The Role and Mechanism of PTPN21 Isoforms in Acute Lymphoblastic Leukemia Escaping NK Cell Killing

Relapse of acute lymphoid leukemia (ALL) is one of the most difficult issues in clinical treatment. There is strong correlation between gene profiles and recurrence in ALL. Previous studies have illustrated the importance of PTPN21 in the development of several kinds of tumors, such as bladder cancer, esophageal cancer, lymphoma etc. PTPN21 is highly expressed in the solid tumors above, which can reflect the extent and prognosis of malignancy. Whether PTPN21 plays a role in the development and progression of leukemia has not been reported

In our previous study, we found that the mRNA expression level of PTPN21 in bone marrow monocytes of ALL patients was increased.Moreover, the ability of cell proliferation was increased after overexpressing PTPN21 in ALL cell lines via lentiviral transfection method.Besides, we designed two kinds of single guide RNA(sgRNA),which located in the exon 2 and exon7 of PTPN21,respectively. We found NALM6,which is one type of PH-ALL cell line ,had two kind of PTPN21 isoforms using PCR method,the longest CDS one(1174aa) and the shortest one(970aa).Using Crispr-Cas9 technology ,we had separately gained the NALM6 clones with PTPN21 knocking out in exon2 site and exon7 site,which stand for the longest isoform(1174aa) knocking out and all isoforms kncoking out ,respectively.We found the cytotoxicity of natural killer (NK) cells to NALM6 with knocking out PTPN21 gene in exon2 site obviously increased(figure 1a).While there was no similar result in the NALM6 clones with knocking out in exon7 of PTPN21(figure 1b). Our results suggested that PTPN21 was involved in the immune escaping process of ALL by NK cells, and the above two types of isoforms of PTPN21 play contary roles in immune killing process in ALL by NK cells. We then tested the expression profile of NK related ligands on the cell surface before and after gene knock-out throgh flow cytometry,such as ULBPs,CD155,CD112,HLA-ABC ect.The result showed after the longest isoform was knocked out from NALM6, The expression of HLA-E,CD112,HLA class I molecules on the cell surface was decreased.Especially, HLAI changed most obviously (figure 1c),and the chaperones of endogenous antigen presenting such as TAP1,TAP2,TAPBP,PSMB8,PSMB9 decreased significantly(figure 1d).we suspect it's the key reason of resulting in the decrease of HLA class I molecules on the cell surface, thus can block the KIR-HLAI signaling axis and improve the killing activity of NK cells.

Together, our data suggest that different PTPN21 isoforms in ALL cells have different roles in mediating immune response by NK. There are mutual checks and balance between isoforms, which provide new idea and therapeutic target for the prevention and treatment of ALL recurrence.

Figure 1:

a-b. The cytotoxicity of natural killer (NK) cells to NALM6 with knocking out PTPN21 gene in exon2.The killing rate =CFSE⁺PI⁺/CFSE⁺. c-d. The cytotoxicity of natural killer (NK) cells to NALM6 with knocking out PTPN21 gene in exon7. The killing rate =CFSE⁺PI⁺/CFSE⁺. e. The expression of HLA class I molecules on the cell surface before and after PTPN21 knock-out in exon2 and exon7,respetively. f.The mRNA expression profiles of the chaperone of endogenous antigen presenting whether PTPN21 knock-out or not in exon2).( _* p=0.05,_**p=0.01,_***p=0.001

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