Among natural killer (NK) cell receptors, the T-cell immunoglobulin and mucin-containing domain (TIM-3) has been associated with both inhibitory and activating functions, depending on context and activation pathway. Ex vivo and in vitro, expression of TIM-3 is inducible and depends on activation stimulus. Here, we report that TIM-3 expression can be downregulated on NK cells under specific conditions. When NK cells are exposed to cancer targets, they synergize with stimulation conditions to induce a substantial decrease in TIM-3 expression on their surface. We found that such downregulation occurs following prior NK activation. Downregulated TIM-3 expression correlated to lower cytotoxicity and lower interferon gamma (IFN-γ) expression, fueling the notion that TIM-3 might function as a benchmark for human NK cell dysfunction.

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NK cell trafficking and migration into solid tumors is often poor. Altered NK cell metabolism in the tumor microenvironment has been correlated with tumor invasiveness, metastasis and cellular dysfunction. Tumorinduced stresses correlate to limited numbers of functional NK cells in solid tumors and, consequently, limited survival. Metabolic impairment of NK cells in cancer is still not fully understood, but the involvement of inhibitory checkpoints-both tumor-associated as well as NK-specificplays a significant role in driving these responses. The metabolic reprogramming events that NK cells undergo in the tumor microenvironment are linked to their ability to support energy metabolism in scenarios of impaired glycolytic fueling and low oxygen. The contributions of inhibitory checkpoints to these events are not entirely known, but are emerging as increasingly significant to the restoration of NK cell function. Here, we discuss our understanding of the role of inhibitory receptors and checkpoints in the metabolic dysfunction of NK cells in solid tumors, as a critical key to the development of new immunotherapies.

BackgroundNatural killer (NK) cells are part of the innate immune system, but are capable of participating in both innate and adaptive immune responses due to their wide range of cytolytic activities, from degranulation, secretion of cytokines to antibody-dependent cell-mediated cytotoxicity. These are possible due to the cells’ ability to recognize self and non-self-entities via the net signal generated from their activating and inhibitory receptors upon engagement. TIM-3 is a part of the NK receptor repertoire, expressed commonly on different lymphocytes. In T cells, TIM-3 is established as an inhibitory marker. However, in NK cells, the role of TIM-3 could be agonistic or antagonistic to NK cytotoxicity based on the disease type and activation status, though limited information is known about its role in cancer and its correlation to NK cell effector functions.MethodsWe measured TIM-3 expression upon activation of human NK cells under various conditions. NK cells were isolated from peripheral blood of healthy donors and expanded either in K562-based feeder media or feeder-free OpTmizerTM media. After expansion, they were co-cultured for 4 hours with patient-derived glioblastoma multiforme cells (GBM43) at effector:target ratios of 2.5:1 and 10:1. To evaluate the effect of TIM-3 expression on NK cells, 7AAD/CFSE killing assays, CD107a degranulation and IFNγ secretion assays were carried out while blocking TIM-3 with neutralizing antibodies. Bioinformatics analysis of GBM patient RNAseq data was carried out to correlate TIM-3 expression with in vivo function, and this analysis is supplemented by phenotyping TIM-3 on NK cells isolated from patient samples in order to infer the role of this receptor in GBM.ResultsWe found that TIM-3 was downregulated on OpTmizerTM -cultured NK cells once exposed to cancer targets, and this correlated to a decreased in NK killing capacity when compared to feeder media-cultured NK cells, where the downregulation was not observed. Culturing NK cells in different derivatives of both media suggested that a combination of serum and cytokines can induce TIM-3 expression change to cancer targets. Flow cytometric assays revealed that while degranulation remained the same, the decreased in cytotoxicity corresponded to a decrease in IFNγ secretion. In GBM patient datasets, TIM-3 expression correlates to high IFN-γ levels and associates with both pro- and anti-tumorigenic functions. Here, we report a new role of TIM-3 in modulating NK functionality by correlating its loss to a loss in NK cell effector functions, and how its expression can be modified by ex vivo activation.ConclusionsTIM-3 expression on NK cells can be induced by ex vivo expansion, and this change in expression could influence NK cytotoxicity and cytokine secretion. Our data suggested that TIM-3 is not necessarily an inhibitory marker in GBM, and more likely to be a status marker or an activation limiter, working in conjunction with other receptors to modulate NK cell anti-tumor responses.Ethics ApprovalThis study was approved by Purdue Intuition’s Ethics Board, approval number [1804020540].

Background Natural killer (NK) cells hold potential as one of the next generation adoptive cell therapy candidates due to their allogenecity, capability to lyse target without prior sensitization, and low risk of host-versus-graft disease. Thus, being able to utilize NK cell receptors to drive their cytotoxicity remains crucial. Additionally, for treatments of solid tumors, NK cells need to retain their viability against the suppressive tumor microenvironment. One method to modulate NK cell immunometabolism is to leverage the surface receptor TIM-3, which lies at the intersection of NK cytotoxic functions and metabolismthe mTOR-associated pathways. Methods NK cells were isolated from healthy adult peripheral blood, and expanded in K562-based feeder media. Flow cytometry was used to phenotype TIM-3 expression and phosphorylation of mTOR, Akt, and ribosomal protein S6 (rpS6). CCK-8 assay was used to assess mTOR inhibition against patient-derived glioblastoma cells (GBM43). NK co-operative activities with mTOR inhibitors against GBM43 were evaluated through target lysis, degranulation, and cytokine secretion. TIM-3 knock-out (TIM-3 KO) NK cells were generated by electroporating CRISPR/Cas9 ribonucleopreotein complexes. Results We first explored the link between TIM-3 expression on NK cells and mTOR proteins, including mTOR itself, rpS6, and Akt, which were linked to TIM-3 regulation in T cells. Previously, we had reported that TIM-3 downregulation was associated with lower IFN-g production. As changes in phosphorylation of rpS6 was linked to lower IFN-g mRNA levels, we hypothesized that TIM-3 expression leads to increased rpS6 phosphorylation, thus encouraging higher IFNg production. Here, we report changes in phosphorylation levels of Akt and rpS6 as consequences of TIM-3 KO, and how these changes can affect NK cell cytotoxicity against GBM43. We also cross-examine these findings using specific pharmacological inhibitors of mTOR, including rapamycin, JR-AB2-011, and torin-1, against mTORC1, mTORC2, and both complexes, respectively, to demonstrate that TIM-3 expression could provide some functional protection against activities these inhibitors through retention of NK cell activity. Lastly, to show that these inhibitions would not compromise NK cell functions, we show GBM43 lysis by NK cells with mTOR inhibitor adjuvants. Conclusions We have found that TIM-3 expression can exert some control over the mTOR pathway on ex vivo NK cells, and this affects NK cells' cytokine production capacity. Since the tumor microenvironment is highly immunosuppressive, the protection against mTOR inhibition by TIM-3 allowed tumorinfiltrating NK cells to remain metabolically and functionally viable, and affords an opportunity to leverage mTOR inhibitors as adjuvants with NK cell therapy against GBM.