|
|
Blood, Vol. 109, Issue 7, 2871-2877, April 1, 2007
Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25– into CD25+ T regulatory cells
Blood Curti et al.
109: 2871
Supplemental materials for: Curti et al, Vol 109, Issue 7, 2871-2877
Files in this Data Supplement:
- Document 1. Supplemental materials (PDF, 17.6 KB)
- Figure S1. Human AML cells constitutively express a functionally active IDO protein (JPG, 38.1 KB)
-
(A) RT-PCR analysis of 13 representative AML samples. (B) Immunocytochemistry for IDO protein was performed in IDO– and IDO+ AML cells at RT-PCR. (C) Functional enzymatic activity. L-tryptophan and L-kynurenine concentrations were assessed by HPLC in 72-hour supernatants of IDO+ and IDO– AML cell cultures. Starting L-tryptophan concentration was 25 µM. Results represent the mean ± SD of 3 independent experiments. *P=.03, IDO– versus IDO+ AML samples. (D) Allogeneic MLRs using IDO– and IDO+ AML cells as stimulators and purified CD3+ T cells as responders in the presence and absence of 1-MT (1000 µM). APC/T-cell ratio equals 1:10. Results are expressed as stimulation index and report the mean ± SD of 7 independent experiments.
- Figure S2. Total CD4+ T cells do not proliferate in response to AML cells (JPG, 37.8 KB)
-
Highly purified CD4+ T cells were labeled with CFSE and then cultured with IDO-expressing AML cells in the presence and absence of IL-2 (100 IU/mL). Flow cytometry analysis of CFSE-labeled CD4+CD25+ T cells before and after culture with AML cells is depicted. Results are representative of 3 independent experiments.
- Figure S3. IDO-expressing AML cells induce the conversion of CD4+CD25– into CD4+CD25+ T cells (JPG, 30.8 KB)
-
Highly purified CD4+CD25– T cells were incubated for 7 days with IDO+ and IDO– AML cells. At the end of culture, CD4+ cells were gated and analyzed for the presence of converted CD4+CD25+ cells. Results are representative of 4 independent experiments.
- Figure S4. Converting CD4+CD25+ T cells have a significant proliferation in response to IDO-expressing AML cells (JPG, 28.3 KB)
-
Highly purified CD4+CD25– T cells were labeled with CFSE and then cultured with IDO-expressing AML cells in the presence and absence of IL-2 (100 IU/mL). Flow cytometry analysis of CFSE-labeled CD4+CD25+ T cells before and after culture with AML cells is depicted. Results are representative of 3 independent experiments.
- Figure S5. The conversion of CD4+CD25– to CD4+CD25+T cells is stable (JPG, 47.1 KB)
-
Highly purified CD4+CD25– T cells were cultured with allogeneic IDO-expressing AML cells for 7 days. At the end of culture, converting CD4+CD25+ T cells were purified and then cultured for 5 days in medium alone or in the presence of AML cells with and without 1-MT (1000 µM). Flow cytometry analysis of CD4+CD25+ T cells is depicted. AML cells were derived from the same patients. Results are representative of 3 independent experiments.
- Figure S6. Cell-to-cell contact may be required to induce the conversion of CD4+CD25– cells to CD4+CD25+ cells upon stimulation with IDO-expressing AML cells (JPG, 38.1 KB)
-
Highly purified CD4+CD25– T cells were cocultured at a fixed ratio with AML blast cells (1 AML blast cell to 10 T cell cells) for 7 days in complete culture medium in either the presence or the absence of 1-MT (1000 µM). A Transwell system (MilliCell inserts, 0.4 µM) was used in order to impede intercellular contact between AML blast cells and naive CD4+CD25– T cells while allowing the diffusion of soluble factors. The results are representative of 3 independent experiments.
|
|
