TGF-β is expressed in the cHL microenvironment. (A) Quantitative RT-PCR for TGF-β, IL-4, IL-10, and TG was performed with the specific primers and probes on RNA extracted from the whole LN sections (8 μm, Ai) or on stromal (S) or parenchymal (P) microdissected areas of serial sections (n = 20 cHL: 11 NS and 9 MC) that underwent LCM on the basis of the localization of TG (Aii). After subtracting the C_t value for RPLP0 from the C_t values of the target genes, results were expressed as ΔC_t. Data are the mean ± SD from 14 NS samples (black columns) and 9 MC samples (gray columns in Ai). *P < .001 versus healthy LN (supplemental Figure 1A). (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) isolated from LN, after 24 hours or culture without or with IL-15 (10 ng/mL), in the presence or absence of LN MSCs, anti–TGF-β mAb (5 μg/mL), as indicated. Data are mean ± SD from 3 experiments with CD8⁺ αβ 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) Lymphocytes isolated from 6 HL LN specimens were evaluated for NKG2D expression by indirect immunofluorescence, either freshly isolated (Ci) or cultured for 6 days with rIL-15 (10 ng/mL, Bii) or on coculture with LN MSCs, in the presence of IL-15 (Ciii) and in the presence of IL-15 and of an anti–TFG-β mAb (5 μg/mL) added to the cocultures (Civ). NKG2D was evidenced by a specific mAb followed by PE-GAM, whereas CD8⁺ cells were identified by an APC-conjugated anti–CD8 mAb (C-Di), whereas γδT cells were detected with a combination of anti-Vδ1 and anti-Vδ2 AlexaFluor-647–conjugated mAbs (Dii). Samples were run on a CyAnADP flow cytometer, and results are expressed as log mean red fluorescence intensity (x-axis MFI, a.u.) versus log mean far red fluorescence intensity (for both APC and AlexaFluor-647, y-axis MFI, a.u.), or as MFI (a.u.). (D) Data are mean ± SD from 6 different experiments with CD8⁺ cells (Di) or γδ T cells (Dii) and LN MSCs from 6 different HLs. *P < .001 versus experiments done in the absence of IL-15, LN MSCs, or anti–TGF-β. **P < .001 versus experiments in the presence of IL-15. ***P < .001 versus experiments in the presence of IL-15 and LN MSCs. All results were determined on CD3⁺ gated T cells after simultaneous staining with anti-CD3 mAb (JT3A, IgG2a) and FITC-GAM.