Fig. 3.

DCs can suppress T cell activation through specific depletion of many different amino acids in vitro. LPS-matured (gray bars) or TGF-β + LPS-treated (black bars) bmDCs were cultured overnight with LPS at 37 °C in either complete RPMI medium 1640 or medium with a limiting concentration of the amino acid of interest, with or without a specific enzyme inhibitor (Table S1), as indicated. Control samples of each medium were similarly incubated without any cells (white bars). After removal of DCs by centrifugation and 0.2-μ filtering, fresh medium lacking the amino acid of interest was added 1:1 to each sample. Medium completely lacking the amino acid of interest was used as a negative control for T cell proliferation. Purified A1.RAG1^−/− CD4⁺ T cells were then stimulated by anti-CD3+CD28 beads in each sample of conditioned medium and proliferation measured at 48 h by ³H thymidine incorporation. The arrows indicate relief of suppression by the inhibitors (A–F) and any nonspecific toxicity of the inhibitor (F).