Journal: Communications Biology

Article Title: Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction

doi: 10.1038/s42003-022-03344-6

Figure Lengend Snippet: a qPCR analysis of ltα mRNA transcripts from wt (black bars) salivary glands at day 0, 3 h, 6 h, day 1, day 2, day 5, day 8 and day 15 p.c. Gene expression was normalized to housekeeping gene β-actin and expressed as RQ values relative to day 0 mRNA transcripts results. b CD45+ cells expressing IL13, IL22 or LTα3 at day 2 p.c. (grey bars) and day 5 p.c. (white bars) in wt mice. c Enumeration of LTα + lymphocytes within the CD45+ cells in wt salivary glands at day 5 p.c. by flow cytometry. Representative dot plot of LTα expression in CD45+ cells and its frequency within CD3ε+ and B220+ cells. d – f Graphs showing qPCR analysis of ltα ( d ), ccl19 ( e ) and cxcl13 ( f ) mRNA transcripts from wt (black bars) and Cd3ε −/− (dark grey bars) salivary glands at day 5, 8 and day 15 p.c. g Quantification of ccl19 and cxcl13 mRNA transcripts from wt (black bars) and Ltα −/− (light grey bars) salivary glands at day 5, 8 and 15 p.c. In all cases, gene expression was normalized to housekeeping gene β-actin and expressed as RQ values relative to day 0 mRNA transcripts results. h Immunofluorescence staining in salivary glands from wt and Ltα −/− mice for lymphoid aggregates with CD3ε (red), CD19 (blue) and CXCL13 or CCL21 (green) at day 15 p.c. Scale bar 100 µm. i qPCR analysis of ccl19 and cxcl13 mRNA transcripts from wt (black bars) and Tnfr1/2 −/− (white bars) salivary glands at day, 5, 8 and 15 p.c. Chemokine mRNA transcript results were normalized to β-actin. j Immunofluorescence staining for lymphoid aggregates within infected salivary glands from wt and Tnfr1/2 −/− mice with CD3ε (red) and CD19 (blue) at day 8 p.c. Scale bar 100 µm. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test ( a ) and unpaired t test ( b , d , e , f , g , i ).

Article Snippet: Quantitative real-time PCR was performed as previously described in , using following primers Ccl19 (Mm00839967_g1), Cxcl13 (Mm00444533_m1), Lta (Mm00440229_g1), Ltb (Mm00434774_g1), Tnf (Mm00443258_m1), Tnfrsf1a alias Tnfra (Mm00441883_g1), Icos (Mm00497600_m1), Icosl (Mm00497237_m1) and Actb alias β-actin (Mm01205647_g1) was used as an endogenous control.

Techniques: Gene Expression, Expressing, Flow Cytometry, Immunofluorescence, Staining, Infection, Comparison

Journal: Communications Biology

Article Title: Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction

doi: 10.1038/s42003-022-03344-6

Figure Lengend Snippet: a Representative dot plots showing flow cytometry staining for ICOS expression in wt salivary glands at day 5 p.c. by CD45+ cells. Graph showing percentage of ICOS+ T lymphocytes and NK cells infiltrating salivary glands at day 5 p.c. b viSNE plots of ICOS+ T lymphocytes from infected salivary gland from wt mice at day 5 p.c., analyzed by multicolor flow cytometry. Colours indicate cell expression level of labelled markers (LTα3 and CD4). c , d Flow cytometry analysis of absolute numbers of T cells and LTα-producing T cells in wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. e , f qPCR analysis of ltα and icos mRNA transcripts in FACS sorted CD3+ cells from wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. mRNA transcripts were normalized to housekeeping gene β-actin and presented as RQ values calculated with calibrator day 0 CD45+ cells. g wt mice were left untreated or treated with anti-ICOS blocking antibody from day 0 post salivary gland cannulation and sacrificed at days 5 and 8 p.c. Cannulated Icosl −/− were also sacrificed and analyzed at days 5 and 8 p.c. Representative microphotographs of salivary glands examined by immunofluorescence for CD3ε (red) and CD19 (blue) showing lymphocytic infiltration. Scale bar 100 µm. h qPCR analysis of mRNA obtained from salivary glands at day 5 and 8 p.c. of lymphoid chemokine ccl19 and cxcl13 in anti-ICOS treated wt mice or Icosl −/− mice (both white bars) in comparison to wt mice (black bars). mRNA transcripts were normalized to β-actin mRNA and results are presented as relative quantitation (RQ). i Transcript levels for ccl19, ccl21c and cxcl13 in FACS sorted CD45-EpCAM-CD31-pdpn+ immunofibroblasts from wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. Transcripts levels were normalised to β-actin. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. non-significant, unpaired t test.

Article Snippet: Quantitative real-time PCR was performed as previously described in , using following primers Ccl19 (Mm00839967_g1), Cxcl13 (Mm00444533_m1), Lta (Mm00440229_g1), Ltb (Mm00434774_g1), Tnf (Mm00443258_m1), Tnfrsf1a alias Tnfra (Mm00441883_g1), Icos (Mm00497600_m1), Icosl (Mm00497237_m1) and Actb alias β-actin (Mm01205647_g1) was used as an endogenous control.

Techniques: Flow Cytometry, Staining, Expressing, Infection, Blocking Assay, Immunofluorescence, Comparison, Quantitation Assay

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: OncoImmunology

Article Title: Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

doi: 10.1080/2162402x.2017.1290034

Figure Lengend Snippet: Figure 4. HDCC myeloid cells recruit Foxp3C Tregs in colitis-associated colon carcinogenesis model. (A) Comparison of circulating chemokine levels in DTA¡ (n D 5) and DTAC (n D 6)AOM/DSS tumor-bearing mice sera. (B) Expression of Cxcl13 mRNA from sorted CD45C cells in colon tumor tissue, comparing DTA¡ and DTAC (n D 3 per group). (C) Representative fluorescence staining showing Foxp3C cells in colon tumor tissue in DTA¡ and DTAC AOM/DSS-treated mice (n D 3 per group). Scale bar D 50 mm. (D) Percentages of Foxp3-GFPC cells in colon CD4C cells in AOM/DSS-treated mice with (n D 3) or without (n D 4) rCxcl13 neutralization antibody. (E) Migration of Foxp3C cells in response to Cxcl13. Showing quantitation of migrated Foxp3C cell numbers by flow cytometry after co-culture of 5,000 splenic Foxp3-GFPC cells (from spleens of AOM/DSS-treated Foxp3-GFP mice) in transwell plates with either HDCC or HDC¡ myeloid cells. N D 3 to 5 per group. p < 0.05; p < 0.01; p < 0.001, data are mean § SEM, representing two to three independent experiments. Data were analyzed with two-tailed Student’s t-test (A, B, and D) and Mann–Whitney test (E).

Article Snippet: Recombinant mouse Cxcl13 (Biolegend, San Diego, CA) was injected i.p. at 50 mg/mouse.

Techniques: Comparison, Expressing, Staining, Neutralization, Migration, Quantitation Assay, Cytometry, Co-Culture Assay, Two Tailed Test, MANN-WHITNEY

Journal: OncoImmunology

Article Title: Histidine decarboxylase (HDC)-expressing granulocytic myeloid cells induce and recruit Foxp3+ regulatory T cells in murine colon cancer

doi: 10.1080/2162402x.2017.1290034

Figure Lengend Snippet: Figure 5. HDCC myeloid-derived Cxcl13 augment Foxp3 expression through Stat3 phosphorylation. (A) Mouse experimental protocol. (B and C) Levels of phospho-Stat3 in Foxp3-GFPC cells isolated by flow sorting from colon tumors. (B) Shows representative flow histogram graphs of phospho-Stat3, while (C) shows quantitation of phos- pho-Stat3CFoxp3C cells (n D 4 DTA¡, 5 DTAC, 4 DTAC C HDCC myeloid transfer, 5 DTAC C rCxcl13). (D) Foxp3C cell proliferation measured by BrdU incorporation assay. Foxp3-GFPC cells cultured in vitro with either HDCC or HDC¡ myeloid cells (n D 5 to 8), with a knockdown in some groups of Cxcr5 or Cxcl13 by siRNA in Foxp3-GFPC cells or HDCC myeloid cells, respectively. rCxcl13 was added to Foxp3-GFPC cells cultured without myeloid cells. Cells were isolated from spleens of AOM/DSS- and tamoxifen- treated Foxp3-GFP;Hdc-CreERT2;Rosa26-tdTomato mice. (E and F) Immunofluorescence images (E) and quantification (F) of CD4C splenic T cells co-cultured with either HDCC or HDC¡ splenic myeloid cells. CD4C cells were sorted from AOM/DSS-treated Foxp3-GFP mice and myeloid cells were isolated from AOM/DSS- and tamoxifen- treated Hdc-CreERT2;Rosa26-tdTomato mice. Cxcr5 or Cxcl13 were knocked down by siRNA before co-culture with CD4C cells or HDCC cells, respectively. Cells were stained with PE-conjugated antibody against phospho-Stat3 and (E) show representative staining of Foxp3-GFP and Phospho-Stat3-PE. The Foxp3-GFPC and Phospho- Stat3C cells were counted as shown in (F). (G) BrdU proliferation assay of cultured Foxp3-YFPC splenic cells from AOM/DSS-treated Foxp3-YFP-Cre-Stat3C/C or Foxp3- YFP-Cre-Stat3¡/¡ mice, with or without Cxcl13 for 24 h (n D 5 to 9 per group). p < 0.01; p < 0.001, data are mean § SEM, representing two to three independent experiments. Data were analyzed with one-way analysis of variation (ANOVA) with Dunnett’s post-hoc test (C, D, and G) and Mann–Whitney test (F).

Article Snippet: Recombinant mouse Cxcl13 (Biolegend, San Diego, CA) was injected i.p. at 50 mg/mouse.

Techniques: Derivative Assay, Expressing, Phospho-proteomics, Isolation, Quantitation Assay, BrdU Incorporation Assay, Cell Culture, In Vitro, Knockdown, Co-Culture Assay, Staining, Proliferation Assay, MANN-WHITNEY

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: The following primary antibodies were used for immunohistochemical analysis and immunofluorescence testing: mouse anti-human CD20 (clone L26; Dako, Glostrup, Denmark), rabbit anti-human CD3 (immunoglobulin; Dako), mouse anti-human CD68 (clone KP1; Dako), mouse anti-human CD21 (clone 1F8; Dako), mouse anti-human CD8 (clone 1A5; Novocastra, Newcastle, UK), mouse anti-human CD45RO (clone UCHL1; Dako), mouse anti-human CD69 (clone CH11; Novocastra), and goat anti-human CXCL13 (IgG; R&D Systems, Abingdon, UK).

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: The following primary antibodies were used for immunohistochemical analysis and immunofluorescence testing: mouse anti-human CD20 (clone L26; Dako, Glostrup, Denmark), rabbit anti-human CD3 (immunoglobulin; Dako), mouse anti-human CD68 (clone KP1; Dako), mouse anti-human CD21 (clone 1F8; Dako), mouse anti-human CD8 (clone 1A5; Novocastra, Newcastle, UK), mouse anti-human CD45RO (clone UCHL1; Dako), mouse anti-human CD69 (clone CH11; Novocastra), and goat anti-human CXCL13 (IgG; R&D Systems, Abingdon, UK).

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: The following primary antibodies were used for immunohistochemical analysis and immunofluorescence testing: mouse anti-human CD20 (clone L26; Dako, Glostrup, Denmark), rabbit anti-human CD3 (immunoglobulin; Dako), mouse anti-human CD68 (clone KP1; Dako), mouse anti-human CD21 (clone 1F8; Dako), mouse anti-human CD8 (clone 1A5; Novocastra, Newcastle, UK), mouse anti-human CD45RO (clone UCHL1; Dako), mouse anti-human CD69 (clone CH11; Novocastra), and goat anti-human CXCL13 (IgG; R&D Systems, Abingdon, UK).

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: The following primary antibodies were used for immunohistochemical analysis and immunofluorescence testing: mouse anti-human CD20 (clone L26; Dako, Glostrup, Denmark), rabbit anti-human CD3 (immunoglobulin; Dako), mouse anti-human CD68 (clone KP1; Dako), mouse anti-human CD21 (clone 1F8; Dako), mouse anti-human CD8 (clone 1A5; Novocastra, Newcastle, UK), mouse anti-human CD45RO (clone UCHL1; Dako), mouse anti-human CD69 (clone CH11; Novocastra), and goat anti-human CXCL13 (IgG; R&D Systems, Abingdon, UK).

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: The following primary antibodies were used for immunohistochemical analysis and immunofluorescence testing: mouse anti-human CD20 (clone L26; Dako, Glostrup, Denmark), rabbit anti-human CD3 (immunoglobulin; Dako), mouse anti-human CD68 (clone KP1; Dako), mouse anti-human CD21 (clone 1F8; Dako), mouse anti-human CD8 (clone 1A5; Novocastra, Newcastle, UK), mouse anti-human CD45RO (clone UCHL1; Dako), mouse anti-human CD69 (clone CH11; Novocastra), and goat anti-human CXCL13 (IgG; R&D Systems, Abingdon, UK).

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

Journal: EBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: Fig. 5: hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA11a isoform in tumor cells affects CXCL13 production by TRM cells. a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si- hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student’s t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey’s multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA11a (Si-hMENA11a), or control (Si-CNTR). Results within CD8+ TRM (CD103+CD69+, left panel) or CD4+ TRM (CD103+CD69+, right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Cytometry, Ex Vivo, Isolation

Journal:

Article Title: Villitis of Unknown Etiology is Associated with a Distinct Pattern of Chemokine Up-regulation in the Feto-maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-fetal Graft-versus-Host Disease 1

doi: 10.4049/jimmunol.0803834

Figure Lengend Snippet: Changes in mRNA expression of a subset of chemokines and receptors in VUE placentas. mRNA expressions of CXCL9, CXCL10, CXCL11, CXCL13, CCL4, and CCL5 are higher in VUE placentas compared with those in term control placentas. mRNA of CXCR3 and CCR5 is also expressed at higher levels in VUE placentas than in term control placentas.

Article Snippet: DNase-treated total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT-PCR analyses were performed with TaqMan gene expression assays (CXCL9, Hs00171065_m1; CXCL10, Hs00171042_m1; CXCL11, Hs00171138_m1; CXCL13, Hs00757930_m1; CCL4, Hs99999148_m1; CCL5, Hs00174575_m1; CXCR3, Hs00171041_m1; CXCR5, Hs00173527_m1; CCR5, Hs00152917_m1; Applied Biosystems) using an ABI 7500 Fast real-time PCR system.

Techniques: Expressing, Control

Journal:

Article Title: Villitis of Unknown Etiology is Associated with a Distinct Pattern of Chemokine Up-regulation in the Feto-maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-fetal Graft-versus-Host Disease 1

doi: 10.4049/jimmunol.0803834

Figure Lengend Snippet: Immunofluorescent staining for selected chemokines and their receptors in VUE placentas. Immunofluorescent signals for corresponding chemokines, receptors, and CD Ags are indicated in green (Alexa 488) and red (Alexa 568 or Alexa 594). A–D, Increased immunoreactivity of CXCR9, CXCR10, CXCR13, and CCL5 in VUE placentas. CXCR3+ and CCR5+ leukocytes infiltrate the chorionic villi, expressing their chemokine ligands in VUE placentas. CXCR5 (C) is not found in either VUE placentas or normal term (TIL) placentas. Immunoreactivity of CXCL9 (A), CXCL10 (B), and CXCL13 (C) is localized in CD14+ Hofbauer cells (arrows) and endothelial cells of VUE placentas. CCL5 (D) is positive in some CD14+ Hofbauer cells (not shown) and CD8+ T cells (arrows). Some infiltrating leukocytes were doubly positive for both a chemokine and its receptor (arrowheads). E and F, CD14+ Hofbauer cells in TIL placentas are negative for CXCR3 (E) while being immunoreactive for CCR5 (F). Infiltrating CD8+ T cells and CD14+ Hofbauer cells in VUE foci are positive for CXCR3 and CCR5. The endothelial cells of villous capillaries are also positive for CXCR3.

Article Snippet: DNase-treated total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT-PCR analyses were performed with TaqMan gene expression assays (CXCL9, Hs00171065_m1; CXCL10, Hs00171042_m1; CXCL11, Hs00171138_m1; CXCL13, Hs00757930_m1; CCL4, Hs99999148_m1; CCL5, Hs00174575_m1; CXCR3, Hs00171041_m1; CXCR5, Hs00173527_m1; CCR5, Hs00152917_m1; Applied Biosystems) using an ABI 7500 Fast real-time PCR system.

Techniques: Staining, Expressing

Journal:

Article Title: Villitis of Unknown Etiology is Associated with a Distinct Pattern of Chemokine Up-regulation in the Feto-maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-fetal Graft-versus-Host Disease 1

doi: 10.4049/jimmunol.0803834

Figure Lengend Snippet: Differential mRNA expression of chemokines and receptors in maternal and fetal blood. Maternal blood shows higher mRNA expression of CXCL9 and CXCL13 in VUE cases than in control cases (TIL). CXCL11 and CXCL13 mRNA expression is higher in fetal blood of VUE cases than in control cases.

Article Snippet: DNase-treated total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT-PCR analyses were performed with TaqMan gene expression assays (CXCL9, Hs00171065_m1; CXCL10, Hs00171042_m1; CXCL11, Hs00171138_m1; CXCL13, Hs00757930_m1; CCL4, Hs99999148_m1; CCL5, Hs00174575_m1; CXCR3, Hs00171041_m1; CXCR5, Hs00173527_m1; CCR5, Hs00152917_m1; Applied Biosystems) using an ABI 7500 Fast real-time PCR system.

Techniques: Expressing, Control

Journal:

Article Title: Villitis of Unknown Etiology is Associated with a Distinct Pattern of Chemokine Up-regulation in the Feto-maternal and Placental Compartments: Implications for Conjoint Maternal Allograft Rejection and Maternal Anti-fetal Graft-versus-Host Disease 1

doi: 10.4049/jimmunol.0803834

Figure Lengend Snippet: Chemokine concentrations in maternal and fetal plasma of VUE and acute chorioamnionitis. A, The median concentrations of chemokines CXCL9, CXCL10, and CXCL11 in both maternal and fetal plasma are higher in VUE cases than in term control cases (TIL), respectively. On the other hand, the median concentration of CXCL13 is not different between VUE and control. B, The median fetal plasma concentrations of CXCL9, CXCL10, CXCL11, and CXCL13 are also higher in PTLI cases than in PTL cases. In contrast, maternal plasma concentrations of CXCL9, CXCL10, and CXCL11 are not different between PTL and PTLI cases. CXCL13 concentration is lower in PTLI cases than in PTL cases. C, Comparison between PTL and TIL cases showed that maternal plasma CXCL9 concentration is higher in PTL cases than in TIL cases, whereas CXCL11 concentration is lower in PTL than in TIL cases.

Article Snippet: DNase-treated total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT-PCR analyses were performed with TaqMan gene expression assays (CXCL9, Hs00171065_m1; CXCL10, Hs00171042_m1; CXCL11, Hs00171138_m1; CXCL13, Hs00757930_m1; CCL4, Hs99999148_m1; CCL5, Hs00174575_m1; CXCR3, Hs00171041_m1; CXCR5, Hs00173527_m1; CCR5, Hs00152917_m1; Applied Biosystems) using an ABI 7500 Fast real-time PCR system.

Techniques: Clinical Proteomics, Control, Concentration Assay, Comparison

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: a , Left, UMAP of LN stromal cells by entity. Right, percentages of rFRCs and rBECs (rLN n = 5, FL n = 6, DLBCL n = 8 patients). b , Differentially expressed genes between rLN-derived ( n = 2,363 cells) and DLBCL-derived ( n = 2,983 cells) FRCs (adjusted P < 0.05, log(fold change) > 0.5). c , Homeostatic (top) and inflammatory (bottom) chemokine expression in bulk data . d , Pearson correlation of CXCL13 expression and CIBERSORTx-derived FDC fractions. e , CXCL13 plasma protein levels in FL ( n = 18 patients) and DLBCL ( n = 22 patients). Vertical lines indicate the mean per entity. f , Exemplary rLN and DLBCL mIF images, representative of n = 4 patients per entity. Scale bar, 50 μm. Dashed circles: CD21+ regions. g , mIF-derived CXCL13 and CXCR5 signals averaged across four adjacent pairs of CD21 + follicular and CD21 − extrafollicular regions per sample. h , i , Spatial transcriptomics plots of FL-LN ( h ) and DLBCL-LN ( i ) cores colored by cell type and CXCL13 – CXCR5 ligand–receptor (L–R) score. j , mIF-derived enrichment of CXCL13 + cells per cell type in DLBCL versus rLN/FL samples. Asterisks indicate P < 0.01; exact P values are provided in the source data. k , CXCL13 expression in CD8 + T cells ( n = 21,268 cells) . l , Percentage of CXCR5 + cells within CD3 − fractions measured by flow cytometry (rLN n = 7, FL n = 24, DLBCL n = 18 patients). m , Migrated rLN- and DLBCL-derived B cells in the Transwell assay (mean ± s.d., n = 3 patients per condition). For c and d : tonsil n = 10, FL 1/2/3A n = 145, FL 3B n = 48, DLBCL n = 430 patients. For f , g and j : rLN n = 4, FL n = 5, DLBCL n = 4 patients. P values in a , c , e , g and l : two-sided Wilcoxon rank-sum test. P value in m : two-sided unpaired Welch’s t test. P values in j : two-sided Fisher’s exact test. P values in c , g and j were adjusted using the Benjamini–Hochberg method. Box plots: center line, median; box, interquartile range; whiskers, 1.5× the interquartile range; points, data values. FC, fold change; T tox EM, effector memory cytotoxic T cells; hr, human recombinant.

Article Snippet: Absolute CXCL13 plasma levels were quantified in duplicates using the Human CXCL13 Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Clinical Proteomics, Spatial Transcriptomics, Flow Cytometry, Transwell Assay, Recombinant

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: ( A ) Heatmap showing estimated cell type fractions as determined using CIBERSORTx (CSx) within a large bulk transcriptomics dataset . ( B ) Box plots depicting CSx fractions of exemplary cell types across disease entities. Boxes indicate the median (center line), interquartile range (bounds) and 1.5x interquartile range (whiskers). Data points represent patient samples. P-values were calculated using two-sided unpaired Welch’s t-test and adjusted according to Benjamini-Hochberg. ( C ) Dot plot showing Spearman correlation coefficients of chemokine expression and cell type fractions. Dot sizes represent -log10 FDR-adjusted p-value of correlation tests. ( D ) Scatter plots color-coded by disease entity correlating CXCL12 (left panel) and CCL21 (right panel) expression with CSx-derived FRC factions. P-values were calculated using Pearson correlation. ( E , F ) Bulk RNA-seq datasets , stratified by FDC abundance into high and low samples using maximally selected rank statistics based on CSx fractions, represented in a Kaplan-Meier curve showing overall survival (left panels; log-rank test) and scatter plot of FDC fractions and CXCL13 expression (right panels; Pearson correlation). T PR =T prol . For panels A – D : tonsil n = 10; FL 1/2/3 A n = 145; FL 3B n = 48; DLBCL n = 430 patients. For panel E : FDC-high n = 61; FDC-low n = 44 patients. For panel F : FDC-high n = 302; FDC-low n = 68 patients.

Article Snippet: Absolute CXCL13 plasma levels were quantified in duplicates using the Human CXCL13 Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Transcriptomics, Expressing, Derivative Assay, RNA Sequencing

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: ( A ) Representative triangle-thresholded mIF images showing CD21 (top panels) as well as CXCL13 (bottom panels) signal in rLN (n = 4), FL (n = 5) and DLBCL (n = 4) patient samples. Scale bar indicates 50μm. ( B ) UMAP representation of spatial transcriptomics data colored by cell type (color code depicted in panel C ). ( C ) Heatmap showing scaled expression of key marker gene expression as well chemokines. ( D ) Stacked bar plot of FDC/FRC/rFRC fractions (top panel) and violin plot of CXCL13 expression in these subsets (bottom panel). ( E ) Spatial transcriptomics plots of FL (left panels) and DLBCL (right panels) tissue cores colored by cell type (left panels) alongside magnified views of chemokine expression. ( F ) Heatmap showing scaled expression of key T cell markers across CD8 + T cell populations as measured using mIF. ( G ) Box plot showing per-sample percentage of CXCL13 + cells (as determined using Otsu thresholding) among PD1 + CD8 + effector memory T cells in the mIF dataset. Boxes indicate the median (center line), interquartile range (bounds) and 1.5x interquartile range (whiskers). Data points represent patient samples. P-values were calculated using a two-sided unpaired Welch’s t-test. ( H ) Spearman correlation coefficients of key T cell markers across all CD8 + T cell populations (n = 21,268 cells). ( I ) Volcano plot showing differentially expressed genes comparing PD1 + CD8 + effector memory T cell populations (T TOX EM-II n = 2,018; T TOX EM-III n = 9,208 cells). Labels indicate cluster-identifying genes as well as CXCL13 . Abbreviations: T TOX EM = effector memory cytotoxic T cells. For panels A , F , G : rLN n = 4; FL n = 5; DLBCL n = 4 patients. For panels B – E : FL n = 1; DLBCL n = 1 patient.

Article Snippet: Absolute CXCL13 plasma levels were quantified in duplicates using the Human CXCL13 Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Spatial Transcriptomics, Expressing, Marker, Gene Expression

Journal: Nature Cancer

Article Title: Reprogramming of stroma-derived chemokine networks drives the loss of tissue organization in nodal B cell lymphoma

doi: 10.1038/s43018-026-01136-z

Figure Lengend Snippet: a , UMAP representation of a microarray dataset with homeostatic ( CXCL12 , CXCL13 , CCL19 , CCL21 ) and inflammatory ( CXCL9 , CXCL10 , CXCL11 ) chemokine expression values used as features for dimensionality reduction. Left, UMAP displaying pie charts (within each dot) that represent the k = 20 nearest neighbors, colored according to disease entity. Right, the same UMAP colored according to the mean expression of homeostatic (top) and inflammatory (bottom) chemokines. b , Bulk RNA-seq dataset stratified according to homeostatic chemokine expression into high ( n = 519 patients) and low ( n = 99 patients) groups using maximally selected rank statistics, shown as a dot plot (top) and a Kaplan–Meier curve of overall survival (bottom). The P value was calculated using the log-rank test. c , Forest plot summarizing log 10 -transformed hazard ratios (center), 95% confidence intervals (error bars) and Wald-derived P values estimated from univariate Cox proportional hazards models assessing the association between homeostatic chemokine expression and overall survival across five individual DLBCL bulk datasets , – . d , Same UMAP as in a , colored by CIBERSORTx-derived FDC fractions. e , Bulk RNA-seq dataset stratified by FDC abundance into high ( n = 255 patients) and low ( n = 364 patients) groups using maximally selected rank statistics based on CIBERSORTx fractions, shown as a Kaplan–Meier curve of overall survival (left; log-rank test) and a scatter plot of log-transformed FDC fractions and CXCL13 expression (right; Pearson correlation). f , Forest plot summarizing log 10 -transformed hazard ratios (center), 95% confidence intervals (error bars) and Wald-derived P values estimated from Cox proportional hazards models assessing the association between FDC fraction and overall survival across five individual DLBCL bulk datasets , – . For panels a and d : tonsil n = 10, FL 1/2/3A n = 145, FL 3B n = 48 and DLBCL n = 430 patients. OS, overall survival; HR, hazard ratio.

Article Snippet: Absolute CXCL13 plasma levels were quantified in duplicates using the Human CXCL13 Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Microarray, Expressing, RNA Sequencing, Transformation Assay, Derivative Assay

Journal: JCI Insight

Article Title: EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing T FH cell differentiation and extrafollicular activities

doi: 10.1172/jci.insight.158193

Figure Lengend Snippet: ELISA of ( A ) activin A and ( B ) IL-21 in serum of ME/CFS cases ( n = 351), GWI veterans ( n = 54), and healthy controls ( n = 77). ( C ) Serum CXCL13 ELISA of ME/CFS cases ( n = 351) and healthy controls ( n = 27). ( D ) Comparison of activin A levels between ME/CFS cases positive for anti-herpesvirus dUTPase Abs ( n = 167) versus negative ( n = 184). ( E ) Comparison of IL-21 levels between ME/CFS cases ( n = 347) positive ( n = 157) versus negative ( n = 190) for Abs against the dUTPases from herpesviruses. Dotted line represents the normal range levels for healthy individuals for each cytokine/chemokine. Data represent 3 experiments with mean ± SEM. ( A and B ) **** P < 0.0001 of disease versus control cohorts by 1-way ANOVA Kruskal-Wallis multiple comparisons test, ** P < 0.01 of anti-virus dUTPase Ab–positive versus –negative groups ( D and E ) by 2-tailed Mann-Whitney U test.

Article Snippet: RNA was extracted using TRIzol reagent (Ambion) followed by RNeasy Mini Kit (QIAGEN) and on-column DNase treatment (QIAGEN). cDNA was synthesized using the SuperScript IV First-Strand Synthesis Kit (Invitrogen, Thermo Fisher Scientific), and qRT-PCR was performed on a QuantStudio 6 Flex instrument (Thermo Fisher Scientific) using the following TaqMan mouse gene-specific fluorogenic assays (Applied Biosystems, Thermo Fisher Scientific): Cxcr5 (Mm00432086_m1), Cxcl13 (Mm04214185_s1), Cxcr4 (Mm01996749_s1), Aicda (Mm01184115_m1), Fas (Mm00433237_m1), Pdcd1 (Mm01285676_m1), Cd40 (Mm00441895_m1), Cd40l (Mm00441911_m1), Icosl (Mm00497237_m1), Il21 (Mm00517640_m1), Batf (Mm00479410_m1), Cd80 (Mm01344159_m1), Il27 (Mm00461162_m1), Cdkn1a (Mm00432448_m1), Tnfrsf13b (Mm03047441_m1), Tnfrsf14 (Mm00619237_m1), Egr1 (Mm00656724_m1), Flt3 (Mm00439016_m1), Spib (Mm03048233_m1), Mcl1 (Mm01257351_g1), Btla (Mm00616981_m1), Tnfrsf4 (Mm00442037_g1), Pax5 (Mm00435501_m1), Cd1d2 (Mm00776138_gH), Xbp1 (Mm00457357_m1), Tnfsf13b (Mm00446347_m1), Il15 (Mm00434210_m1), Ebf1 (Mm00432954_m1), Cd38 (Mm00483143_m1), B2m (Mm00437762_m1), and Hsp90ab1 (Mm00833431_g1).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control, Virus, MANN-WHITNEY

Journal: JCI Insight

Article Title: EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing T FH cell differentiation and extrafollicular activities

doi: 10.1172/jci.insight.158193

Figure Lengend Snippet: ( A ) Naive CD4 + T cells were isolated from PBMCs as described above ( and Methods section), cultured in vitro with anti-CD3/anti-CD28 coated beads alone (Beads) or in the presence of DCM (Ctl or EBV dUTPase or HHV-6A dUTPase; 25% vol/vol), and restimulated or not on day 3 with PMA/I. Following treatments, supernatants were examined for IL-21 ( A ) and IFN-γ ( B ) concentrations by ELISA. ( C and D ) ELISA of CXCL13 or IL-10 in supernatants of naive CD4 + T cells stimulated for 5 days by plate-bound anti-CD3 and human recombinant ICOS ligand chimera, in the absence (Neg Ctl) or presence of DCM (Ctl, EBV dUTPase, or HHV6A dUTPase) alone or together with IL-12. Treatment of cells with activin A plus IL-12 was used as the positive control, and cells stimulated with beads only were used as the negative control. Data represent the mean ± SEM of n = 6–8 ( A , C , and D ) and n = 4 ( B ) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 of treatments versus negative Ctl or beads by 1-way ANOVA Kruskal-Wallis multiple comparisons test with Dunn’s correction.

Article Snippet: RNA was extracted using TRIzol reagent (Ambion) followed by RNeasy Mini Kit (QIAGEN) and on-column DNase treatment (QIAGEN). cDNA was synthesized using the SuperScript IV First-Strand Synthesis Kit (Invitrogen, Thermo Fisher Scientific), and qRT-PCR was performed on a QuantStudio 6 Flex instrument (Thermo Fisher Scientific) using the following TaqMan mouse gene-specific fluorogenic assays (Applied Biosystems, Thermo Fisher Scientific): Cxcr5 (Mm00432086_m1), Cxcl13 (Mm04214185_s1), Cxcr4 (Mm01996749_s1), Aicda (Mm01184115_m1), Fas (Mm00433237_m1), Pdcd1 (Mm01285676_m1), Cd40 (Mm00441895_m1), Cd40l (Mm00441911_m1), Icosl (Mm00497237_m1), Il21 (Mm00517640_m1), Batf (Mm00479410_m1), Cd80 (Mm01344159_m1), Il27 (Mm00461162_m1), Cdkn1a (Mm00432448_m1), Tnfrsf13b (Mm03047441_m1), Tnfrsf14 (Mm00619237_m1), Egr1 (Mm00656724_m1), Flt3 (Mm00439016_m1), Spib (Mm03048233_m1), Mcl1 (Mm01257351_g1), Btla (Mm00616981_m1), Tnfrsf4 (Mm00442037_g1), Pax5 (Mm00435501_m1), Cd1d2 (Mm00776138_gH), Xbp1 (Mm00457357_m1), Tnfsf13b (Mm00446347_m1), Il15 (Mm00434210_m1), Ebf1 (Mm00432954_m1), Cd38 (Mm00483143_m1), B2m (Mm00437762_m1), and Hsp90ab1 (Mm00833431_g1).

Techniques: Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Negative Control

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 1 | CXCL13 expression in subpopulation of liver cells and its regulation by toll-like receptor (TLR) ligands. (A) Comparison of CXCL13 mRNA expression in Kupffer cell (KC), hepatic stellate cell (HSC), liver sinusoidal endothelial cell (LSEC), and primary human hepatocyte (PHH). Different subpopulation of liver cells were isolated from non-hepatitis B virus-infected livers. The total RNA of these cells was extracted and the expression of CXCL13 mRNA was determined by real-time RT-PCR. (B) Comparison of CXCL13 mRNA expression in KC after stimulation with TLR1–9 ligands. Isolated KC were stimulated with different TLR1–9 ligands for 6 h and then harvested for RNA extraction. CXCL13 mRNA expression was determined by real-time RT-PCR. The stimulus and concentration used as below: TLR1/2 agonist: Pam3CSK4 (4 μg/ml), TLR2 agonist: HKLM (108 cells/ml), TLR3 agonist: poly I:C (50 μg/ml), TLR4 agonist: LPS (30 μg/ml), TLR5 agonist: FLA (2 μg/ml), TLR6/2 agonist: FSL1 (1 μg/ml), TLR7 agonist: imiquimod (20 μg/ml), TLR8 agonist: ssRNA40 (10 μg/ml), TLR9 agonist: CpG (31.8 μg/ml). Data are representative with mean ± SEM, and determined by Mann–Whitney U test.

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Comparison, Isolation, Virus, Infection, Quantitative RT-PCR, RNA Extraction, Concentration Assay, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 2 | Toll-like receptor (TLR) 3 agonist-induced expression of CXCL13 in liver cells from hepatitis B virus (HBV)-positive and non-HBV-infected patients. Comparison of the basal and TLR3 agonist-induced expression of CXCL13 in Kupffer cell (KC) (A), hepatic stellate cell (HSC) (B), liver sinusoidal endothelial cell (LSEC) (C), and primary human hepatocyte (PHH) (D). Different subpopulation of liver cells were isolated from either Non-HBV or HBV-positive subjects and stimulated with or without TLR3 agonist for 6 h. Total RNA was extracted and then the CXCL13 mRNA expression to β-actin were determined by real-time RT-PCR. Data are representative with mean ± SEM, and determined by Mann–Whitney U test and Wilcoxon’s signed-ranks test.

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Virus, Infection, Comparison, Isolation, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 3 | CXCL13 expression in serum samples from chronic hepatitis B (CHB) patients and its correlation with viral parameters. (A) Comparison of serum CXCL13 concentration in healthy control (HC) and CHB patients with chronic hepatitis B virus (HBV) infection [divided into immune activation (IA), IT, and IC groups]. Serum CXCL13 concentrations were measured by specific enzyme-linked immunosorbent assay kit. Data are representative with median, and determined by Mann–Whitney U test. (B) The correlation between serum CXCL13 concentrations and HBV DNA (log10 copies/ml) in IA patients, including the baseline of CHB patients received telbivudine therapy. (C) The correlation between serum CXCL13 concentrations and alanine aminotransferase (ALT) levels in IA patients. (D) The correlation between serum CXCL13 concentrations and the age in IA patients.

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Comparison, Concentration Assay, Control, Virus, Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 4 | Longitudinal analysis of serum CXCL13 concentrations of chronic hepatitis B (CHB) patients during telbivudine therapy. (A) Comparison of serum CXCL13 concentrations at baseline and at 12, 24, and 52 weeks after starting telbivudine therapy in all 55 hepatitis B virus e antigen-positive CHB patients. Data are representative with median, and determined by Mann–Whitney U test and Kruskal–Wallis H test. (B) Comparison of serum CXCL13 concentrations between complete response (CR) group and non-complete response (NCR) group at indicated time points. Data are representative with median, and determined by Mann–Whitney U test. (C) Repeated measures analysis of temporal dynamics of serum CXCL13 concentrations between CR group and NCR group at indicated time points. The paired Student’s t-test was used to compare individual values at different time points. (D) Receiver-operating characteristic (ROC) curves for prediction of CR after telbivudine therapy, including CXCL13 concentrations at week 12, log10 HBV DNA copies/ml of week 0 − week 12, and combination factors above.

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: Comparison, Virus, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 5 | CXCL13-mediated chemotaxis effect on CXCR5+ cells in vitro. (A) Schematic diagram of chemotaxis assay. A 1 × 106 fresh-isolated peripheral blood mononuclear cells were added into the upper chamber, and different concentration of rh-CXCL13 or median alone was added into lower chamber. After 2–6 h, the cells in the lower chamber were harvested and analyzed. (B) Comparison of the numbers of CXCR5+ cell counting in chronic hepatitis B (CHB) patients with different CXCL13 concentration in Transwell assay. Data are representative with mean ± SEM, and determined by Mann–Whitney U test. (C) Comparison of the numbers of CXCR5+ cell counting in CHB patients with different incubation time in Transwell assay. Data are representative with mean ± SEM, and determined by paired Student’s t-test.

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: Chemotaxis Assay, In Vitro, Isolation, Concentration Assay, Comparison, Cell Counting, Transwell Assay, MANN-WHITNEY, Incubation

Journal: Frontiers in immunology

Article Title: Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

doi: 10.3389/fimmu.2017.00323

Figure Lengend Snippet: Figure 6 | In situ expression of CXCL13 in the ectopic germinal center (GC) of liver tissue from chronic hepatitis B (CHB) patients. (A) Immunohistochemical staining of CXCL13 expression in liver tissue samples with or without ectopic GC from CHB patients and healthy control (HC). Representative slides were shown with different magnifications as indicated. (B) Immunohistochemical staining of CXCL13+, CD4+, CD19+, CXCR5+, CD38+, and CD68+ cells in serial sections of liver tissue samples with ectopic GC from four representative CHB patients. Co-localization of CXCL13 expression was observed with CD68+ KCs, CXCR5+ CD4+ T cells, and CXCR5+CD19+ B cells in the ectopic GC (magnification ×400, plotting scale equal to 50 μm).

Article Snippet: HBV DNA was measured using the COBAS TaqMan HBV Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) (17), which has a detection limit of 12 IU/ml (1 IU/ml = 5.82 copies/ml). enzyme-linked immunosorbent assay (elisa) The concentration of CXCL13 was quantitated in duplicate wells using a commercial human CXCL13 ELISA kit (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions.

Techniques: In Situ, Expressing, Immunohistochemical staining, Staining, Control

Journal: Immunity

Article Title: Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation

doi: 10.1016/j.immuni.2017.11.020

Figure Lengend Snippet: Microbiota-Independent Local 7α,25-OHC Production Is Necessary for Colonic CP and ILF Formation (A) Ch25h , Cyp7b1 , Hsd3b7 , and Gpr183 mRNA expression in the colon of human CD2 GFP transgenic mice (n = 3). mRNA expression was compared in micro-dissected CPs and ILFs (CPs-ILFs) versus micro-dissected lamina propria. (B) Number of CPs, ILFs, Peyer’s patches, and colonic patches in Ch25h +/+ and Ch25h −/− mice (n = 3–6). (C) Ch25h , Cyp7b1 , Hsd3b7 , Gpr183 , and Ltb mRNA expression in the colon of germ-free and specific-pathogen-free (SPF) mice (n = 6). (D) Ccl20 and Cxcl13 mRNA expression in the intestine of germ-free and SPF mice (n = 6). Data are represented as means ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by Student’s t test. Data are representative of or combined from two experiments. See also Figure S7 .

Article Snippet: TaqMan Assay: Mouse Cxcl13 , Life Technologies , Mm00444534_m1.

Techniques: Expressing, Transgenic Assay

Journal: Immunity

Article Title: Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation

doi: 10.1016/j.immuni.2017.11.020

Figure Lengend Snippet:

Article Snippet: TaqMan Assay: Mouse Cxcl13 , Life Technologies , Mm00444534_m1.

Techniques: Control, In Vivo, Recombinant, Cell Stimulation, Reverse Transcription, cDNA Synthesis, Purification, Transgenic Assay, Quantitative RT-PCR, Software, TaqMan Assay