Figure 6.
Granulocytic MDSCs are expanded in the blood of patients and potently inhibit T-cell responses. (A) Flow cytometric analysis of fresh patient PBMC by FSC vs SSC showed the presence of large granular cells (G-MDSCs), shown in blue; percentage of G-MDSCs to total PBMCs isolated is shown. A representative healthy donor is shown for comparison. (B) The percentage of G-MDSC is plotted with the mean compared with the mean from 10 healthy donors. A 2-tailed Mann-Whitney test was used to compare groups. (C) Nuclear morphology of G-MDSCs (low density) and neutrophils (high density) revealed characteristic band cell/neutrophil morphology by hematoxylin and eosin staining at ×100 magnification. (D) Flow cytometric analysis of the G-MDSC revealed expression of CD11b, CD15, CD16, CD33 but no expression of HLA II. Isotype controls (gray-filled histogram) are shown. (E) Luminex assays for GM-CSF, IL-1β, IL-6, and TNF-α were performed upon patient plasma and compared with healthy donors. All patients had higher concentrations of these cytokines in their plasma than the healthy donors. Dashed horizontal line indicates the limit of accurate detection. (F) Luminex assay was used to determine the concentration of arginase-1 in the plasma of CAEBV and EBV-HLH patients, and healthy donors. Dashed horizontal line indicates the limit of accurate detection. (G) Inhibition of T-cell proliferation by MDSCs was measured following coculture of T cells with decreasing numbers of G-MDSCs (T:G-MDSC range 1:1 to 1:0.125). The percentage T-cell proliferation in the presence of G-MDSCs is plotted relative to T cells alone. Data shown are an average of 3 patients. (H) The inhibition of T-cell proliferation by G-MDSCs from patients and healthy donors at a T-cell:G-MDSC ratio of 1:1 is plotted. Proliferation is shown relative to control (T cells stimulated in the absence of MDSCs). In panels G-H, a Kruskal-Wallis test was used to compare groups. In panels B, E, and F, each patient is represented by a unique symbol as follows: patient 1 = +, patient 2 = ×, patient 3 = ○, patient 4 = △, and patient 5 = □.

Granulocytic MDSCs are expanded in the blood of patients and potently inhibit T-cell responses. (A) Flow cytometric analysis of fresh patient PBMC by FSC vs SSC showed the presence of large granular cells (G-MDSCs), shown in blue; percentage of G-MDSCs to total PBMCs isolated is shown. A representative healthy donor is shown for comparison. (B) The percentage of G-MDSC is plotted with the mean compared with the mean from 10 healthy donors. A 2-tailed Mann-Whitney test was used to compare groups. (C) Nuclear morphology of G-MDSCs (low density) and neutrophils (high density) revealed characteristic band cell/neutrophil morphology by hematoxylin and eosin staining at ×100 magnification. (D) Flow cytometric analysis of the G-MDSC revealed expression of CD11b, CD15, CD16, CD33 but no expression of HLA II. Isotype controls (gray-filled histogram) are shown. (E) Luminex assays for GM-CSF, IL-1β, IL-6, and TNF-α were performed upon patient plasma and compared with healthy donors. All patients had higher concentrations of these cytokines in their plasma than the healthy donors. Dashed horizontal line indicates the limit of accurate detection. (F) Luminex assay was used to determine the concentration of arginase-1 in the plasma of CAEBV and EBV-HLH patients, and healthy donors. Dashed horizontal line indicates the limit of accurate detection. (G) Inhibition of T-cell proliferation by MDSCs was measured following coculture of T cells with decreasing numbers of G-MDSCs (T:G-MDSC range 1:1 to 1:0.125). The percentage T-cell proliferation in the presence of G-MDSCs is plotted relative to T cells alone. Data shown are an average of 3 patients. (H) The inhibition of T-cell proliferation by G-MDSCs from patients and healthy donors at a T-cell:G-MDSC ratio of 1:1 is plotted. Proliferation is shown relative to control (T cells stimulated in the absence of MDSCs). In panels G-H, a Kruskal-Wallis test was used to compare groups. In panels B, E, and F, each patient is represented by a unique symbol as follows: patient 1 = +, patient 2 = ×, patient 3 = ○, patient 4 = △, and patient 5 = □.

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