| Autor: |
Wu, Chunjing, Spector, Sydney A., Theodoropoulos, George, Nguyen, Dan J. M., Kim, Emily Y., Garcia, Ashley, Savaraj, Niramol, Lim, Diane C., Paul, Ankita, Feun, Lynn G., Bickerdike, Michael, Wangpaichitr, Medhi |
| Rok vydání: |
2023 |
| DOI: |
10.6084/m9.figshare.23167883 |
| Popis: |
Additional file 1: Supplementary Figure S1A. Figure S1A. Gating strategy used to identify NK and CD8+T cells;viable cells were gated based on FSC-A versus Fixable Viability Dye;cells were gated on CD45+ and further divided into CD3- and CD3+ fractions;CD56+ and NKG2D+ cells were selected from the CD3- fraction;CD8+ and NKG2D+ cells were selected from CD3+ fraction. Gating strategy used to identify human-regulator T cells.Viable cells were gated based on FSC-A versus Fixable Viability Dye.Cells were gated on CD45+CD127low and CD4+CD25+ were gated.CD4+ and intracellular FoxP3+ were selected from CD4+CD25+ population. Gating strategy used to identify human-MDSC cells.Viable cells were gated based on FSC-A versus Fixable Viability Dye.Cells were gated on CD45+ and CD14-and HLA-DRlow were gated.CD11b+ and CD33+ were selected from the CD14-HLA-DRlow population. Note: Fluorescence minus onewas used to identify gating boundaries. Supplementary Figure S1B. Gating strategy used to identify mouse-regulator T cells.Viable cells were gated based on CD45+ versus Fixable Viability Dye.CD4+CD25+ were gated from CD45+live.CD4+ and intracellular FoxP3+ cells were selected from CD4+CD25+ population. Gating strategy used to identify mouse-CD8+ T cells.Viable cells were gated based on CD45+ versus Fixable Viability Dye.CD3+CD8+ were gated from CD45+live.NKG2D+and CD8+ cells were selected from CD3+CD8+population. Gating strategy used to identify mouse-NK-cells.Viable cells were gated based on CD45+ versus Fixable Viability Dye.CD3-CD11b+ were gated from CD45+live.NKG2D+ and CD49b+ cells were selected from CD3-CD11b+population. Gating strategy used to identify mouse-MDSC cells.Viable cells were gated based on CD45+ versus Fixable Viability Dye.F4/80low and Ly6C+were gated from CD45+live.CD11b+ and Gr1+ were selected from the F4/80low and Ly6C+population. Note: Fluorescence minus onewas used to identify gating boundaries. Figure S2. IDO-mediated KYN production from CR cells suppressed immunomodulatory profile.Immunoblot of IDO1, IDO2, and TDO2. Resistant cells were treated with either IDO1 inhibitor or shRNA targeting IDO1.Detection of amino acid KYN and TRP in culture supernatants of F vs FC. The concentration of amino acids is measured using Amino Acid Analyzer Biochrome30+.Immune profile assayed by flow cytometry. Using the same experimental co-condition as Fig. 1C, IDO1 inhibition significantly enhanced percent of NKG2D on NK cellsand percent of NKG2D on CD8+cells, but significantly suppressed Tregand MDSCfrequencies in CD45+ lymphocytes.The indicated cytokines are quantified in culture supernatants by LEGENDplex™ bead-based immunoassay. The panel below indicated that anti-NKG2D blocking antibodies blunt the effect of IDO1 inhibition. Note: To detect the MDSC population, cells were activated by PHA+IL2 instead of antiCD2/28+IL2. In all the experiments, data presented as mean ± SEM of 3 independent experiments and were analyzed using one-way ANOVA followed by Tukey with *P |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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