Figure 2.

l-arginine synthesis is necessary for l-citrulline-mediated T cell functions. (A–C) Lymphocytes from Asl^flox/flox;Tie2-cre mice were isolated and stained with carboxyfluorescein succinimidyl ester (CFSE). Cells were stimulated with α-CD3 and α-CD28 in R-free RPMI supplemented with 1 mM l-arginine (black), 1 mM l-citrulline (gray), or neither amino acid added (white) for 72 h. CFSE dilution of CD3⁺CD4⁺ T cells was analyzed by flow cytometry. Data are displayed as representative histograms (A), mean percent of divided cells (B), and proliferation index (C) as defined in the methods. Data are combined from four experiments. (D–H) Lymphocytes from Asl^flox/flox;Tie2-cre mice were polarized under T_Ø, T_H1, or T_H17 polarizing conditions for 5 days in the indicated culture conditions (see Materials and Methods). Following restimulation, cells were stained for intracellular cytokines and analyzed by flow cytometry. Cytokine-producing cells are represented in the graphs by single-positive, mean frequency of cytokine-expressing cells (E, G), and mean fluorescence intensity of the indicated cytokine (F, H). Data are combined from three experiments. Error bars, SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.