Advertisement

Regulation of human auto- and alloreactive T cells by indoleamine 2,3-dioxygenase (IDO)–producing dendritic cells: too much ado about IDO?

Peter Terness, Jing-Jing Chuang, Thomas Bauer, Lucian Jiga and Gerhard Opelz

Data supplements

  • Supplemental materials for: Terness et al, Vol 105, Issue 6, 2480-2486

    Files in this Data Supplement:

    • Figure S1. T-cell regulatory property of dendritic cells pretreated with TNF-α and IFN-γ. (PDF, 40 KB) - Mature Dendritic Cells (mDCs) of 8 healthy donors, in which IDO synthesis was stimulated by an enhancer (1000U/ml TNF-) in addition to IFN- (1000U/ml), were co-cultured at decreasing ratios with autologous anti-CD3-stimulated T cells for 3 days. The control consisted of mDCs without IDO-inducer. Proliferation was assessed by adding [3H]thymidine. Single values represent the percentage of maximum stimulation (=100%). The difference between the T-cell stimulatory capacity of native DCs and IDO-DCs was statistically not significant.
    • Figure S2. Complete suppression of T-cell proliferation by dendritic cells pretreated with protein synthesis inhibitor. (PDF, 24 KB) - Mature Dendritic Cells of 3 healthy donors were loaded with MBP, treated for 30 min. with a selective protein synthesis inhibitor (=PI; 100µg/ml) and extensively washed. Vital staining with subsequent FACS analysis showed unimpaired cell viability. PI-treated as well as untreated DCs were coincubated at a ratio of 1:10 with H. saimiri-transformed MBP-specific T cells (clone ES-BP8T). DCs and T cells shared at least one HLA-DR antigen. Controls consisted of MBP-DCs or T cells only. Cell proliferation was measured after 4 days by [3H]thymidine incorporation. Data represent mean $#177; SD and are expressed as a percentage of the positive control values (= 100%). PI-treated DCs completely suppress the T-cell response, showing that suppression below basic proliferation of transformed T cells is possible.

Article Figures & Data

Figures

  • Figure 1.

    Phenotypic characteristics of monocyte-derived mature DCs. mDCs were generated from blood monocytes as described. Nonadherent cells were stained with fluorochrome-conjugated mouse antihuman monoclonal antibodies. FACScan data show the cell surface expression of the indicated marker (gray area) or isotope controls (bold lines) with a life gate from one representative experiment of DCs matured with CD40L + LPS.

  • Figure 2.

    Expression of IDO-specific transcripts in nonadherent CD123+/CCR6+ DCs from healthy donors and MS patients before and after treatment with IFN-γ. Immature DCs (iDCs) obtained from healthy donors (A) or MS patients with or without MBP loading (B) were cultured overnight with medium containing maturation factors, supplemented or not with IFN-γ. Total RNA was isolated and reverse transcribed into cDNA. PCR was performed using IDO-specific primers, and the products were analyzed by agarose gel electrophoresis. The positive control (+) consisted of material extracted from IDO transgene expressing 293 cells, whereas negative control “a” (-a) was water instead of RNA for reverse transcription and negative control “b” (-b) was water instead of cDNA template for PCR; M indicates DNA molecular marker. One representative example for healthy donors and MS patients is shown. A faint IDO band occurred in 2 of 9 tested DCs.

  • Figure 3.

    Kynurenine and tryptophan concentrations measured in the culture medium of DCs. iDCs were matured with CD40L + LPS without or with IFN-γ (± 1-methyl-tryptophan). iDCs of MS patients received MBP in addition to maturation factors. After 36 hours, the cell culture supernatants were harvested and the concentrations of kynurenine and tryptophan were measured by HPLC. The chromatograms (top rows of A and B) show the total free kynurenine (Kyn) and tryptophan (Trp) in cell-culture supernatants of DCs—without (left) and with (right) IFN-γ treatment—from one representative healthy donor (A) and MS patient (B). The mean ± SD concentrations of tryptophan and kynurenine of DCs from 7 healthy donors and 10 MS patients are shown in the bottom rows of panels A and B.

  • Figure 4.

    Effect of IFN-γ–treated DCs on anti-CD3 antibody-activated autologous and allogeneic peripheral lymphocytes of healthy donors. Untreated or IFN-γ–treated mDCs generated with CD40L + LPS were coincubated for 3 days with anti-CD3 antibody-stimulated autologous blood lymphocytes at a ratio of 1:10 (n = 7) (A) or at increasing ratios (n = 6) (B). Alternatively, DCs were cocultured for 5 days with allogeneic lymphocytes at a ratio of 1:10 (n = 8) (C) or at increasing ratios (n = 5) (D). Proliferation was assessed by adding [3H]thymidine (1 μCi [0.037 MBq]/well) during the last 18 hours of culture. The positive control consisted of DCs plus anti-CD3 antibody-activated autologous or allogeneic lymphocytes, and the negative control consisted of lymphocytes and DCs alone. Single values represent mean ± SD and are expressed as a percentage of the positive control values (= 100%) (mean stimulation: autologous, 23 000 ± 6000 cpm; allogeneic, 27 800 ± 8000 cpm). The difference between the T-cell stimulatory capacity of native DCs and IDO DCs was statistically not significant in experiment A and borderline significant (P = .026) in experiment C.

  • Figure 5.

    Effect of IFN-γ–treated DCs on MBP-specific T-cell proliferation of MS patients. mDCs were generated with CD40L + LPS, treated with IFN-γ, loaded with MBP, and cocultured with autologous peripheral lymphocytes obtained from MS patients (n = 10) (A). Alternatively, IFN-γ–treated MBP-DCs (± 1-MT) of HLA-DR compatible healthy donors were coincubated with H saimiri–transformed MBP-specific T cells (clone ES-BP8T) at a ratio of 1:10 (n = 8) (B) or at increasing ratios (n = 5) (C). For experiment A, controls consisted of MBP-loaded DCs plus autologous peripheral lymphocytes, MBP-DCs only, or lymphocytes only. For experiment B, controls consisted of MBP-loaded (without IFN-γ) or -unloaded DCs (± IFN-γ) plus ES-BP8T cells, ES-BP8T cells only, or MBP-loaded DCs only. Cell proliferation was measured after 4 days. Data represent mean ± SD and are expressed as a percentage of the positive control values (= 100%) (mean stimulation for A: 11 000 ± 5600 cpm and for B-C: 19 200 ± 5500 cpm). No statistically significant difference between the T-cell stimulatory capacity of native DCs and IDO DCs was noted in experiment A. IDO DCs significantly suppressed the T-cell response in experiment B (P = 10-4).