Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I. If self-MHC is down-regulated or absent, lack of inhibition triggers “missing self” killing. ...NK cells developing in the absence of MHC class I are hypo-responsive, demonstrating that MHC class I molecules are required for NK-cell education. Here, we show that the number and the type of MHC class I alleles that are present during NK-cell education quantitatively determine the frequency of responding NK cells, the number of effector functions in individual NK cells, and the amount of interferon-γ production in NK cells of specific Ly49 subsets. A relationship between the extent of inhibitory signals during education and functional responsiveness was corroborated by an enhanced probability of NK cells expressing more than one inhibitory receptor for a single host self–MHC class I allele to degranulate after activation. Our data suggest that the capacity of an individual NK cell to respond to stimulation is quantitatively controlled by the extent of inhibitory signals that are received from MHC class I molecules during NK-cell education.
The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) ...molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell’s surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.
DNAX accessory molecule-1 (DNAM-1, also known as CD226) is an activating receptor expressed on subsets of natural killer (NK) and T cells, interacts with its ligands CD155 or CD112, and has co-varied ...expression with inhibitory receptors. Since inhibitory receptors control NK-cell activation and are necessary for MHC-I-dependent education, we investigated whether DNAM-1 expression is also involved in NK-cell education. Here we show an MHC-I-dependent correlation between DNAM-1 expression and NK-cell education, and an association between DNAM-1 and NKG2A that occurs even in MHC class I deficient mice. DNAM-1 is expressed early during NK-cell development, precedes the expression of MHC-I-specific inhibitory receptors, and is modulated in an education-dependent fashion. Cd226
mice have missing self-responses and NK cells with a normal receptor repertoire. We propose a model in which NK-cell education prevents or delays downregulation of DNAM-1. This molecule endows educated NK cells with enhanced effector functions but is dispensable for education.
NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions ...are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.
An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype ...and functions of NK cells from tumour-infiltrated (TILN) and tumour-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56dim CD57dim CD69+CCR7+KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients, the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared with healthy subjects, and blood NK cells also express the receptors for CCL2 and IL-6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset.
Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells ...with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.
Checkpoint inhibitors have been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients demonstrate a durable clinical response. PD-L1 scoring is ...currently the only biomarker measure routinely used to select patients for immunotherapy, but its predictive accuracy is modest. The aim of our study was to evaluate a proteomic assay for the analysis of patient plasma in the context of immunotherapy. Pretreatment plasma samples from 43 NSCLC patients who received anti-PD-(L)1 therapy were analyzed using a proximity extension assay (PEA) to quantify 92 different immune oncology-related proteins. The plasma protein levels were associated with clinical and histopathological parameters, as well as therapy response and survival. Unsupervised hierarchical cluster analysis revealed two patient groups with distinct protein profiles associated with high and low immune protein levels, designated as “hot” and “cold”. Further supervised cluster analysis based on T-cell activation markers showed that higher levels of T-cell activation markers were associated with longer progression-free survival (PFS) (p < 0.01). The analysis of single proteins revealed that high plasma levels of CXCL9 and CXCL10 and low ADA levels were associated with better response and prolonged PFS (p < 0.05). Moreover, in an explorative response prediction model, the combination of protein markers (CXCL9, CXCL10, IL-15, CASP8, and ADA) resulted in higher accuracy in predicting response than tumor PD-L1 expression or each protein assayed individually. Our findings demonstrate a proof of concept for the use of multiplex plasma protein levels as a tool for anti-PD-(L)1 response prediction in NSCLC. Additionally, we identified protein signatures that could predict the response to anti-PD-(L)1 therapy.
Escape of tumor cells from cell-intrinsic barrier mediated by tumor suppressors and cell-extrinsic barrier mediated by the immune system is crucial for tumorigenesis. Growing evidence suggests that ...reactivation of tumor suppressor function or restoration of anticancer immunity is promising strategy for anticancer therapy due to their high potential to combat cancer. p53, a key tumor suppressor, represses tumorigenesis by eliciting growth arrest, apoptosis or senescence in cancer cells. Here, we unravel that, apart from these cell-autonomous effects, p53 activates the innate immune response against cancer cells. Our results show that pharmacological reactivation of p53 can stimulate the expression of ULPB2, a ligand for NK cell activating receptor NKG2D in human tumor cells of different origin, which enhance the susceptibility of tumor cells to NK cell-mediated killing. The molecular mechanism controlling ULPB2 expression by p53 is neither ATM/ATR- nor caspase-dependent. Using several approaches, we identified p53 as a direct transcriptional regulator of ULBP2 and found a p53 response element within ULBP2 gene, which confers the p53 regulation. Furthermore, we demonstrated that demethylation of p53-binding region within ULBP2 gene was required for p53-dependent induction of ULPB2, which can be achieved via repression of DNA methyltransferases (DNMTs) by p53. This molecular evidence for the direct control of immunosurveillance by p53 links tumor suppressor activation to innate immune stimuli and provides a possibility to integrate cell-extrinsic and -intrinsic defenses against tumorigenesis by pharmacological activation of p53, which may increase the probability to achieve a durable therapeutic success.
Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK‐cell subsets ...and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti‐CTLA‐4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL‐15 stimulation improved NK cell‐mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56bright and CD56dim NK subsets and increased serum concentrations of the cytokines IL‐10, IL‐8 and TNF‐α. After tremelimumab treatment, the ratio between the CD56bright and CD56dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM‐3+CD8+ T‐cell frequency, high DNAM‐1+CD56dim NK‐cell frequency and high expression levels of NKp46 on the CD56dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.
What's new?
Treatment options for malignant mesothelioma (MM) have not significantly improved over the past four decades. A better understanding of the immune response in MM might guide new treatment strategies. In this study, the authors found evidence that natural killer (NK) cells can infiltrate, recognize, and kill MM cells. However, MM patients have distinct alterations in NK, CD8+ T‐cell, and cytokine profiles. Some of these alterations correlated with prognosis, or were affected by treatment with tremelimumab.
Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial immune phenotypes in the mutational and ...clinicopathological background of non–small cell lung cancer (NSCLC).
We established a multiplexed fluorescence imaging pipeline to spatially quantify 13 immune cell subsets in 359 NSCLC cases: CD4 effector cells (CD4-Eff), CD4 regulatory cells (CD4-Treg), CD8 effector cells (CD8-Eff), CD8 regulatory cells (CD8-Treg), B-cells, natural killer cells, natural killer T-cells, M1 macrophages (M1), CD163+ myeloid cells (CD163), M2 macrophages (M2), immature dendritic cells (iDCs), mature dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs).
CD4-Eff cells, CD8-Eff cells and M1 macrophages were the most abundant immune cells invading the tumour cell compartment and indicated a patient group with a favourable prognosis in the cluster analysis. Likewise, single densities of lymphocytic subsets (CD4-Eff, CD4-Treg, CD8-Treg, B-cells and pDCs) were independently associated with longer survival. However, when these immune cells were located close to CD8-Treg cells, the favourable impact was attenuated. In the multivariable Cox regression model, including cell densities and distances, the densities of M1 and CD163 cells and distances between cells (CD8-Treg–B-cells, CD8-Eff–cancer cells and B-cells–CD4-Treg) demonstrated positive prognostic impact, whereas short M2–M1 distances were prognostically unfavourable.
We present a unique spatial profile of the in situ immune cell landscape in NSCLC as a publicly available data set. Cell densities and cell distances contribute independently to prognostic information on clinical outcomes, suggesting that spatial information is crucial for diagnostic use.
•Immune cells showed specific infiltration patterns within the tumour microenvironment.•Infiltration of lymphocytes and plasmacytoid dendritic cells are associated with good prognosis.•Distances of immune cells to each other or to tumour cells reveal an independent prognostic impact.
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