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. 2018 Oct 16;49(4):725-739.e6.
doi: 10.1016/j.immuni.2018.08.015. Epub 2018 Oct 9.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus

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Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus

Scott A Jenks et al. Immunity. .
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Abstract

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Keywords: Autoimmunity; CD11c; Systemic Lupus Erythematous; T-bet; TRAF5; Toll-like receptor 7; effector; human B cells; plasma cells.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. IgD-CD27-DN B cells in SLE.
A. B cell populations in SLE and HCD. Parental CD19+ populations defined by IgD and CD27: IgD-CD27+ (switched memory; SWM); IgD+CD27+ (unswitched memory; USW); IgD+ CD27-(naïve; NAV); IgD-CD27-(double negative; DN cells). Mean ± SD shown for HCD n=21, SLE1 n=40, SLE2 n=50, Welch’s t-test. B. In HCD most DN B cells are CXCR5+ (DN1 cells) with a minor fraction (DN2 cells) lacking CXCR5 and expressing high amounts of CD19. Representative examples of large DN2 cells expansions in SLE (n=8), where they become the largest DN fraction. C. CXCR5-, CD19 bright DN2 cells are CD21. D. DN2 cells are CD11c bright. E. A higher proportion of DN cells are DN2 cells in SLE patients than HCD (Welch’s t-test). F. The frequency of DN2 cells in SLE cohorts, rheumatoid arthritis (RA, n=15), primary Sjögren’s syndrome (pSS, n=11), and scleroderma patients (SCD, n=21). Dotted line indicates the mean of the HCD frequency plus 2 SD (4.01%). G. Absolute number of DN2 cells/μl in SLE patients from cohort 2 with (n=11) or without (n=21) DN2 cell expansion.
Figure 2.
Figure 2.. Phenotype of SLE DN2 B cells.
A. Surface staining of SLE B cells for the indicated markers. DN2 cells (shaded) are compared to NAV (solid line) and SWM (dotted line) B cells. B. Representative (n=8) flow cytometry from active SLE. NAV and DN cells are separated based on CXCR5 and CD19 into DN1 cells, DN2 cells, rNAV (IgD+, CXCR5+, CD19+), and aNAV (IgD+, CXCR5−, CD19 bright). MTG is positive for both DN2 cells and aNAV (open) but negative for rNAV and DN1 cells (filled). C. aNAV and DN2 cells are CD24−CD38−, MTG+, and CD11c bright SLE and HCD. D. Frequency of IgA+, IgG3+, and non-IgG3 IgG+ for the indicated subsets in HCD (n=5) and SLE (n=4) patients (Mean ± SD) (Welch’s t-test). E. IgG+ DN2 cells have lower surface IgG than DN1 cells or SWM in both HCD and SLE (Mean ± SD). F. Unlike HIV CD21-DN cells, SLE DN2 cells do not express FCRL4. On the left, representative (n=5) flow profiles comparing CD21 and FCRL4 for HIV and SLE DN cells, followed by the percent of CD21-DN cells that are FCRL4+ for HCD (5), HIV (8), and SLE (6) patients. On the right, the mean fluorescence intensity of FCRL5 staining is shown for different B cell populations from SLE patients (filled; n=5) and HCD (open; n=5) (Mean ± SD, n=7). G. Proximal BCR signaling is intact in DN2 cells as indicated by BLNK phosphorylation fold increase after anti-IgG stimulation. Values are shown for non-responding (IgA+) or responding (IgA-) cells (n= 5; *= p value relative to responding SWM; †=p value relative to non-responding total DN, repeated measure 1-way ANOVA).
Figure 3.
Figure 3.. Clinical and demographic correlates of patients with expanded DN2 cells.
A. African American (AA) patients (n=58, open symbol) have significantly higher proportions of DN2 cells relative to non-AA patients (n=32, closed symbol) (Mean ± SD, Welch’s t-test). B. Lack of correlation between the DN2 cell frequency of CD19+ B cells and age in SLE cohorts. The percentage of DN2 is shown on a log2 scale. C. Flow profile of a 5-year-old female lupus patient in whom most CD19+ cells are DN2 cells and aNAV. D. Positive correlation between DN2 cell frequency and high disease activity using Spearman’s r coefficient (left panel). Median SLEDAI is plotted against the percentage of DN2 cells with the dotted line indicating the cut-off for expanded DN2 cells (left panel). Middle panel: SLEDAI in patients with or without DN2 cell expansion. Right panel: frequency of DN2 cells in patients with active lupus nephritis (n=22), patients with low disease activity (n=33) and HCD (n=21). E. Patients with expanded DN2 cells are more likely to be treated with prednisone (PDN). The percentage of patients treated is shown in black and untreated in white for patients with high or low (L) DN2 cell frequencies. P value is shown for Fischer’s exact test. Mycophenolate mofetil (MMF); hydroxychloroquine (HCQ); azathioprine (AZA); cyclophosphamide (CYC). F. Correlation between serum interferon activity and percent of DN2 cells (Spearman’s r coefficient). Right panel: Patients with increased serum IFN activity had significantly higher DN2 frequencies. G. Serum anti-RNA antibody concentrations in patients with low (L, n=33) or high (H, n=25) frequencies of DN2 cells. The dotted line is the mean OD for HCD serum (Mean ± SD, Welch’s t-test). H. The percentage of samples that are positive (black) for each of the indicated serologies in patients with a high (H) and low (L) frequency of DN2 cells (Fischer’s exact test); I. Anti-Smith-D and anti-RNP-70 serum antibody titers from patients from cohort 1 measured by LIPS assay correlate with the percentage of DN2 cells (Spearman’s r coefficient). Please also see Figure S1.
Figure 4.
Figure 4.. DN2 cells gene expression is distinct from other B cell populations.
A. Number of differentially expressed genes (DEG), between each B cell subset of pooled HCD and SLE samples. B. Analysis of principal components accounting for 47% of the variance between B cell subsets. Patients groups and cell subsets are indicated. * identifies NAV B cells from the lupus nephritis patient (SLE3), with a large fraction of aNAV cells. C. Heat map of select DEG that differentiate B cell subsets. Samples are clustered based in Euclidian distance. In SLE-3 (*), NAV cells cluster with the DN2 cells. Genes are grouped by expression pattern as shown in the legend. D. DN2 cells highly express TBX21 and ZEB2. The top panel represents FPKM (Fragments Per Kilobase of transcript per Million mapped reads) RNA expression values for TBX21 and ZEB2. Middle: representative (n=4) histogram depiction of T-bet expression by flow cytometry; Bottom: T-bet MFI (n=4, Mean ± SD). E. FPMK values of TRAF5 and ZEB1 showing uniquely low expression in DN2 cells (top). Conversely, TCF7 is expressed only by SWM and DN1 while BACH2 is expressed only by rNAV (bottom). F. Network diagram of select genes up regulated (left) or down regulated (right) in DN2 cells. Transcription factors are green octagons, genes are pink ovals. Arrows represent that motifs for that TF are enriched in a gene and arrows pointing to a TF indicate differential expression of that TF. Please also see Figure S2, S3, S4, and S5.
Figure 5.
Figure 5.. Transcriptional and functional characterization of SLE DN2 cells as precursors of autoantibody producing plasma cells.
A. GSEA analysis of RNA-Seq data. DN2 cells are enriched in IRF4 target genes expressed in PC relative to SWM cells. B. Flow cytometry histograms demonstrate higher expression in DN2 cells and aNAV cells of SLAMF7, an IRF4 target gene highly expressed by PC. C. RNA-Seq analysis shows that DN2 cells express more IRF4 and PRDM1 but less IRF8 and ETS1 than SWM B cells. D. BLIMP1 measurement by flow cytometry. aNAV and DN2 cells have higher expression than other B cells (n=4, Mean ± SD, repeated measure 1-way ANOVA). E. DN2 cells and aNAV cells express more IRF4 than rNAV and DN1 cells and less IRF8 than other B cell subsets and a lower IRF4/IRF8 ratio in both HCD (n=5) and SLE(n=5). A representative SLE example is shown on the left and quantification of IRF4 and IRF4/IRF8 ratio is shown on the right (repeated measure 1-way ANOVA). F. DN2 cells differentiate into IgG ASC despite little expansion. Replicate wells of day 7 expansion (left), IgG concentration (middle), and IgG concentration normalized to cell number (right). Replicate cultures are shown for 1 out of 4 patients (Mean ± SD, Welch’s t-test). G. The frequency of IgG producing spots does not significantly differ between DN1 cells, DN2 cells, and SWM (n=4). H. Frequency of IgG ASC measured by ELISPOT of B cell subsets stimulated with or without IL-21 and IL-2. Replicate ELISPOT wells are shown for 1 out of 2 patients. I. Quantitation of autoantibody production by LIPS assay. DN2 cells cultured as before produce anti-Smith, anti-Ro, and anti-RNP autoantibodies in amounts comparable with SWM cells. Results are shown for three patients as light units normalized to IgG concentration. Serum autoantibody concentrations for the same time point are shown above. Please also see Figure S6 and S7.
Figure 6.
Figure 6.. TLR7 signaling and responsiveness is enhanced in DN2.
A. CD25 expression is increased over baseline (filled) by trimerized CD40L stimulation (blue line) in rNAV but not DN2 cells. In contrast TLR7 stimulation by R848 (red line) increased CD25 expression in both rNAV and DN2 cells. One example out of two experiments is shown. B. The percentage of dual positive phospho-ERK and phospho-MAPKp38 positive CD11c+ DN cells is increased after R848 stimulated (bottom) relative to unstimulated (top) and to SWM, rNAV, and DN1 B cells. C. Fluorescence intensity of phospho-ERK and phospho-MAPKp38 is higher in DN2 cells and aNAV cells after R848 stimulation. Histogram coloring is based on median fluorescence intensity as shown by the scale bars. D. Relative induction of pERK (n=5) and pMAPKp38 (n=10), expressed as the fold increase in MFI of R848 stimulated samples, is higher in DN2 cells and aNAV cells. Although induced pY-ERK phosphorylation is lower in HCD B cells (n=7), DN2 and aNAV cells still have significantly higher phosphorylation (repeated measure 1-way ANOVA). E. In SLE patients HLA-DR (n=6) and CD86 (n=5) expression is higher at baseline in DN2 cells (green) than NAV (blue) or SWM (red) but this difference is further enhanced after TLR7 stimulation. Expression of inhibitory receptors CD32b and CD72 is reduced by R848 stimulation in DN2 cells (blue) but not NAV B cells (green) (n=5). HCD B cells demonstrated similar changes except for CD72, which did not decrease in either rNAV or DN2 cells. Red asterisks indicate significant differences from SWM; blue asterisks indicate significant differences from NAV (repeated measure 1-way ANOVA).
Figure 7.
Figure 7.. TLR7 and cytokine stimulation induce differentiation of naïve B cells into aNAV, DN cells and PC.
A. TLR7 and IFNγ (left) but not IL-4 (right) induce aNAV, DN2 cells, and PC differentiation from HCD resting naïve (rNAV) B cells in an IL-21 dependent manner. Cells from cultures featuring IFNγ and IL-21 reproduce the in vivo phenotype of aNAV and DN2 cells with CD11c, T-bet, and FCRL5 upregulation (bottom left). B. IL-21 is required for day 7 PC development within the IFNγ cultures from HCD rNAV cells. Similar results were obtained with SLE B cells (not shown). C. rNAV cells from both SLE patients and HCD stimulated with TLR7 agonist and cytokines differentiate into PC, DN2 cells, and aNAV B cells with equal efficiency (representative examples; n=5). D. Kinetics of the development of aNAV and DN2 cells from rNAV cultures from HCD (left). Frequencies of aNAV and DN2 cells in the non-PC fraction of day 7 HCD and SLE cultures (right). E. Direct differentiation into DN2 cells starting from rNAV or aNAV in 3 day cultures using similar stimulation as before (left and middle). aNAV cells generated significantly more DN2 cells in either HCD (n=1) or SLE (n=3) than rNAV. Day 5 cultures started with aNAV cells (2 HCD and 2 SLE; right) contained significantly higher frequencies of PC (Mann Whitney test). F. DN2 cell cultures have an equivalent ability to generate PC as SWM and DN1 cell cultures and are superior to rNAV cultures (HCD= open circles; SLE = shaded circles). Representative examples are shown for HCD and SLE (n=4). TLR7 stimulation, tested by excluding R848 was essential for DN2 cell to PC differentiation (tested in SLE only; bottom left). When normalized to cell number the IgG output of DN2 cell cultures were equivalent to SWM and DN1 cell cultures and superior to rNAV in both HCD and SLE cells (bottom right; Welch’s t-test). G. LIPS analysis of day 7 culture supernatants from two patients with Ro, SmD, and RNP autoantibody titers.

Comment in

  • Double-negative B cells.
    Bernard NJ. Bernard NJ. Nat Rev Rheumatol. 2018 Dec;14(12):684. doi: 10.1038/s41584-018-0113-6. Nat Rev Rheumatol. 2018. PMID: 30367167 No abstract available.

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