TNFR1 Contributes to Activation-Induced Cell Death of Pathological CD4+ T Lymphocytes During Ischemic Heart Failure

Figures

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  Figure 1. T Cells Activated During HF Are Proinflammatory (A) Representative image-stream pictures showing TNF-α and TNFR1 in splenic CD4+ T cells. (B) Representative flow contour plots showing cardiac TNFR1+ CD4+ T cells in HF and sham mice (left) and their group quantitation at 1 day, 3 days, 1 week, 2 weeks, and 8 weeks postsurgery (right). (C) Representative flow contour plots showing TNF-α in cardiac CD4+ T cells at 3 days post-MI or sham surgery. Group quantitation for the frequency of cardiac TNF-α+ CD4+ T cells (left) and TNF-α mean fluorescence intensity in CD4+ T cells (right) at 3 days (D) and 8 weeks (E) post-MI or sham surgery. (F) Representative flow cytometry scatterplots showing the expression of TNF-α in CD4+ TNFR1− and CD4+ TNFR1+ T cells in the failing hearts and (G) its group quantitation (frequency and MFI). N = 5-10 in each group in B. Data were normalized using logarithmic transformation in B and analyzed using 2-way analysis of variance with the Bonferroni post hoc test. To avoid overcrowding of data, especially at early time points, individual data points are not shown. Data in DandE were analyzed using the unpaired Student’s t-test, whereas the paired Student’s…

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  Figure 2. TNFR1 Neutralization Promotes Pro-Survival Signaling (A) Representative flow histograms showing the proliferation of CD4+ T cells either nonstimulated or stimulated with anti-CD3/CD28 antibodies in the absence and presence of different concentrations of neutralizing anti-TNFR1 antibody and (B) their group quantitation. (C) Representative flow histograms for Bcl-xL expression in proliferated T cells treated with different concentrations of neutralizing anti-TNFR1 antibody (left), and fold change (right) with respect to nonstimulated (N.S.) control. (D) Representative flow histograms for TNF-α expression in proliferated T cells treated with different concentrations of anti-TNFR1 antibody (left) and fold change (right) with respect to stimulated control. (E) Representative Western blots and (F) densitometric ratio of phospho- to total-NFκB (p65) (normalized to β-actin) in stimulated T cells in the absence and presence of 10 μg/mL anti-TNFR1 antibody. Experiments were repeated 4 to 5 times with 3 to 4 replicates in each, and the average of each experiment is shown as individual points. Data were analyzed using 1-way analysis of variance, and P values were adjusted using the false…

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  Figure 3. TNFR1−/− Improves Survival of HF-Activated T Cells (A) Representative flow scatterplots for donor CD45.2+ CD4+ T cells isolated either from WT or TNFR1−/− mice in the blood of CD45.1+ mice at 2 days and 1, 2, and 14 weeks and (B) their group quantitation at 2 days and 1, 2, 4, 8, and 14 weeks post-AT. (C) Representative flow scatterplots and (D) group quantitation for donor CD45.2+ CD4+ T cells in the spleens of CD45.1 recipient mice at 14 weeks. Group quantitation for (E) donor-derived CD45.2+ CD4+ Bcl-xL+ and (F) CD45.2+ CD4+ CD44high CD62L+ cells at 14 weeks post-AT. The study was repeated 2 times (n = 3-5 in each study), and data from a representative experiment are shown. Data in B were analyzed using repeated measures 2-way analysis of variance, and P values were adjusted using the false discovery rate. Data in D, E, andF were analyzed using the unpaired Student’s t-test. AT = adoptive transfer; WT = wild type; other abbreviations as in Figure 1.

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  Figure 4. TNFR1 Does Not Regulate Pathogenicity of HF-Activated T Cells (A) Group quantitation for end-systolic and end-diastolic volumes (ESV and EDV), and ejection fraction (EF) of CD45.1+ recipient mice at 14 weeks post-AT of sham or HF-activated splenic CD45.2+ CD4+ T cells isolated either from WT or TNFR1−/− mice. (B) Tibia normalized heart and LV weights of recipient mice at 14 weeks post-AT. The study was repeated 2 times (n = 2-5 in each study), and cumulative data from both the experiments are shown. Data in AandB were analyzed using 2-way analysis of variance with the Tukey post hoc test. LV = left ventricular; other abbreviations as in Figures 1 and 3.

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  Figure 5. TNFR1−/− Promotes Proliferation of HF-Activated T Cells (A) Representative flow contour plots for HF-activated WT or TNFR1−/− donor CD45.2+ CD4+ T cells in the hearts of CD45.1+ recipient mice and (B) their group quantitation for the total number of cells (left) and their frequency (right) with respect to total cardiac CD4+ T cells at 14 weeks. (C) Representative flow scatterplots showing ki67+ cells in CD45.2+ CD4+ T cells infiltrated into the hearts of CD45.1 recipient mice and (D) their group quantitation (frequency [left] and ki67 MFI [right]) at 14 weeks post-AT. Donor-derived CD45.2+ CD4+ Ki67+ T cells in the (E) blood and (F) spleen of recipient CD45.1 mice at 14 weeks post-AT. Studies were repeated 2 times (n = 2-5 in each), and cumulative data are shown. Ki67 MFI and splenic cell counts are representative from 1 experiment. Data were analyzed using 2-way analysis of variance with P values adjusted using the false discovery rate in BandD (left) and the unpaired Student’s t-test in D (right), E, andF.