IRSs are involved in the negative regulation of sHLA-mediated apoptosis.

The NK cell clone Cl.S2 (CD8^bright CD94⁺KIR2D⁻, A-C) or the NK cell clone Cl.209 (CD8^bright CD158b⁺ [GL183⁺], D-F) was incubated with the following reagents: medium (nil), sHLA-I (4 μg/mL) alone or after masking of either CD8 (sHLA-I + OKT8, A, or sHLA-I + astra 102, D) or CD94 (sHLA-I + HP-3B1, A) or CD158b (sHLA-I + GL183, D) with specific mAbs, or with anti-CD94 mAb (HP-3B1, A), or anti-CD158b mAb (GL183, D). Some samples were treated with anti-CD8 mAb (OKT8, A, or astra 102, D) alone or in combination with either anti-CD94/NKG2 or anti-CD158b mAb followed by 4-per-cell GAM-coated magnetic beads to induce cross-linking of the indicated molecules (OKT8-XL, OKT8-XL + HP-3B1-XL, A; astra 102-XL, astra 102-XL + GL183-XL, D). Results are expressed as the percentage of annexin V⁺ PI⁻ cells. (B,E) Aliquots of SN derived from cells incubated for 48 hours with medium (nil), or sHLA-I (B,E), or HP-3B1 mAb (B) or GL183 mAb (E) were analyzed for the presence of FasL by enzyme-linked immunosorbent assay. In some samples, cells were exposed to sHLA-I after incubation with anti-CD94/NKG2 (sHLA-I + HP-3B1, B) or with anti-CD158b (sHLA-I + GL183, E) mAbs. Results are expressed as nanogram per milliliter of FasL. (C,F) NK cell clone Cl.S2 (C) or Cl.209 (F) was labeled with Fura-2 and treated with anti-CD8 mAb (OKT8, C, astra102, F), or with anti-CD8 plus either anti-CD94/NKG2 (OKT8-XL + HP-3B1-XL, C) or anti-CD158b (astra 102-XL + GL183-XL, F) mAb. The cross-linking (XL) of the corresponding surface molecules was achieved by adding 20 μg/mL GAM as indicated. Results are expressed as [Ca⁺⁺]_i nM and are representative of 4 independent experiments by using 4 different NK cell clones with a comparable surface phenotype.