![Fig. 2. Soluble HLA-I–specific alleles induce NK cell apoptosis upon interaction with activating forms of either killer Ig-like receptor (KIR) or C-type lectin inhibitory receptor (CLIR). / (A) One-dimensional PAGE under nonreducing/nondenaturing conditions and silver staining of sHLA-I molecules used to induce apoptosis of NK cells. These molecules were isolated from culture supernatant of either untransfected (lane 1: sHLA non-A, -B, -C, and -G [that is, putative sHLA-E and -F]) or HLA-I allele–transfected (lane 2, Cw3; lane 3, Cw4; lane 4, A2; lane 5, sHLA-G) 721.221 cells 62829 by precipitation with ammonium sulfate, low-medium pressure chromatography, strong anionic and strong cationic ion exchange, and gel filtration and purified by affinity chromatography on anti–HLA class I mAb W6/32 coupled to cyanogen bromide. Molecular weights are indicated on the left. (B-E) NK cell clones selected for the expression of activating isoforms of CD158a ([B] clone 262; [D] clone 255) or CD158b ([C] clone A1.25) or CD94/NKG2 ([E] clone 45) were incubated for 48 hours with their specific sHLA-I ligand (sHLA-Cw4 for CD158a; sHLA-Cw3 for CD158b; sHLA non-A, -B, -C, and -G [that is, putative HLA-E and -F] for CD94/NKG2 complex, respectively), and percentage of apoptotic cells was determined by labeling cells with FITC-conjugated annexin V. In some experiments, covering of activating receptor (CD158a, CD158b, CD94/NKG2) was obtained by preincubating NK cells with specific anti-CD158a or CD158b or CD94 mAb. NK cell apoptosis induced by activating receptor was also achieved upon cross-linking of these receptors with specific mAb (1 μg/mL) followed by GAM-coated beads (4 per cell) ([A,C] CD158a-XL; [B] CD158b-XL; [D] CD94-XL). nil and CD54-XL:NK cells incubated with medium alone or upon cross-linking of CD54. In panel C, either covering of CD8 or its optimal cross-linking was obtained as for activating receptor to determine the role of CD8 in NK cell clones bearing activating receptor. Results are expressed as the percentage of apoptotic cells (FITC–annexin V+ but PI−) and are representative of 3 independent experiments using 3 different NK cell clones for each activating receptor.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/100/12/10.1182_blood-2002-04-1284/5/h82323474002.jpeg?Expires=1735543751&Signature=TZzW-uvz0jCDsc6KWilz7qHNBfjvK5ZbBnYQfc5nmc-QfAFlDhkcRh~c4y2qXOeDmghukecabpTfUhMbjE-NauOd5ov8blpfog3G82bm59XVkyDIpyAZmTbgtcIIFjOgwTi~z4KHzJQTWCA6ed17PO~hY6sp81VfOjP92hPzVlXlEyb3abgNZElOqs01QGtKK2bOdvE4oRRcOna1aRFoMPL9lrflhUtEMnZPXD-LUgoGD6BIFd4kAKwK~PrXhWgtHoADTSvPpivPpzCjWmHVd7IOU7uJeGLEta3hkJe1kahfwLMZLZ7JROrN0bVzAw-t~xM5rRiAHBihz2eG~Rbyug__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Soluble HLA-I–specific alleles induce NK cell apoptosis upon interaction with activating forms of either killer Ig-like receptor (KIR) or C-type lectin inhibitory receptor (CLIR).
(A) One-dimensional PAGE under nonreducing/nondenaturing conditions and silver staining of sHLA-I molecules used to induce apoptosis of NK cells. These molecules were isolated from culture supernatant of either untransfected (lane 1: sHLA non-A, -B, -C, and -G [that is, putative sHLA-E and -F]) or HLA-I allele–transfected (lane 2, Cw3; lane 3, Cw4; lane 4, A2; lane 5, sHLA-G) 721.221 cells 6,28 29 by precipitation with ammonium sulfate, low-medium pressure chromatography, strong anionic and strong cationic ion exchange, and gel filtration and purified by affinity chromatography on anti–HLA class I mAb W6/32 coupled to cyanogen bromide. Molecular weights are indicated on the left. (B-E) NK cell clones selected for the expression of activating isoforms of CD158a ([B] clone 262; [D] clone 255) or CD158b ([C] clone A1.25) or CD94/NKG2 ([E] clone 45) were incubated for 48 hours with their specific sHLA-I ligand (sHLA-Cw4 for CD158a; sHLA-Cw3 for CD158b; sHLA non-A, -B, -C, and -G [that is, putative HLA-E and -F] for CD94/NKG2 complex, respectively), and percentage of apoptotic cells was determined by labeling cells with FITC-conjugated annexin V. In some experiments, covering of activating receptor (CD158a, CD158b, CD94/NKG2) was obtained by preincubating NK cells with specific anti-CD158a or CD158b or CD94 mAb. NK cell apoptosis induced by activating receptor was also achieved upon cross-linking of these receptors with specific mAb (1 μg/mL) followed by GAM-coated beads (4 per cell) ([A,C] CD158a-XL; [B] CD158b-XL; [D] CD94-XL). nil and CD54-XL:NK cells incubated with medium alone or upon cross-linking of CD54. In panel C, either covering of CD8 or its optimal cross-linking was obtained as for activating receptor to determine the role of CD8 in NK cell clones bearing activating receptor. Results are expressed as the percentage of apoptotic cells (FITC–annexin V+ but PI−) and are representative of 3 independent experiments using 3 different NK cell clones for each activating receptor.
