Innate lymphoid cells: NK and cytotoxic ILC3 subsets infiltrate metastatic breast cancer lymph nodes.

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.

Specific Patterns of Blood ILCs in Metastatic Melanoma Patients and Their Modulations in Response to Immunotherapy.

Immunotherapy targeting immune checkpoint receptors brought a breakthrough in the treatment of metastatic melanoma patients. However, a number of patients still resist these immunotherapies. Present on CD8+T cells, immune checkpoint receptors are expressed by innate lymphoid cells (ILCs), which may contribute to the clinical response. ILCs are composed of natural killer (NK) cells, which are cytotoxic effectors involved in tumor immunosurveillance. NK cell activation is regulated by a balance between activating receptors that detect stress molecules on tumor cells and HLA-I-specific inhibitory receptors. Helper ILCs (h-ILCs) are newly characterized ILCs that secrete cytokines and regulate the immune homeostasis of tissue. We investigated the modulation of blood ILCs in melanoma patients treated with ipilimumab. Circulating ILCs from metastatic stage IV melanoma patients and healthy donors were studied for their complete phenotypic status. Patients were studied before and at 3, 6, and 12 weeks of ipilimumab treatment. A comparison of blood ILC populations from donors and melanoma patients before treatment showed changes in proportions of ILC subsets, and a significant inverse correlation of CD56dim NK cells and h-ILC subsets was identified in patients. During treatment with ipilimumab, percentages of all ILC subsets were reduced. Ipilimumab also impacted the expression of the CD96/TIGIT/DNAM-1 pathway in all ILCs and increased CD161 and CTLA-4 expression by h-ILCs. When considering the response to the treatment, patients without disease control were characterized by higher percentages of CD56bright NK cells and ILC1. Patients with disease control displayed larger populations of activated CD56dimCD16+ DNAM-1+ NK cells, while anergic CD56dimCD16-DNAM-1- NK cells were prominent in patients without disease control. These results provide original findings on the distribution of ILC subsets in advanced melanoma patients and their modulation through immunotherapy. The effects of ipilimumab on these ILC subsets may critically influence therapeutic outcomes. These data indicate the importance of considering these innate cell subsets in immunotherapeutic strategies for melanoma patients.

BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis.

BACKGROUND: Targeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine-threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a BRAFV600E/K mutation. However, acquired resistance occurs in the majority of patients, leading to relapse. Immunotherapies that activate immune cytotoxic effectors induce long-lasting responses in 30% of patients. In that context, combination of targeted therapies with immunotherapy (IT) is a promising approach. We considered boosting natural killer (NK) cell tumor immunosurveillance, as melanoma cells express stress-induced molecules and activate NK cell lysis. METHODS: Here we have generated vemurafenib (a BRAF inihibitor)-resistant (R) cells from BRAFV600E SK28 and M14-sensitive (S) melanoma cell lines and investigated how resistance interferes with immunogenicity to NK cells. We determined the levels of several soluble molecules including NK ligands in 61 melanoma patients at baseline and 6 months M post-treatment with targeted therapies or immunotherapies. RESULTS: Vemurafenib resistance involved activation of p-AKT in SK28R and of p-MEK/p-ERK in M14R cells and was accompanied by modulation of NK ligands. Compared with S cells, SK28R displayed an increased expression of natural killer group 2 D (NKG2D) receptor ligands (major histocompatibility complex class (MHC) I chain-related protein A (MICA) and UL16-binding protein 2 (ULBP2)) whereas M14R exhibited decreased ULBP2 . SK28R and M14R cells induced higher NK degranulation and interferon gamma secretion and were more efficiently lysed by donor and patient NK cells. SK28R showed increased tumor necrosis factor-related apoptosis-inducing ligand receptor II (TRAIL-RII) expression and TRAIL-induced apoptosis, and TRAIL-induced apoptosis of M14R was decreased. Combined BRAF/MEK inhibitors abrogated the growth of SK28S, M14S, and M14R cells, while growth of SK28R was maintained. BRAF/MEK inhibition attenuated NK activity but R cell lines activated polyfunctional NK cells and were lysed with high efficiency. We investigated the relationship of soluble NK ligands and response to treatment in a series of melanoma patients. Soluble NKG2D ligands known to regulate the receptor function have been associated to cancer progression. Serum analysis of patients treated with target therapies or IT indicates that soluble forms of NK ligands (MICA, B7H6, programmed cell death ligand 1, and carcinoembryonic antigen cell adhesion molecule 1) may correlate with clinical response. CONCLUSION: These results support strategies combining targeted therapies and NK-based immunotherapies.

Two-dimensional dynamic evaluation of natural killer cell-mediated lysis of adherent target cells.

Cell-mediated cytotoxicity is a major function of cytotoxic lymphocytes. The cytotoxic function of immune effectors requires accurate and sensitive quantification as tumor immunotherapies are actively developed to treat growing types of tumors. Various methods have been developed to quantify this function. A first approach consists in measuring the externalization of LAMP-1 (lysosomal associated membrane 1), CD107a molecules transitory expressed at the cell surface by degranulating cytotoxic cells and is determined by flow cytometry. The focus of the chapter concerns the second approach that quantifies target cell lysis resulting from the close contact interaction with cytotoxic Natural Killer (NK) lymphocytes. For long time, target cell lysis was evaluated by chromium release assay, requiring use of radioactive labeled salt (51Cr) and specific devices not compatible with repeated tests performed for immunomonitoring of patients. Other methods include fluorimetric and bioluminescence assays. Monitoring immune cell-mediated lysis of adherent targets by the dynamic measure of cell proliferation using the Real time cell analyzer (RTCA) is a good alternative. The test relies on sensitive dynamic measure of cell index values during their interactions with cytotoxic immune effectors. The cell index increasing with cell proliferation rapidly drops in presence of immune cells. The lysis is quantified in reference with targets alone and expressed as percentages of lysis curves. Moreover, the dynamic monitoring of target cell index allows the evaluation of drugs, cytokines, mAbs on target cell lysis. Here we describe a robust and sensitive method for quantification of immune cell-mediated lysis of adherent targets.

NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment.

The antitumor functions of NK cells are regulated by the integration of positive and negative signals triggered by numerous membrane receptors present on the NK cells themselves. Among the main activating receptors, NKG2D binds several stress-induced molecules on tumor targets. Engagement of NKG2D by its ligands (NKG2D-Ls) induces NK cell activation leading to production of cytokines and target cell lysis. These effects have therapeutic potential as NKG2D-Ls are widely expressed by solid tumors, whereas their expression in healthy cells is limited. Here, we describe the genetic and environmental factors regulating the NKG2D/NKG2D-L pathway in tumors. NKG2D-L expression is linked to cellular stress and cell proliferation, and has been associated with oncogenic mutations. Tumors have been found to alter their to NKG2D-L expression as they progress, which interferes with the antitumor function of the pathway. Nevertheless, this pathway could be advantageously exploited for cancer therapy. Various cancer treatments, including chemotherapy and targeted therapies, indirectly interfere with the cellular and soluble forms of NKG2D-Ls. In addition, NKG2D introduced into chimeric antigen receptors in T- and NK cells is a promising tumor immunotherapy approach.