Understanding the matrix: collagen modifications in tumors and their implications for immunotherapy.

Tumors are highly complex and heterogenous ecosystems where malignant cells interact with healthy cells and the surrounding extracellular matrix (ECM). Solid tumors contain large ECM deposits that can constitute up to 60% of the tumor mass. This supports the survival and growth of cancerous cells and plays a critical role in the response to immune therapy. There is untapped potential in targeting the ECM and cell-ECM interactions to improve existing immune therapy and explore novel therapeutic strategies. The most abundant proteins in the ECM are the collagen family. There are 28 different collagen subtypes that can undergo several post-translational modifications (PTMs), which alter both their structure and functionality. Here, we review current knowledge on tumor collagen composition and the consequences of collagen PTMs affecting receptor binding, cell migration and tumor stiffness. Furthermore, we discuss how these alterations impact tumor immune responses and how collagen could be targeted to treat cancer.

Mechanosensing regulates tissue repair program in macrophages.

Tissue-resident macrophages play important roles in tissue homeostasis and repair. However, how macrophages monitor and maintain tissue integrity is not well understood. The extracellular matrix (ECM) is a key structural and organizational component of all tissues. Here, we find that macrophages sense the mechanical properties of the ECM to regulate a specific tissue repair program. We show that macrophage mechanosensing is mediated by cytoskeletal remodeling and can be performed in three-dimensional environments through a noncanonical, integrin-independent mechanism analogous to amoeboid migration. We find that these cytoskeletal dynamics also integrate biochemical signaling by colony-stimulating factor 1 and ultimately regulate chromatin accessibility to control the mechanosensitive gene expression program. This study identifies an "amoeboid" mode of ECM mechanosensing through which macrophages may regulate tissue repair and fibrosis.

Leukocyte-associated immunoglobulin-like receptor-1 blockade in combination with programmed death-ligand 1 targeting therapy mediates increased tumour control in mice.

Collagen expression and structure in the tumour microenvironment are associated with tumour development and therapy response. Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a widely expressed inhibitory collagen receptor. LAIR-2 is a soluble homologue of LAIR-1 that competes for collagen binding. Multiple studies in mice implicate blockade of LAIR-1:collagen interaction in cancer as a promising therapeutic strategy. Here, we investigated the role of LAIR-1 in anti-tumour responses. We show that although LAIR-1 inhibits activation, proliferation, and cytokine production of mouse T cells in vitro, tumour outgrowth in LAIR-1-deficient mice did not differ from wild type mice in several in vivo tumour models. Furthermore, treatment with NC410, a LAIR-2-Fc fusion protein, did not result in increased tumour clearance in tested immunocompetent mice, which contrasts with previous data in humanized mouse models. This discrepancy may be explained by our finding that NC410 blocks human LAIR-1:collagen interaction more effectively than mouse LAIR-1:collagen interaction. Despite the lack of therapeutic impact of NC410 monotherapy, mice treated with a combination of NC410 and anti-programmed death-ligand 1 did show reduced tumour burden and increased survival. Using LAIR-1-deficient mice, we showed that this effect seemed to be dependent on the presence of LAIR-1. Taken together, our data demonstrate that the absence of LAIR-1 signalling alone is not sufficient to control tumour growth in multiple immunocompetent mouse models. However, combined targeting of LAIR-1 and PD-L1 results in increased tumour control. Thus, additional targeting of the LAIR-1:collagen pathway with NC410 is a promising approach to treating tumours where conventional immunotherapy is ineffective.

Sensing context: Inhibitory receptors on non-hematopoietic cells.

Similar to immune cells, non-hematopoietic cells recognize microbial and endogenous threats. Their response to these stimuli is dependent on the environmental context. For example, intact intestinal epithelium expresses pattern recognition receptors (PRRs) but should tolerate commensal bacteria, while damaged epithelium should respond promptly to initiate an immune response. This indicates that non-hematopoietic cells possess mechanisms to sense environmental context and regulate their responses. Inhibitory receptors provide context sensing to immune cells. For instance, they raise the threshold for activation to prevent overzealous immune activation to harmless stimuli. Inhibitory receptors are typically studied on hematopoietic cells, but several of these receptors are expressed on non-hematopoietic cells. Here, we review evidence for the regulation of non-hematopoietic cells by inhibitory receptors, focusing on epithelial and endothelial cells. We explain that inhibitory receptors on these cells can sense a wide range of signals, including cell-cell adhesion, cell-matrix adhesion, and apoptotic cells. More importantly, they regulate various functions on these cells, including immune activation, proliferation, and migration. In conclusion, we propose that inhibitory receptors provide context to non-hematopoietic cells by fine tuning their response to endogenous or microbial stimuli. These findings prompt to investigate the functions of inhibitory receptors on non-hematopoietic cells more systematically.

Macrophages transfer mitochondria to sensory neurons to resolve inflammatory pain.

The current paradigm is that inflammatory pain passively resolves following the cessation of inflammation. Yet, in a substantial proportion of patients with inflammatory diseases, resolution of inflammation is not sufficient to resolve pain, resulting in chronic pain. Mechanistic insight into how inflammatory pain is resolved is lacking. Here, we show that macrophages actively control resolution of inflammatory pain remotely from the site of inflammation by transferring mitochondria to sensory neurons. During resolution of inflammatory pain in mice, M2-like macrophages infiltrate the dorsal root ganglia that contain the somata of sensory neurons, concurrent with the recovery of oxidative phosphorylation in sensory neurons. The resolution of pain and the transfer of mitochondria requires expression of CD200 receptor (CD200R) on macrophages and the non-canonical CD200R-ligand iSec1 on sensory neurons. Our data reveal a novel mechanism for active resolution of inflammatory pain.

Collagen Fragments Produced in Cancer Mediate T Cell Suppression Through Leukocyte-Associated Immunoglobulin-Like Receptor 1.

The tumor microenvironment (TME) is a complex structure comprised of tumor, immune and stromal cells, vasculature, and extracellular matrix (ECM). During tumor development, ECM homeostasis is dysregulated. Collagen remodeling by matrix metalloproteinases (MMPs) generates specific collagen fragments, that can be detected in the circulation of cancer patients and correlate with poor disease outcome. Leukocyte-Associated Immunoglobulin-like Receptor-1 (LAIR-1) is an inhibitory collagen receptor expressed on immune cells in the TME and in the circulation. We hypothesized that in addition to ECM collagen, collagen fragments produced in cancer can mediate T cell immunosuppression through LAIR-1. Our analyses of TCGA datasets show that cancer patients with high tumor mRNA expression of MMPs, collagen I and LAIR-1 have worse overall survival. We show that in vitro generated MMP1 or MMP9 collagen I fragments bind to and trigger LAIR-1. Importantly, LAIR-1 triggering by collagen I fragments inhibits CD3 signaling and IFN-γ secretion in a T cell line. LAIR-2 is a soluble homologue of LAIR-1 with higher affinity for collagen and thereby acts as a decoy receptor. Fc fusion proteins of LAIR-2 have potential as cancer immunotherapeutic agents and are currently being tested in clinical trials. We demonstrate that collagen fragment-induced inhibition of T cell function could be reversed by LAIR-2 fusion proteins. Overall, we show that collagen fragments produced in cancer can mediate T cell suppression through LAIR-1, potentially contributing to systemic immune suppression. Blocking the interaction of LAIR-1 with collagen fragments could be an added benefit of LAIR-1-directed immunotherapy.

Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction.

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1. LAIR-2 is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 expressed on immune cells, and that LAIR-2 releases LAIR-1-mediated immune suppression. Analysis of public human datasets shows that collagens, LAIR-1 and LAIR-2 have unique and overlapping associations with survival in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 increases human T cell expansion and effector function in vivo in a mouse xenogeneic-graft versus-host disease model. In humanized mouse tumor models, NC410 reduces tumor growth that is dependent on T cells. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a novel strategy for cancer immunotherapy for immune-excluded tumors.

Identification of a novel conserved signaling motif in CD200 receptor required for its inhibitory function.

The inhibitory signaling of CD200 receptor 1 (CD200R) has been attributed to its NPxY signaling motif. However, NPxY-motifs are present in multiple protein families and are mostly known to mediate protein trafficking between subcellular locations rather than signaling. Therefore, we investigated whether additional motifs specify the inhibitory function of CD200R. We performed phylogenetic analysis of the intracellular domain of CD200R in mammals, birds, bony fish, amphibians and reptiles. Indeed, the tyrosine of the NPxY-motif is fully conserved across species, in line with its central role in CD200R signaling. In contrast, P295 of the NPxY-motif is not conserved. Instead, a conserved stretch of negatively charged amino acids, EEDE279, and two conserved residues P285 and K292 in the flanking region prior to the NPxY-motif are required for CD200R mediated inhibition of p-Erk, p-Akt308, p-Akt473, p-rpS6 and LPS-induced IL-8 secretion. Altogether, we show that instead of the more common NPxY-motif, CD200R signaling can be assigned to a unique signaling motif in mammals defined by: EEDExxPYxxYxxKxNxxY.

Transcriptome of airway neutrophils reveals an interferon response in life-threatening respiratory syncytial virus infection.

BACKGROUND: Neutrophils are the most abundant cell type infiltrating the airways during severe respiratory syncytial virus (RSV) infection. Their exact role in disease pathophysiology remains enigmatic. Therefore, we determined genome-wide RNA expression profiles of local and systemic neutrophils in RSV bronchiolitis to provide further insight into local neutrophil biology. METHODS: We performed a single-center analysis, in 16 infants, admitted to the pediatric intensive care unit with severe RSV bronchiolitis. Neutrophils were isolated from blood and tracheobronchial aspirates (sputum). After low input RNA sequencing, differential expression of genes was determined followed by gene set analysis. RESULTS: Paired transcriptomic analysis of airway versus blood neutrophils showed an inflammatory phenotype, characterized by NF-kB signaling and upregulated expression of IL-6 and interferon pathways. We observed distinct expression of neutrophil activation genes (TNFSF13B, FCER1G). DISCUSSION: Our data indicate that airway neutrophils regulate their function at the transcriptional level in response to viral infection. It also suggests that local interferon drives the neutrophil response of severe RSV bronchiolitis.

Leukocyte Associated Immunoglobulin Like Receptor 1 Regulation and Function on Monocytes and Dendritic Cells During Inflammation.

Inhibitory receptors are crucial immune regulators and are essential to prevent exacerbated responses, thus contributing to immune homeostasis. Leukocyte associated immunoglobulin like receptor 1 (LAIR-1) is an immune inhibitory receptor which has collagen and collagen domain containing proteins as ligands. LAIR-1 is broadly expressed on immune cells and has a large availability of ligands in both circulation and tissues, implicating a need for tight regulation of this interaction. In the current study, we sought to examine the regulation and function of LAIR-1 on monocyte, dendritic cell (DC) and macrophage subtypes, using different in vitro models. We found that LAIR-1 is highly expressed on intermediate monocytes as well as on plasmacytoid DCs. LAIR-1 is also expressed on skin immune cells, mainly on tissue CD14+ cells, macrophages and CD1c+ DCs. In vitro, monocyte and type-2 conventional DC stimulation leads to LAIR-1 upregulation, which may reflect the importance of LAIR-1 as negative regulator under inflammatory conditions. Indeed, we demonstrate that LAIR-1 ligation on monocytes inhibits toll like receptor (TLR)4 and Interferon (IFN)-α- induced signals. Furthermore, LAIR-1 is downregulated on GM-CSF and IFN-γ monocyte-derived macrophages and monocyte-derived DCs. In addition, LAIR-1 triggering during monocyte derived-DC differentiation results in significant phenotypic changes, as well as a different response to TLR4 and IFN-α stimulation. This indicates a role for LAIR-1 in skewing DC function, which impacts the cytokine expression profile of these cells. In conclusion, we demonstrate that LAIR-1 is consistently upregulated on monocytes and DC during the inflammatory phase of the immune response and tends to restore its expression during the resolution phase. Under inflammatory conditions, LAIR-1 has an inhibitory function, pointing toward to a potential intervention opportunity targeting LAIR-1 in inflammatory conditions.

Functional categories of immune inhibitory receptors.

The human genome encodes more than 300 potential immune inhibitory receptors. The reason for this large number of receptors remains unclear. We suggest that inhibitory receptors operate as two distinct functional categories: receptors that control the signalling threshold for immune cell activation and receptors involved in the negative feedback of immune cell activation. These two categories have characteristic receptor expression patterns: 'threshold' receptors are expressed at steady state and their expression remains high or is downregulated upon activation, whereas 'negative feedback' receptors are induced upon immune cell activation. We use mathematical models to illustrate their possible modes of operation in different scenarios for different purposes. We discuss how this categorization may impact the choice of therapeutic targets for immunotherapy of malignant, infectious and autoimmune diseases.

LAIR-1 Limits Neutrophilic Airway Inflammation.

Neutrophils are crucial to antimicrobial defense, but excessive neutrophilic inflammation induces immune pathology. The mechanisms by which neutrophils are regulated to prevent injury and preserve tissue homeostasis are not completely understood. We recently identified the collagen receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1 as a functional inhibitory receptor on airway-infiltrated neutrophils in viral bronchiolitis patients. In the current study, we sought to examine the role of LAIR-1 in regulating airway neutrophil responses in vivo. LAIR-1-deficient (Lair1-/-) and wild-type mice were infected with respiratory syncytial virus (RSV) or exposed to cigarette smoke as commonly accepted models of neutrophil-driven lung inflammation. Mice were monitored for cellular airway influx, weight loss, cytokine production, and viral loads. After RSV infection, Lair1-/- mice show enhanced airway inflammation accompanied by increased neutrophil and lymphocyte recruitment to the airways, without effects on viral loads or cytokine production. LAIR-1-Fc administration in wild type mice, which blocks ligand induced LAIR-1 activation, augmented airway inflammation recapitulating the observations in Lair1-/- mice. Likewise, in the smoke-exposure model, LAIR-1 deficiency enhanced neutrophil recruitment to the airways and worsened disease severity. Intranasal CXCL1-mediated neutrophil recruitment to the airways was enhanced in mice lacking LAIR-1, supporting an intrinsic function of LAIR-1 on neutrophils. In conclusion, the immune inhibitory receptor LAIR-1 suppresses neutrophil tissue migration and acts as a negative regulator of neutrophil-driven airway inflammation during lung diseases. Following our recent observations in humans, this study provides crucial in-vivo evidence that LAIR-1 is a promising target for pharmacological intervention in such pathologies.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

The pro-tumor effect of CD200 expression is not mimicked by agonistic CD200R antibodies.

Tumor-infiltrating immune cells can impact tumor growth and progression. The inhibitory CD200 receptor (CD200R) suppresses the activation of myeloid cells and lack of this pathway results in a reduction of tumor growth, conversely a tumorigenic effect of CD200R triggering was also described. Here we investigated the role of CD200R activation in syngeneic mouse tumor models. We showed that agonistic CD200R antibody reached tumors, but had no significant impact on tumor growth and minor effect on infiltration of immune myeloid cells. These effects were reproduced using two different anti-CD200R clones. In contrast, we showed that CD200-deficiency did decrease melanoma tumor burden. The presence of either endogenous or tumor-expressed CD200 restored the growth of metastatic melanoma foci. On the basis of these findings, we conclude that blockade of the endogenous ligand CD200 prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, whereas agonistic anti-CD200R has no effect on tumor development.

Antitumor Activity of TLR7 Is Potentiated by CD200R Antibody Leading to Changes in the Tumor Microenvironment.

Stimulation of Toll-like receptor 7 (TLR7) activates myeloid cells and boosts the immune response. Previously, we have shown that stimulation of the inhibitory CD200 receptor (CD200R) suppresses TLR7 signaling and that the absence of CD200R signaling leads to a decreased number of papillomas in mice. Here, we investigated the effects of agonistic anti-CD200R on the antitumor activity of a TLR7 agonist (R848) in a syngeneic mouse tumor model. Intratumoral administration of R848 inhibited the growth of the CT26 colon carcinoma and simultaneously decreased CD200R expression in tumor-infiltrating immune cells. The antitumor effects of R848 were potentiated by anti-CD200R. Successfully treated mice were resistant to rechallenge with the same tumor cells. However, the immediate antitumor effects were independent of lymphocytes, because treatment efficacy was similar in wild-type and Rag1tm1Mom mice. Administration of R848, particularly in combination with anti-CD200R, changed the phenotype of intratumoral myeloid cells. The infiltration with immature MHC-II+ macrophages decreased and in parallel monocytes and immature MHC-II- macrophages increased. Combined treatment decreased the expression of the macrophage markers F4/80, CD206, CD86, CD115, and the ability to produce IL1ß, suggesting a shift in the composition of intratumor myeloid cells. Adoptively transferred CD11b+ myeloid cells, isolated from the tumors of mice treated with R848 and anti-CD200R, inhibited tumor outgrowth in recipient mice. We conclude that administration of agonistic anti-CD200R improves the antitumor effects of TLR7 signaling and changes the local tumor microenvironment, which becomes less supportive of tumor progression. Cancer Immunol Res; 6(8); 930-40. ©2018 AACR.

Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging.

A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.

Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets.

OBJECTIVE: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif-containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif-containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1-deficient mice. APPROACH AND RESULTS: Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1-deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI-specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI-FcRγ-chain and integrin αIIbß3 in megakaryocytes because of enhanced Src family kinase activity. CONCLUSIONS: Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein-coupled receptors.

Immune checkpoints and rheumatic diseases: what can cancer immunotherapy teach us?

The recent success of immune checkpoint blockade in cancer therapy illustrates the importance of the inhibitory receptors cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) in the regulation of antitumour immune responses. However, blocking signalling by these inhibitory immune checkpoint receptors is also associated with substantial inflammatory effects that can resemble autoimmune responses, which is consistent with the role of these receptors in protecting the host from excessive inflammation. The human genome encodes over 300 inhibitory receptors, which represent as many opportunities to modulate inflammation in a disease-specific and tissue-specific manner. We argue that rheumatologists and oncologists should join forces to study these inhibitory immune molecules. An improved understanding of these immune checkpoints will enable both fields to make progress in exploiting inhibitory immune receptors therapeutically. In this Review, we discuss data from studies reporting the adverse inflammatory effects of cancer therapies that target immune checkpoints. We discuss the potential implications of these findings on the biological understanding of autoimmune rheumatic diseases and highlight therapeutic strategies that could be used to target inhibitory receptors for the treatment of these conditions.

Fine-tuning neutrophil activation: Strategies and consequences.

In spite of their important role in host defense, neutrophils can also cause severe morbidity and mortality. Neutrophils have an extensive armory necessary to eradicate pathogens, but neutrophil infiltration and activation also induces major tissue injury associated with acute and chronic inflammatory disorders. Here, we review neutrophil anti-microbial functions and discuss their individual contribution to disease pathogenesis. Furthermore, we provide an overview of the anti-inflammatory drugs that can dampen neutrophil transmigration, elastase activity, and the production of reactive oxygen species which are already in clinical trials. The discovery of potential inhibitors of the release of neutrophil extracellular trap is still in its infancy. Here, we discuss small molecule inhibitors and inhibitory receptors that show promising results in reducing neutrophil extracellular trap formation in vitro and in vivo and discuss the advantages and drawbacks of inhibiting the release of neutrophil extracellular traps as a therapeutic treatment. Specific suppression of neutrophil extracellular trap formation, preferably while other antimicrobial functions are preserved, would be an ideal approach to treat neutrophilic inflammation, since it prevents acute as well as chronic neutrophil-associated pathology.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.