KIR2DS1 induces uptake of CCR7 and acquisition of migratory properties in NK cells interacting with C2⁺ cell transfectants. (A) Freshly isolated CD56^dull NK cells (from a representative C1/C1 KIR2DS1⁺ donor) contained a subset of KIR2DS1⁺/KIR2DL1⁻ cells that did not express CCR7 (upper panels). The level of expression (median values) of CCR7 and HLA-E molecules on 221 cells transfected with either HLA-Cw3 (C1 epitope) or HLA-Cw4 (C2 epitope) is also shown (lower panels). (B) The same NK cells as in (A) were incubated for 15 minutes at 37°C with either 221-Cw3 or 221-Cw4 transfectants, in the absence or presence of specific anti–HLA-I mAbs (6A4). Then cells were harvested and stained with anti-CD16 (to distinguish NK cells from the 221 cells), different anti-KIRs (to select and gate on the KIR2DS1⁺/KIR2DL1⁻ cell subset), and anti-CCR7 mAbs for cytofluorimetric analysis. Percentages of CCR7⁺ cells are indicated. (C) Freshly isolated NK cells from 4 C1/C1 KIR2DS1⁺ donors were incubated alone (NK) or with 221-Cw3 or 221-Cw4 cells, in the absence or presence of anti–HLA-I mAbs (6A4). After 15 minutes at 37°C, NK cells were analyzed for their migratory properties in response to CCL19/CCL21 chemokines. The average of 4 independent experiments is shown. The histogram refers to the migration index of NK cells after incubation. (D) A representative KIR2DS1⁺ NKG2A⁺ NK-cell clone (from a C1/C1 donor) was incubated at 37°C with untransfected 221 cells or 221-Cw3 or 221-Cw4 transfectants in the absence or presence of anti–HLA-I (6A4) or anti-NKG2A mAbs. After 15 minutes, NK cells were harvested and stained with anti-CD56 and anti-CCR7 mAbs for cytofluorimetric analysis. The percentages of CCR7⁺ cells are indicated. (E) The average of 6 independent experiments of KIR2DS1⁺ or KIR2DS1⁻ NK-cell clones incubated with 221-Cw3 or 221-Cw4 is shown. NK clones (6 for each type) were derived from 3 different C1/C1 donors. All NK-cell clones expressed NKG2A as the only inhibitory receptor. Co-incubation was performed in the absence or presence of anti–HLA-I (6A4), anti-KIR, or anti-NKG2A mAbs, as indicated. The histogram represents the percentage of CCR7⁺ NK cells after incubation with 221 cell transfectants. (F) The same KIR2DS1⁺ or KIR2DS1⁻ NK-cell clones, which had been incubated alone (NK) or with 221 cell transfectants in the absence or presence of anti–HLA-I (6A4) or anti-NKG2A mAbs, were analyzed for their migratory properties in response to CCL19/CCL21 chemokines. NK cells incubated with K562 were used as a control. The average of 6 independent experiments is shown. The histogram refers to the migration index of NK cells after coculture. (G) The relationship between the migration index and the CCR7 expression on 3 representative KIR2DS1⁺ NKG2A⁺ NK-cell clones (from 3 C1/C1 donors) was analyzed according to a linear regression model (R = 0.93) and confirmed by analyzing the Pearson correlation coefficient (=0.93). Shown are NK alone (empty rhombus); NK plus 221-Cw3 (black circle), NK plus 221-Cw4 (black triangle), NK plus 221-Cw4 in the presence of anti-NKG2A mAbs (gray cross), and NK plus 221-Cw4 in the presence of anti–HLA-I mAbs (6A4) (gray square). *P < .05; **P < .01.