Determination of soluble HLA-G and HLA-A, -B, and -C molecules in pregnancy

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

“…The purity of sHLA-I molecule preparations was analyzed by 1-dimensional polyacrylamide gel electrophoresis (PAGE) under nonreducing/nondenaturing (Figure 2A) or reducing/denaturing conditions (not shown) followed by silver staining. 28,29 721.221 untransfected cells appeared to be faintly stained with W6/32 mAb followed by PE-conjugated GAM antiserum; thus, in accordance with previous reports, [30][31][32][33][34][35][36] it is conceivable that the sHLA-I derived from these cells was represented, at least in part, by HLA-E and HLA-F molecules. 37 …”

“…Each clone was analyzed in a killing assay using the Fc␥R ϩ P815 murine mastocytoma cell line in the presence of mAb recognizing either KIRs or C-type lectin inhibitory receptors at the effector-target ratio (E/T) of 20:1 or 2:1 to identify clones with inhibiting or activating forms of these HLA-I receptors, respectively. [1][2][3][10][11][12][13]22 Soluble HLA-I antigen preparations Soluble HLA-A2, -Cw3, and -Cw4 were prepared from culture supernatant of HLA-I-A Ϫ , -B Ϫ , -C Ϫ , and -G Ϫ 721.221 cells transfected with the corresponding HLA-I alleles 6,28,29 while soluble HLA non-A, -B, -C, and -G was prepared from culture supernatant of untransfected 721.221 cells by precipitation with ammonium sulfate, low-medium pressure chromatography, strong anionic and strong cationic ion exchange, and gel filtration as described 28,29 and purified by affinity chromatography on anti-HLA class I mAb W6/32 (10 mg/mL) coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia). The purity of sHLA-I molecule preparations was analyzed by 1-dimensional polyacrylamide gel electrophoresis (PAGE) under nonreducing/nondenaturing (Figure 2A) or reducing/denaturing conditions (not shown) followed by silver staining.…”

“…6,28,29 Standard curve was obtained using progressive dilutions of recombinant FasL from Alexis (Leufelfingen, Switzerland). Results were expressed as mean Ϯ SD of triplicate wells.…”

“…In some experiments cells were incubated with anti-CD158a or anti-CD158b or anti-CD94 or anti-CD54 mAb for 30 minutes at 4°C, washed, and either used in apoptotic assays (to analyze the effect of the covering of a given activating receptor) or further incubated for different times with 4-per-cell GAM-coated magnetic beads 6,28,29 (to obtain the optimal cross-linking of a given activating receptor). Early apoptotic events were evaluated by annexin V labeling method, and viable apoptotic cells were differentiated from necrotic cells by flow cytometry after PI staining of nonpermeabilized cells.…”

Soluble HLA class I molecules induce natural killer cell apoptosis through the engagement of CD8: evidence for a negative regulation exerted by members of the inhibitory receptor superfamily

“…This phenomenon may play a role in the regulation of antigen-specific immune responses by inducing programmed cell death of effector T lymphocytes. [13][14][15][16][18][19][20][21][22][23][24] However, it is not clear whether sHLA-I delivers an apoptotic signal by interacting with CD8 only or through the simultaneous engagement of CD8 and TCR.…”