Figure 2.

T cells in a NET-rich environment exhibit functional exhaustion. (A, B) Following I/R, T cells infiltrating the TME express reduced T cell cytokine levels; the changes return to sham levels after treatment with DNAse. Representative flow cytometry plots comparing CD4⁺ (A) or CD8⁺ (B) T cells in IR vs IR + DNAse are shown. (C, D) Following I/R, T cells in the TME have altered metabolic function measured by TMRE, Mitotracker, Bodipy and NBDG. These metabolic effects on T cell metabolism are reversed in animals treated with I/R + DNAse; Representative flow plots are shown comparing No I/R vs I/R and I/R vs I/R + DNAse. (E) T cells from mice that underwent No I/R, I/R or I/R + DNAse treatment were cultured in vitro. Proliferation was assessed following staining with a CFSE proliferation dye and 5 days of stimulation with anti-CD3/anti-CD28 beads. (F, G) Following I/R T cells infiltrating the TME express higher levels of exhaustion markers and lower levels of cytokines, these effects are reversed with DNAse. T cells in the spleen are unaffected by liver I/R or DNAse administration. (bar graphs represent the mean +/- SEM of three individual experiments performed in duplicates or triplicates: *p < 0.05/**p < 0.01). ns, not significant.