Soluble HLA-I and CD8 engagement induce intracellular calcium increases in NK cells.

(A-C) The NK cell clone S2 (CD8^bright) was labeled with Fura-2 and fluorescence monitored in real time on a spectrofluorometer LS50B at 37°C. (A) Baseline, [Ca⁺⁺]_ioscillations in the absence of any addition. (B,C) Calcium mobilization induced by sHLA-I (4 μg/mL, B) before or after pretreatment with anti-CD8 mAb (OKT8, C) to avoid the interaction of sHLA-I with CD8 antigen (CD8 masking). (D-I) The NK cell clones S4 (CD8^dull, D-F) and S2 (CD8^bright, G-I) were treated with the anti-CD8 mAb astra102 (E,H) or the anti-CD16 mAb NK54 (F,I). Cross-linking of CD8 or CD16 was achieved by adding 20 μg/mL GAM. (D,G) Cells treated with anti-CD54 mAb (14D12D2) matched for isotype followed by GAM, as negative control. (M,N) CD8 cross-linking (M) or addition of 4 μg/mL sHLA-I (N) were performed in the presence of 2 mM calcium chelator EGTA. An excess of calcium (4 mM of CaCl₂) was added (on 250 seconds) (right arrows) after the addition of GAM or sHLA-I (left arrows). (L) Calcium oscillations in the presence of EGTA without any stimulus (baseline). Arrows in each histogram indicate the addition of either GAM (D-I,M) or sHLA-I (B,C,N); the addition of CaCl₂ is indicated in panels M and N by the second arrow on the right. Results are expressed as [Ca⁺⁺]_i nM and are representative of 3 independent experiments performed by using NK cell clones from 3 different healthy donors.