Journal of Immunology Research / 2020 / Article / Fig 5

Research Article

Circulating Neutrophils of Nonalcoholic Steatohepatitis Patients Show an Activated Phenotype and Suppress T Lymphocytes Activity

Figure 5

Proliferation and activation of CD4+ and CD8+ T cells cocultured with PMNs. (a–d) In all experiments, PBMCs obtained from healthy donors, NAFL, and NASH patients were stimulated with aCD3/aCD28 and cultured in the presence or in absence of autologous PMNs for 96 h. The capacity of PMN to suppress T cells was evaluated by measuring proliferating T cells (CFSE dilution). (a) Top panels: frequency calculated by flow cytometry analysis as the percentage of CD4+ (left) and CD8+ (right) T cell proliferation is measured in healthy donors (), NAFL (), and NASH () by CFSE assay. Each dot in the graph represents a single subject. (b) The expression of activation marker (CD25) on T cells was analyzed by flow cytometry, after 96 h of cultured with PMNs. Dot plots show the percentage of inhibition of CD25 MFI [] on CD4+ (left) and CD8+ (right) T cells in healthy donors (), NAFL (), and NASH (). Each dot in the dot-plot (a and b) represents one subject/experiment. Each subject has been studied in an independent experiment. Plots are representative for two independent experiment with one sample per experiment. (c–d) After 96 h of coculture with PMNs, T cells were stained with H2DCF-DA probe for identifying intracellular production of ROS, and Annexin-V/Live Dead for their vitality. (c) Histograms show the percentage of H2DCFDA (ROS) MFI on gated CD4+ (left) and CD8+ (right) T cells in healthy donors (), NAFL (), and NASH (). (d) Frequency of cell death CD4+ (left) and CD8+ (right) T cells in healthy donors (), NAFL (), and NASH (). Survival is defined as the percentage of Annexin V, Live Dead double negative cells. In all histograms reported (c and d), for each subgroup the distribution of each subject/experiment is showed and results are expressed as . Each dot represents one subject/experiment. Each subject has been studied in an independent experiment. In all experiments (a–d), statistics were performed by Mann-Whitney unpaired test, 2-tailed. value, , , , , ns: nonsignificant.
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