Blocking PD1 prevents T-cell-promoted fibrosis in myocardial infarction

Fig. 1. PD1+ T cells are enriched and activated in the infarcted mouse heart. a Violin plot of Pdcd1 (encoding PD1) expression in different types of non-myocytes in hearts subjected to MI surgery. b Volcano plot of genes upregulated in cells isolated from MI hearts compared with those isolated from sham hearts at 7 days post-MI; log2FC > 0.5, P-value_adj < 0.05. c Comparison of Pdcd1 gene expression between MI hearts and sham hearts. d scRNA-seq analysis of CD3+ T cells isolated from murine hearts 7 days post-MI. Violin plots depict mPdcd1 expression levels across six major T-cell subsets: naive CD8+ T cells, naive CD4+ T cells, effector CD8+ T cells, effector CD4+ T cells, Tregs, and γδ T cells. e Flow cytometry-mediated quantification of PD1 expression levels across different T-cell subsets in murine hearts 7 days post-MI. f Flow cytometry results showing the percentage of PD1+ cells among CD3+ T cells, CD4+ T cells, and CD8+ T cells in the mouse heart at 3, 7, and 28 days after MI; n = 6–7 in each group. g Flow cytometry results showing the percentage of PD1 expression on CD3+ T cells in the peripheral blood of healthy donor (n = 14), AS (n = 62), CHD (n = 116), and STEMI patients (n…

Fig. 2. Loss of PD1 expression alleviates cardiac fibrosis accompanied by enhanced cardiac function. a GSEA of KEGG signaling pathways in PD1+ T cells vs PD1− T cells. b, c Western blot analysis of ECM proteins, including collagen I, periostin, and α-SMA, in fibroblasts co-cultured with PD1+ T cells and PD1− T cells, which were isolated from the mouse heart. Quantitative bar graphs of fibrotic proteins are displayed in c; n = 6 in each group. d–g Analyses of left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), systolic volume (LVSV), and diastolic volume (LVDV) in the WT group and PD1−/− group before MI and at 3 days and 28 days after MI; n = 6 mice per group. h Representative images of Sirius red staining showing the degree of fibrosis in the hearts of WT mice compared with those of PD1−/− mice after MI injury. i Quantification of scar size in the WT group compared with the PD1−/− group after MI injury; n = 6 mice per group. j, k Picrosirius red staining showing different degrees of fibrosis in the remote area of MI hearts from the WT and PD1−/− groups, respectively. The fibrosis data are summarized in k; n = 6 mice per group. Scale bar, 100 μm. l, m…

Fig. 3. PD1+ T cells induce fibroblast activation independent of the PD-L1 pathway. a Violin plot of Cd274 expression in different types of non-myocytes in hearts subjected to MI surgery. b, c Flow cytometry showing the frequency of PD-L1 expression in isolated cardiac fibroblasts from sham, 3-, 7- and 28-day post-MI hearts; n = 3–6 in each group. d Immunostaining image showing the location of PD-L1 (red) and vimentin (green) in the infarcted heart on Day 3, 7, 28 after MI. Scale bar, 100 μm. e, f Expression of extracellular matrix proteins, including extra domain A (EDA)-fibronectin, periostin, and α-SMA, in fibroblasts harvested from WT and PD-L1−/− hearts and co-cultured with PD1+ T cells; n = 3 in each group as indicated. The summary data are plotted in f. g–j LVEF, LVFS, LVSV and LVDV measured by 2-dimensional echocardiography in each experimental group, including WT, PD-L1−/−, WT with PD1+ T-cell infusion and PD-L1−/− with PD1+ T-cell infusion at 3 days and 28 days following MI; n = 6 in the WT group, n = 7 in the PD-L1−/− group, n = 6 in the WT + PD1+ T-cell group and n = 7 in the PD-L1−/− + PD1+ T-cell group. k Representative images of picrosirius red-stained heart sections from…

Fig. 4. PD1+ T cells induce cardiac fibroblast activation through the CXCL9/CXCR3 axis. a Volcano plot of the differentially abundant cytokines between PD1+ T cells and PD1− T cells according to the P-value and fold change. b Heatmap of significantly differentially expressed inflammatory cytokines (CXCL9, ICAM-1, and IL-2) between PD1+ T cells and PD1− T cells. c Relative quantification of CXCL9 expression in the cytokine array between PD1+ T cells and PD1− T cells; n = 4 in each group. d, e Western blot showing the expression of fibrotic proteins in neonatal cardiac fibroblasts treated with recombinant cytokine CXCL9 or the control. Summary data are plotted in e. f, g Representative western blot analyses showing the expression of fibrosis-associated proteins (fibronectin, collagen I, α-SMA and periostin) and TGF-β/Smad signaling molecules in mouse cardiac fibroblasts after 48 h of treatment. The summary data are plotted in g. h–k In the mouse model, CXCR3 inhibitors were given to WT mice to block the CXCL9/CXCR3 pathway (300 µg/mouse every 3 days until sacrifice), and an isotype was used as the control. The LVEF, LVFS, LVSV, and LVDV were measured by 2-dimensional echocardiography in…

Fig. 5. PD1 blockade alleviates cardiac fibrosis and improves cardiac function. a Study design for WT mice treated with an anti-PD1 inhibitor or anti-IgG antibody post-MI. b Representative M-mode images of WT mice after anti-PD1 inhibitor or IgG administration post-MI. c–f Evaluation of cardiac function by echocardiography revealed the LVEF, LVFS, LVSV, and LVDV in mice subjected to PD1 antibody injection before MI and every 2 days at 200 µg/mouse to 28 days, with isotype injection used as the control group; n = 6 mice per group. g, h Representative images of Sirius red-stained heart sections from each experimental group. Summary data of scar size are plotted in h; n = 6 in each group. i Representative images of interstitial fibrosis in the remote area after MI in each group. j Fibrosis quantification results are plotted as a percentage of the fibrotic area (mean with SEM); n = 6 per group. k Study design for WT mice receiving anti-PD1 inhibitor or anti-IgG injection post-MI. l Representative M-mode images of WT mice after anti-PD1 inhibitor or IgG injection post-MI. m–p Cardiac function evaluated by echocardiography revealed the LVEF, LVFS, LVIDs, and LVIDd in mice subjected to PD1…

Fig. 6. Nivolumab moderates cardiac fibrosis in infarcted non-human primate hearts. a, b MI was surgically induced in cynomolgus monkeys, followed by treatment with saline or nivolumab (10 mg/kg, two doses on the day before surgery and 14 days after MI). c–e Cardiac MRI assessments were conducted 3 days and 1 month after MI and treatment and were used to calculate the change (Δ) in the LVEF, ESV and EDV between the two time points (saline group, n = 6; nivolumab group, n = 6). f Summary of infarct size data determined by cardiac MRI assessments in the control group and nivolumab group. g Heart tissue sections from apex to base were collected at 1 month. h, i Representative images of interstitial fibrosis in non-infarcted areas in the heart from each experimental group. Summary data on fibrosis percentage are plotted in i; n = 6 in each group. Scale bar, 200 μm. j, k Expression of fibrosis-related proteins in heart tissues harvested from monkey hearts in each group; n = 6 in each group as indicated. Two-group comparisons were performed using an unpaired two-tailed Student’s t-test.

Fig. 7. Prognosis of MI patients with different layers of PD1+ T cells. a Association between cardiovascular risk status and PD1+ T-cell proportions. b Comparison of PD1+ T-cell proportions by age stratification. c Echocardiographic analysis revealed that patients with lower PD1+ T-cell proportions (< 20%) exhibited greater improvement in EF from baseline to Day 30 and Day 90 post-MI. d Functional recovery by PD1+ T-cell levels in the low-risk and high-risk cohorts (Day 90 post-MI). e Functional recovery by PD1+ T-cell levels in different age stratification cohorts (Day 90 post-MI). The proportion of PD1+ T cells increased from healthy donors to patients with STEMI, and 20% served as a cut-off value to distinguish coronary artery disease (CAD, including STEMI) patients from healthy controls or AS patients.