FIGURE 2. Fasting/Feeding Differentially Regulate CD4⁺ T Cell Activation and Differentiation.

a-c, The dot-line plots show relative cell populations (percentiles, n=19 biologically independent subjects, Wilcoxon two-sided paired analysis). a, Representative flow plots of activated CD4⁺ T cells (CD38⁺HLADR⁺) comparing PBMCs (baseline, 24-hr. fasting, and refed states). b, Comparing relative cell population frequencies with specific CD4⁺ T cell surface markers. The plots show increased Th1 (CXCR3⁺CCR6⁻), Th17 (CXCR3⁻CCR6⁺), and Th2 (CD294⁺) surface markers on refed PBMCs. c, Comparison of intracellular cytokine markers. The plots show increased (percentiles) of IFNγ⁺ and IL-17⁺ in refed cells (n=19 biologically independent subjects). d-e, ELISA measurement of cytokine release after CD4⁺ T cell activation. Bar graphs (mean ± SEM with value of each subject, n=13, duplicate experiments, two-sided, Wilcoxon paired analysis after normalization). d, Release of IFN, IL-17, IL-22, e, IL-5 and IL-13 from baseline, 24-hr. fasted and refed CD4⁺ T cells. f, Following Th-subpopulation differentiation IFNγ, IL-5 and IL-17 secretion were measured from Th1, Th2 cells and Th17 cells respectively. Bar graphs (mean ± SEM with data points for each subject, n=5 duplicate experiments, two-sided, ratio paired Student’s t-test). g, Relative mRNA levels of Th1 (TBX21), Th2 (GATA3) and Th17 (RORC) canonical TFs in fasted vs. refed CD4⁺ T cells. Bar graphs represent mean ± SEM with each value 3–4 replicates (n=10 biological distinct subjects, two-sided paired Student’s t-test).

Supplementary Data1for Fig.2 shows flow cytometry gating strategy.