Journal: The Journal of Experimental Medicine

Article Title: Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers

doi: 10.1084/jem.20111449

Figure Lengend Snippet: DTx-mediated selective FDC ablation disrupts B cell follicle architecture despite retention of CXCL13. Unimmunized CD21-DTR chimeras were control (Ctrl) or DTx treated 2 d before analysis of spleen (spl) and pLN sections. (A) Immunofluorescence analysis for BP3 + stromal cells, CD35 + FDCs, and IgD + follicular B cells. (B) In situ hybridization for Cxcl13 mRNA with serial sections stained for B220 and CD35 or for Cxcl13 and CD35. (C) Quantitative RT-PCR analysis for Cxcl13 , Cr1 (CD35), Mfge8 , and Tnsf13B (BAFF) mRNA in pLNs, spleen, and mLNs, normalized to Hprt mRNA. Data are representative of four experiments (mean ± SEM). Statistical analysis was performed with the two-tailed unpaired Student’s t test. *, P < 0.05; n.s., not significant. (D) Immunohistochemical analysis for CXCL13 and CD3 or B220 in serial sections of spleen and pLNs. Data in A, B, and D are representative of at least three experiments (at least two mice of each type per experiment). Bars: (A [left] and B [left and middle]) 200 µm; (A and D, right) 100 µm; (B [right] and D [left]) 50 µm.

Article Snippet: Cryosections of 7 μm were fixed and stained immunohistochemically as previously described ( ) with the following first antibodies: PE-conjugated anti-IgD (11-26c.2a; BD), biotin-conjugated anti-CD35 (8C12; BD), rat anti–mouse fibroblast (ER-TR7; Novus Biologicals), rabbit anti–mouse Collagen IV (Abcam), rat anti–mouse B220 (RA-6B2; BD), biotin-conjugated anti–T/B cell activation antigen or Ly77 (GL7; BD), biotin conjugated anti–mouse CD3e (145-2C11; BD), goat anti–mouse CXCL13 (R&D Systems), goat anti–mouse CCL21 (R&D Systems), rat anti-CD169 (Ser4; P. Crocker, University of Glasgow, Glasgow, Scotland, UK), rat anti–mouse madcam-1 (MECA-367; BD), rat anti–mouse FDC (FDC-M1; BD), and FITC-conjugated rat anti–mouse CD16/32 (FcγRII/III; UCSF Hybridoma Core).

Techniques: Control, Immunofluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Two Tailed Test, Immunohistochemical staining

Journal: Arthritis and rheumatism

Article Title: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

doi: 10.1002/art.23966

Figure Lengend Snippet: Figure 1. CXCL13 expression/production in synovial fluid T cells. A, Real-time polymerase chain reaction for CXCL13 mRNA expression in synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) and healthy donors (n). CXCL13 expression levels are normal- ized to -actin expression levels. Values are the mean and SEM results from 3 patients or healthy donors per group. B, CXCL13 mRNA relative quantification (RQ) of the T cell fractions (solid bars) isolated from RA SFMCs, the respective unfractionated SFMCs (shaded bars), and the T cell–depleted fractions (open bars) for patient samples SF5, SF7, and SF8. Values are the mean and SEM results from triplicate reactions. C, Double immunofluorescence staining for CD3 and CXCL13 on cytospin slides of the isolated T cell fractions. CXCL13 protein with a Golgi-like staining pattern is visualized in 1 of the 3 CD3 cells. D, CXCL13 protein expression analysis in the supernatant of isolated synovial fluid T cell cultures from 3 patients (each line represents an individual patient) at 36 hours and 60 hours. Circles and error bars represent the mean SEM results for triplicate cultures for each sample at each time point. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.

Article Snippet: Sections were then permeabilized with 0.3% saponin solution (Sigma-Aldrich) in TBS, incubated with goat anti-human CXCL13 antibodies (IgG; R&D Systems), and with biotinylated secondary antibodies, followed by rhodamine red X–conjugated streptavidin.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Isolation, Double Immunofluorescence Staining, Staining

Journal: Arthritis and rheumatism

Article Title: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

doi: 10.1002/art.23966

Figure Lengend Snippet: Figure 2. Localization and phenotypic characterization of CXCL13 CD3 cells in rheumatoid synovial tissue. A, Double immunohisto- chemistry for CD3 and CXCL13 in the sublining (left). Right, Higher magnification view of the boxed area on the left. Inset (right), Representative double immunostaining results for CD3–goat IgG. Arrow indicates a double-positive cell. Note the presence of CXCL13 T cells in unorganized areas of lymphoid infiltration and in contact with the lining layer. B, Consecutive sections showing double immu- nohistochemistry for CD3 and CXCL13 (left) and in situ hybridization for CXCL13 (right) in sublining areas enriched for CXCL13CD3 cells (arrows). C, Consecutive sections showing double immunohisto- chemistry for CD3 and CXCL13 (left) and single immunohistochem- istry for CD20 (right). Inset, Higher-magnification view of a CXCL13 T cell. Note the presence of CXCL13CD3 cells in B cell–rich areas. Asterisks indicate CXCL13CD3 cells coexisting with a CXCL13 T cell (arrow) in the same aggregate. D, Three-color immunofluorescence staining for CXCL13CD3CD45RO/CD69/ CD8 on synovial tissue sections, showing colocalization with CD45RO but not with CD8. Some of the CXCL13CD3 cells also express CD69. Arrows indicate triple-positive cells. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.

Article Snippet: Sections were then permeabilized with 0.3% saponin solution (Sigma-Aldrich) in TBS, incubated with goat anti-human CXCL13 antibodies (IgG; R&D Systems), and with biotinylated secondary antibodies, followed by rhodamine red X–conjugated streptavidin.

Techniques: Immunohistochemistry, Double Immunostaining, In Situ Hybridization, Immunofluorescence, Staining

Journal: Arthritis and rheumatism

Article Title: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

doi: 10.1002/art.23966

Figure Lengend Snippet: Figure 3. Quantification/phenotypic characterization of synovial fluid (SF) CXCL13CD3 cells. SF mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) and healthy donors were separated by density-gradient centrifugation and immunostained as described in Patients and Methods. A, Double immunofluorescence staining for CXCL13 (red) and markers of specific T cell subsets (green) on cytospin preparations of SFMCs. Note the presence of CXCL13 in the cytoplasm of CD3CD4CD45RO cells. Counterstaining of the cell nuclei with 4,6-diamidino-2-phenylindole is shown in blue. B, Four-color fluorescence-activated cell sorting (FACS) analysis of SFMCs stained for CXCL13, CD3, CD4, and CD45RO. Middle and bottom, Plots representing the profiles of the gate (top), which was set to select CXCL13 cells. Gates were set based on negative controls with isotype-matched irrelevant antibodies. C, Representative FACS plots showing the profile of CXCL13 staining in CD3CD4CD45RO gated cells from normal donor (ND) PBMCs and from paired RA PBMCs and RA SFMCs obtained by synchronized collection. Data from 6 independent normal donors and 6 patients per group are shown in the graph (right). The horizontal line represents the median. FSC forward scatter.

Article Snippet: Sections were then permeabilized with 0.3% saponin solution (Sigma-Aldrich) in TBS, incubated with goat anti-human CXCL13 antibodies (IgG; R&D Systems), and with biotinylated secondary antibodies, followed by rhodamine red X–conjugated streptavidin.

Techniques: Gradient Centrifugation, Double Immunofluorescence Staining, Fluorescence, FACS, Staining

Journal: Arthritis and rheumatism

Article Title: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

doi: 10.1002/art.23966

Figure Lengend Snippet: Figure 4. Differentiation phenotype of SF CXCL13 T cells. SFMCs from RA patients with active joint involvement were separated by density-gradient centrifugation and immunostained as described in Patients and Methods. A and B, Left, Representative FACS profiles of triple stainings for colocalization of CXCL13CD3CCR7 (A), and CXCL13CD3CXCR5 (B). Gates were set based on negative controls with isotype-matched irrelevant antibodies. Right, Represen- tative double immunofluorescence staining on cytospin preparations for CXCL13 (red) and CD27 (A) or CXCL13 and BCL6 (B). Coun- terstaining of the cell nuclei was performed using 4,6-diamidino-2- phenylindole. Arrowheads show CXCL13 single-positive cells, while the arrows indicate CXCL13CD27 (A) and CXCL13–BCL6 (B) double-positive cells. C, Percent of each studied marker in CXCL13CD3 cells. Values are the mean SEM results obtained from 7 patients (CD69), 4 patients (CCR7 and CXCR5), 9 patients (CD27), and 8 patients (BCL6). See Figure 3 for definitions. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.

Article Snippet: Sections were then permeabilized with 0.3% saponin solution (Sigma-Aldrich) in TBS, incubated with goat anti-human CXCL13 antibodies (IgG; R&D Systems), and with biotinylated secondary antibodies, followed by rhodamine red X–conjugated streptavidin.

Techniques: Gradient Centrifugation, Double Immunofluorescence Staining, Marker

Journal: Arthritis and rheumatism

Article Title: Mature antigen-experienced T helper cells synthesize and secrete the B cell chemoattractant CXCL13 in the inflammatory environment of the rheumatoid joint.

doi: 10.1002/art.23966

Figure Lengend Snippet: Figure 5. CXCL13 modulation by immunologic activation of synovial T cells. A, Time course analysis of CXCL13 protein secretion in the supernatant of synovial fluid mononuclear cell (SFMC) cultures. Circles and error bars represent the mean SEM results for triplicate cultures of 3 samples from 3 different patients. B, Time course analysis of CXCL13 mRNA expression in SFMC cultures. Values are the mean and SEM results for 3 samples from 3 different patients analyzed in triplicate. C, Fluorescence-activated cell sorter (FACS) analysis. After 36 hours of culture with medium alone (resting), plate-bound CD3–CD28 antibodies, or autologous SF (aSF), SFMCs were collected and analyzed by FACS. Plots show representative profiles of CXCL13 staining in CD3-gated cells. Note that a minor proportion of CD3 cells show a high forward scatter (FSC) signal, probably attributable to residual cell clustering following culture. Gates were set on negative controls with isotype-matched irrelevant antibodies. D, Results of the analysis for all patients. The y-axis indicates the percentage of CXCL13 cells in total CD3-gated cells. Values are the mean and SEM results for 3 independent samples from 3 different patients. The input column shows the results of the analysis performed in each sample before culture. P 0.01 by one-way repeated-measures analysis of variance and by Student-Newman-Keuls test for pairwise multiple comparisons. RQ relative quantification; RA rheumatoid arthritis.

Article Snippet: Sections were then permeabilized with 0.3% saponin solution (Sigma-Aldrich) in TBS, incubated with goat anti-human CXCL13 antibodies (IgG; R&D Systems), and with biotinylated secondary antibodies, followed by rhodamine red X–conjugated streptavidin.

Techniques: Activation Assay, Expressing, Fluorescence, Staining, Quantitative Proteomics