Pgam1 Regulate Adaptive NK Cells in Anti-Viral and Anti-Tumor Response

Abstract

Natural killer (NK) cells are the innate immune cells that play a role in the anti-viral infections and tumor cells. The adaptive NK cells are characterized with expression of the HLA-E-specific CD94/NKG2C activating receptor. The reconstitution of NKG2C+ adaptive NK cells post HSCT is much faster in NKG2C homozygous(NKG2C wt/wt) donors than NKG2C heterozygous donors (NKG2C wt/del), which resulted in lower incidence of CMV infection. In this study, we aimed to find out the mainly regulators of adaptive NK cells between NKG2C wt/wt and NKG2C wt/del donors. RNA-sequencing analysis were performed. Results shown that NK cells from NKG2C wt/wt donors constant upregulated glycolysis and biosynthesis-related genes, while downregulated in TGF-beta signaling and FOXO signaling pathway(Figure A and B). In order to determine the role of glycolysis in adaptive NK cells, upregulated glycolysis related genes were assessed by q-PCR, and PGAM1 is considered as the most important gene in the expansion of adaptive NK cells(Figure C). PGAM1 is upregulated upon NK cells stimulation via cytokine or target cells, which is related to the CD107a expression. HKB99 is an allosteric inhibitor for PGAM1, which is considered as a potential therapeutic target for many cancers nowadays. NK cells were added with HKB99 in vitro for 12h hours to evaluate the anti-K562 function. After that, Glycolysis is inhibited with the decreasing of ECAR while the OCR is not affected (FigureD). The adding of PGAM1 inhibitor resulted in decreasing of CD107a expression in NK cells, especially in the adaptive NK cells. NK cells activation signaling pathway is also inhibited, including the phosphorylation of mTORC1 protein S6, Syk, zap70, and the PLC-gamma. The PI3K/AKT is inhibited after the PGAM1 inhibition. After that,supplementation of pyruvate is efficient to rescue the inhibition caused by PGAM1 inhibitor. 5mM pyruvate adding can restore the cytokine release of NK cells and the phosphorylation of S6 and syk/zap70(Figure E). PGAM1-NCR-cre mice was constructed in which PGAM1 is knock out in NK cells specifically. In KO group, the ratio of NK cells and the maturation of NK cells are decreased. Upon cytokine stimulation, the activator receptors expression is less in KO mice. Besides, PGAM1 deficiency NK cells exhibited glucose uptake deficiency and disadvantages in proliferation both in vivo and in vitro (Figure F). The differentiation (CD11b+CD27-) and activation marker (DNAM1, Ly49H) are decreased in the competing transplantation mouse model, while the exhaustion markers such as PD-1 and NKG2A is upregulated. Further, MCMV infection models shown that PGAM1 deficiency mice with lower ratio of Ly49H+ adaptive NK cells at the early stage of infection, while Ly49G2 and Ly49C+ cells are not affected(Figure G). Further, Ly49H+ adaptive NK cells shown less expression of Ki67 in PB-NK cells, with the activating markers CD25 is decreased. Higher MCMV load induced inflammation is occurred in PGAM1 deficiency NK cells. Anti-tumor effect was also evaluated. The apoptosis of YAC-1 cells is decrease in coincidence with CD107a expression decreasing. MLL-AF9 leukemia mice model shown in day 18, the Ly49H+ adaptive NK cells in PGAM1 deficiency mice is less than WT mice, and the tumor burden is higher and accompanied with shorter survival under tumor circumstances(Figure H). RNA seq analysis shown compared to WT mice, the downregulated genes in PGAM1 deficiency NK cells are enriched in leukocyte activation and innate immune response also interfered the cytokine-mediated signaling pathway. At last, we monitored expression of PGAM1 and the function of NK cells in patients whose CMV is reactive post HSCT. PGAM1 is elevated post CMV infection, which is correlated with CD107a secretion. ROC curve suggests that PGAM1 may act as a predictor for refractory recurrence CMV infection in patients post HSCT(Figure I). In summary, our studies shown the glycolysis is effectively support the faster adaptive NK cells expansion and CMV response. PGAM1-mediated glycolysis is significant for NK cells anti-tumor and anti-viral function.