Fig. 5. AMPK:mTOR signaling axis and lactate transporter MCT1 are critical regulators of tolerogenic DC.

A Heatmap and clustering analysis of gMFI expression profiles for DC-lineage markers over time. Average expression from three donors is represented. Protein synthesis levels, Percentual SCENITH parameters, donor label, and DC differentiation stages are annotated along with expression profiles for signaling factors and calculated mTOR:AMPK phosphorylation ratio. B Kinetic phosphorylation levels for p-AMPK and p-mTOR across DC differentiation and their trajectory overlaps are depicted. Barplots with mean ± SE represent changes in calculated p-mTOR:p-AMPK ratios (N = 3). C gMFI expression values of p-AMPK and p-mTOR in control (black), vitd3+dexa (purple) and vitd3 (orange)-treated DC across maturation stages are shown (N = 4). Lines connect data points from an individual donor. D Boxplots represent changes in calculated p-mTOR:p-AMPK ratios between control (black), vitd3+dexa (purple) and vitd3 (orange)-treated DC (N = 5 donors) across maturation timeline. Lines connect data points from an individual donor. E Bar graphs with mean ± SE represent gMFI expression values and F box plots represent Glucose and lactate measurements for control and vitd3-tol mDC treated with Vehicle (DMSO), Rapamycin (1 μM), Dorsomorphin (3.75 μM) and BAY8002 (80 μM) (N = 3). Diagrams of pathway inhibitor targets and timeline for inhibitor treatment are depicted. Multiple comparisons statistical significance in B–F was calculated via one-way ANOVA with Tukey’s post-hoc test. For all panels, P-values are represented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****P ≤ 0.0001. p-values < 0.05 were considered statistically significant (ns). Box plots indicate second and third quantile (box), median (horizontal line) and 1.5× the interquartile range (whiskers). Source data are provided as a Source Data file.