Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A and B) LV tissue sections from non-HF or nonischemic HF human subjects were stained for CXCR3 or isotype control by IHC (representative CXCR3+ leukocyte–shaped cells are indicated with red arrows) (A) and quantified in multiple fields of view using a 40× objective (B). (C and D) LV tissue sections were stained for DAPI (blue), CD3 (red), and CXCR3 (green) by immunofluorescence (C), and the number of cells showing colocalization were quantified in multiple fields of view per section using a 40× objective (D). Scale bar: 100 μm. n = 2 control, 3 HF. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (E and F) CD4+ T cells isolated from the LV tissue of mice 4 weeks after Sham or TAC surgery were analyzed (E) and quantified (F) for surface CXCR3 and LFA-1 expression within the CD4+ gate by flow cytometry. n = 3 Sham, 7 TAC. Error bars represent mean ± SEM (**P < 0.01; Mann-Whitney unpaired U test). (G–J) Chemokine and cytokine mRNA levels in the LV of WT mice at different time points after surgery were determined by qPCR for Cxcl9 (G), Cxcl10 (H), Ifng (I), and Tnfa (J). n = 4 Sham, 5 TAC 1 weeks (w); 5 Sham, 9 TAC 2w; 6 Sham, 8 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Staining, Immunofluorescence, MANN-WHITNEY, Isolation, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A and B) Representative flow cytometry plots (A) and quantification (B) of CD4+ T cells isolated from the mLNs of WT mice at the indicated times after Sham and TAC surgery, indicating surface CXCR3 and LFA-1 expression within the CD4+ gate. Relative values to Sham 1 week (w) are indicated. n = 3 Sham, 3 TAC 1w; 4 Sham, 4 TAC 2w; 3 Sham, 4 TAC 4w. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C–F) Histogram representation (C and E) and quantification (D and F) of LFA-1 expression on CXCR3+CD4+ and CXCR3–CD4+ T cells in the mLNs of WT mice 4 weeks after TAC surgery (C and D) and in peripheral blood of nonischemic HF patients (E and F). n = 7 TAC mice, 20 nonischemic HF patients. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney unpaired U test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Flow Cytometry, Isolation, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: CD4+ T cells were isolated from the mLN of WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. (A and B) CD62LloCD44hi effector CD4+ T cells were identified by flow cytometry (A) and quantified (B). (C and D) CD4+ T cells recruited to the LV were identified by IHC (C) and quantified (D) per LV section. n = 4 Sham, 4 TAC WT; 3 Sham, 5 TAC Cxcr3–/–. Scale bars: 100 μm. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (E–H) The indicated gating strategy shown for WT TAC (E) was used to quantify the total cell number per LV of CD4+ T cells (F), CD11b+ myeloid cells (G), and CD11b+MerTK+CCR2+ recruited macrophages (H) by flow cytometry in WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. n = 3 Sham, 3 TAC WT; 6 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test). (I–K) mRNA levels in the LV of WT and Cxcr3–/– mice at 4 weeks after surgery was determined by qPCR for Cxcl9 (I), Cxcl10 (J), and Ifng (K). n = 9 Sham, 10 TAC WT; 3 Sham, 5 TAC Cxcr3–/–. Error bars represent mean ± SEM (*P < 0.05, **P < 0.01; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Isolation, Flow Cytometry

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: LV tissue sections were isolated from WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries. IHC was used to determine perivascular fibrosis (A) (quantified in C) as well as interstitial fibrosis (B) (quantified in D) by Picrosirius red staining. Scale bars: 100 μm. (E and F) Mean cardiomyocyte area was quantified by wheat germ agglutinin (WGA) IHC of LV tissue sections (E) (as shown in F). n = 4 Sham, 5 TAC WT; 3 Sham, 5 TAC Cxcr3–/– mice. Scale bars: 50 μm. Error bars represent mean ± SEM (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (G) The relative LV mRNA expression of the α and β myosin heavy chain isoforms were determined by qPCR to assess pathological cardiomyocyte hypertrophy. n = 3 Sham, 3 TAC WT; 3 Sham, 5 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: Isolation, Staining, Expressing

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: (A–C) Transthoracic short axis M mode images of the mid LV were acquired by echocardiography (A) of WT and Cxcr3–/– mice, 4 weeks after Sham and TAC surgeries to quantify fractional shortening (B) and ejection fraction (C). n = 5 Sham, 7 TAC WT; 5 Sham, 7 TAC Cxcr3–/– mice. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (D–F) Intraventricular hemodynamic measurements were acquired by a pressure volume transducer catheter to quantify maximum LV pressure (D), as well as dP/dt max (E) and dP/dt min (F) as parameters of cardiac contractility and relaxation, respectively. n = 3 Sham, 3 TAC WT; 3 Sham, 4 TAC Cxcr3–/–. Error bars represent mean ± SEM (*P < 0.05; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques:

Journal: JCI Insight

Article Title: CXCR3 regulates CD4 + T cell cardiotropism in pressure overload–induced cardiac dysfunction

doi: 10.1172/jci.insight.125527

Figure Lengend Snippet: Naive WT and Cxcr3–/– T cells were differentiated in vitro to Th1 cells and treated with either PMA (50 ng/ml), CXCL9 (100 ng/ml), or CXCL10 (100 ng/ml) for 5 minutes at 37°C to induce integrin LFA-1 activation, prior to perfusion over immobilized ICAM-1–coated coverslips at 1 dyne/cm2 in a parallel plate flow chamber. (A and B) Representative images (A) and quantification (B) of real-time videos of WT Th1 cell adhesion to ICAM-1 after treatment with anti–LFA-1 function-blocking antibody or IgG isotype control (20 μg/ml 30 minutes at 37°C). Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 5 independent experiments, analysis of 6 different fields of view per experiment (*P < 0.05, ***P < 0.001; 1-way ANOVA with Bonferroni post hoc test). (C and D) Representative images (C) and quantification (D) of real-time videos of Cxcr3–/– Th1 cells. Scale bars: 100 μm. Statistical comparisons are indicated compared with untreated cells. n = 3 independent experiments, analysis of 6 different fields of view per experiment. Error bars represent mean ± SEM (***P < 0.001; 1-way ANOVA with Bonferroni post hoc test).

Article Snippet: Immunofluorescence was performed using anti-CXCR3 (LSBio) (1:100 dilution) and anti-CD3 (Agilent Dako, clone F7.2.38) (1:50 dilution) overnight at 4°C, followed by incubation with Alexa Flour 488–conjugated anti-rabbit (Invitrogen, catalog A-11008) and Alexa Flour 568–conjugated anti-mouse secondary antibodies (Abcam, catalog ab175473).

Techniques: In Vitro, Activation Assay, Blocking Assay