Figure 6.

Requirement for Extracellular Alanine in T Cell Mitogenesis

(A) Dose-dependent proliferation of primary T cells in response to exogenous alanine. Primary human CD4+ T cells were stained with CFSE, stimulated with CD3/CD28 Dynabeads in media supplemented with alanine at the concentrations indicated, and analyzed by flow cytometry after 120 hr (green filled histograms). Peaks are labeled by division number, and unstimulated cells were included as a control (black dotted lines). Representative data from three independent experiments are shown.

(B) Dose-dependent inhibition of primary T cell proliferation by MeAIB. Primary human CD4+ T cells were stimulated with CD3/CD28 Dynabeads in media supplemented with alanine and MeAIB at the concentrations indicated. Viable cells were enumerated using CytoCount beads after 72 hr and numbers expressed as a fraction of the maximum.

(C) Regulation of free intracellular alanine pool by System A-dependent alanine uptake. Primary human CD4+ T cells were expanded, rested, and re-stimulated for 48 hr with CD3/CD28 Dynabeads. Cells were then resuspended in media supplemented with alanine at the concentrations indicated in the presence or absence of MeAIB. Abundance of free intracellular alanine at baseline and 60 min is expressed as a fraction of the maximum. Mean values and 95% confidence intervals are shown for data obtained in triplicate. No difference in cell size was observed between 0 and 0.5 mM alanine (Figure S7D, left panel).

(D–F) Reconstitution of free intracellular alanine pool by extracellular alanine. Washed cells prepared as in (C) were resuspended in media supplemented with 5.6 mM 13C6-glucose and 0.5 mM 15N-alanine (D) in the presence or absence of MeAIB. Abundances of labeled and unlabeled free intracellular alanine (E) and supernatant lactate (F) at the indicated time points are expressed as a fraction of the maximum. Mean values and 95% confidence intervals are shown for data obtained in triplicate. No difference in cell size was observed in the presence or absence of MeAIB (Figure S7D, right panel).

(G) Defective proliferation of primary T cells depleted of SNAT1 by HIV-1. Primary human CD4+ T cells were stained with CellTrace Violet, stimulated with CD3/CD28 Dynabeads, infected with the indicated NL4-3 Vpu 2_87 HIV-1 viruses at an MOI of 3, and analyzed by flow cytometry after 120 hr (violet filled histograms). Peaks are labeled by division number, and unstimulated cells are included as a control (black dotted lines). Representative data for infected (p24+) and uninfected (p24−) cells are shown. Mean percent of infected cells in each generation from four independent experiments are depicted as stacked columns. Error bars indicate SEM. ^∗∗p < 0.01.

See also Figure S7.