Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Effector machinery of CD57 +/ −CD45RA +/ − or CD57 +/ − CXCR5 +/ − CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Identification of CD57 + CXCR5 + T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57 + CD4 T cells in GCs. (C) Localization of CD57 + CXCR5 + CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, In Situ, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Chemotactic responses (A) and chemokine receptor expression (B) by CD57 +/2 CXCR5 +/ − CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, Chemotaxis Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Spontaneous B cell helper activity of CD57 +/ − CXCR5 +/ − CD4 T cells. Sorted CD57 + CXCR5 + , CD57 − CXCR5 + , and CXCR5 − CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Phenotype and effector function of circulating CXCR5 + T cells. (A) Surface phenotype of circulating naive or CXCR5 +/ − memory T cells. **Significant differences between CXCR5 + and CXCR5 − memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5 +/ − T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5 + T cells after T cell receptor activation. Naive (IgD + ) or memory (IgD − ) B cells (2 × 10 4 ) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10 3 , 5 × 10 3 , 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 10 5 ) of autologous T cells (CXCR5 − CD45RA + , CXCR5 + CD45RA − , or CXCR5 − CD45RA − ). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activation Assay, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Relation of CXCR5 expression with clinicopathological parameters in 158 cases of SACC.

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Clinical significance of CXCR5 expression in SACC patients. ( A ) Immunohistochemical staining of CXCR5 in normal salivary gland (NSG), SACC without PNI (No-PNI), far away from nerve of SACC with PNI (Far-nerve), nerve invasion front of SACC with PNI (Nerve-front). ‘N’ represented nerve. (Bar: upper, 50 μm; lower, 20 μm) ( ** p < 0.01, *** p < 0.001). ( B ) Kaplan-Meier survival analysis on SACC patients with different expression status of CXCR5. The overall survival time for patients without or with low CXCR5 expression was longer than those with high CXCR5 expression (log-rank test, p < 0.0001).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Cox multivariate regression analysis of overall survival in SACC patients.

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: The expression of CXCR5 in normal salivary gland and SACC with different PNI status.

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Silencing CXCR5 attenuates migration, invasion and PNI in SACC cells. ( A ) Immunofluorescence and qRT-PCR analysis showed that CXCR5 expression was dramatically inhibited by siRNA treatment. ( B – D ) CXCR5 silence with siRNA or CXCR5 blockade with anti-CXCR5 antibody impeded migration, invasion and PNI of SACC-LM cells, (Bar: 100 μm). ( E ) Immunofluorescence and qRT-PCR analysis showed that CXCR5 level was significantly increased by CXCR5-overexpressing plasmid. ( F – H ) CXCR5 overexpression enhanced migration, invasion and PNI capacity of SACC-LM cells, (Bar: 100 μm) ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Migration, Immunofluorescence, Quantitative RT-PCR, Expressing, Plasmid Preparation, Over Expression

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Schwann-like cell differentiation is involved in CXCR5 induced PNI in SACC cells. ( A ) CXCR5 silence with siRNA or CXCR5 blockade with anti-CXCR5 antibody downregulated the expression of Schwann cell markers, including S100A4, p75NTR, GFAP by qRT-PCR analyses. ( B ) CXCR5 overexpression upregulated the expression of S100A4, p75NTR, GFAP in SACC-LM cells. ( C ) CXCR5 silence with siRNA or CXCR5 blockade with anti-CXCR5 antibody resulted in the transformation of SACC-LM cells from spindle-like fibroblastic cellular morphology to epithelial plasticity with little pseudopodia, (Bar: 50 μm). ( D ) The heatmap of 8 differentially expressed miRNAs in SACC-LM cells transfected with control or si-CXCR5 on a scale from blue (low) to red (high). ‘Blue’ represents low expression, and ‘red’ represents high expression. ( E ) The microarray data of 8 differentially expressed miRNAs in the si-CXCR5 and control SACC-LM cells was confirmed by qRT-PCR. For qRT-PCR, U6 was used to normalize the Ct values. ( F ) CXCR5 overexpression upregulated the expression of miR-187 in SACC-LM cells, ( * p < 0.05, ** p < 0.01).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Cell Differentiation, Expressing, Quantitative RT-PCR, Over Expression, Transformation Assay, Transfection, Control, Microarray

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: CXCR5 promotes the expression of Schwann cell markers by inhibiting miR-187. ( A ) An RNA-FISH assay was conducted to determine the level of miR-187 in normal salivary gland (NSG), SACC without PNI (No-PNI), far away from nerve of SACC with PNI (Far-nerve), nerve invasion front of SACC with PNI (Nerve-front). ‘N’ represented nerve, (Bar: 50 μm). ( B ) The binding sites on S100A4 3′UTR for miR-187 and the mutant sites. ( C ) Luciferase reporter analysis was carried out to determine the interaction of miR-187 with S100A4. ( D ) qRT-PCR analysis showed that reduced expression of Schwann cell hallmarks mediated by CXCR5 silence could be partially reversed by miR-187 inhibitor. ( E ) Inhibitory migratory, invasive and PNI ability in SACC-LM cells mediated by CXCR5 silence could be reversed by S100A4 overexpression. ( F ) Repressive migratory, invasive and PNI ability in SACC-LM cells induced miR-187 overexpression was alleviated by S100A4 overexpression, ( * p < 0.05, ** p < 0.01).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Quantitative RT-PCR, Over Expression

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: Inhibition of CXCR5 suppresses PNI in SACC xenograft model. ( A ) Representative images on hindlimb function of the mouse and HE staining were presented. ( B ) qRT-PCR analysis showed that anti-CXCR5 antibody group mice showed reduced expression of S100A4, p75NTR and GFAP compared with saline group mice. ( C ) Immunohistochemical staining showed that anti-CXCR5 antibody group mice showed reduced expression of S100A4, p75NTR and GFAP compared with saline group mice. The expression of S100A4, p75NTR, GFAP was higher at nerve invasion frontier than that in tumors far away from nerve in PNI groups, ‘N’ represented nerve, (Bar: 50 μm) ( ** p < 0.01, *** p < 0.001).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Inhibition, Staining, Quantitative RT-PCR, Expressing, Saline, Immunohistochemical staining

Journal: Aging (Albany NY)

Article Title: CXCR5 induces perineural invasion of salivary adenoid cystic carcinoma by inhibiting microRNA-187

doi: 10.18632/aging.203097

Figure Lengend Snippet: CXCR5 expression intrinsically connects with the level of Schwann cell markers in SACC specimens. ( A ) Immunohistochemical staining of Schwann cell markers, including S100A4, p75NTR and GFAP in normal salivary gland (NSG), SACC without PNI (No-PNI), far away nerve of SACC with PNI (Far-nerve), nerve invasion front of SACC with PNI (Nerve-front). ‘N’ represented nerve, (Bar: 20 μm). ( B ) Correlation of CXCR5 with Schwann cell markers at nerve invasion frontier of SACC patients. CXCR5 expression was positively correlated with the expression of S100A4, p75NTR and GFAP at nerve invasion frontier of SACC ( ** p < 0.01).

Article Snippet: 1.0 μg/ml of anti-human CXCR5 antibody (R&D Systems) was added to 6-well plates after SACC cells were seeded [ ].

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Cytotoxic CD8 + T cells may be drivers of tissue destruction in Sjögren’s syndrome

doi: 10.1038/s41598-022-19397-w

Figure Lengend Snippet: The most prominent CD4 + T cell subsets in SS tissues are Th1 cells and Tfh cells. ( a ) Representative multi-color immunofluorescence image of CD3 (red), CD19 (green) and DAPI (blue) staining in a pSS lesion (patient number 2). ( b ) Absolute numbers of CD3 + T cells, CD19 + B cells in pSS (n = 10). Paired t test used to calculate p-value. ( c ) Representative multi-color staining showing each CD4 + T cell subset. [Th1: CD4+ (red) T-bet+ (light blue)] [Th2: CD4+ (red) GATA3+ (yellow)] [Th17: CD4+ (red) RORγ+ (white)] [Tfh: CD4+ (red) CXCR5+ (green)] [Treg: CD4+ (red) Foxp3+ (orange)] [CD4+ CTLs: CD4+ (red) GZMA+ (green)] (d) Relative proportions of Th1, Th2, Th17, Tfh, Treg and CD4+ CTL subsets in tissues from pSS (n = 13), secondary SS (n = 7) and IgG4-RD patients (n = 6). Error bars represent mean ± SEM. *p < 0.05.

Article Snippet: Specimens were incubated with primary antibodies specific for the following proteins: anti-CD3 (dilution; 1:100) (clone: A045229-2; DAKO), anti-CD4 (1:250) (clone: CM153A; Biocare Medical), anti-CD19 (1:200) (clone: SKU310; Biocare Medical), anti-T-bet (1:500) (clone: ab150440; Abcam), GATA3 (1:800) (clone: CM405A; Biocare), Rorc (1:400) (clone: ab212496; Abcam), CXCR5 (1:400) (clone: MAB190; R&D Systems), Foxp3 (1:200) (clone: 98377; Cell Signaling Technology), anti-CD8 (1:2000) (clone: ab85792; Abcam), anti-CD68 (1:200) (clone: ab955; Abcam), anti-cleaved caspase-3 (1:400) (clone: 9664; Cell Signaling Technology), anti-GZMA (1:800) (clone: LS-C312742; LSBio), anti-pan-CK (1:200) (clone: ab27988; Abcam), anti-AQP5 (1:500) (clone: ab92320; Abcam), anti-HLA-DR (1:1000) (clone: ab20181; Abcam), anti-NKp46 (1:600) (clone: MAB1850; R&D Systems), anti-Fas (1:200) (clone: 4233; Cell Signaling Technology), anti-FasL (1:200) (clone: LS-B4195-50; LSBio), α-SMA (1:400) (clone: 19245; Cell Signaling Technology) and anti-ICOS (1:400) (clone: 89601S; Cell Signaling Technology).

Techniques: Immunofluorescence, Staining

Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet: Surface antibody master mix, titrated for staining of up to 3 M cells per condition

Article Snippet: Anti-Human CD185/CXCR5 (RF8B2)-153Eu—100 Tests (1:100) , Fluidigm , Cat#3153020B.

Techniques: Staining

Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet:

Article Snippet: Anti-Human CD185/CXCR5 (RF8B2)-153Eu—100 Tests (1:100) , Fluidigm , Cat#3153020B.

Techniques: Recombinant, Saline, Staining, Blocking Assay, Isolation, Software, Cell Counting, Cytometry

Journal: Infection and Immunity

Article Title: Single-Cell RNA Sequencing Reveals Unique Alterations in the Immune Panorama and Treg Subpopulations in Mice during the Late Stages of Echinococcus granulosus Infection

doi: 10.1128/iai.00029-23

Figure Lengend Snippet: Differential analysis of Tfh and B cell subpopulations between E. granulosus -infected and uninfected mice. (A to F) Changes in Tfh cells. (A) t-SNE plots of Tfh cell distribution in different samples. (B) Bubble plot of Tfh cell marker gene expression distribution. (C) Tfh2 top 10 differential gene expression peak plots. (D) Immunofluorescence combined with RNA fluorescence in situ hybridization results. DAPI, CD4, and CXCR5 represent immunofluorescence stains. Cxxc5 and Spock2 represent RNA probes. The white arrows indicate triple-positive cells. The statistical plot’s horizontal coordinate represents the group, and the vertical coordinate represents the mean fluorescence intensity value of the positive region ( n = 3 views). (E) qRT-PCR statistics of Cxxc5 ( n = 3). (F) Volcano plot of differential gene expression of Tfh2 cells in infected SP versus uninfected SP. The number in parentheses represents the number of genes that meet this screening condition. (G) t-SNE plots of B cell distribution in different samples. (H) Plot of the proportion of cells in different B cell subpopulations in the four samples. (I) Heat map of top 10 marker gene expression in each B cell subpopulation. (J) t-SNE plot of B cell subpopulation distribution in all samples after annotation. (K) Volcano plot of B4 differential gene expression in infected PB versus uninfected PB. (L) Bubble plot of B cell subpopulation marker gene expression distribution; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For the CD4 primary antibody, a PBS solution containing BSA (Servicebio, G5001) and a dilution of the primary antibody (CD4, 1:100; CD25, 1:100 [Servicebio, GB11612]; CXCR5, 1:200 [ABclonal, A8950]) were added and incubated at 4°C overnight.

Techniques: Infection, Marker, Gene Expression, Immunofluorescence, Fluorescence, In Situ Hybridization, Quantitative RT-PCR