NK Cells Suppress Hyperfunction of CTLs Via ATM-Mediated NKG2D/NKG2DLs Activation in Severe Aplastic Anemia

Object: The activating receptor NKG2D on the surface of NK cells in peripheral blood of patients with severe aplastic anemia (SAA) was significantly increased, and NK cells may be activated during the pathogenesis of SAA. However, the immunomodulatory effect of NK cells on CTL cells remains unclear. ATM protein is a serine/threonine protein kinase mainly involved in cell cycle regulation, DNA damage recognition and repair. Activation of ATM may be the key to regulating the expression of NKG2DLs in T cells stimulated by superantigens. By detecting the changes of NKG2DLs in CTL cells of SAA patients under the regulation of ATM, we explored the immunoregulatory effect of NK cells on autologous CTL cells through the signaling axis NKG2D/NKG2DLs.

Methods: We used flow cytometry, RT-qPCR and Western Blot to detect the expression of NKG2DLs (MICA, ULBP1, ULBP3) in CTL cells of untreated SAA patients, remission SAA patients and healthy controls under the intervention of CD3/CD28 antibody and ATM agonist (chloroquine phosphate). NK and CTL cells from SAA patients were co-cultured in vitro, and the function of CTL under the regulation of ATM was detected.

Results: Under the intervention of CD3/CD28 antibody and chloroquine phosphate, the level of MICA in CTL cells detected by flow cytometry in untreated SAA patients was higher than that in convalescent SAA patients and healthy controls (Fig.A). RT-qPCR detection of mRNA levels of NKG2DLs (MICA, ULBP1 and ULBP3) in CTL cells of untreated SAA patients was also higher than that of remission SAA patients and healthy controls (Fig.B). When ATM agonist (chloroquine phosphate), ATM inhibitor (KU55933), and chloroquine phosphate + KU55933 were used to intervene T cells, Western Blot detected the correlation between ATM phosphorylation and NKG2DLs (MICA, ULBP1) expression(Fig.D). We co-cultured CTL cells from SAA patients with different interventions in vitro with autologous NK cells. The expression of IFN-γ in CTL cells in the ATM agonist group was significantly lower than that in the ATM inhibition group, and the apoptosis rate was higher than that in the ATM inhibition group and the control group (Fig.C). There was no significant difference in the expression of perforin and granzyme B. In addition, NK cell apoptosis did not differ among the groups.The expression levels of free MICA and ULBP3 in the co-culture supernatant were detected by ELISA, and the ATM agonist group was higher than the inhibition group(p<0.05).

Conclusion: Activation of ATM increases the expression of NKG2DLs in CTL cells of SAA patients, which promotes the inhibitory effect of NK cells on CTL cells. Our study further confirms that ATM can regulate the relationship between NK cells and CTL cells in SAA disease, and inhibit the hyperactive state of CTL cells.

No relevant conflicts of interest to declare.