Generation of a Novel Regulatory NK Cell Subset from Peripheral Blood CD34+ Progenitors Promoted by Membrane-Bound IL-15
Figure 8
NKPSG cells secrete immuno-regulatory factors.
(A) Detection of IL-10 production by three-weeks-old NKPSG cells using : RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. ELISA assay. Conventional PB-NK cells and BM-MSC were used as negative controls while LPS- activated PB-macrophages were used as positive control. Western blot. recombinant IL-10 was used as positive control. Flow cytometry. Detection of membrane bound IL-10 is represented with continuous black line. Isotype control (shadowed peak). (B) Detection of IL-21 expression on three-weeks-old NKPSG cells using: RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. Western blot. IL-21 expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for IL-21. HL-60 cell line and recombinant IL-21 were used as positive controls. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. (C) Detection of HLA-G expression on three-weeks-old NKPSG cells using: Western blot. HLA-G expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for HLA-G. M8-HLA-G1 cell line was used as positive control. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. Flow cytometry. Detection of surface and intracellular expression of HLA-G on NKPSG cells is represented with continuous black line. Isotype control (shadowed peaks). The data are representative of three separate experiments. Confocal microscopy. As negative controls, NKPSG cells were incubated with Isotype-matched IgG1-FITC.