Hydroxychloroquine Sensitizes Chronic Myeloid Cell to γ9δ2T Cell Mediated Lysis Independent of Autophagy
Authors: Han Biqing , Zhao Yanmin , Lin Yu , Wang Limengmeng , Wang Binsheng , Tie Ruxiu , Long Yan , Zhang Mingming , Huang He 
Published: October 2019

Abstract
Introduction
Tyrosine kinase inhibitor (TKI) has greatly improved survival in CML. However, relapse remains a great challenge because of the persistent of leukemia stem cells. Recently autophagy is revealed to be a mechanism of drug resistance of leukemia stem cells. Targeting autophagy in the treatment of CML has been highlighted. Hydroxylchloroquine (HCQ), the only autophagy inhibitor approved for clinical use, has drawn more and more attention. Combining TKI with HCQ, primary CML stem cells can be almost completely eliminated in vitro. What's more, a phaseⅡ clinical research is going on in the University of Glasgow. HCQ as autophagy inhibitor can also augment the anticancer activity of some chemotherapeutics such as histone deacetylase inhibitor, IFNα etc and overcome drug resistance in CML.
HCQ has offered the hope that CML may be cured. But seldom do we know whether HCQ exerts anticancer effects through other mechanisms besides autophagy inhibition. Especially the mechanisms underlying the ability of HCQ to modulate the immune visibility of CML are unknown. In our research, we revealed HCQ can sensitize CML to γδT cell mediated lysis through ULBP4 translocation, which proved to be autophagy independent.
Results
To test the cytotoctity of γδT cells to CML cells. CML cell lines K562 ( imatinib sensitive) and K562/GO1( imatibin resistant) were firstly exposed to nontoxic HCQ, then cocultured with γδT cells from peripheral blood of healthy donors. Compared to untreated cells, pretreated K562 and K562/GO1 were more sensitive to γδT cell mediated lysis. The mortality increased from 35.9% to 47.1% in K562 (P<0.05) and from 31.7% to 46.6% in K562/GO1 (P<0.05) at effecter target ratio 20:1. To evaluate the activation of γδT cells , we tested the degranulation of γδT cells co-cultured with treated or untreated CML. Consistent with the cytotoxity assay,compared to untreated CML HCQ pretreated CML accelerated γδT cells degranulation from 12.45% to 23.05(P<0.05) in K562 and from 18.3% to 28.2% (P<0.05) in K562/GO1 respectively. And we also confirmed this in primary CML stem cells (CD34+) from 3 CML patients. To test whether HCQ sensitized CML to γδT cells by autophagy inhibition, we detected ATG protein LC3 P62 and Bclin1 as well as autophagosome in CML. HCQ treatment can induce the accumulation of LC3Ⅱ P62 and autophagosome in a time dependence manner, which means HCQ can block autophagy in CML. We further conducted an ATG7 knockdown assay by shRNA to block autophagy in CML. ATG7 knockdown can significantly block autophagy in CML, but without effects on CML sensitivity to γδT cells. Moreover, after ATG7 knockdown, HCQ can still sensitize CML to γδT. This implied an autophagy independence manner of HCQ in sensitizing CML to γδT cell mediated lysis.
Next we detected the expression of NKG2D ligands on CML cytomembrane after HCQ exposure, which are important for cancer cell immune recognition by γδT cells. Among all NKG2D ligands including MICA/B ULBP1 2 3 4 5, we found only ULBP4 was up regulated after HCQ treatment depend on exposure time. The fact is ULBP4 doesn't express on the cytomembrane of K562 and K562/GO1. After exposure to HCQ for 4 8 and 12 hours, the cells expressed ULBP4 on cytomenbrane raised from 0.84% to 68.14% 87% and 86.4% in K562 (P<0.001) and raised from 1.2% to 54.6% 84.3% and 87.1% in K562/GO1(P<0.001). To test whether HCQ sensitize CML to γδT due to the ULBP4 upregulation on cytomembrane, blocking NKG2D mAb were used to block the binding of NKG2D with its ligands. After that, HCQ exposure can't augment the sensitivity of CML to γδT cells. This means HCQ sensitize CML to γδT cells through up-regulating ULBP4 on cytomembrane.
To know how HCQ unregulates ULBP4 on cytomembrane. We checked the mRNA and protein of ULBP4, and found HCQ did not influence the transcription and translation of ULBP4. We further tested the expression of ULBP4 both in CML cytomembrane and cytoplasm, found ULBP4 exists in cytoplasm and doesn't express on cytomembrane untill HCQ exposure. Immunofluorescene showed that HCQ treatment induce ULBP4 transfer from cytoplasm to cytomembrane.
Conclusion
Our data shows the first time HCQ can sensitize CML to γδT cell mediated lysis in an autophagy independence manner. In CML, ULBP4 intracellular retention may be a way of immune escape. HCQ can induce ULBP4 translocation from cytoplasm to cytomenbrane through which sensitizes CML to γδT cell mediated lysis.

Disclosures
No relevant conflicts of interest to declare.