Fig 1: Gene-function validation in hypoxic PH mice.C57BL/6 mice exposed to hypoxia (10% O2) for 4 weeks exhibited higher RVSP (a) and RVHI (b) compared to that of mice in normoxia (21% O2) (n = 10 for per group, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). c Representative images of hematoxylin and eosin (H&E) staining of lung tissues from mice under normoxic or hypoxic condition. Scale bars, 20 μm. d Assessment of pulmonary vascular remodeling by determination of the ratio of the media cross-sectional area to the total vessel cross-sectional area in each group (n = 10 for per group, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). e The expression of Prf1, Spon2, Tbx21, Cd28 and Cd3e in lung tissues of the mice under normoxic or hypoxic condition at mRNA levels (n = 7–8 for per group, *p < 0.05, **p < 0.01; Student’s t test. Data were presented as mean ± SD). f–i Representative images of immunoblottings and the quantification of the expression of Cd28 (g), Spon2 (h) and Tbx21 (i) in lung tissues of the control mice and hypoxic PH mice at protein levels (n = 8 for per group, **p < 0.01, ***p < 0.001; Student’s t test. Data were presented as mean ± SD). j Immunoblottings for the expression of Cd28, Tbx21 and Spon2 in RAW264.7 monocytes/macrophages in response to CoCl2 (150 μmol/L) or vehicle for 24 h. k Immunoblottings for the expression of CD28, TBX21 and SPON2 in THP-1 monocytes in response to CoCl2 (150 μmol/L) or vehicle for 24 h. l Quantification of the expression of Cd28 (CD28), Tbx21 (TBX21) and Spon2 (SPON2) in RAW264.7 and THP-1 cells (n = 4 for per group, *p < 0.05, **p < 0.01; Student’s t test. Data were presented as mean ± SD). m Representative images of immunoblottings of Jurkat Clone E6-1 cells transfected with CD28 siRNA (siCD28) or control siRNA (siControl). n Viability of human PASMCs co-cultured with or without CD28-silencing or control Jurkat cells for 48 h (n = 5 for per group, *p < 0.05, **p < 0.01; one-way ANOVA, Tukey’s post-hoc test. Data were presented as mean ± SD). PH pulmonary hypertension, RVSP right ventricular systolic pressure, RVHI right ventricular hypertrophy index, PASMCs pulmonary arterial smooth muscle cells. Prf1 Perforin 1, Spon2, Spondin 2, Tbx21 t-box transcription factor 21.