CD1d-dependent immune suppression mediated by regulatory B cells through modulations of iNKT cells

Abstract

Regulatory B cells (Breg) express high levels of CD1d that presents lipid antigens to invariant natural killer T (iNKT) cells. The function of CD1d in Breg biology and iNKT cell activity during inflammation remains unclear. Here we show, using chimeric mice, cell depletion and adoptive cell transfer, that CD1d-lipid presentation by Bregs induces iNKT cells to secrete interferon (IFN)-γ to contribute, partially, to the downregulation of T helper (Th)1 and Th17-adaptive immune responses and ameliorate experimental arthritis. Mice lacking CD1d-expressing B cells develop exacerbated disease compared to wild-type mice, and fail to respond to treatment with the prototypical iNKT cell agonist α-galactosylceramide. The absence of lipid presentation by B cells alters iNKT cell activation with disruption of metabolism regulation and cytokine responses. Thus, we identify a mechanism by which Bregs restrain excessive inflammation via lipid presentation.

Fig. 1

α-GalCer-iNKT-cell-dependent amelioration of arthritis requires B cells. a Mean clinical score of wild-type (WT) mice (left) and μMT mice (right) that received α-GalCer or vehicle alone following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (μMT ± α-GalCer n = 14 per group, WT ± α-GalCer n = 20 per group, one of three experiments is shown). b Representative flow cytometry plots and bar charts showing the frequency and number of splenic iNKT cells in μMT and WT mice that received α-GalCer or vehicle alone (n = 5 per group, one of three experiments is shown). c Representative histograms and bar chart showing median fluorescence intensity (MFI) of Ki-67 in splenic iNKT cells from α-GalCer- or vehicle-treated μMT and WT mice (n = 3 per group, one of three experiments is shown). d Representative histograms and bar chart showing MFI of PLZF in splenic iNKT cells from α-GalCer- or vehicle-treated μMT and WT mice (n = 3 per group, one of three experiments is shown). e Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺ splenic iNKT cells from α-GalCer- or vehicle-treated μMT and WT mice (n = 4 per group, one of three experiments is shown). f Representative histograms and bar chart showing MFI of PLZF in hepatic iNKT cells from α-GalCer- or vehicle-treated μMT and WT mice (n = 3 per group, one of two experiments is shown). g Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺ hepatic iNKT cells from α-GalCer- or vehicle-treated μMT and WT mice (n = 4 per group, one of three experiments is shown). b, c Analyzed on day 3 post disease onset, d–g analyzed at 16 h post disease onset. Data are mean ± s.e.m. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (a two-way analysis of variance (ANOVA), b–g one-way ANOVA)

Fig. 2

B cell depletion abrogates the protective effect of α-GalCer on arthritis. a Left, schematic showing the experimental design for B cell depletion. Right, bar charts showing the frequency and number of splenic CD19⁺ B cells in B cell-depleted and isotype-control-treated WT mice prior to α-GalCer or vehicle administration (n = 5 per group, one of three experiments is shown). b Mean clinical score of isotype-control-treated WT mice (left) and B cell-depleted WT mice (right) that received α-GalCer or vehicle alone following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (n = 5 per group, one of two experiments is shown). c Representative flow cytometry plots and bar charts showing the frequency and number of splenic iNKT cells in B cell-depleted and isotype-control-treated WT mice that received α-GalCer or vehicle alone (n = 5 per group, one of two experiments is shown). d Representative histograms and bar chart showing MFI of PLZF in splenic iNKT cells in B cell-depleted and isotype-control-treated WT mice that received α-GalCer or vehicle alone (n = 3 per group, one of two experiments is shown). e Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺ splenic iNKT cells in B cell-depleted and isotype-control-treated WT mice that received α-GalCer or vehicle alone (n = 4 per group, one of two experiments is shown). c Analyzed on day 3 post disease onset; d, e analyzed at 16 h post disease onset. Data are mean ± s.e.m. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (a Student’s t test, b two-way ANOVA, and c–e one-way ANOVA)

Fig. 3

B cell-specific CD1d is critical for α-GalCer-iNKT cell amelioration of arthritis. a Mean clinical score of B-WT chimeric mice (left) and B-Cd1d^−/− chimeric mice (right) that received α-GalCer or vehicle alone following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (B-Cd1d^−/− ± α-GalCer n = 5 per group, B-WT ± α-GalCer n = 4 per group, one of three experiments is shown). b Representative flow cytometry plots and bar charts showing the frequency (top) and number (bottom) of splenic IFN-γ⁺ and IL-17⁺ CD4⁺ T cells in B-Cd1d^−/− and B-WT chimeric mice that received α-GalCer or vehicle alone (n = 3 per group, one of two experiments is shown). c Representative flow cytometry plots and bar charts showing the frequency and number of splenic iNKT cells in B-Cd1d^−/− and B-WT mice that received α-GalCer or vehicle alone (n = 3 per group, one of two experiments is shown). d Representative histograms and bar chart showing MFI of Ki-67 in splenic iNKT cells from α-GalCer- or vehicle-treated B-Cd1d^−/− and B-WT mice (n = 3 per group, one of two experiments is shown). e Representative histograms and bar chart showing MFI of PLZF in splenic iNKT cells from α-GalCer- or vehicle-treated B-Cd1d^−/− and B-WT mice (n = 3 per group, one of two experiments is shown). f Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺PLZF⁺ splenic iNKT cells from α-GalCer- or vehicle-treated B-Cd1d^−/− and B-WT mice (n = 4 per group, one of two experiments is shown). g Representative histograms and bar charts showing MFI of T-bet and GATA3 in splenic iNKT cells from α-GalCer- or vehicle-treated B-Cd1d^−/− and B-WT mice (n = 3 per group, one of two experiments is shown). h Representative immunofluorescence showing the localization of splenic NK1.1⁺CD3⁺ T cells and IgD⁺ B cells in B-Cd1d^−/− and B-WT chimeric mice that received α-GalCer or vehicle alone. Arrowheads point to NK1.1⁺CD3⁺ cells. Bar, 100 μm, inset bar, 5 μm (n = 3 per group, one of two experiments is shown). i Mean clinical score of B-Il10^−/− chimeric mice (right) and B-WT chimeric mice (left) that received α-GalCer or vehicle alone following induction of arthritis; the Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (B-Il10^−/− ± α-GalCer n = 5 per group, B-WT ± α-GalCer n = 6 per group, one of two experiments is shown). b Analyzed on day 7 post disease onset, c, d, h analyzed on day 3 post disease onset, and e–i analyzed at 16 h post disease onset. Data are mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (a, i two-way ANOVA, b–g one-way ANOVA)

Fig. 4

B cell inability to present α-GalCer affects iNKT cell expression of genes involved in cytokine responses and metabolism. a Scatter plot showing fold-change relationships between common expressed genes (n = 5797) in iNKT cells from B-Cd1d^−/− and B-WT mice that received α-GalCer or vehicle alone following induction of arthritis (data sets from RNA-seq). Color gradient indicates the degree of difference in fold-change values between iNKT cells in B-Cd1d^−/− and B-WT mice following α-GalCer treatment. b Pathway enrichment analysis of pathways (right) showing enrichment among genes expressed differentially in iNKT cells from B-Cd1d^−/− and B-WT mice following α-GalCer treatment. It is presented as net pathway perturbation in iNKT cells from B-Cd1d^−/− mice compared to B-WT mice. Negative values mean inhibited in iNKT cells from B-Cd1d^−/−, positive values mean activated in iNKT cells from B-Cd1d. c Heat maps showing the expression of genes involved in glycolysis (top) and fatty acid oxidation (bottom) by iNKT cells from B-Cd1d^−/− and B-WT mice that received α-GalCer or vehicle alone. d Heat map showing the expression of cytokine genes by iNKT cells from B-Cd1d^−/− and B-WT mice that received α-GalCer or vehicle alone. e, f Volcano plots showing fold changes of differentially expressed genes associated with e NKT1 and NKT2 phenotypes, and f PLZF transcriptional regulation, between iNKT cells from B-Cd1d^−/− and B-WT mice following α-GalCer treatment, plotted against p values

Fig. 5

α-GalCer-mediated amelioration of arthritis is partially dependent on IFN-γ production by iNKT cells. a Top, mean clinical score of anti-IFN-γ-treated and isotype-control-treated WT mice that received α-GalCer or vehicle alone following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee. Bottom, bar chart showing the suppression of knee swelling in anti-IFN-γ-treated and isotype-control-treated WT mice that received α-GalCer, compared to anti-IFN-γ-treated and isotype-control-treated WT mice that received vehicle alone (anti-IFN-γ-treated WT ± α-GalCer n = 5 per group, isotype-control-treated WT ± α-GalCer n = 4 per group, one of two experiments is shown). b Representative flow cytometry plots (top) and bar charts (bottom) showing the frequency and number of splenic IFN-γ⁺ and IL-17⁺ CD4⁺ T cells in anti-IFN-γ-treated and isotype-control-treated WT mice that received α-GalCer or vehicle alone (n = 3 per group, one of two experiments is shown). c Representative flow cytometry plots (top) and bar charts (bottom) showing the frequency of T-bet⁺, ROR-γt⁺, and GATA3⁺ CD4⁺ T cells in μMT and WT splenocyte cultures with α-GalCer and isotype control or α-GalCer and anti-IFN-γ-blocking antibodies (μMT n = 3, WT n = 6, one of two experiments is shown). d Representative flow cytometry plots (top) and bar chart (bottom) showing the frequency of IFN-γ⁺CD4⁺ T cells following coculture with in vivo α-GalCer-stimulated iNKT cells (or alone), isolated from the spleens of arthritic μMT and WT mice in the presence of IFN-γ-neutralizing or isotype-control antibodies (n = 4 per condition, one of two experiments is shown). e Bar charts showing the frequency of T-bet⁺ (left) and ROR-γt⁺ (right) CD4⁺ T cells, isolated from the spleens of arthritic WT mice, following stimulation with increasing concentrations of IFN-γ (n = 3 per condition, one of two experiments is shown). b Analyzed on day 3 post disease onset. Data are mean ± s.e.m. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (a two-way ANOVA, b–e one-way ANOVA)

Fig. 6

T2-MZP Bregs mediate suppression via iNKT cells and require CD1d. a Top, schematic showing the experimental design. Bottom, bar chart showing suppression of knee swelling in Cd1d^−/− and WT mice that received T2-MZP B cells, isolated from the spleens of WT mice on day 7 post disease onset, compared to Cd1d^−/− and WT mice that received no transfer (Cd1d^−/− n = 11, WT n = 16, one of two experiments is shown). b Representative flow cytometry plots (left) and bar charts (right) showing the frequency and number of splenic IFN-γ⁺ and IL-17⁺ CD4⁺ T cells in Cd1d^−/− and WT mice that received T2-MZP B cells, isolated from the spleens of WT mice on day 7 post disease onset, or Cd1d^−/− and WT mice that received no transfer (n = 3 per group, one of two experiments is shown). c Top, schematic showing the experimental design. Bottom, mean clinical score of WT mice that received T2-MZP B cells, isolated from the spleens of Jα18^−/− or WT mice on day 7 post disease onset, or WT mice that received no transfer following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (Jα18^−/− T2-MZP transfer n = 4, WT T2-MZP transfer n = 3, no transfer n = 4, one of two experiments is shown). d Representative flow cytometry plots (top) and bar charts (bottom) showing the frequency and number of splenic IFN-γ⁺ and IL-17⁺CD4⁺ T cells in WT mice that received T2-MZP B cells, isolated from the spleens of Jα18^−/− or WT mice on day 7 post disease onset, or WT mice that received no transfer (n = 4 per group, one of two experiments is shown). e Top, schematic showing the experimental design. Bottom, mean clinical score of WT mice that received T2-MZP B cells, isolated from the spleens of Cd1d^−/− or WT mice on day 7 post disease onset, or WT mice that received no transfer following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (n = 6 per group, one of two experiments is shown). f Representative flow cytometry plots (top) and bar charts (bottom) showing the frequency and number of splenic IFN-γ⁺ and IL-17⁺CD4⁺ T cells in WT mice that received T2-MZP B cells, isolated from the spleens of Cd1d^−/− or WT mice on day 7 post disease onset, or WT mice that received no transfer (n = 4 per group, one of two experiments is shown). g Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺ splenic iNKT cells in WT mice that received T2-MZP B cells, isolated from the spleens of Cd1d^−/− or WT mice on day 7 post disease onset, or WT mice that received no transfer (n = 4 per group, one of two experiments is shown). b, d, f, g Analyzed on day 3 post disease onset. Data are mean ± s.e.m. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^••p < 0.01, and ^•••p < 0.001 (a, c, e two-way ANOVA; b, d, f one-way ANOVA)

Fig. 7

MZ B cells are dispensable for α-GalCer-mediated amelioration of arthritis. a Mean clinical score of control mice (left) or MZ B cell-depleted mice (right) that received α-GalCer or vehicle alone following induction of arthritis. The Y axis shows the percentage of swelling in antigen-injected knee compared to control knee (α-GalCer-treated n = 5 per group, vehicle-treated n = 6 per group, one of two experiments is shown). b Representative flow cytometry plots and bar charts showing the frequency and number of splenic iNKT cells in MZ B cell-depleted mice and control mice that received α-GalCer or vehicle alone (n = 3 per group, one of two experiments is shown). c Representative histograms and bar chart showing MFI of Ki-67 in splenic iNKT cells from α-GalCer- or vehicle-treated MZ B cell-depleted mice and control mice (n = 3 per group, one of two experiments is shown). d Representative histograms and bar chart showing MFI of PLZF in splenic iNKT cells from α-GalCer- or vehicle-treated MZ B cell-depleted mice and control mice (n = 3 per group, one of two experiments is shown). e Representative flow cytometry plots and bar chart showing the frequency of IFN-γ⁺ splenic iNKT cells from α-GalCer- or vehicle-treated MZ B cell-depleted mice and control mice (n = 4 per group, one of two experiments is shown). b, c Analyzed on day 3 post disease onset; d, e analyzed at 16 h post disease onset. Data are mean ± s.e.m. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (a two-way ANOVA; b–e one-way ANOVA)

Fig. 8

Regulatory B cell development and in vitro suppressive capacity are not impaired in iNKT-cell-deficient mice. a Representative flow cytometry plots and bar charts showing the frequency and number of splenic IL-10-eGFP⁺CD19⁺ B cells from IL-10-eGFP mice that received α-GalCer or vehicle alone following induction of arthritis (α-GalCer-treated group n = 4, vehicle-treated group n = 3, one of two experiments is shown). b Representative flow cytometry plots and bar charts showing the frequency and number of splenic IL-10⁺CD19⁺ B cells in Jα18^−/−, Cd1d^−/−, and WT mice following arthritis (Jα18^−/− n = 5, Cd1d^−/−n = 8, and WT n = 9, one of two experiments is shown). c Bar chart showing IL-10 production by B cells isolated from the spleens of arthritic Jα18^−/−, Cd1d^−/−, and WT mice following stimulation with methylated BSA (mBSA), anti-CD40, or mBSA+anti-CD40 as measured by ELISA (n = 9 per group, one of three experiments is shown). a–c Cells isolated on day 7 post disease. Data are mean ± s.e.m. (a, c two-way ANOVA; b one-way ANOVA)