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. 2024 Sep 23;8(9):e70005.
doi: 10.1002/hem3.70005. eCollection 2024 Sep.

Characterization of myeloid-derived suppressor cells in the peripheral blood and bone marrow of patients with chronic idiopathic neutropenia

Nikoleta Bizymi  1   2 , Athina Damianaki  1   2 , Nikoletta Aresti  1   2 , Anastasios Karasachinidis  1   2 , Zacharenia Vlata  3 , Matthieu Lavigne  4 , Emmanuel Dialynas  4 , Niki Gounalaki  4 , Irene Stratidaki  4 , Grigorios Tsaknakis  1   2 , Aristea Batsali  1   2 , Irene Mavroudi  1   2 , Maria Velegraki  1   2 , Ioannis Sperelakis  5 , Charalampos Pontikoglou  1   2 , Panayotis Verginis  6   7 , Helen A Papadaki  1   2
Affiliations

Characterization of myeloid-derived suppressor cells in the peripheral blood and bone marrow of patients with chronic idiopathic neutropenia

Nikoleta Bizymi et al. Hemasphere. .
No abstract available

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow cytometric analysis of MDSC subsets, quantitative analysis of PB and BM MDSC subsets in CIN patients and healthy subjects, T‐cell suppression assay for testing the MDSC suppressive capacity, and distinct pattern of gene expression between CIN patients and normal controls. (A) The graphs show the gating strategy for the identification of the MDSC populations. We first gated the live cells based on the Forward Scatter (FSC) and Side Scatter (SSC) properties (not shown). For the quantification of the polymorphonuclear CD11b+/CD33+/HLA‐DR–/low/CD15+ MDSCs (PMN‐MDSCs) (upper graphs), analysis was initially performed in the CD15+ cells gated on the live cells (Gate A), followed by analysis in the HLA‐DR–/low cells gated on the CD15+ cells (Gate B) and finally in the CD11b+/CD33+ positive cells gated on the CD15+HLA‐DR–/low cells (Gate C). For the quantification of the monocytic CD11b+/CD33+/HLA‐DR–/low/CD14+ MDSCs (M‐MDSCs) (lower graphs), we performed the same analysis following the initial gating in the CD14+ cells gated on the live cells. Using this back‐gating strategy, results were expressed as a proportion (%) of CD11b+/CD33+/HLA‐DR–/low/CD15+ and CD11b+/CD33+/HLA‐DR–/low/CD14+ MDSCs in the live PBMCs. The same gating strategy was followed for the quantification of PMN‐MDSCs and M‐MDSCs in the BMMC fraction. (B) The graphs show the gating strategy for the sorting of the PMN‐MDSC and M‐MDSC populations from the PBMC fraction. We initially gated the live cells based on the FSC and SSC properties (not shown). Gating was then performed in the CD33+/HLA‐DR–/low cell compartment gated on the live cells (left graph), followed by gating and sorting of the CD11b+/CD15+ cells (middle graph) and CD11b+/CD14+ cells (right graph) gated on the previous cell compartment. (C) Characteristic pictures that show CFSE staining of sorted T cells on day 3 of culture. The graph on the left depicts normal T‐cell proliferation without the presence of MDSCs, the middle graph shows the decreased T‐cell proliferation in the presence of MDSCs from a representative healthy control, and the graph on the right the decreased T‐cell proliferation in the presence of MDSCs from a representative CIN patient. (D) Characteristic pictures that show CFSE staining of CD5+ PBMCs from a healthy individual on Day 3 of culture. The graph on the left depicts the T‐cell proliferation in PBMC cultures containing MDSCs, while the graph on the right shows the T‐cell proliferation in PBMC cultures depleted of MDSCs (i.e., following depletion of CD33+ cells). The box plots in graphs (E) and (F) show the median (horizontal line) and the 25% and 75% percentiles of the distribution of the proportions of PMN‐MDSCs (graph E) and M‐MDSCs (graph F) detected by flow cytometry in the PBMC fraction of CIN patients (n = 102) and healthy controls (n = 77). The respective box plots in graphs (G) and (H) depict the proportions of PMN‐MDSCs and M‐MDSCs in the BMMC fraction of patients (n = 37) and healthy controls (n = 8). The whiskers indicate the minimum and maximum values of the respective distributions. Comparisons have been performed using the nonparametric Wilcoxon signed ranks test for paired samples, the nonparametric Mann–Whitney U test for nonpaired data, and the statistically significant p values are shown. (I) Percentage of “reads” associated with the gene expression sequence having the highest number for each sample. (J) Pivot cluster heatmap of our data that shows the differences in the gene expression between patients and controls. In the heatmap, each row in the y‐axis represents one gene and each column in the x‐axis represents one case. NB22, NB23, and NB24 represent M‐MDSCs from healthy controls, NB6 and NB8 represent PMN‐MDSCs from healthy controls, NB18, NB25, NB27, and NB30 represent M‐MDSCs from CIN patients, while NB10 and NB12 represent PMN‐MDSCs from CIN patients. (K) Networks of enriched terms from the upregulated (upper part) and downregulated (lower part) genes in CIN MDSCs colored by cluster, where nodes that share the same cluster are typically close to each other. The network was visualized using Cytoscape (cytoscape.org) through Metascape (metascape.org). ANC, absolute neutrophil count; BM, bone marrow; BMMC, BM mononuclear cells; CFSE, carboxy‐fluorescein succinimidyl ester; CIN, chronic idiopathic neutropenia; Con, control; MDSC, myeloid derived suppressor cells; M‐MDSC, monocytic MDSC; Pat, patient; PB, peripheral blood; PBMC, PB mononuclear cells; PMN‐MDSC, polymorphonuclear MDSC.
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