Fig. 1 |. NAD(P)H and FAD autofluorescence imaging reveals metabolic differences between quiescent and activated T cells.

(a) Representative optical redox ratio, NAD(P)H τ_m, and FAD τ_m images (4 images selected out of 202 images acquired from 6 different donors with similar results) of quiescent (columns 1, 3) and activated (columns 2, 4) CD3⁺ (rows 1–3) and CD3⁺CD8⁺ (row 4–6) T cells from two different donors. Scale bar is 20 μm. Cell size (b), optical redox ratio (c), NAD(P)H τ_m (d), FAD τ_m (e), NAD(P)H α₁ (f), and FAD α₁ (g) of quiescent and activated CD3⁺ and CD3⁺CD8⁺ T cells. Black circles represent mean of all data (6 donors), triangles (donors A [dark red], B [medium red], and F [light red]) represent data from female donors, squares (donors C [dark blue], D [medium blue], and E [light blue]) represent data from male donors. Each colour shade represents data from an individual donor. Data are mean +/− 99% CI. Horizontal lines indicate statistical comparisons, Stars (*** p<0.001) indicate statistical comparisons at the cellular level (n=4,877 biologically independent CD3⁺ T cells from 6 donors and n=3,478 biologically independent CD3⁺CD8⁺ T cells from 6 donors) from a two-sided logistic regression, generalized linear model using an α significance level of 0.05. Carets (B: ˆˆ p=0.001, ˆ=0.016; C: ˆ p=0.011, ˆˆ p=0.002; D: ˆ p=0.015, ˆˆ p=0.002; E: ˆ p=0.022; F: ˆˆ p=0.008, ˆˆˆ p<0.001; G: ˆˆ p=0.002, ˆ p=0.021) indicate significance at the donor level, n=6 biologically independent donors. Aggregated cellular data was compared using a double-sided paired t-test. (h-j) Cellular respiration increases in activated T cells. The oxygen consumption rate (OCR; panel H) and extracellular acidification rate (ECAR, panel I) are increased in activated bulk CD3⁺ and isolated CD3⁺CD8⁺ T cells. The ratio of OCR to ECAR (J) is significantly decreased in activated bulk CD3⁺ and isolated CD3⁺CD8⁺ T cells as compared with that of quiescent T cells. *** p<1*10⁻⁵, horizontal lines indicate statistical comparisons, double-sided, Student’s t-test, n=6 wells/group CD3⁺CD8⁺ isolation, n=12 wells/group CD3⁺ isolation (1 donor). Mean +/− 95% CI. Error bars smaller than the symbol for the mean are not shown.