Journal: American Journal of Surgical Pathology

Article Title: Angioimmunoblastic T-Cell Lymphoma and Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

doi: 10.1097/pas.0000000000001646

Figure Lengend Snippet: FIGURE 2. Histopathology of the LN with composite lymphoma from patient no. 2. A and B, The LN architecture was effaced by a diffuse lymphoid infiltrate with vaguely nodular appearance, and presence of a few incomplete fibrous bands and small foci of ischemic necrosis (hematoxylin and eosin). C, The lymphoid proliferation is richly vascularized and composed of polymorphic cells rich in large blasts, mixed with smaller, intermediate and small cells with irregular nuclei, associated with eosinophils and some histiocytes (hematoxylin and eosin). D, High magnification of the boxed areas in (A) showed a monotonous population of small mature lymphocytes admixed to some prolymphocytes and paraimmunoblasts (hematoxylin and eosin). Immunostains for CD20 (E), CD5 (F), and CD23 (G) highlighted small lymphoid cells. H, The atypical cells in the polymorphous areas were CD4+, CXCL13+ (I), and PD1+ (J).

Article Snippet: Antibodies against the following antigens were used: CD3 (clone 2GV6; Ventana Medical Systems), CD20 (clone L26; Novocastra, Newcastle, UK), CD79a (clone JCB117, DakoCytomation; Agilent Technologies, Santa Clara, CA), CD2 (clone AB75; Novocastra), CD4 (clone SP35; Ventana Medical Systems), CD5 (clone SP19, Spring Bioscience; Ventana Medical Systems), CD7 (clone CBC37; DakoCytomation), CD8 (clone C8/144B; DakoCytomation), CD10 (clone 56C6; Novocastra), CD19 (clone BT51E; Novocastra), CD21 (clone 1F8; DakoCytomation), CD23 (clone 1B12; Cell Marque, Rocklin, CA), CD30 (clone Ber-H2; DakoCytomation), CD43 (clone DF-T1; DakoCytomation), CD56 (clone CD564; Novocastra), CD138 (clone BA38; Bio-Rad, Hercules, CA), BCL2 (Clone E17; Cell Marque), BCL6 (GI191E/A8; Cell Marque), TIA-1 (clone 2G9A10F5; Beckman Coulter, Brea, CA), granzyme B (clone GrB-7; Monosan, Uden, The Netherlands), CXCL13 (Polyclonal Goat IgG; R&D Systems, Minneapolis, MN), PD1 (polyclonal goat IgG; R&D Systems), ICOS (rabbit polyclonal; Spring Bioscience), TCRb-F1 (clone 8A3; Thermo Fisher Scientific, Waltham, MA), Ki-67 (clone MIB-1; DakoCytomation), kappa, lambda, immunoglobulin (Ig) A, IgG, IgM, IgD (polyclonal rabbit; DakoCytomation).

Techniques: Histopathology

Journal: Transplantation Direct

Article Title: CXCL13 Is an Indicator of Germinal Center Activity and Alloantibody Formation Following Transplantation

doi: 10.1097/TXD.0000000000001247

Figure Lengend Snippet: CXCL13 expressed in graft-DLNs correlates with GC alloreactivity and indicates DSA formation following transplantation. (A) B6 mice were transplanted with BALB/c (allogeneic) or b6 (syngeneic) skin grafts and draining lymph nodes collected for flow cytometric and PCR analysis. Mice were also serially bled, and serum collected for CXCL13 and antibody analysis. (B) Serum CXCL13 levels in naïve and syngeneic or allogeneic skin-grafted mice 10 d posttransplant. (C) IL-21 and CXCL13 mRNA expression in graft-DLNs 5 d after primary and secondary skin grafts. (D) IL-21 and CXCL13 mRNA expression in sorted murine Tfh (PD1 hi CXCR5 + ) cells compared with CD4 + non-Tfh (CD44 hi CXCR5 – ) cells. (E) Serum CXCL13 levels over time relative to graft-DLN Tfh cell (green), GC B cell (blue), and DSA (orange) kinetics. Summary data represent mean (SE) and are representative of at least 2 independent experiments with a total of 5–10 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; DLN, draining lymph node; DSA, donor-specific antibody; GC, germinal center; Tfh, T follicular helper.

Article Snippet: For murine serum samples, the mouse CXCL13/BLC/BCA-1 DuoSet kit (R&D Systems DY470) or an in-lab assay using anti-mouse CXCL13 (R&D MAB470) as a capture antibody and R&D anti-mouse CXCL13 biotinylated antibody (BAF470) as a detection antibody were used.

Techniques: Transplantation Assay, Expressing

Journal: Transplantation Direct

Article Title: CXCL13 Is an Indicator of Germinal Center Activity and Alloantibody Formation Following Transplantation

doi: 10.1097/TXD.0000000000001247

Figure Lengend Snippet: Human CXCL13 correlates with GC-like Tfh:B-cell reactivity in vitro. (A) Lymph node–derived human lymphocytes were sorted into CXCR5 – T and CXCR5 + Tfh cells and cultured in vitro with enriched B cells for 5 d. Cocultured cells were collected for flow cytometric and PCR analysis, and culture supernatants were collected for antibody assessment. (B) Representative flow plots and summary data of plasmablast (MHCII + CD27 hi CD38 + B cells) formation. (C) Summary data of IgG antibody production as measured by ELISA (450 nm OD). (D) Summary data of coculture IL-21 and CXCL13 mRNA expression. (E) Representative flow plots and summary data of CXCL13 production by CD4 + T cells. Summary data represent mean (SE) and are representative of 2 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; Tfh, T follicular helper.

Article Snippet: For murine serum samples, the mouse CXCL13/BLC/BCA-1 DuoSet kit (R&D Systems DY470) or an in-lab assay using anti-mouse CXCL13 (R&D MAB470) as a capture antibody and R&D anti-mouse CXCL13 biotinylated antibody (BAF470) as a detection antibody were used.

Techniques: In Vitro, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Transplantation Direct

Article Title: CXCL13 Is an Indicator of Germinal Center Activity and Alloantibody Formation Following Transplantation

doi: 10.1097/TXD.0000000000001247

Figure Lengend Snippet: CXCL13 levels correlate with the development of alloantibodies in kidney transplant recipients. (A) Serum CXCL13 levels in HC (n = 19) and kidney transplant recipients with (DSA+, n = 15) and without (ST, n=8) de novo DSA. DSA+ samples were collected from patients within 2 wk of developing de novo DSA. (B) Serum CXCL13 levels (red line) and cumulative HLA antibodies (black bars) over time in 3 kidney transplant recipients with de novo DSA formation. Serum CXCL13 levels in 3 separate healthy controls (C) and 3 separate ST recipients without DSA (D) over time. Summary data represent mean (SE). * P < 0.05, *** P < 0.001. CXCL13, chemokine (C-X-C motif) ligand 1; DSA, donor-specific antibody; HC, healthy controls; ST, stable transplant.

Article Snippet: For murine serum samples, the mouse CXCL13/BLC/BCA-1 DuoSet kit (R&D Systems DY470) or an in-lab assay using anti-mouse CXCL13 (R&D MAB470) as a capture antibody and R&D anti-mouse CXCL13 biotinylated antibody (BAF470) as a detection antibody were used.

Techniques:

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