenic B cells were the source of these antibodies, IL-10^+ve and IL-10^−ve splenic B cells were sorted seven days after the administration of ACs (as shown in Figure S1Ci–ii in Supplementary Material). These cells were cultured in vitro with a proliferation inducing ligand (APRIL), IL-4, and CpGB to stimulate antibody secretion. After 10 days, supernatants were harvested and tested for the specificity of secreted immunoglobulin. Only IL-10^+ve splenic B cells secreted significant quantities of IgM (Figure 3C, i) but not IgG (data not shown). The antibodies secreted into the supernatants were of similar specificities to those antibodies seen in the serum of mice injected with ACs (Figure 3C, ii); confirming that IL-10^+ve regulatory B cells respond to AC expressed neoantigens by secreting NAbs. To further assess the specificity of these regulatory cells, IL-10^+ve and IL-10^−ve splenic B cells were sorted from mice that had been given an infusion of ACs 7 days earlier. As noted before, IL-10^+ve B cells had the markers of B1a B cells (Figure S3B in Supplementary Material). B cells were then fused with the SP2/0 cell line to form hybridomas (as illustrated in Figures S3C,D in Supplementary Material). Individual clones were tested for reactivity to a variety of epitopes found on ACs and within the NAb repertoire. Again, only IgM secreted by the hybridomas originating from the IL-10^+ve (but not IL-10^−ve) splenic B1a cells, showed cross-reactivity with a range of epitopes expressed on ACs, particularly MDA-LDL, Ox-LDL, and the autoantigens Ro, La, histones, and Smith (Sm) protein (Figure 3D). Hence, ACBregs that respond to ACs are B1a cells that secrete both IL-10 and IgM with specificities that resemble NAbs.

Natural antibodies augment the clearance of ACs (43) and IgM secreted by ACBregs preferentially bound to ACs, while the antibodies derived from IL-10^−ve splenic B cells did not (Figure 4A, i–ii; Figures S4A,B in Supplementary Material). The phagocytosis of ACs by BMDM, that had bound ACBreg derived IgM, was also significantly increased (Figure 4A, iii), as expected (44). BMDMs are particularly sensitive to the effects of IL-10, which is largely responsible for the anti-inflammatory response, acting via the IL-10 receptor and increasing signaling through the JAK1/STAT3 cascade (45). In contrast, TLR7/8 stimulation with R848 induces BMDMs to secrete TNFα. We noted that, in distinction to FOB cells, the coculture of B1a B cells with BMDMs, where both cell types had been activated with R848, resulted in a significant decrease in the amount of TNFα secreted into the culture medium (Figure 4B, i), as well as the percentage of macrophages (F4/80⁺CD19⁻) positive for IC TNFα (Figure 4B, ii–iii). Supernatants transferred from R848 activated B1a B cells to similarly activated macrophages also diminished TNFα production (Figure 4B, i–iii), as well as the activation marker CD86 (Figure 4C). IL-10 secreted by activated B1a cells (Figure 4D) was required to prevent TNFα secretion, as evidenced by the loss of macrophage TNFα suppression in the presence of anti-IL-10 antibodies (Figure 4E, i–ii).

The neuroinflammatory disease model EAE is driven by both activated, antigen-specific T cells and macrophages. We next asked if activated B1a could limit auto-immune mediated inflammation. Mice were immunized with MOG peptide emulsified in CFA (MOG/CFA) to induce EAE. PerC B1a cells and splenic FOB cells were stimulated with the TLR7/8 ligand R848 for 48 h in vitro and either 10⁵ B1a cells or 10⁵ FOB cells were injected intravenously 3–4 days following immunization with MOG/CFA. Despite activating the B1a cells with a proinflammatory stimulus (via TLR7/8 with R848), clinical disease severity was significantly reduced (Figure 5A). On day 20 the spinal cords of control FOB cell-treated mice (FOB) also contained a greater number of total cells compared to those given B1a cells (Figure 5B), though cell numbers in the spleen and draining lymph nodes were unchanged (data not shown). The spinal cords were further analyzed for CD4^+ve and CD19^+ve cells. Compared to B1a-treated mice, the percentage of CD4^+ve T cells in the spinal cords of mice given control FOB cells was more than six times higher, while CD19^+ve B cell infiltration was approximately halved in B1a-treated mice (Figure 5C, i–ii). Lymphocytes from the spleen, draining lymph nodes and spinal cord were re-stimulated with MOG peptide for 72 h and the cytokines IL-17, IFN-γ, and GM-CSF quantified by ELISA. In keeping with the reduced inflammation seen clinically, B1a-treated mice generated significantly less proinflammatory cytokines than control FOB cell-treated mice in all organs tested, but particularly the spinal cords (Figure 5D, i–iii). IC cytokine staining confirmed that a source of these cytokines was the CD4^+ve T cell, which again showed a significantly lower production of IL-17, IFN-γ, and GM-CSF in B1a-treated mice (Figure 5D, iv–vii). This confirms that activated B1a B cells are able to suppress autoimmune mediated inflammation when administered after the initiation of EAE.