A CD4+ T cell population expanded in lupus blood provides B cell help through interleukin-10 and succinate

Abstract

Understanding the mechanisms underlying autoantibody development will accelerate therapeutic target identification in autoimmune diseases such as systemic lupus erythematosus (SLE)¹. Follicular helper T cells (T_FH cells) have long been implicated in SLE pathogenesis. Yet a fraction of autoantibodies in individuals with SLE are unmutated, supporting that autoreactive B cells also differentiate outside germinal centers². Here, we describe a CXCR5^-CXCR3⁺ programmed death 1 (PD1)^hiCD4⁺ helper T cell population distinct from T_FH cells and expanded in both SLE blood and the tubulointerstitial areas of individuals with proliferative lupus nephritis. These cells produce interleukin-10 (IL-10) and accumulate mitochondrial reactive oxygen species as the result of reverse electron transport fueled by succinate. Furthermore, they provide B cell help, independently of IL-21, through IL-10 and succinate. Similar cells are generated in vitro upon priming naive CD4⁺ T cells with plasmacytoid dendritic cells activated with oxidized mitochondrial DNA, a distinct class of interferogenic toll-like receptor 9 ligand³. Targeting this pathway might blunt the initiation and/or perpetuation of extrafollicular humoral responses in SLE.

Figure 1. Ox mtDNA induces a unique pDC phenotype.

a, Cytokine profile of pDCs activated for 24 h with media, CpGB, CpGA or Ox mtDNA (n=7 independent experiments). b, Gene expression profile of pDCs in response to CpGA or Ox mtDNA (n=3 independent experiments). c, Gene expression profile of Th0, CpA and Ox mtDNA CD4⁺ T cell (n=3 independent experiments). d, e Cytokine profile (d) and proliferation (e) of Th0, CpGA or Ox mtDNA CD4⁺ T cells upon reactivation with CD3/CD28 (n=3 independent experiments). f, g MtROS production by CpGA or Ox mtDNA CD4⁺ T cells was assessed by flow cytometry (f, n=3 independent experiments) or by immunofluorescence microscopy (g, one representative of three independent experiments). Scale bar = 7 μm. h, Intracellular (left) and extracellular (right) succinate levels in CpGA or Ox mtDNA CD4⁺ T cells (n=5 independent experiments). Shown are mean ± s.e.m.; statistical analysis by nonparametric one-way ANOVA (a-e) and two-tailed nonparametric unpaired t-test at 95% CI (f, h).

Figure 2. Ox mtDNA CD4⁺ T cells help B cells through IL10 and succinate.

a, Percentage of IgD⁻ CD27⁺ and CD27⁺ CD38⁺ B cells upon co-culture with CpGA or Ox mtDNA CD4⁺ T cells (n=3 independent experiments). Representative flow cytometry density plots are also shown. b, IgM and IgG levels in the supernatants from CpGA or Ox mtDNA CD4⁺ T cells and naïve B cell co-cultures (n=6 independent experiments). c, Immunoblot analysis of succinate receptor (SUCNR1) expression by purified human B cells subsets (n=3 independent experiments). d, IgM and IgG levels in the supernatants from Ox mtDNA CD4⁺ T cell and naïve B cell co-cultures in the presence of anti-SUCRN1 (n=3 independent experiments). e, IgM and IgG levels in the supernatants from co-cultures of Ox mtDNA CD4⁺ T cells, generated in the presence of isotype control or anti-PD1 antibody, and naïve B cells (n=3 independent experiments). Shown are mean ± s.e.m.; statistical analysis by nonparametric one-way ANOVA (b) and two-tailed nonparametric unpaired t-test at 95% CI (a, d, e).

Figure 3. Memory CXCR5⁻ CXCR3⁺ PD1^hi CD4⁺ T cells represent the blood counterpart of Ox mtDNA CD4⁺ T cells.

a, Percentage of CXCR3⁻ PD1^hi, CXCR3⁺ PD1^hi and CXCR3⁺ PD1^low CD4⁺ T cells in the blood CD45RA⁻ CXCR5⁻ compartment of healthy controls (n=13), SLE patients (n=27) or JDM patients (n=6). b-c, IgG levels (b, n=12 independent experiments) and CD20/CD38 expression (c, n=4 independent experiments) on naïve B cells co-cultured with CXCR3⁻ PD1^hi CD4⁺ T cells, CXCR3⁺ PD1^hi CD4⁺ T cells, CXCR3⁺ PD1^low CD4⁺ T cells or Tfh cells. d, Cytokine profile of sorted CXCR3⁺ PD1^hi CD4⁺ T cells and Tfh cells (n=18 independent experiments). e-f, Succinate (e, n=12 independent experiments) and mtROS (f, n=5 independent experiments) levels in CXCR3⁺ PD1^hi CD4⁺ T cells and Tfh cells. g, IgG levels in the supernatants from co-cultures of CXCR3⁺ PD1^hi CD4⁺ T cells or Tfh cells and naïve B cells (n=8 independent experiments). h, Top: principal component analysis (PCA) of RNA-seq data corresponding to genes differentially expressed between CXCR3⁺ PD1^hi CD4⁺ T cells and Tfh cells (n=7 independent experiments). Bottom: volcano plot of up-regulated genes in each population (DESeq2, Wald test, adjusted p-value <0.05 and fold change >2). i, Heat map of differentially expressed transcripts in CXCR3⁺ PD1^hi CD4⁺ T cells and Tfh cells (n=7 independent experiments). j, Top: PCA of ATAC-seq data on peaks differentially accessible between CXCR3⁺ PD1^hi CD4⁺ T cells (n=4 independent experiments) and Tfh cells (n=2 independent experiments). Bottom: chromatin sites with differential accessibility. Plot indicates the number of opening/closing chromatin peaks in CXCR3⁺ PD1^hi CD4⁺ T cells when compared to Tfh cells (EdgeR, adjusted p-value < 0.05 and fold change >2). Shown are mean ± s.e.m.; statistical analysis by nonparametric one-way ANOVA (a-c; g) and two-tailed nonparametric unpaired t-test at 95% CI (d-f).

Figure 4. IL10⁺ IFNγ⁺ ROS⁺ PD1⁺ CD4⁺ T cells accumulate in PLN lesions.

a, Pearson correlation analysis between the frequency of SLE blood CXCR3⁺ PD1^hi CD4⁺ T cells and serum immunoglobulin IgG and IgA levels (n=14 biologically independent samples). b, Representative flow cytometry density plot (left) and percentage of CD19⁺CD21⁻CD11c⁺ B cells (ABCs) among CD3⁻ CD19⁺ cells (right) in the blood of healthy donors (n=6) or SLE patients (n=25). c, Pearson correlation analysis between the frequency of blood CXCR3⁺ PD1^hi CD4⁺ T cells and ABCs. d, Percentage of CXCR3⁺ PD1^hi CD4⁺ T cells in blood of SLE patients with nephritis Class II (n=4 biologically independent samples), Class III/IV (PLN; n=15 biologically independent samples) or without kidney disease (n=20 biologically independent samples). e, Representative immunofluorescence microscopy of CD3, IFNγ and IL10 staining in the kidney of a class IV LN section. The percentage of CD3⁺ IFNγ⁺ IL10⁺; CD3⁺ IFNγ⁺ IL10⁻ and CD3⁺ IFNγ⁻ IL10⁺ cells is also shown (n=10 class III/IV PLN samples; 5 dHPF [digital High Power Field] per sample). f, Representative immunofluorescence microscopy of CD3, Nytrotyrosine and IL10 staining in the kidney of a class IV LN section. The percentage of CD3⁺ IL10⁺ Nytrotyrosine⁺ and CD3⁺ IL10⁻ Nytrotyrosine⁺ cells is also shown (n=5 class III/IV PLN samples; 4 dHPF per sample). g, Representative immunofluorescence microscopy of CD3, IL10 and CD20 staining in the kidney of a class IV LN section. The percentage of CD3⁺ IL10⁺ and CD3⁺ IL10⁻ cells adjacent to CD20⁺ B cells is also shown (n=5 class III/IV PLN samples; 4 dHPF per sample). Scale bar = 10 μm. Shown are mean ± s.e.m.; statistical analysis by nonparametric one-way ANOVA (d) and two-tailed nonparametric unpaired t-test at 95% CI (Welch’s correction b; f-g).