Figure 1.

The arginine precursor citrulline preserves human T cell proliferation under low arginine concentrations and this correlates with the induction of argininosuccinate synthase (ASS). (A) Scheme showing the two-step conversion of citrulline to arginine by the enzymes ASS and argininosuccinate lyase (ASL). (B–G) Primary human CD3⁺ T cells were isolated from blood of healthy donors by negative selection and stimulated with anti-CD3/anti-CD28-tagged beads for 96 h in the presence of the indicated arginine concentrations and either no further supplement or (B–D) 1 mM citrulline (Cit) or (F,G) 1 mM argininosuccinate (ASA). T cell proliferation was determined (B) by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester staining (one representative experiment of four is shown) and (C,F) by the incorporation of [³H]thymidine over 16 h (C: n = 15–30 from 5 to 10 different donors; F: n = 21 from 7 donors). (D,G) IFN-γ secretion was detected in T cell supernatants by ELISA (D: n = 8–24 from 3 to 8 donors; G: n = 9–12 from 4 donors). (E) ASS, ASL, and ERK expressions were analyzed in Western blot experiments. Shown is one representative blot of three. Columns represent mean ± SD in percentage of the mean respective value obtained from cells from the same donor stimulated in the presence of 100 µM arginine (C: 12,811 ± 4,173 cpm, D: 1,913 ± 1,421 pg/mL IFN-γ) or 1,000 µM arginine (denoted as Arg+; F: 43,355 ± 29,672 cpm, G: 1,858 ± 2,321 pg/mL IFN-γ). Statistical calculations were performed with one-way analysis of variance and Tukey’s post hoc test (***p < 0.001, **p < 0.01, n.s.: p ≥ 0.05).