Fig 2: Lymphocytes make up the main immune population within digested GCT.Viable single tumor cell suspension and PBMCs from healthy donors were analyzed using polychromatic flow cytometry and progressive gating strategy. (A) Representative staining with CD3, CD4, CD8, CD25, CD45, and FOXP3 used to quantify helper (CD4+), cytotoxic (CD8+), and regulatory (Tregs) (CD4+CD25+FOXP3+) T cells in a GCT sample. (B–D) Representative staining with CD11b, HLA-DR, CD11c, Lineage, CD14, CD15, and CD33 used to identify the myeloid populations in a GCT sample. Tumor-associated macrophages (TAMs)/monocytes were separated from DC based on CD14 expression (C). Myeloid-derived suppressor cells (MDSC) were separated as eMDSC based on Lineage, HLA-DR, CD11b, and CD33 markers (B), whereas PMN-MDSC were characterized as CD15+CD14–CD11b+ (D). Proportions of tumor-infiltrating immune cells in GCT were quantified as percentage of total cell suspension. (E) Percentages of CD4+ T cells, CD8+ T cells, and Tregs compared with total tumor cell suspension. (F) Comparison of PD1-expressing T cells in the GCT vs. PBMCs. (G) CD8+ T cells/Tregs ratio in GCT vs. PBMCs. (H) Percentage of TAMs/monocytes, DC, PMN-MDSC, and eMDSC of total tumor cell suspension. Mean ± SEM is shown. Each dot represents a patient (n = 7) or healthy control PBMCs (n = 3).

Fig 3: Analysis of the effect of chrysin treatment on human hematopoietic cell engraftment and reconstitution in NOG mice. (A) Transplantation strategy with uncultured or ex vivo expanded human CD34+CD38−CD45RA−CD90+ cells. Uncultured cells were transplanted into NOG mice on day 0 and cultured cells derived from 300 input cells were transplanted in to NOG mice on day 4. (B) Levels of human cell engraftment in peripheral blood (PB) of NOG mice at 4, 8, or 12 weeks post-transplantation. (n = 5 mice per group) (C) Levels of the engrafting human cells (hCD45+) in the injection side bone marrow (IF) and the opposite side bone marrow (BM) at 16 weeks post-transplantation. Number of mice at different engraftment levels (< 20%; 20%~30%; > 30%) in each group were shown. (D) Levels of the reconstituting myeloid (hCD45+ CD33+) and lymphoid (hCD45+ CD19+) in the injection side and the opposite side bone marrow at 16 weeks post-transplantation. (E) Representative flow cytometric profiles of the engrafting human CD45+ cell and myeloid, lymphoid, T lymphocyte (hCD45+ CD3+), natural killer cell (NK, hCD45+ CD56+), megakaryocyte (Mk, hCD45− CD41a+), and erythroid (E, hCD45− CD235a+) reconstitution in BM 16 weeks post-transplantation. All data represent the means ± SD. Compared with uncultured group unless specified, *p < 0.05, ***p < 0.001, ****p < 0.0001 and ns, no significance by two-tailed unpaired t-test.●, ■, ▲ and ♦ represent a single data point in Uncultured (Uncult.), Con, SR1 and C2968 groups respectively in (B, D). ●, ■, ▲ and ♦ represent a single data point of IF in Uncult., Con, SR1 and C2968 groups respectively while ○, □, Δ and ◊ represent those of BM in (C).

Fig 4: (a) Flow cytometry was used for gating HLA‐DR‐CD11b+CD15+ CD33+ LDNs. Data were shown as proportions of LDNs in whole blood cells. (b) Variation of LDN ratios and counts during different GBS courses. (c) Association between low‐density neutrophils (LDNs) and disease severity. Pearson's correlation was used to analyze the relevance of LDN counts (upper) and LDN ratios (lower) to the Medical Research Council sum score (*** p < .001)

Fig 5: Human peripheral blood MDSC phenotype in IPF and gating strategy for flow cytometry. Freshly isolated whole blood leukocytes or PBMCs were stained with and analyzed by flow cytometry. (A) A representative set of flow cytometry gating strategy. The cells were first gated for HLA-DR−/lo, and then total MDSCs were defined from the HLA-DR−/lo population as the CD11b+CD33+ fraction. The MDSC population was subsequently classified into two distinct subpopulations, namely, G-MDSCs (CD14−CD15+) and M-MDSCs (CD14+CD15−), by further analysis for CD14 and CD15 expression. (B) The MDSCs (HLA-DR−/loCD11b+CD33+) were further gated for CD33hi and CD33lo followed by analysis for CD14 or CD15 expression. MDSC, myeloid-derived suppressor cell; IPF, idiopathic pulmonary fibrosis; PBMCs, peripheral blood mononuclear cells.