mouse cxcl16 antibody mab503  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: CXCR6–CXCL16 axis promotes NT CD4 TRM establishment. (A) Box plot showing the expression of Cxcr6 mRNA among CD4 TRM of the NT and lungs. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (B and C) Box plot showing the expression of Cxcr6 mRNA among different cell clusters of lungs and NT. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by Wilcoxon rank sum test with Benjamini–Hochberg correction. (C) Volcano plot for differential expressed genes in the NT in comparison with the lungs of the Th17 cluster. The dotted lines indicate fold change and adjusted P value cutoffs. (D) Bar plot with individual data points for the expression of CXCR6 (each median fluorescence intensity [MFI] normalized to mean MFI from CD4 TEM iv + of NT) in CD4 TRM and iv + CD4 TEM from the lungs and NT of naïve mice and PR8 IAV-infected mice (30 dpi) as indicated. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (E and F) Expression of CXCR6 on OT-II CD4 TRM of NT and lung on day 30 following PR8-OVA infection as indicated. (E) Representative histograms for CXCR6 expression from OT-II CD4 TRM of NT and lung are shown. (F) Bar plot with individual data points showing the expression of CXCR6 (each MFI normalized to mean MFI of NT OT-II CD4 TRM). The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (G–K) Frequency of different cell populations within different organs derived from WT and Cxcr6 −/− BM chimeric mice on day 30 after infection with PR8 IAV as indicated. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots showing the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs. (I) Bar plot with individual data points for the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. (J) Bar plot with individual data points for the percentage of I-A b NP 306–322 tetramer-specific WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. Samples that had <7 OT-II CD4 TRM were excluded from the analysis. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (K) Bar plot with individual data points showing the percentage of Cxcr6 −/− and WT CD4 T cells in different organs of the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (L) A representative microscopic image of CXCL16 expression (magenta) and OT-II CD4 T cells (red and green) in the olfactory epithelium of the murine NT. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Scale bar: 100 μm for main image and 50 μm for the inset. (M–O) Antigen-specific CD4 TRM in the lungs and NT of mice treated with isotype control or anti-CXCL16 antibody on day 10 after PR8 IAV infection. (M) Schematic representation of the experimental setup. (N) Representative flow cytometry plots indicating the percentage of I-A b NP 306–322 tetramer-specific CD4 TRM are shown. (O) Bar plot with individual data points indicating the absolute number of I-A b NP 306–322 tetramer-specific CD4 TRM. The experiment was performed twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test.

Article Snippet: Mouse CXCL16 antibody (MAB503) was purchased from R&D Systems and was dissolved in 1× PBS.

Techniques: Expressing, Two Tailed Test, Comparison, Fluorescence, Infection, Derivative Assay, Flow Cytometry, Olfactory, Isolation, Control

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: Additional characterization of experimental models, related to Figs. 6, 7, and 8. (A) Percentage of CD45 + donor cells (both Cxcr6 −/− and WT) and recipient cells in the blood of BM chimera on day 67 following BM transplantation. Left panel: Bar plot with individual data points for the percentage of donor cells and recipient cells. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by one-way ANOVA, with Tukey’s multiple comparison test. Right panel: A representative flow cytometry plot showing the percentage of donor cells and recipient cells. (B) Bar plot with individual data points showing the percentage of donor and recipient cells among CD4 T cells in different organs in the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Representative microscopic image of CXCL16 expression (magenta) in the olfactory epithelium of the murine NT is shown. Two negative controls for CXCL16 staining showing NT stained (1) with α-rabbit secondary IgG Texas red only and (2) stained with isotype control and α-rabbit secondary IgG Texas red. Hoechst is indicated in grey. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Top images are stitched to show the whole NT. Scale bar: 500 μm for top panels and 100 μm for bottom. (D) Gating strategy to identify CD4 TEM (CD4 + CCR7 − CD45RA − ), CD4 TCM (CD4 + CCR7 + CD45RA − ), naïve CD4 Tc (CD4 + CCR7 + CD45RA + ), CD4 TEMRA(CD4 + CCR7 − CD45RA + ), and CD4 TRM (CD4 + CCR7 − CD45RA − CD69 + ) in NT and blood. (E) RORγt + CD4 + T cells in the PPs of WT C57BL/6 mice, CD4 cre Rorc fl/wt , and CD4 cre Rorc fl/fl mice. Left panel: Bar plot with individual data points showing the frequency of RORγt + CD4 + T cells among all CD3 + T cells. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the percentage of RORγt + CD4 + T cells in different groups. (F) Microscopy images (magnified) for TUNEL + cells in the nasal septum (respiratory region) from CD4 cre Rorc fl/wt and CD4 cre Rorc fl/fl mice (same as ). Scale bar: 50 μm. (G and H) Microscopy for TUNEL + cells and viral titer (TCID 50 /g) from organs of mice infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The mice were treated with isotype or IL-17a/f antibody. (G) Bar plot with individual data points showing number of TUNEL + cells in the nasal septum (respiratory region) on day 4 after X31 IAV infection. The experiment was repeated twice. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. (H) Viral titer (TCID 50 /g) from NT and lungs on day 3 after X31 IAV infection. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

Article Snippet: Mouse CXCL16 antibody (MAB503) was purchased from R&D Systems and was dissolved in 1× PBS.

Techniques: Transplantation Assay, Comparison, Flow Cytometry, Infection, Expressing, Olfactory, Staining, Control, Isolation, Two Tailed Test, Microscopy, TUNEL Assay

Journal: Journal of Neuroinflammation

Article Title: NLRP3 activation induces BBB disruption and neutrophil infiltration via CXCR2 signaling in the mouse brain

doi: 10.1186/s12974-025-03468-6

Figure Lengend Snippet: NLRP3-activated neutrophil-induced CXCL1 and CXCL2 expression reduces extracellular Claudin-5 and VE-Cadherin on brain endothelial cells. ( A ) Total RNA was isolated from neutrophils in the bone marrow of mice, and Cxcl1 , Cxcl2 , Mmp2 , and Mmp9 expression levels were analyzed using quantitative real-time PCR (qPCR). ND: not detectable. ( B ) ELISA analysis for CXCL1 and CXCL2 in the neutrophils. ( C ) Representative scatter plots and percentage of dead cells and early apoptotic cells in the brain endothelial cells. ( D ) Representative histograms and mean fluorescence intensity (MFI) of extracellular Claudin-5 (left), intracellular ZO-1 (center), and extracellular VE-Cadherin (right) in brain endothelial cells. ( E ) Experimental scheme for co-culturing neutrophils and bEnd.3 cells. Neutrophils were isolated from the bone marrow of control and active mutant mice. bEnd.3 cells were then stimulated with the isolated neutrophils in the presence or absence of anti-CXCL1 or anti-CXCL2 blocking antibodies. ( F ) Representative histograms and mean fluorescence intensity (MFI) of Claudin-5 (upper panels) and VE-Cadherin (lower panels). Data represent the results of at least three independent experiments. Mean values are shown with error bars representing the SEM. ns: non-significant. * P < 0.05, ** P < 0.01

Article Snippet: In some experiments, bEnd.3 cells were incubated with mouse neutrophils (1 × 107 cells/well) [ ] for 24 h in the presence or absence of anti-mouse CXCL1 (0.2 μg/mL, 48415, R&D Systems, MN, USA) or anti-mouse CXCL2 (2 μg/mL, 40605, R&D Systems, MN, USA) antibodies to block CXCL1 or CXCL2 [ ].

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Fluorescence, Control, Mutagenesis, Blocking Assay

Journal: Journal of Neuroinflammation

Article Title: NLRP3 activation induces BBB disruption and neutrophil infiltration via CXCR2 signaling in the mouse brain

doi: 10.1186/s12974-025-03468-6

Figure Lengend Snippet: CXCL1 and CXCL2 are produced by astrocytes, pericytes, neutrophils, and endothelial cells during neuroinflammation. Immunofluorescence staining of the cerebral cortex region in coronal brain sections from ( A and B ) LPS-injected WT mice and ( C and D ) PBS-injected WT mice. Representative fluorescent images of ( A and C ) CXCL1 and ( B and D ) CXCL 2 in astrocytes, pericytes, neutrophils, and endothelial cells in the brain. Fluorescence images were acquired by staining for GFAP, NG2, LysM, or CD31 (green), CXCL1 or CXCL2 (red), and nuclei (blue). Scale bar: 10 μm. Data represent the results from the cortex region, based on the analysis of at least 15 brain slices per group across three independent experiments. Quantification of ( E ) CXCL1 and ( F ) CXCL2 levels in these cells. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: In some experiments, bEnd.3 cells were incubated with mouse neutrophils (1 × 107 cells/well) [ ] for 24 h in the presence or absence of anti-mouse CXCL1 (0.2 μg/mL, 48415, R&D Systems, MN, USA) or anti-mouse CXCL2 (2 μg/mL, 40605, R&D Systems, MN, USA) antibodies to block CXCL1 or CXCL2 [ ].

Techniques: Produced, Immunofluorescence, Staining, Injection, Fluorescence