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Supporting Information Fig. S1. Gating strategy used in flow cytometric analyses. Co-cultured lymphocytic and myelocytic cells were initially gated according to side scatter (SSC) and forward scatter (FSC) properties. The cells were re-gated according to staining with a myeloid marker (designated as fluorescence channel (FL1)). Then, the expression of several surface molecules on myeloid leukemia cells and/or T cells was analyzed on FL2 or FL3.

Supporting Information Fig. S2. Immunophenotypic analysis of HL-60 and iHL- 60 cells was performed for the determination of FAB category. (A) Representative flow cytometry histograms and (B) quantification of the immunophenotypic markers used in AML diagnosis and classification are given. Overlay graphs are shown with specific mAb staining (filled histograms) and with isotype-matched control staining (open histograms). Data were obtained from five independent experiments and are shown as mean ± SD (Student's t-test, **P<0.01).

Supporting Information Fig. S3. Upregulation of B7-2 and HLA-DR expression on iHL-60 cells. The percentage of HLA-DR and/or B7-2 positive cells was determined at different time points upon incubation with PMA. Data were obtained from three independent experiments and are shown as mean ± SD.

Supporting Information Fig. S4. Secretion of common proinflammatory cytokines from myeloid leukemia cells. The presence of cytokines in the supernatants obtained from HL-60 or iHL-60 cells, and their counterparts co-cultured for 20 h with CD4+ T cells with or without anti-CD3 mAb was studied by ELISA array. CD13+ myeloid leukemia cells were back-sorted from the co-cultures and incubated alone for another 20 h. Samples obtained from three independent experiments were pooled and used in the arrays. The graphical output of optical densities (OD) and corrected absorbance values higher than negative standard are shown. Considering the negative and positive standard readings, the results with high OD values are typed in bold. (bs, supernatants collected from the cells after back-sorting from cocultures).

Supporting Information Fig. S5. Purified CD4+ T cells were cultured for 20 h with (red bars) or without (green bars) suboptimal anti-CD3 mAb stimulation and the expression of CD25, CD69, and CD154 activation markers were determined. Data were obtained from three independent experiments and are shown as mean ± SD.

Supporting Information Fig. S6. Secretion of the cytokines related to helper T cell differentiation was studied by ELISA array. Following 20 h co-culturing with HL-60 or iHL-60 cells in the presence of anti-CD3 mAb, T cells were back-sorted, incubated alone for another 20 h and supernatants were collected. Samples obtained from three independent experiments were pooled and used in the arrays. The graphical output of optical densities and corrected absorbance values higher than negative standard are shown.

Supporting Information Fig. S7. CD4+ T cell responses in the co-cultures established with myeloid leukemia cells. Purified CD4+ T cells were co-cultured with AML cell lines HL-60, iHL-60, U-937, THP-1, or KG-1 cells at 2:1 ratio with suboptimal anti-CD3 mAb stimulation. The expression of CD25, CD69, and CD154 activation markers were determined on T cells at 20 h of incubation. T cell proliferation was determined at 96 h by flow cytometric CFSE assay. Representative histograms are shown.

Supporting Information Fig. S8. Median fluorescence intensity (MFI) levels of B7-2 expressed on HL-60 or iHL-60 cells upon 20 h co-culturing with CD4+ T cells. Data were obtained from four independent experiments and are shown as mean ± SD (Student's t-test; **p < 0.01).

Supporting Information Fig. S9. Schematic demonstration of the study design and major findings. 1- Costimulatory support for CD4+ T cells was provided especially by B7-2+ subpopulation in HL-60 and to higher extend by iHL-60 myeloid leukemia cells. 2- Co-culturing with activated helper T cells influence the expression of B7 family molecules. The expression of B7-H2 was decreased whereas B7-DC and B7-H1 were upregulated. 3- Following the co-culturing, iHL-60 cells gained immunosuppressive character and hampered helper T-cell responses through PD-1 ligation.

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