Fig 2: The FDC mAb CNA.42 recognizes FBXO2. (A) Immunoprecipitation (IP) and characterization of the CNA-42 binding protein. (A-I) Western blotting of total cell lysate (a), negative control (agarose beads only) and the CNA.42-immunoprecipitated proteins (c and d, 1.5 and 6 uls/lane respectively) from tonsillar single cell suspension probed with CNA-42. A single band is detectable at 120 Kd. (A-II) the reactivity of the CNA.42 mAb on the HuProt™ human proteome microarray showing subarray 9-1 of array 1300017931 (used for the CNA.42) with fluorescence detection at 633 nm excitation (a) and 543 nm excitation (b). (a) Staining with biotinylated anti-GST and Streptavidin-647. Rows 1-28 show generic staining of the GST-tagged immobilized human proteins, among them FBXO2 in row 11. (b) Probing with CNA.42 and Cy3 labeled anti-mouse IgM shows one hit, the human protein FBXO2 in the subarray. (A-III) Western blotting of tonsillar lysates with FBXO2 and CNA-42-specific antibodies recognize 120 Kd bands in the lysates [CNA.42 BP = CNA.42 binding protein]. (B) In situ hybridization of FBXO2 mRNA (green) showing intracellular signal in tonsillar CD21+ FDC reticula (red). (C) Western blotting of lysates from the CAN.42 expressing CEM cell line using mAb CAN.42 and anti FBXO2. CEM were untreated or treated either with Accell human FBXO2 siRNA (1 uM), or non-targeting control (NTC). GAPDH is used as a loading control. Compared to untreated cells, densitometric analysis with Image J indicates that FBXO2 siRNA-treated cells expressed 50% (*) and 35% (**) less FBXO2 and CNA.42, respectively.

Fig 3: Regulation of synovial stromal cell gene expression by PDGF-BB and TNFa/LTß. RA synovial stromal cells expressing Thy-1 on the cell membrane and displaying intracellular aSMA labeling (A) were treated with 300 ng/ml PDGF-BB or 100 ng/ml TNF-a + 100 ng/ml LT-aß and the fold change in gene expression compared to untreated cells was calculated by Livak's DD equation. (B–I) Show the fold change in PDGFR-ß, CSGP4 (NG2), ACTA2 (aSMA), TNFRSF1A (TNF-R), THY1, CR1 (CD35), CR2 (CD21L), Fc?RIIB (CD32), and TNF-aR, respectively. Error bars represent the SEM of three different cell cultures.

Fig 4: Retention of peptidyl citrulline on synovial FDCs and correlation of the FDC gene module with synovial pathotypes and cellular infiltrates. (A) Colocalization of peptidyl citrulline with FDCs in the synovial tissues (A-I), and tonsillar control (A-II). (A-III,IV) peptidyl citrulline trapping on CD21+ synovial FDCs but not CD21-/aSMA+ myofibroblasts (A-III), and colocalization of PAD4 with CD21+ synovial FDCs and peptidyl citrulline [(A-IV), insets: Upper intracellular PAD4, Lower, extracellular PAD4]. Scale bars, overlays and single channels are shown with the relevant panels. (B) Correlation of the FDC module genes with synovial pathotypes and cellular infiltrates. (B-I) Heatmap showing hierarchical clustering of the synovial FDC module gene expression (variance stabilizing transformed counts) against different synovial pathotypes. Upper tracks show histological scores for CD3, CD20, CD68L (lining), CD68SL (sublining), and CD138 and overall pathotype. Tracks on the left-hand side indicate the relationships between the FDC genes expression across all the pathotypes. (B-II) Correlation of the synovial FDC gene module with synovial plasma cells (CD138), B cells (CD20), and T cells (CD3). r = Spearman rho correlation coefficient, p = significance value.

Fig 5: Immunohistochemical localization of human synovial and tonsillar FDCs in the perivascular space vs. established follicles. (A) CNA.42 distribution in the tonsil (A-I) and RA synovium (A-II). Labeling of human FDCs with the mAb CNA.42 (red) colocalizes with an Ab specific for the FDC-restricted CD21 long isoform (CD21L, green) in FDC reticula. The white bracket borders extra-reticular FDCs that are CNA.42+/CD21L-. (B) RA synovial CNA.42+ cells (red) are: (B-I) associated with CD20+ B cells (green) and do not colocalize with CD45 (blue). The inset in the lower left corner shows the synovial intima with CNA.42+ fibroblasts (red/white arrow) arranged side by side with CD45+ intimal macrophages (green) in the lining layer. (B-II) directly related to the CD31+ BVs (blue) within CD20+ B cell follicles (green), and (B-III) directly located in juxtaposition with the CD31+ (blue)/PNAd+ (red) HEVs (white rectangle). The white circle borders established CNA.42+ FDC reticulum (green) detached/distant from the vascular walls. (C) Early FDC markers in the RA synovial perivascular space: (C-I) Synovial labeling with the FDC marker CNA.42 together with the pericyte markers NG2, aSMA, and PDGFR-ß shows that CNA.42 is expressed by cells immediately adjacent to the pericyte layer of the BVs (white arrows). (C-II) The CNA.42+/PDGFR-ß+ early FDCs also express the fibroblasts marker Thy-1 (white rectangle) which is similarly expressed on the BV-associated type-1 pericytes (dashed while circle) but not on the dissociated type-2 pericytes (complete white circle) (C-III). (C-IV) Pericyte-derived differentiating FDCs progressively acquire CNA.42 and CD21/CR2 expression. NG2+ pericytes (green) are marked by a star (*); and differentiating NG2+ (green)/CNA.42+ (blue) and CNA.42+ (blue)/CD21+ (red) FDCs are indicated by dashed and complete white boxes, respectively. (D) Co-labeling of the FDC marker CNA.42 and the fibroblast marker Thy-1 in established follicles (mature FDCs) of RA synovial ELS (D-I) and tonsils (D-II). The fibroblast marker is significantly lost in mature FDCs populating established CD20+ B cell follicles. (D-III) Co-localization of CNA.42 (Blue), Thy-1 (green), and PDGFR-ß (red) in the follicular (F) and inter-follicular areas. The dotted oval demarcates the loss of PDGFR-ß and Thy-1 expression at the follicular border. Single channel, dual and triple overlays are shown in the different panels. Scale bare = 100 um.