Down-regulation of NKG2D on effector cells by TGF-β produced by LN MSCs from HL. NKG2D expression was evaluated by indirect immunofluorescence on coculture of CD8⁺αβ (Ai,Ci,Di) and γδ T lymphocytes (Aii,Cii,Dii) with LN MSCs, in the presence of IL-15 (10 ng/mL) and in the absence or presence of an anti–TGF-β mAb (5 μg/mL) added to the cocultures (A). NKG2D was evidenced by a specific mAb followed by PE-GAM. Samples were run on a CyAnADP flow cytometer, and results are expressed as log MFI (a.u.) versus number of cells (A) or as MFI (a.u., C-D). Results are shown as overlay histograms of negative control (gray thin line) and NKG2D mAb reactivity (black thick line, as indicated by the arrows). (B) Quantitative RT-PCR for NKG2D was performed with the specific primers and probes on RNA extracted from CD8⁺ αβ (dark gray columns) and γδ T cells (light gray columns), after 24 hours or culture without or with IL-15 (10 ng/mL) and/or anti–TGF-β mAb (5 μg/mL), in the presence or absence of LN MSCs, as indicated. Data are mean ± SD from 3 experiments with CD8⁺ αβ T cells and 3 with γδ T cells. *P < .001versus experiments done in the absence of LN MSCs and IL-15. **P < .001 versus experiments in the presence of IL-15. ***P < .001 versus experiments in the presence of IL-15 and LN MSCs. (C) Kinetics of NKG2D intensity of expression (MFI, a.u.) in CD8⁺ αβ (Ci) and γδT lymphocytes (Cii) cocultured with LN MSCs in the presence of IL-15 or in medium alone (medium). (D) NKG2D intensity of expression (MFI, a.u.) in CD8⁺ αβ (Di) and γδ T lymphocytes (Dii) cocultured with LN MSCs at different ratios (as indicated) in the presence of IL-15 or in medium alone (basal). Data are mean ± SD from 6 experiments performed with 6 different LN MSCs. (E) TGF-β in the cytoplasm (Ei, 1 representative experiment) and in the supernatant of cultured LN MSCs (Eii, 4 different LN MSCs). (Ei) For cytoplasmic staining, cells were fixed and permeabilized before addition of anti–TGF-β mAb, followed by FITC-GAM. Samples were run on CyAnADP flow cytometer, and results are expressed as MFI versus number of cells. (Eii) TGF-β1 was measured in SN recovered from LN MSC cultures after 24 hours, on treatment for 1 hour of each SN with 1N HCl followed by 1N NaOH, with a TGF-β1 specific ELISA kit. Results were normalized to a standard curve and expressed as picograms per milliliter.