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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

From: PD-1 signaling affects cristae morphology and leads to mitochondrial dysfunction in human CD8+ T lymphocytes

Fig. 2

PD-1 ligation impairs mainly CD8+ T cell metabolism. a KEGG signaling pathways with the highest scores significantly enriched in the 578 transcripts selected by LRT. b Lactate production in hCD8+ T cells stimulated 48 h with TCTRL, TACT and TACT + PD1 beads. c Lactate production in hCD8+ T cells stimulated 48 h with TACT + PD1 beads containing the indicated amounts of PD-L1-Fc. d-f hCD8+ T cells were stimulated with beads as in b and analyzed with SeaHorse. Basal extracellular acidification rate (ECAR; D), basal O2 consumption rate (OCR; e), and basal OCR/ECAR ratio (f). g OCR obtained during mitochondrial stress test in cells stimulated as in b, performed by injection of oligomycin, the mitochondrial uncoupler FCCP, and the electron transport chain inhibitors antimycin A/rotenone. h-j Maximal OCR obtained after FCCP injection (h), spare respiratory capacity (SRC; i) calculated as the difference between maximal and basal OCR, and relative proton leak (j) determined as OCR after oligomycin and subsequent injection of rotenone plus antimycin A. k-n hCD8+ T cells were stimulated with beads as in b, treated with etomoxir or vehicle and analyzed with SeaHorse, using palmitate as substrate. Basal OCR with vehicle (solid) or with etomoxir (hatched) (k), FAO-specific OCR from data in k (l), maximal OCR after FCCP injection in vehicle or etomoxir-treated cells (m), FAO-specific maximal OCR calculated from m (n). o Representative immunoblots for CPT1A and β-actin (loading control) in CD8+ T cells stimulated as indicated. p Densitometric analysis of immunoblots as in o. The CPT1A/β-actin ratio is shown, with the value for TCTRL cells as reference (n = 3 donors). Data are mean ± SEM from six (b, d-j), four (k-n) or three (p) donors; for c, data are mean ± SD representative of one donor from two. ** p < 0.01, * p < 0.05, Kruskal-Wallis with Dunn’s post-hoc test for multiple comparisons (b, d-f, h-k, m), two-tailed Student’s t-test (l, n), or two-way ANOVA with Newman-Keuls post-hoc test for multiple comparisons (p)

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