sHLA-I induces transcription of Fas-L mRNA, Fas-L secretion, and apoptosis by Fas-FasL interaction.

(A,B) Indirect immunofluorescence analysis of the NK cells clone Cl.77.12 (CD8^bright) for surface (A) or cytoplasmic (B) expression of FasL. Left histograms in each panel represent cells stained with an unrelated isotype matched mAb followed by GAM-PE. Data are representative of results obtained analyzing 10 different NK cell clones. (C,D) FasL concentration (nanogram per milliliter) in culture SN (C) or NK cell apoptosis (annexin-V⁺ PI⁻, D) were evaluated at different time points after incubation of NK cells with medium alone (nil) or with 4 μg/mL sHLA-I or the anti-CD8 mAb (OKT8, 1 μg/mL) followed by 4-per-cell GAM-coated magnetic beads (OKT8-XL) to achieve CD8 cross-linking, or sHLA-I (4 μg/mL) after covering CD8 with anti-CD8 mAb (sHLA-I plus OKT8) or with anti-CD54 mAb as control mAb. (E) Normalized aliquots of total RNA (10 μg), isolated from the NK-1 population, were incubated for 3 hours with medium alone (lane 1) or anti-CD8 mAb (OKT8, 1 μg/mL) followed by 4-per-cell GAM-coated magnetic beads (lane 2) or with 4 μg/mL sHLA-I (lane 3), or sHLA-I (4 μg/mL) after covering of CD8 with anti-CD8 mAb (lane 4) and amplified by PCR with the specific primers for β-actin or FasL, as indicated, and PCR products were size-fractionated by agarose electrophoresis. Bands corresponding to FasL (F) and β-actin (G) were subjected to densitometric analysis, and results are expressed in pixel number (au). (H) NK cells (clone 35.6, CD8^bright) were either untreated or pretreated with the apoptosis-blocking anti-Fas mAb (ZB4) and incubated with medium alone (nil), or with the apoptosis-inducing anti-Fas mAb (CH-11, Fas-XL) or OKT8 followed by 4-per-cell GAM-coated magnetic beads (OKT8-XL) or 4 μg/mL sHLA-I. Similar results were obtained with 4 additional CD8^bright NK cell clones.