LAG3 is a Central Regulator of NK Cell Cytokine Production

Abstract

25Natural killer (NK) cells are innate effectors, which play a crucial role in controlling 26 viral infections. Administration of IFN-α has shown promising results as a therapeutic, 27 controlling HIV, and chronic viral hepatitis. However the downstream mechanisms by which 28 IFN-α mediates its anti-viral effects is largely unknown. In this investigation, we evaluated 29 the impact of IFN-α on peripheral blood NK cells from healthy donors. High dimensional 30 flow cytometry analysis of NK cell surface receptors following exposure to IFN-α showed an 31 increased expression of the check point inhibitor LAG3. Further characterization revealed 32 that LAG3 was expressed in a subset of NK cells with high expression of activation and 33 maturation markers. Assessment of metabolic pathways showed that LAG3+ NK cells had 34 enhanced rates of glycolysis and glycolytic capacity, suggesting that it is a primed effector 35 subset with enhanced glucose metabolism. Inhibition of LAG3 on NK cells using antibody in 36 vitro resulted in a profound increase in secretion of cytokines IFN-γ, TNF-α, MIP-1α and 37 MIP-1β, without affecting the cytotoxic activity. Taken together, these results showed that 38 LAG3 is a negative regulator of cytokine production by mature NK cells. 39 40 Introduction: 41 2 The plasmacytoid (pDC)-Natural Killer (NK) cell axis acts as a first line of defence by the 42 host against viral infections. pDCs secrete type I interferons upon recognizing virus-43 associated pathogen associated molecular patterns (PAMPs) through pattern recognition 44 receptors (PRRs). Type I interferons (IFN-I) bind to IFN (IFN-/) receptors on the surface 45 of NK cells to prime, activate and initiate mechanisms for the destruction of infected cells. 46 The subsequent stimulation of the IFN signalling pathway results in the increased expression 47 65 expression of LAG3, an inhibitory receptor, commonly associated with T cell exhaustion in 66 chronic infections and cancer. To date, the function of LAG3 in NK cells is not well 67 understood due to contrasting results from mouse and human NK cells. While studies using 68 knockout mouse models suggested a positive role for LAG3 in NK cell cytotoxicity, these 69 could not be successfully reproduced in human NK cells (10,11). High dimensional flow 70 cytometry analysis in these studies revealed that LAG3 expression marks a subset of mature 71 activated NK cells. Metabolic assays additionally showed that NK cells expressing LAG3 72 have enhanced glycolytic activity. Blockade of LAG3 enhanced cytokine production by 73 activated NK cells without altering their cytotoxicity. Taken together, these results 74 demonstrate that LAG3 is a negative regulator of cytokine production and a potential 75 therapeutic target for chronic viral infections. 76 77 78 3 Materials and methods: 79 Isolation and stimulation of NK cells: Blood samples from healthy volunteer donors were 80 obtained after approval by the National Healthcare Group Domain Specific Review Board, 81 Singapore (NHG DSRB Ref: 2000/0...

Artificial intelligence-based analysis for immunohistochemistry staining of immune checkpoints to predict resected non-small cell lung cancer survival and relapse

“…The function of LAG-3 on NK cells was not well investigated and controversial. However, a factual finding was that inhibition of LAG-3 could increase the secretion of interferon-gamma (IFN-γ), tumor necrosis factoralpha (TNF-α), macrophage inflammatory proteins-1 alpha (MIP-1α), MIP-1β, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (44). In this way, the expression of LAG-3 could collaborate with the inhibition of NK cell functions from KIR2D.…”

Enhancing a Natural Killer: Modification of NK Cells for Cancer Immunotherapy

“…However, how relevant they are for NK cells remains to be investigated and the significance of LAG-3 expression in NK cells remains largely unclear [ 70 ]. Recently, it was shown that LAG-3 is preferentially expressed on CD56 dim NK cells after IFN-α treatment and acts specifically as a negative regulator of cytokine production without compromising cytotoxic function [ 93 ]. Another candidate, TNF receptor family member, OX40 is expressed on leukemic blasts in a substantial percentage of patients with AML and promotes activation and proliferation of T cells.…”

Targeting NK Cell Inhibitory Receptors for Precision Multiple Myeloma Immunotherapy

“…A more recent analysis of the status of LAG-3 on the NK cell surface following exposure to IFN-α demonstrated an increased expression of LAG3 ( 239 ). This paper proposes more studies on the impact of other cytokines on these IRs, and questions whether a single cytokine or a group of them cooperate and upregulate the IR, and/or one cytokine triggers one or array of other chemokines.…”

“…Further characterization showed that LAG3 was expressed in the NK cells populations that show high expression of activation and maturation markers. Additionally, in-vitro LAG3 blocking on NK cells using mAb led to an increase in the production of cytokines IFN-γ, TNF-α, MIP-1α and MIP-1β, without affecting the cytotoxic activity, which suggests that LAG3 is a negative regulator of cytokine production by mature NK cells ( 239 ).…”

“…Specifically, studies have shown that expression of ligands such as PVR, PD-L1 can be enhanced by chemotherapy and/or IFN-γ ( 191 , 192 , 243 ). Similarly, NK cell contact with malignant MM cells was shown to enhance expression of PD-1 and CD94 by a process called trogocytosis ( 67 , 214 ) and LAG-3 due to the exposure to the IFN-α ( 239 ). Recent studies also show a shift in NK cell populations during MM disease progression from MGUS to active MM ( 66 , 67 ).…”