Uptake of CCR7 by KIR2DS4⁺ NK cells is induced upon recognition of certain HLA-C alleles

Abstract

The KIR2DS4 receptor is the oldest KIR2DS expressed by human NK lymphocytes. The specificity of recognition of this receptor for various HLA class I alleles has been demonstrated; however it remains poorly understood whether these interactions may result in the activation of some specific functions in NK cells. Here, we examined the functional outcome of the KIR2DS4/HLA class I interaction by the use of an alternative functional system based on the ability of KIR2DS4 to regulate the mechanism of trogocytosis by NK cells. We demonstrate that KIR2DS4 can induce the uptake of CCR7 by KIR2DS4(+) NKG2A(+) NK cell clones after interacting with CCR7(+) target cells expressing HLA-Cw4 and HLA-Cw6 alleles. However this interaction is not always sufficient to override the inhibition generated by NKG2A expressed on the same NK cells. The recognition of HLA-Cw4 was confirmed by experiments of cytotoxicity against HLA-C-transfected cells. We also show that, different from resting NK cells, the acquisition of CCR7 in response to IL-18 cannot occur in IL2-activated NK cells because of a marked downregulation in their IL-18Rα expression. As a consequence trogocytosis represents the major mechanism by which KIR2DS4(+) activated NK cells acquire the expression of this chemokine receptor.

Figure 1

De novo expression of CCR7 in NK cell clones upon interaction with transfected 221 cell lines: comparison between KIR2DS4⁺ and KIR2DS4⁻ NK cell populations. (a) Expression profile of HLA-C and HLA-E molecules (left panels) and of CCR7 receptor (right panel) on 221 cells or 221 cells transfected with HLA-Cw3 or HLA-Cw4 or HLA-Cw6 is shown. (b) KIR2DS4⁻ NKG2A⁺ or KIR2DS4⁺ NKG2A⁺ NK cell clones were incubated with untransfected 221 cells or 221 cells transfected with HLA-Cw3 or HLA-Cw4 or HLA-Cw6 alleles. NK cell clones (6 for each type) were derived from one B haplotype donor. Coincubation was performed in the absence or presence of anti-NKG2A mAbs, as indicated. The histogram represents the percentage of CCR7⁺ NK cells after incubation with untransfected or HLA-transfected 221 cells. The average of 6 independent experiments and standard deviation (mean ± SD) are indicated. ^∗∗ p < 0.01; ^∗ p < 0.05. (c) KIR2DS4⁻ NKG2A⁺ or KIR2DS4⁺ NKG2A⁺ NK cell clones (from one B haplotype donor) were incubated with 221-Cw4 cells. 3 KIR2DS4⁻ NKG2A⁺ and 3 KIR2DS4⁺ NKG2A⁺ NK cell clones characterized by low NKG2A-mediated inhibition (on the left) and 3 KIR2DS4⁻ NKG2A⁺ and 3 KIR2DS4⁺ NKG2A⁺ characterized by high NKG2A-mediated inhibition (on the right) are represented. The average of 6 independent experiments and standard deviation (mean ± SD) are indicated. ^∗∗ p < 0.01; ^∗ p < 0.05. (d) A representative KIR2DS4⁺ NKG2A⁺ NK cell clone (derived from one B haplotype donor) was incubated at 37°C with CCR7⁺ untransfected 221 cells or 221 cells transfected with HLA-Cw4. After 30 minutes, NK cells were harvested and stained with anti-CD56 and anti-CCR7 mAbs for cytofluorimetric analysis. The percentages of CCR7⁺ cells are indicated in the upper-right corners. (e) KIR2DS4⁺ NKG2A⁺ NK cell clones derived from the one B haplotype donor were incubated with untransfected 221 cells or 221 cells transfected with HLA-Cw3 or HLA-Cw4 or HLA-Cw6 alleles. Coincubation was performed in the absence or in the presence of mAbs to the indicated molecules. The histogram represents the percentage of CCR7⁺ NK cells after incubation with untransfected or HLA-transfected 221 cells. The average of 6 independent experiments and standard deviation (mean ± SD) are indicated. ^∗ P < 0.05. (f) KIR2DS4⁺ NKG2A⁺ NK cell clones derived from one A haplotype donor (upper panel) and one B haplotype donor (lower panel) (3 for each haplotype) were incubated with untransfected 221 cells or 221 cells transfected with HLA-Cw3 or HLA-Cw4 or HLA-Cw6 alleles. The average of 3 independent experiments and standard deviation (mean ± SD) are indicated. ^∗ p < 0.05.

Figure 2

Analysis of the IL-18Rα surface expression on resting or activated NK cells: functional implications. (a) Freshly isolated (NK) and IL-2 activated (BULK) NK cells from one B haplotype donor were cultured in the presence of exogenous IL-18 for 18 hours and then analyzed for CCR7 expression. Analysis of CD83 expression on NK cells was used as a positive control of the effect mediated by IL-18. The values reported in the upper-right corners indicate the percentage of CD56⁺ CCR7⁺ or of CD56⁺ CD83⁺ NK cells. A representative of 10 independent experiments performed using different donors is shown. (b) Freshly isolated or short-term (18 h) activated NK cells (NK) from one B haplotype donor were analyzed for the expression of IL-18Rα in comparison with long-term IL-2 activated (BULK) NK cells form the same donor. BULK NK cells were also treated for 18 h with either IL-18 or IL-12, after washing the cells, to remove IL-2 from the supernatant and then analyzed for the expression of IL-18Rα. The average of 6 independent experiments and standard deviation (mean ± SD) are indicated. ^∗∗ p < 0.01. (c) A representative B haplotype donor is shown for the expression of IL-18Rα on both resting and BULK NK cells. (d) The histogram plots refer to the expression of IL-18Rα on both resting and BULK NK cells of a representative B haplotype donor after gating on KIR2DS4⁺ NK cells. The dashed line represents the isotype control.

Figure 3

Comparison between KIR2DS4⁺ and KIR2DS4⁻ NK cell clones in the killing of transfected 221 cell lines. (a, b) The cytolytic activity of KIR2DS4⁻ NKG2A⁺ and KIR2DS4⁺ NKG2A⁺ NK cell clones (derived from a B haplotype donor) was analyzed against untransfected or Cw3-, Cw4-, or Cw6-transfected 221 cells in the absence or in the presence of mAbs to the indicated molecules. The E/T ratio used was 1 : 1. The average of 4 independent experiments and standard deviation (mean ± SD) are indicated. ^∗∗ p < 0.01; ^∗ p < 0.05.