Impact of a Functional NKG2D Genetic Variant on Lymphoma Susceptibility and NK Cell Immune Response to Epstein Barr Virus

Background and Methods: NKG2D is an activating receptor expressed by NK cells and various T cell subsets that plays important roles in virus immunity and cancer immune-surveillance. The genetic variant rs1049174 in the NKG2D gene generates two well-defined genotypes: the LNK (low natural killer) and HNK (high natural killer). Notably, rs1049174 occurs within a targeting site for miR-1245, a negative regulator of NKG2D expression, where the LNK allele is more efficiently targeted by miR-1245 than the allele HNK, and thus NK cells from individuals with the LNK genotype display lower NKG2D expression and less efficient NKG2D-mediated functions than NK cells with the HNK genotype. We hypothesized that rs1049174 may influence lymphoma susceptibility given the critical role of immunosurveillance in lymphoma development. We investigated the rs1049174 genotype distribution in a cohort of 679 Vietnamese patients with lymphoma, including 601 non-Hodgkin lymphoma (NHL), 78 Hodgkin lymphoma (HL), and 618 healthy individuals. The potential association of the NKG2D gene variant rs1049174 with the immune responses of NK cells against EBV-infected B cells was also studied. The rs1049174 genotypes were carried out using the TaqMan method, and comparisons were made using chi-square or two-sided Fisher's exact tests. Significance was assessed at the 0.05 level.

Results: The ages of lymphoma patients tested for rs1049174 ranged from 16 to 92 years, with a mean of 53 years, and 58% were male. Compared to the genotype frequency in healthy individuals (HNK 35%), the HNK genotype in lymphoma patients was significantly less frequent (HNK 27%, p = 0.01). This was true even when the patients were categorized into NHL (n = 78, HNK = 27%, p = 0.01) and NHL (n = 610, HNK = 28.2%, p = 0.02), although the association between HNK genotypes was not significant among patients with diffuse large B cell lymphoma (n = 280, HNK = 29.2%, p = 0.07). Notably, among the 310 EBV+ lymphomas in this cohort, the HNK genotype in lymphoma patients was significantly less frequent (HNK 24.2%, p = 0.005) compared with healthy controls, suggesting a link between EBV-induced carcinogenesis and the rs1049174 genotypes.

To assess the NKG2D-mediated immune response in EBV-mediated carcinogenesis, we used flow cytometry time kinetic studies to evaluate the expression of NKG2D ligands (NKG2D-Ls) on the surface of B cells during the in vitro transformation process induced by EBV infection. Among the NKG2D-Ls tested (MICA/B, ULBP1, ULBP2, ULBP3, and ULBP5), we observed that ULBP3 and, to some extent, MICA/B were upregulated in EBV-infected B cells, reaching their highest expression level by day 7 after infection, while gradually decreasing their expression to become almost undetectable by three weeks after EBV infection. In cytotoxicity studies, freshly isolated autologous NK cells recognized and eliminated a considerable fraction of EBV-infected B cells, and these effects were attenuated by the prior exposure of NK cells to an anti-NKG2D blocking antibody, supporting the involvement of the NKG2D axis in NK cell immunity to EBV-infected B cells. Importantly, NK cells from individuals with the HNK genotype were significantly more effective than NK cells with the LNK genotype in eliminating their corresponding autologous EBV-infected B cells and more efficiently preventing their transformation into lymphoblastoid cells.

Conclusions: The NKG2D variants may influence cancer immunosurveillance, including NK cell immunity against EBV infection, and thus determine susceptibility to EBV-associated malignancies.