Fig. 6. Discovery and validation of an age-associated mitochondrial decline in CD4⁺ T cells.

(A) Top left: Positive LEA associations with donor age projected on the t-SNE as in Fig. 5A [colored by −log₁₀(P)]. Middle left: LEA associations of mitochondrial electron transport projected on the t-SNE. Bottom left: Bright-field single-cell intensity projected onto the t-SNE. Median value of overlapping data points is calculated, and color is assigned accordingly. Points are plotted in order of intensity, with the lowest intensity on top. Right: Heatmap overview of all significantly enriched pathways in positive age-associated T cells [−log₁₀(P) > 5]. ATP, adenosine 5′-triphosphate; NIK, NFκB–inducing kinase; ER, endoplasmic reticulum; MHC, major histocompatibility complex. (B) Comparison of the significance in difference in bright-field intensity of positively versus negatively associated immune cells per population [with an association cutoff of −log₁₀(P) > 1.3]. Bar plots show the −log₁₀(P) * sign of the change {1 – [median(positive enrichment) / median (negative enrichment)]}. (C) Negative and positive age associations with cellular T cell phenotypes and donor age in an independent validation cohort of 15 healthy individuals calculated by LEA (left and middle). t-SNE depicts a total of 5000 cells per donor. Right: Comparison of differences in bright-field intensity of positively versus negatively age-associated CD4⁺ T cells [with an association cutoff of −log₁₀(P) > 1.3]. (D) Mitochondrial content (as measured by MitoTracker) of CD4⁺ T cells decreases with increased bright-field (BF) intensity. Bar plots display the mean MitoTracker intensity of CD4⁻ T cells per well per 10-percentile bins of bright-field intensity within each well. Mean and SDs across 10 repeat wells with a combined total of n = 78,095 CD4⁺ T cells are shown. P values are from a two-tailed t test of all replicate wells per bin against those of the brightest bright-field (right most) bin. ***P < 0.001.