Galectin-9 Drives Functional Exhaustion Of TIM-3+ Natural Killer Cells In Head And Neck Squamous Cell Carcinoma

Chapter 1 Immune phenotypes of NK cells and NK subsets in patients with HNSCCObjectives:Head and neck squamous carcinoma(HNSCC)is a malignant tumor derived from epithelial cells of the upper respiratory gastrointestinal tract.Monoclonal antibody therapies based on blockade of inhibitory checkpoint receptors(ICRs)have been made an important achievement in activating T cells for the anti-tumor responses.However,a small population of HNSCC patients benefits from this antibody blockade therapy.Therefore,investigating other immune cells in anti-tumor responses of immunotherapy will be a new strategy for cancer treatment.Natural killer(NK)cells are innate immune cells with characteristics of effector cells that can be involved in the immune responses against tumors by directly killing target cells and producing cytokines.This chapter therefore aims to explore the immune phenotype and function of NK cells and NK subsets in peripheral blood lymphocytes(PBL)and tumor infiltrating lymphocytes(TIL)of HNSCC tumor microenvironment.Methods:A total of 13423 NK cells from peripheral blood and matched tumor tissues of 29 HNSCC patients were obtained,descended and sub-clustered using scRNAseq(single cell RNA sequencing)technology.Twelve NK clusters were visualized on UMAP plots,and highly expressed genes in each cluster was performed.The proportion of NK cells in PBL and TIL were separately calculated and differentially expressed genes(DEGs)were analyzed according to the sample source of NK scRNAseq.Multi-panel flow cytometry was utilized to detect the expression of NK cell surface receptors,such as CD 16,NKG2D,CD69,NKG2A and KIR2DL3,as well as cytotoxic markers Perforin and Granzyme B in PBL and TIL of HNSCC patients.Results:The scRNAseq identified diverse cell populations in the TME of HNSCC patients by visualizing a landscape UMAP plots.Transcriptome profiles of NK cells were clustered into twelve subgroups,where ten NK cell subgroups from PBL with highly expressing NKG7,FGFBP2,SPON2,CST7 and other genes that related to cell activation,cytotoxicity and mitochondrial metabolism genes.The two NK cell clusters from TIL are associated with cell homing,tissue residence,and immune recruitment.NK cell proportion were significantly higher in PBL than in TIL in scRNAseq data.DEGs analysis showed that NK cells have significant differences between PBL and TIL in immune characteristics,and genes in TIL NK cell exhibits a more complex transcriptome profile than that in PBL NK cell.Flow cytometry shows that CD3-CD56+NK cells were significantly decreased in TIL compared to PBL.NK cell subsets display a significant decrease in the expression frequency of CD56dim CD16+ NK subsets in TIL compared to that in PBL,while CD56bright CD16and CD56dim CD 16dim/-NK subsets were significantly increased in TIL compared to PBL.NK cell-associated functional markers were reshaped in CD56dim CD16+ NK subset.Conclusion:Tumor-infiltrating NK cells of HNSCC exhibit functional suppressed status such as reduced proportion,decreased activation,and attenuated cytotoxicity that were contributed by decreased function of CD56dim CD16+ NK subsets.Chapter 2 Upregulated TIM-3 expression marks inhibitory status in tumor-infiltrating NK cells of HNSCCObjectives:NK cells will be dysfunctional once they exposure to the tumor context.In the first section,we demonstrated that tumor-infiltrating NK cells from HNSCC patients exhibit functionally suppressed features.The anti-tumor effector of NK cells depends on the balance of activating and inhibiting receptor signaling.In addition to the specific surface receptors of NK cells,dysregulation of checkpoint receptor signaling also leads to NK cell dysfunction.Therefore,identifying checkpoint receptors with therapeutic potent as NK cell therapeutic targets would improve the current immunotherapy strategies.This section aims to explore the distributions and expressions of ICRs in circulating and tumor-infiltrating NK cells in HNSCC patients,which will provide new insight for NK-targeted therapy in HNSCC patients.Methods:Expression profiles of key ICRs gene transcript levels in NK cells were depicted by scRNAseq from HNSCC patients.ICR genes were analyzed using fraction of cells and average gene expression of each patient.Flow cytometry determined protein expression of ICRs in NK cells from PBL and TIL.Flow cytometry then determined the expression of CD 16,NKG2D,CD69,NKG2A,Perforin and Granzyme B in PBL and TIL to identify the ICRs’function in NK cells.Results:NK cell scRNAseq data in HNSCC patients showed that LAG3 and HAVCR2 were highly expressed in the UMAP,and there were significant differences in expression between PBL and TIL.NK cell scRNAseq of ICRs genes for each patient showed that HAVCR2 and LAG3 genes were expressed at prominent levels in both fraction of cells and average geneexpression,and there were significant differences of expression between PBL and TIL NK cells.Flow cytometry exhibits that TIM-3 was significantly higher than other ICRs and was significantly upregulated in TIL NK cells.CD 16,NKG2D,Perforin and Granzyme B were significantly decreased in TIM-3+NK populations in TIL compared to that in PBL.No change of those markers was observed in TIM-3-NK populations.Inhibitory receptors,NKG2A and KIR2DL3,did not show significant changes between TIM-3+and TIM-3-NK populations in both TIL and PBL.Conclusion:TIM-3 is the dominantly expressed ICR on NK cells of HNSCC patients,and its upregulation on tumor-infiltrating NK cells is associated with NK cells dysfunction.Chapter 3 TIM-3+NK cells display an exhausted status after Galectin-9 ligationObjectives:Our study has been confirmed that upregulated TIM-3 expression on tumor-infiltrating NK cells inhibits NK cell function.Like PD-1/PD-L1 ligation,TIM-3 was found to have multiple potential ligands for binding to its surface,including Galectin-9,HMGB1,Ceacam1,and PS.However,the interaction of TIM-3 and its ligands on NK cells is poor understand.Therefore,this study aims to evaluate these potential TIM-3 ligands to identify specific ligand that bind to TIM-3,and identify which ligand was involved in NK cell dysfunction in the context of the HNSCC TME,which will provide a better understanding on potential applications for TIM-3 blocking in NK cell-mediated immunotherapy in solid tumors.Methods:Peripheral blood NK cells from HNSCC patients were sorted by human NK cell magnetic beads.NK cells from HNSCC patients were stained using CFSE and were co-cultured with different TIM-3 ligands in IL-2 condition.CFSE expression was determined for the proliferative capacity of NK cells by flow cytometry.NK cells were pre-treated with four different ligands,and their killing ability were determined by measuring the number of viable K562 cells at different effector/target cell ratios.NK cell-conditioned supernatants were collected after ligand ligation and were determined for cytokines secretion by Luminex assay.Flow cytometry was performed to detect key NK markers after Galectin-9 ligation.Results:Among these four TIM-3 ligands,only Galectin-9 ligation significantly inhibits the proliferation on CFSE-labeled NK cells.Galectin-9 also specifically inhibits the killing of NK cells targeting K562 cells.Multi-cytokines immunoassay for NK cell-conditioned supernatants showed that TIM-3 ligands are unable to disturb cytokine release of NK cells.Flow cytometry demonstrated that functional markers of TIM-3+NK populations are altered after Galectin-9 ligation,including decreased Ki-67,CD 16,NKG2D,Perforin and Granzyme B expression.Conclusion:Galectin-9 is the TIM-3 homologous ligand that inhibits peripheral blood NK cells in HNSCC patients.Galectin-9 exhibits inhibition of TIM-3+NK cell population effector functions,including NK cell proliferation,activation and cytotoxic functions.However,there was no significant alteration in the cytokine secretion function of NK cells.Chapter 4 Galactin-9/TIM-3 interaction suppresses IL-2/STAT5 signaling in NK cellsObjectives:In the above study we found that Galectin-9 inhibits the effector function of NK cells by TIM-3 binding.However,the molecular mechanism of TIM-3 in NK cells remains unclear.Therefore,this chapter aims to investigate and characterize the role of Galectin-9/TIM-3 ligation in NK cell downstream signaling,which will be providing a new strategy of combination of ICR blocking therapy and targeting the signaling pathway in NK cells.Methods:The scRNAseq of NK cells from PBL and TIL of HNSCC was used to analyze the differences between TIM-3+and TIM-3-gene sets in the using enrichment analysis of the gene set through first top 100 DEGs.TIM-3siRNA was transfected into NK92 cells by Nucleofector electroporation system,and the surface expression of TIM-3 after transfection was detected by flow cytometry.Expression of downstream STAT5 and pSTAT5 were analyzed by western blotting after human recombinant Glaectin-9 or PBS treated TIM-3siRNA and NT(blank siRNA control)NK cells.Results:In comparison with the gene set enrichment analysis in TIM-3-NK cells,the gene set of TIM-3+NK cells exhibiting significant enrichment of the IL-2/STAT5 pathway in PBL,but it showed enrichment deficiency in tumor TIM-3+NK cells,suggesting that TIM-3+NK cells may be functionally impaired in the TME due to IL-2/STAT5 signaling deficiency.TIM-3siRNA and NT-transfected NK92 cells were treated with Galectin-9 or PBS in IL-2 stimulation,Galectin-9 was found to significantly inhibited phosphorylation of STAT5,a downstream signaling protein of IL-2,in TIM-3 normally expressing NK cells(Y694),while it had no effect on TIM-3siRNA NK cells.Conclusion:TIM-3+NK cells show a loss of IL-2/STAT5 signaling enrichment in tumor tissue.Galectin-9 inhibits IL-2-mediated STAT5 activity in NK cells in a TIM-3-dependent manner.