Susceptibility of MICA-129-expressing targets to NK cell-mediated killing

To further establish the functional relevance of the MICA-129 dimorphism for killing of target cells, the susceptibility of the two L-MICA-129 variants to killing by IL-2-stimulated human LAK and MACS-isolated NK cells was analyzed usingChromium-51 (⁵¹Cr) release assays. A representative experiment is depicted in Figure 3.12. A showing the specific cell lysis of MICA-negative L-con cells and one clone of MICA-129Met and MICA-129Val-expressing L cells by LAK cells at different effector to target (E:T) ratios. K562 cells, which are known to be highly susceptible to killing by human NK cells, were included as positive control. The results demonstrate that MICA-expressing cells were effectively killed by human LAK cells in contrast to the vector-only transfected L-con cells, which were largely resistant to killing indicating that the lysis of the MICA-positive L transfectants was MICA-dependent.

The MICA expression intensity on the target cells as well as the binding of a recombinant NKG2D-Fc protein was always determined in parallel to the cytotoxicity assays (Figure 3.12 A, box). In this experiment, the L-MICA-Met clone exhibited besides an increased NKG2D binding also an increased specific cell lysis compared to the MICA-Val clone, which however expressed more MICA.

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200:1 100:1 50:1 25:1 12.5:1 6.3:1 3.1:1

A

200:1 100:1 50:1 25:1 12.5:1 6.3:1 3.1:1

Specificlysis [%]

Lower MICA expression quartile Upper MICA expression quartile

E:T ratio

Figure 3.12: Cytotoxicity of NK cells in response to the MICA-129Met and MICA-129Val isoforms significantly varies and depends on the MICA expression intensity.

(A) A representative experiment is shown demonstrating the specific cytotoxic activity of human LAK cells stimulated for 4 days with IL-2 (100 U/ml) against L-MICA-129Met, L-MICA-129Val and L-con cells as negative control. K562 cells were included as positive control. The means of specific lysis of triplicates ± standard deviation (SD) at different effector:target (E:T) ratios (200:1 to 3:1) are shown as measured in a ⁵¹Cr release assay. The MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein to the target cells was always determined in parallel by flow cytometry and the MFIs for the experiment shown here are indicated in the box. This individual experiment is included in the summary shown in panel B and D. (B) The mean of relative lysis ± SD of the before mentioned target cells by IL-2-stimulated LAK cells was determined in 9 independent experiments using 10 different clones of each MICA-129 variant (n = 84). (C) The mean of

84 relative lysis ± SD of target cells by MACS-isolated IL-2-activated NK cells was determined in 3 independent experiments using 6 different clones of each MICA-129 variant (n ≥ 11). The percentage of specific lysis of K562 cells at the highest E:T ratio (200:1 for LAK cells or 20:1 for NK cells) was adjusted to 100% in each test and the relative lysis of the target cells by various effector cells at different E:T ratios was calculated. (D) Based on a linear statistical model, the MICA expression intensity was integrated in the calculation of the expected relative lysis of the target cells expressing the MICA-129Met or Val variant by LAK cells at different E:T ratios for the lower and upper MICA expression quartile (MFI: 43.92 or 40.45 and 94.23 or 102.12 for the MICA-129Met or MICA-129Val variant, respectively).

For determination of the relative cell lysis, killing of K562 cells at an E:T ratio of 200:1 was set to 100% in every experiment and used to calculate the relative lysis of the other targets as depicted in Figure 3.12 B. This graphical representation, which does not include the MICA expression of the target cells, showed no difference in lysis between the two MICA variants for all performed experiments using LAK cells as effectors. However, with adjustment to the MICA expression intensity, the statistical analysis revealed that the relative lysis of the MICA-129Val variant expressing L cells was significantly reduced compared to cells expressing the Met variant (n = 84, P = 0.0044, ANOVA, -13.0 % at an E:T ratio of 200:1). Importantly, the MICA expression intensity had even a negative influence on killing for targets expressing the MICA-129Met variant, (P = 0.0083, ANOVA, regression coefficient slope -0.0834,). In contrast, killing increased with expression intensity of the MICA-129Val variant on target cells (slope 0.1257, P < 0.0001). Based on a linear statistical model adjusted for the MICA expression intensity, Figure 3.12 D depicts the expected relative cell lysis of MICA-129Met/Val-expressing target cells with low or high expression levels (lower MICA expression quartile with a MFI of 43.92 or 40.45 and upper MICA expression quartile with a MFI of 94.23 or 102.12 for the MICA-129Met or MICA-129Val variant, respectively). These data show that high MICA expression intensities are associated with lower killing of cells expressing the MICA-129Met than MICA-129Val variant.

Instead of using human LAK cells, containing between 10% to 15% NK cells, we also used purified IL-2-stimulated NK cells comprising 95% to 98% of NK cells (Figure 3.12 C). Similar results were obtained with isolated NK cells but with reduced values for the relative cell lysis of MICA-expressing L targets and L-con cells suggesting a better activation of NK cells by IL-2 in a co-culture with other PBMCs or an involvement of a further cell population. Since γδ T cells also express NKG2D on their cell surface and do not necessarily need additional stimulation of other activating receptors to be activated (Girardi et al., 2001; Whang et al., 2009), this type of T cells might be also involved in killing of the target cells.

In summary, the MICA-129Met variant elicited stronger NK cell cytotoxicity at lower MICA expression intensities compared to the MICA-129Val variant. Notably, cytotoxic effector function increased with the MICA expression intensity of the 129Val variant, whereas high expression of the

MICA-85 129Met variant even decreased cytotoxic activity of NK cells. Thus, the Met variant is overall a stronger trigger of NK cell cytotoxicity but the Val variant gains advantages at high MICA expression intensities on target cells.