Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked

Abstract

Background: There is limited understanding of the dysregulation of the innate immune system in multiple myeloma (MM). We analysed the expression of the activating receptor NKG2D on NK cells and T cells of MM patients and investigated the impact of soluble versus membrane-bound NKG2D ligands on the expression of NKG2D.

Design: NKG2D expression on NK cells and CD8+ alphabeta T cells from patients with MM or monoclonal gammopathy of uncertain significance and healthy controls was examined flow-cytometrically. Sera from patients and controls were analysed for soluble NKG2D ligands (sNKG2D ligands).

Results: Significantly fewer NK cells and CD8+ alphabeta T cells from patients expressed NKG2D compared to healthy controls (NK cells: median 54% interquartile range (IQR) 32-68 versus 71% IQR 44-82%, P = 0.017, CD8+ alphabeta T cells: median 63% IQR 52-81 versus 77% IQR 71-90%, P = 0.018). The sNKG2D ligand sMICA was increased in patients [median 175 (IQR 87-295) pg/ml] versus controls [median 80 (IQR 32-129) pg/ml, P < 0.001], but levels of sMICA did not correlate with NKG2D expression on effector cells. To elucidate the mechanism of NKG2D down-regulation, we incubated lymphocytes from healthy donors in the presence of sNKG2D ligands or in co-culture with MM cell lines. sNKG2D ligands in clinically relevant concentrations did not down-regulate NKG2D expression, but co-culture of effector cells with myeloma cells with high surface expression of NKG2D ligands reduced NKG2D expression significantly.

Conclusions: These results indicate that MM is associated with a significant reduction in NKG2D expression which may be contact-mediated rather than caused by soluble NKG2D ligands.

Fig. 1

a Flow-cytometrical analysis of expression of NKG2D on NK cells and CD8+ αβ T cells of healthy controls and patients with MGUS, low disease burden, progressive disease and newly diagnosed disease. The first line depicts the gating strategy of NK cells (CD3–CD56+), CD8+ αβ T cells (CD8++γδTCR-) and γδ T cells (γδTCR+) and representative dot plots of the NKG2D expression of the respective cell populations. The second line depicts representative examples of NK cells and the third line representative examples of CD8+ αβ T cells of 17 controls, 12 patients with MGUS, 17 patients with low disease burden, 8 patients with progressive disease and 12 newly diagnosed patients. The grey line represents the isotype control, the black line the stained sample; b Median percentage of NKG2D-expressing NK cells and CD8+ αβ T cells from patients and controls (left plot) as well as median mean fluorescence intensity of NKG2D expression of NK cells and CD8+ αβ T cells from patients and controls. The line depicts the median, the box the interquartile range and the whiskers the 95% confidence interval. Patients: NK cells: percentage NKG2D+ cells median 54% IQR 32–68, MFI median 1,461 IQR 1,268–1,765; CD8+ αβ T cells: percentage NKG2D+ cells median 63%, IQR 52–81%, MFI median 1,780 IQR 1,559–1,967. Controls: NK cells: percentage NKG2D+ cells median 71% IQR 44–82%, MFI median 1,491 IQR 1,348–1,686; CD8+ αβ T cells: percentage NKG2D+ cells median 77%, IQR 71–90%, MFI median 1,828 IQR 1,767–2,249. *P < 0.05

Fig. 2

a Soluble MICA and MICB in the sera of healthy controls and patients with MGUS or MM detected by ELISA as described in the methods section. The line depicts the median, the box the interquartile range and the whiskers the 95% confidence interval. sMICA: patients median 175 (IQR 87–295) pg/ml, controls median 80 (IQR 32–129) pg/ml (**P < 0.001), sMICB: patients median 66 (IQR 15–128) pg/ml, controls 42 (IQR 7–111) pg/ml (P = 0.8); b Soluble ULBP1, ULBP2 and ULBP3 in the sera of controls and patients as described above. sULBP1: patients median 448 (IQR 0–1,125) pg/ml, controls median 0 (IQR 0–640) pg/ml, sULBP2: patients median 0 (IQR 0–1,202) pg/ml, controls median 0 (IQR 0–501) pg/ml, sULBP3: patients median 265 (IQR 0–552) pg/ml, controls median 56 (IQR 0–473) pg/ml, P > 0.05 for all sULBP; c Level of soluble MICA depending on current stage of disease. MGUS monoclonal gammopathy of uncertain significance, LD Low Disease, PD Progressive Disease, ND Newly Diagnosed. The line depicts the median, the box the interquartile range and the whiskers the 95% confidence interval. MGUS: median 107, IQR 84–156 pg/ml, low disease burden: median 189, IQR 96–312 pg/ml, progressive disease: median 121, IQR 26–437 pg/ml, newly diagnosed: median 232, IQR 147–331 pg/ml, P < 0.001 Kruskall–Wallis

Fig. 3

a Concentration-dependent NKG2D down-regulation by sMICA after culture and flow-cytometrical analysis as described in the methods section. CM complete medium. Mean ± standard error of the mean of four independent experiments with effector cells from four different healthy donors is depicted. *P < 0.05; b No effect on NKG2D-expression of NK cells and CD8+ T cells was seen when effector cells from healthy donors where incubated with varying concentrations of patient serum. Here, a representative flow-cytometrical analysis of lymphocytes from a healthy donor cultured in the presence of varying concentrations of patient serum as described in the methods section is shown. In this example, the sMICA concentration in the serum was 317 pg/ml

Fig. 4

a Culture of effector cells in the presence of supernatants with varying concentrations of sNKG2D ligands as described in the method section does not change NKG2D expression significantly. CM complete medium, SN supernatant. One representative example showing flow-cytometrical analysis of NKG2D-expression on NK cells and CD8+ αβ T cells of 4 independent experiments is shown; b Co-culture with leukaemia cells (K562) leads to down-regulation of NKG2D on NK cells and CD8+ αβ T cells whereas the presence of the supernatant does not change NKG2D expression. SN supernatant. One representative example of 6 independent flow-cytometrical analyses is shown; c Co-culture with myeloma cells (RPMI 8226) as described in the “Patients and methods” leads to down-regulation of NKG2D on NK cells and CD8+ αβ T cells. One representative example of eight independent flow-cytometrical analyses is shown

Fig. 5

a Differential effect of coculturing effector cells with different tumour cell lines in different tumour cell: NK cell ratios is analysed flow-cytometrically. CM complete medium. Here, the mean ± standard error of the mean of four independent experiments is shown. *P < 0.05, **P < 0.01 compared to CM; b Cytotoxic activity against K562. Purified NK cells cells after 24 h co-culture with RPMI 8226 at 1:1 were evaluated in a standard chromium release assay at various E:T. Representative example (triplicates ± standard deviation) of two independent experiments; c Antibodies against NKG2D ligands inhibit down-regulation of NKG2D. CM complete medium. Mean ± standard error of the mean of four independent flow-cytometrical analyses is depicted. *P < 0.05 **P < 0.01 compared to CM