Fig 2: Increased immunoglobulin production and antibody diversity in HS skin and complement activation.Box-and-whisker plots of BCR CDR3 expressions (A). The y axis shows normalized log-transformed BCR CDR3 expression. The x axis represents patient group. In all cases there were more BCR CDR3 sequences detected in HS skin compared with control healthy skin. Box-and-whisker plots of BCR gene segment expression. The y axis shows normalized log-transformed BCR gene segment expression. The x axis represents patient group. The Shannon diversity index for BCR CDR3 gene segment is plotted on the y axis. The x axis represents patient group. HS skin had a significantly more diverse BCR repertoire (B). Beta diversity–based principal coordinates analysis (PCoA) of BCR CDR3 sequences. Sample matrix was generated using Jaccard dissimilarities, and respective profiles were compared by PCoA. Each color represents 1 patient group, HS (red) and control (blue). This analysis revealed clear separation for ? and ? light chains but not Ig heavy chain (C) Hierarchical clustering of expressed TCR V/J gene segment expression. Heatmaps by clonal abundance across sample sets. Note good separation of HS from controls based upon clonal abundances in BCR ? and ? repertoires. Components of the complement pathway (C1q) and breakdown products of activated complement components (C3b, C4d) were increased in HS skin, particularly in the deeper layers of the skin (n = 3) (scale bar: 100 µm) (D). Complement receptors, CR1 and CR2, were increased in the deeper layers of HS, along with IgG1 immune complex deposition (n = 3) (scale bar: 100 µm) (E). Immunofluorescence of B cells (CD20) and plasma cells (CD138) showed primary localization of TNF to the plasma cell population in HS skin (n = 3) (scale bar: 50 µm) (F). For A and B, the bold vertical line represents the median, and the upper and lower limits of the box represent the interquartile range (IQR). The whiskers represent 1.5× IQR.

Fig 3: Lymphoid-specific NR4A subset genes correlate with RA synovial tissue pathotype and increase in blood during RA flare(A) Boxplots of histology by tertile demonstrating correlation with CD83 (Padj lymphoid versus myeloid = 3.0e–02; Padj myeloid versus fibroid = 3.4e–02; Padj lymphoid versus fibroid = 3.3e–07), GPR183 (Padj lymphoid versus myeloid = 5.4e–03; Padj myeloid versus fibroid = 4.6e–02; Padj lymphoid versus fibroid = 4.1e–08) and LY9 (Padj lymphoid versus myeloid = 1.4e–07; Padj myeloid versus fibroid = 8.0e–01; Padj lymphoid versus fibroid = 5.0e–07). p values were calculated by linear regression models.(B) Correlation heatmap showing spearman correlations of CD3, CD20, CD138, CD68L, and CD68SL histology scores and ultrasound biopsy joint parameters (ST, synovial thickness; PD, power Doppler) at the biopsy joint (ultrasound ST/PD BJ) against CD83, GPR183, LY9, and NR4A1-3 expression. The stars represent the significance of the correlation coefficient: *p < 0.05, **p < 0.01, ***p < 0.001. The plots and p values were analyzed from Lewis et al. (2019).(C) Relative expression of marker genes (see STAR Methods for details of calculation) from the synovial B cell subpopulations in the weeks before and after flare (Orange et al., 2020). See Table S2 for list of overlapping genes. Top panel shows the mean RAPID3 disease activity for the four donors studied. The dashed vertical line marks the time at flare. See also Figure S9.

Fig 4: NR4A1 is expressed by B cells and plasma cells in synovium ELS(A) Synovial serial sections were stained with four antibody combinations, as indicated on the top of each image. The dashed yellow line outlines a GC containing CD20+IgD-PCNA+ centroblasts. Green and white boxes mark the area magnified in the right images to show details of cellular morphology and subcellular location of NR4A1. Corresponding single-color images for CD20, CD3, CD138, and NR4A1 are shown on the right. # and * indicate location of cropped areas. Green arrows point to NR4A1+CD20+ B cells, yellow arrows depict NR4A1+CD3+ T cells, and white arrows show areas with significant accumulation of NR4A1+CD20+CD138+ plasmablasts on the periphery of secondary B cell follicles in the synovium. Scale bars, 100 µm in (100× magnification pictures).(B) Bar graph represents mean of percent NR4A1+ B cells in ex vivo sample from NC blood (n = 5), RA blood (n = 5), and RA synovial fluid (n = 6).(C) Example dot plots showing the distribution of NR4A1+ B cells from synovial fluid. See also Figure S8A.(D) Bar graphs display distribution (%) of NR4A1+ or NR4A1- B cells across B cells subsets: naive, double-negative (DN), unswitched memory (USM), switched memory (SM) (this gate may include PC), and plasma cell (PC) from synovial fluid (n = 7). PC is defined as CD19+IgD-CD27hiCD38hi.(E) Bar graphs display percent of naive, DN, USM, SM, and PC that are NR4A1+.(F) Histogram of NR4A1 expression by naive, USM, SM, DN, and PC, from synovial fluid (SynF) compared with blood naive and NR4A1 fluorescent-minus-one control (FMO). Right line graphs show NR4A1 geometric mean fluorescent intensity (gMFI) in naive, USM, SM, DN, and PC from synovial fluid (n = 6). Each line represents an individual sample.(G) Dot plots display NR4A1+ and NR4A1- B cell distribution across B cell subsets from synovial tissue. Histogram represents NR4A1 expression by B cell subsets gated using CD38 and IgD. Line plot shows NR4A1 gMFI in different B cell subsets from synovial tissue (n = 1). In (B), (D), and (E), error bars are SEM. Statistical test was non-parametric one-way ANOVA and Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01.(H) NR4A1 expression in B cells from PBMC treated with anti-Ig(A + M + G) for 4 h. Scatterplots represent percent NR4A1+ B cells after treatment (S) compared with untreated control (U). **p < 0.01 by paired t test. See also Figures S8B and S8C.