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. 1999 May 3;189(9):1363–1372. doi: 10.1084/jem.189.9.1363

Figure 3.

Figure 3

Figure 3

Elimination kinetics of tryptophan in cocultures and expression of IDO by MCSF-derived Møs. (A) MCSF-derived Møs were cultured for 24 h with autologous T cells, either with (•) or without (▪) anti-CD3 mAb. The medium was then replaced with fresh medium and supernatant from replicate cultures harvested at the times shown. Tryptophan concentration was assayed spectrofluorometrically as described in Materials and Methods. (B) IFN-γ–inducible IDO mRNA in MCSF-derived Møs. RT-PCR showing IDO expression in MCSF-derived Møs before (lane 4) and after (lanes 1–3) activation for 24 h with recombinant IFN-γ. Starting RNA for the reverse transcriptase reaction in lanes 1–3 was from 20,000, 2000, and 200 activated Møs, respectively, and from 20,000 unactivated Møs in lane 4. Lane 5 shows amplification of human IDO plasmid template giving the expected 182-bp product. (C) HPLC analysis of Mø culture supernatants showing degradation of tryptophan and production of kynurenine. MCSF-derived Møs were preactivated for 24 h with IFN-γ to induce IDO expression, and then the spent medium was replaced 90:10 with fresh medium. Trace 1 shows the analysis of supernatant immediately after adding fresh medium (time 0); trace 2 shows the conditioned medium 24 h later. The number of Møs in these experiments was kept low so that some tryptophan would be detectable at the end of the assay. The traces shown represent the portion of the elution gradient between 28 and 42% acetonitrile (minutes 7.00–10.50), during which kynurenine (K) and tryptophan (T) appeared. The peak labels are positioned at the points at which the purified standards eluted, which were within ±3 s of the corresponding sample peak. Compounds present in culture medium that also absorbed at OD254 (unlabeled peaks) were readily resolved from tryptophan and kynurenine, and the T and K peaks were confirmed by mass spectroscopy (see Materials and Methods). The experiment shown used purified Møs activated with recombinant ligands; identical results were obtained when Møs were activated in coculture with T cells plus mitogen. One of four experiments is shown.