B7H6 is the predominant activating ligand driving natural killer cell-mediated killing in patients with liquid tumours: evidence from clinical, in silico, in vitro, and in vivo studies.
Natural killer (NK) cells are a subset of innate lymphoid cells that are inherently capable of recognizing and killing infected or tumour cells. This has positioned NK cells as a promising live drug for tumour immunotherapy, but limited success suggests incomplete knowledge of their killing mechanism. NK cell-mediated killing involves a complex decision-making process based on integrating activating and inhibitory signals from various ligand-receptor repertoires. However, the relative importance of the different activating ligand-receptor interactions in triggering NK killing remains unclear.
We employed a systematic approach combining clinical, in silico, in vitro, and in vivo data analysis to quantify the impact of various activating ligands. Clinical data analysis was conducted using massive pan-cancer data (n = 10,595), where patients with high NK cell levels were stratified using CIBERSORT. Subsequently, multivariate Cox regression and Kaplan-Meier (KM) survival analysis were performed based on activating ligand expression. To examine the impact of ligand expression on NK killing at the cellular level, we assessed surface expression of five major activating ligands (B7H6, MICA/B, ULBP1, ULBP2/5/6, and ULBP3) of human tumour cell lines of diverse origins (n = 33) via flow cytometry (FACs) and their NK cell-mediated cytotoxicity on by calcein-AM assay using human primary NK cells and NK-92 cell lines. Based on this data, we quantified the contribution of each activating ligand to the NK killing activity using mathematical models and Bayesian statistics. To further validate the results, we performed calcein-AM assays upon ligand knockdown and overexpression, conjugation assays, and co-culture assays in activating ligand-downregulated/overexpressed in liquid tumour (LT) cell lines. Moreover, we established LT-xenograft mouse models to assess the efficacy of NK cell targeting toward tumours with dominant ligands.
Through the clinical analysis, we discovered that among nearly all 18 activating ligands, only patients with LT who were NK cell-rich and specifically had higher B7H6 level exhibited a favorable survival outcome (p = 0.0069). This unexpected dominant role of B7H6 was further confirmed by the analysis of datasets encompassing multiple ligands and a variety of tumours, which showed that B7H6 exhibited the highest contribution to NK killing among five representative ligands. Furthermore, LT cell lines (acute myeloid leukemia (AML), B cell lymphoma, and T-acute lymphocytic leukemia (ALL)) with lowered B7H6 demonstrated decreased susceptibility to NK cell-mediated cytotoxicity compared to those with higher levels. Even within the same cell line, NK cells selectively targeted cells with higher B7H6 levels. Finally, LT-xenograft mouse models (n = 24) confirmed that higher B7H6 results in less tumour burden and longer survival in NK cell-treated LT mice (p = 0.0022).
While NK cells have gained attention for their potent anti-tumour effects without causing graft-versus-host disease (GvHD), thus making them a promising off-the-shelf therapy, our limited understanding of NK killing mechanisms has hindered their clinical application. This study illuminates the crucial role of the activating ligand B7H6 in driving NK cell killing, particularly in the context of LT. Therefore, the expression level of B7H6 could serve as a prognostic marker for patients with LT. Moreover, for the development of NK cell-based immunotherapy, focusing on increasing the level of B7H6 on its cognate receptor, NKp30, could be the most effective strategy.
This work was supported by the National Research Council of Science & Technology (NST) grant (CAP-18-02-KRIBB, GTL24021-000), a National Research Foundation grant (2710012258, 2710004815), and an Institute for Basic Science grant (IBS-R029-C3).
Lee S
，Chae SJ
，Jang IH
，Oh SC
，Kim SM
，Lee SY
，Kim JH
，Ko J
，Kim HJ
，Song IC
，Kim JK
，Kim TD
... -
《EBioMedicine》
Differential impact of TIM-3 ligands on NK cell function.
The transmembrane protein T-cell immunoglobulin and mucin-domain containing molecule 3 (TIM-3) is an immune checkpoint receptor that is expressed by a variety of leukocyte subsets, particularly in the tumor microenvironment. An effective TIM-3-targeting therapy should account for multiple biological factors, including the disease setting, the specific cell types involved and their varying sensitivities to the four putative TIM-3 ligands (galectin-9, phosphatidylserine, high mobility group protein B1 and carcinoembryonic antigen cell adhesion molecule 1), each of which engages a unique binding site on the receptor's variable immunoglobulin domain. The primary objectives of this study were to assess the prevalence and function of TIM-3+ natural killer (NK) cells in patients with head and neck squamous cell carcinoma (HNSCC), determine whether the four TIM-3 ligands differentially affect TIM-3+ NK cell functions, identify the most immunosuppressive ligand, and evaluate whether targeting ligand-mediated TIM-3 signaling enhances NK cell effector functions.
Single-cell RNA sequencing and flow cytometry were used to study the prevalence, phenotypes and function of TIM-3+ NK cells in HNSCC patient tumors and blood. In vitro killing, proliferation and cytokine production assays were implemented to evaluate whether the four TIM-3 ligands differentially modulate TIM-3+ NK cell functions, and whether disruption of TIM-3/ligand interaction can enhance NK cell-mediated antitumor effector mechanisms. Finally, The Cancer Genome Atlas survival analysis and digital spatial profiling were employed to study the potential impact of etiology-associated differences on patients with HNSCC outcomes.
We demonstrate that TIM-3 is highly prevalent on circulating and tumor-infiltrating NK cells. It co-expresses with CD44 and marks NK cells with heightened effector potential. Among the four putative TIM-3 ligands, galectin-9 most consistently suppresses NK cell-mediated cytotoxicity and proliferation through TIM-3 and CD44 signaling, respectively, but promotes IFN-γ release in a TIM-3-dependent manner. Among patients with HNSCC, an elevated intratumoral TIM-3+ NK cell gene signature associates with worse outcomes, specifically in those with human papillomavirus (HPV)+ disease, potentially attributable to higher galectin-9 levels in HPV+ versus HPV- patients.
Our findings underscore the complex functional impact of TIM-3 ligand signaling, which is consistent with recent clinical trials suggesting that targeting TIM-3 alone is suboptimal as an immunotherapeutic approach for treating malignancies.
Wang J
，Li H
，Kulkarni A
，Anderson JL
，Upadhyay P
，Onyekachi OV
，Arantes LMRB
，Banerjee H
，Kane LP
，Zhang X
，Bruno TC
，Bao R
，Ferris RL
，Vujanovic L
... -
《Journal for ImmunoTherapy of Cancer》
Construction of self-driving anti-αFR CAR-engineered NK cells based on IFN-γ and TNF-α synergistically induced high expression of CXCL10.
Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells.
To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer.
Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo.
RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue.
This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer.
He M
，Ao X
，Yang Y
，Xu Y
，Liu T
，Ao L
，Guo W
，Xing W
，Xu J
，Qian C
，Yu J
，Xu X
，Yi P
... -
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