Fig 1: 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.
Fig 2: Tregs in Human Fetal Skin Demonstrate an Effector Memory PhenotypeTwenty-three weeks g.a. fetal torso skin and healthy adult torso skin samples were analyzed for 22 markers using mass cytometry.(A) PCA plot of Tregs (cluster C, Figure 2) and CD4+ Tconv (clusters A & B, Figure 2) from 23 week fetal skin versus adult skin based on relative expression of key markers as assessed by mass cytometry.(B–E) Graphs showing median expression intensity (m.e.i.) of (B) Foxp3, (C) CD25, (D) PD-1, and (E) CTLA4 by mass cytometry on fetal versus adult skin Tregs.(F and G) Cells were isolated from 17–23 weeks g.a. fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (F) Percentage of CD45RO+ Tregs in skin by age and (G) accompanying representative flow plots.(H) Representative histogram and (i) quantification of Nur77 MFI on fetal skin CD45RO+ versus CD45RA+ Tregs.(J) Nur77 in fetal skin CD45RA+ CD8+, CD4+ Tconv, and Tregs.Each point in (A)–(E) represents data from an individual donor. Points in (F), (I), and (J) point represent data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3: A novel Nr4a1/Ear2 axis mediated anti-inflammatory signaling in TLR4 deficient BMDMs. A) Different macrophage/monocyte clusters’ maker genes expression in LPS-stimulated BMDMs in vitro between Tlr4flox/flox and Tlr4fl/fl/LysM-cre group. B) Gene expression in BMDMs, with or without Nr4a1 siRNA (siNr4a1) or Ctrl siRNA (siCtrl) pre-treatment, under LPS stimulation for 6 h. C) ELISA analysis of BMDMs EAR2 protein expression with or without siNr4a1 or siCtrl pre-treatment, under 500 ng mL-1 LPS stimulation for 48 h. D) Direct binding of Nr4a1 subunit on Ear2 promoter region in BMDM under LPS stimulation shown by ChIP assay. E) Gene expression in BMDMs, with or without Ear2 siRNA (siEar2) or siCtrl pre-treatment, under LPS stimulation for 6 h.
Fig 4: Subsets of Fetal Skin CD4+ Tconv and CD8+ T Cells Display a Memory Phenotype and Demonstrate Capacity for IFN? ProductionCells were isolated from second trimester fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry.(A) Representative plots demonstrating CD45RO expression by fetal CD8+ T cells (gated on live CD3+CD8+CD4neg) and CD4+ Tconv (gated on live CD3+CD4+CD8negFoxp3negCD25lo).(B and C) Percentage of CD45RO+ (B) CD8+ T cells and (C) CD4+ Tconv in fetal versus adult skin.(D–F) Representative histogram (D) and quantification (E and F) of Nur77 MFI on fetal skin CD45RO+ versus CD45RA+ CD4+ Tconv and CD8+ cells.(G–J) Percentage of CD4+ Tconv producing (G) IL-17A, (H) IL-13, and (I) IFN? after PMA/ionomycin re-stimulation, and (J) percentage of IFN?-producing CD8+ T cells.Each point in (B)–(G) represents data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 5: B cells in NR4A+ clusters display somatic hypermutation (SHM) and clonal expansion(A) Violin plots display SHM rate, averaged across detected chains, in each B cell cluster obtained from single-cell BCR sequencing. Each cluster’s SHM rate was tested against naive(i) using linear mixed models, with p < 0.05 displayed. See also Figures S2E and S2F.(B) Scatterplots display the associations between percent SHM and expression level of CD27, NR4A1, NR4A2, and IGHD in NR4A+, memory and plasma cell (combined PC(i) and PC(ii)) clusters. Blue lines display a linear mixed model fit. p values are from a linear mixed effect model that adjusts for sample and number of genes detected. Only values of p < 0.05 are displayed.(C) Number of cells with >97% DNA identity of the heavy-chain CDR3. The amino acid sequence of each putative clone is shown on the left.(D) Heavy-chain BCR data were superimposed on cluster t-SNE plot. “None” (gray) indicates cells from which BCR was not recovered, “Recovered” (black) indicates cells from which BCR was recovered, “Expanded” (blue) shows cells with clonality in =2 cells, “CARHWRGKKPFDSW” (red) indicates cells with a shared clone recovered from both NR4A+ and plasma cells. See also Figure S2D.
Supplier Page from Thermo Fisher Scientific for Nur77 Antibody PE