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. 2019 Oct 3;4(19):e127716. doi: 10.1172/jci.insight.127716

Figure 7. Metabolic alterations in B7-H4–/– versus WT donor T cells during GVHD.

Figure 7

Lethally irradiated B6 recipients were infused with 107 WT BALB/c BM cells plus 6 × 106 CTV-labeled or unlabeled WT BALB/c or B7-H4–/– purified T cells. Mice were sacrificed on day 5 after BMT, and experiments were performed as described. (A) Splenocytes were analyzed by flow cytometry for intracellular expression of GLUT1 in undivided (CTVhi) and divided (CTVlo) donor T cells. (B) Extra-cellular acidification rate (ECAR) of purified donor T cells was measured after addition of glucose, oligomycin, and 2-deoxyglucose (2-DG). Basal glycolysis was measured after addition of glucose, and glycolytic capacity was measured after addition of oligomycin. The graph represents pooled data from 5 time points to measure glycolysis and pooled data from 4 time points to measure glycolytic capacity. (C) Oxygen consumption rate (OCR) of purified donor T cells was measured after addition of oligomycin, FCCP, and rotenone plus antimycin A. T cells from naive WT BALB/c mice (n = 4) were included as control. (D) Basal OCR (resting OCR – rotenone plus antimycin A OCR) was measured before addition of oligomycin, and maximal OCR was measured after addition of FCCP subtracting nonmitochondrial OCR (rotenone plus antimycin A OCR). The graph represents pooled data from 3 time points to measure basal OCR or maximal OCR. Splenic donor T cells were analyzed by flow cytometry for BoDipy (E) or CD36 (F) expression. (G) Splenocytes were also analyzed by flow cytometry for intracellular expression of CPT1a in undivided and divided donor T cells. (H and I) OCR of purified donor T cells was measured after addition of oligomycin, FCCP, etomoxir (Eto), and rotenone plus antimycin A. (I) Percent inhibition was calculated using pooled data from 3 time points after addition of etomoxir. (A and E–G) Data are representative of 5 mice per group from 2–3 independent experiments. (B–D, H, and I) Data are representative of 12 mice per group from 2–3 independent experiments. MFI, mean fluorescence intensity. Data represent mean ± SEM, and P values were calculated by 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.