CAR-NK Cells: From Natural Basis to Design for Kill.

Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an 'off-the-shelf product' because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.

NK cell receptor and ligand composition influences the clearance of SARS-CoV-2.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

The role of NK cells in rheumatoid arthritis.

OBJECTIVE: Natural killer (NK) cells are part of the innate immune system which not only provides a primary response to pathogenic conditions but can also play an important regulatory role in immune responses. Furthermore, these cells can influence immune responses by affecting other involved cells. Human NK cells can be classified as CD56dim and CD56bright; the former demonstrates mostly cytotoxic effects, while the latter comprises mostly tolerant or regulatory NK cells. These cells participate in the immunopathogenesis of rheumatoid arthritis (RA) and their role remains still unclear. METHODS: We searched PubMed/MEDLINE and Scopus databases to review and analyze relevant literature on the impact of NK cells in the pathogenesis of RA. RESULTS: Although the percentage of NK cells increases in peripheral blood of RA patients compared to healthy individuals, the cytotoxic function of these cells is impaired. It is demonstrated by reduced "perforin+ NK cells" and decreased per-cell lytic function. These cytotoxic NK cells may control the pathogenic bone absorptive function of osteoclasts by directly targeting these cells. CONCLUSION: Collectively, the evidence collected in the current review emphasizes the possible protective role of CD56dim NK cells in the pathogenesis of RA.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

MyD88 is an essential regulator of NK cell-mediated clearance of MCMV infection.

The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.

Natural killer (NK) cell-based immunotherapies and the many faces of NK cell memory: A look into how nanoparticles enhance NK cell activity.

Natural killer (NK) cells are lymphocytes able to exert potent antitumor and antiviral functions by different means. Besides their classification as innate lymphoid cells (ILCs), NK cells exhibit memory-like and memory responses after cytokine preactivation, viral infections and hapten exposure. Multiple NK cell-based immunotherapies have been developed and are currently being tested, including the possibility to translate the NK cell memory responses into the clinic. Nevertheless, still there is a need to improve these therapies, especially for the treatment of solid tumors, and nanotechnology represents an attractive option to increase NK cell effector functions against transformed cells. In this article, we review the basis of NK cell activity, the diversity of the NK cell memory responses and the current NK cell-based immunotherapies that are being used in the clinic. Furthermore, we take a look into nanotechnology-based strategies targeting NK cells to modulate their responses for effective immunotherapy.

Interaction between HLA-G and NK cell receptor KIR2DL4 orchestrates HER2-positive breast cancer resistance to trastuzumab.

Despite the successful use of the humanized monoclonal antibody trastuzumab (Herceptin) in the clinical treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, the frequently occurring drug resistance remains to be overcome. The regulatory mechanisms of trastuzumab-elicited immune response in the tumor microenvironment remain largely uncharacterized. Here, we found that the nonclassical histocompatibility antigen HLA-G desensitizes breast cancer cells to trastuzumab by binding to the natural killer (NK) cell receptor KIR2DL4. Unless engaged by HLA-G, KIR2DL4 promotes antibody-dependent cell-mediated cytotoxicity and forms a regulatory circuit with the interferon-γ (IFN-γ) production pathway, in which IFN-γ upregulates KIR2DL4 via JAK2/STAT1 signaling, and then KIR2DL4 synergizes with the Fcγ receptor to increase IFN-γ secretion by NK cells. Trastuzumab treatment of neoplastic and NK cells leads to aberrant cytokine production characterized by excessive tumor growth factor-ß (TGF-ß) and IFN-γ, which subsequently reinforce HLA-G/KIR2DL4 signaling. In addition, TGF-ß and IFN-γ impair the cytotoxicity of NK cells by upregulating PD-L1 on tumor cells and PD-1 on NK cells. Blockade of HLA-G/KIR2DL4 signaling improved the vulnerability of HER2-positive breast cancer to trastuzumab treatment in vivo. These findings provide novel insights into the mechanisms underlying trastuzumab resistance and demonstrate the applicability of combined HLA-G and PD-L1/PD-1 targeting in the treatment of trastuzumab-resistant breast cancer.

Immunopathology of Chronic Hepatitis B Infection: Role of Innate and Adaptive Immune Response in Disease Progression.

More than 250 million people are living with chronic hepatitis B despite the availability of highly effective vaccines and oral antivirals. Although innate and adaptive immune cells play crucial roles in controlling hepatitis B virus (HBV) infection, they are also accountable for inflammation and subsequently cause liver pathologies. During the initial phase of HBV infection, innate immunity is triggered leading to antiviral cytokines production, followed by activation and intrahepatic recruitment of the adaptive immune system resulting in successful virus elimination. In chronic HBV infection, significant alterations in both innate and adaptive immunity including expansion of regulatory cells, overexpression of co-inhibitory receptors, presence of abundant inflammatory mediators, and modifications in immune cell derived exosome release and function occurs, which overpower antiviral response leading to persistent viral infection and subsequent immune pathologies associated with disease progression towards fibrosis, cirrhosis, and hepatocellular carcinoma. In this review, we discuss the current knowledge of innate and adaptive immune cells transformations that are associated with immunopathogenesis and disease outcome in CHB patients.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

NK Cells in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a prevalent chronic airway disease with varied frequencies of acute exacerbations, which are the main cause of morbidity and mortality of the disease. It is, therefore, urgent to develop novel therapies for COPD and its exacerbations, which rely heavily on understanding of the pathogenesis and investigation for potential targets. Current evidence indicates that natural killer (NK) cells play important roles in the pathological processes of COPD. Although novel data are revealing the significance of NK cells in maintaining immune system homeostasis and their involvement in pathogenesis of COPD, the specific mechanisms are largely unknown. Specific and in-depth studies elucidating the underlying mechanisms are therefore needed. In this review, we provided a brief overview of the biology of NK cells, from its development to receptors and functions, and outlined their subsets in peripheral blood and lungs. Then we reviewed published findings highlighting the important roles played by NK cells in COPD and its exacerbations, with a view of providing the current state of knowledge in this area to facilitate related in-depth research.

Pelvic endometriosis and natural killer cell immunity.

Endometriosis is a chronic disease that commonly affects women in their reproductive age. It has been reported that the infertility due to endometriosis is largely caused by pelvic adhesion, oocyte damage, and so on. There are several causes of endometriosis including bacterial infections, immunological abnormalities, epigenetics, and aberrant DNA methylation. The natural killer (NK) cells present in the peritoneal fluid express CD16 and CD56. They also express NK cell inhibitory receptors and activating receptors and usually work to eliminate endometrial cells in the retrograde menstruation. However, in women with endometriosis, the changes in these receptors and production of cytokines by NK cells cause the onset and progression of endometriosis. In this review, we have focused on the role of NK cells in pelvic endometriosis and presented the immunological abnormalities in endometriosis including the possibility of future treatment.

Non-KIR NK cell receptors: Role in transplantation of allogeneic haematopoietic stem cells.

Natural killer (NK) cells are of major significance in patients after allogeneic haematopoietic stem cell transplantation (HSCT). They are the first subset of lymphocytes to appear in peripheral blood after transplantation and play an important role in the immune responses against cancer and viral infections. The function of NK cells is controlled by various surface receptors, of which type I integral proteins with immunoglobulin-like domains (killer-cell immunoglobulin-like receptors, KIRs) have been the most extensively studied. The present review focuses on less studied NK cell receptors, such as type II integral proteins with lectin-like domains (CD94/NKG2, NKG2D), natural cytotoxicity receptors (NCRs), immunoglobulin-like transcripts (ILTs) and their ligands. Their potential role in patients with haematological disorders subjected to HSC transplant procedure in the context of post-transplant complications such as viral reactivation and acute graft-versus-host disease (GvHD) will be presented and discussed.

Functionally Relevant Differences in Plasma Fatty Acid Composition and Expression of Cytotoxic and Inhibitory NK Cell Receptors between Healthy Young and Healthy Elder Adults.

(1) Background: In the healthy ageing, NK cell number is not modified; however, their spontaneous cytotoxicity decreases. We postulated that the age-dependent decline in metabolic activities might be responsible for this effect. (2) Methods: The fatty acid profile of 30 healthy young males (23 ± 4 years old, BMI 22.1 ± 1.3) and 30 older males (63 ± 5 years old, BMI 22.9 ± 2.5) donors were evaluated along with the expression of killing (KR) and inhibitory NK receptors (KIR) at basal level and after cultivation with fatty acids for 24 h. (3) Results: Significantly higher levels of oleic (p < 0.01), arachidonic (p < 0.001), lignoceric (p < 0.001), and nervonic acids (p < 0.0001) and significantly lower levels of docosapentaenoic and docosahexaenoic acids (p < 0.01) were found in elders as compared to young adults. At basal levels, significant (p < 0.005) differences in KR and KIR expression were encountered; 12/16 antigens. Treatment of cells with saturated fatty acids or arachidonic acid (AA) significantly enhanced KR expressions (p < 0.001). AA treatment decreased inhibitory KIR expression while docosahexaenoic, and eicosapentaenoic acid increased them. (4) Conclusions: Changes in fatty acids blood levels, and KR and KIR expression in NK cell, are age-dependent. Supplementation of NK cells with eicosapentaenoic or docosahexaenoic acid enhanced inhibitory KIR receptors' expression which may improve their cell function.

The role of senescent T cells in immunopathology.

The development of senescence in tissues of different organs and in the immune system are usually investigated independently of each other although during ageing, senescence in both cellular systems develop concurrently. Senescent T cells are highly inflammatory and secrete cytotoxic mediators and express natural killer cells receptors (NKR) that bypass their antigen specificity. Instead they recognize stress ligands that are induced by inflammation or infection of different cell types in tissues. In this article we discuss data on T cell senescence, how it is regulated and evidence for novel functional attributes of senescent T cells. We discuss an interactive loop between senescent T cells and senescent non-lymphoid cells and conclude that in situations of intense inflammation, senescent cells may damage healthy tissue. While the example for immunopathology induced by senescent cells that we highlight is cutaneous leishmaniasis, this situation of organ damage may apply to other infections, including COVID-19 and also rheumatoid arthritis, where ageing, inflammation and senescent cells are all part of the same equation.

Expression of NK cell receptor ligands in primary colorectal cancer tissue in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome.

INTRODUCTION: Natural killer (NK) cells and natural killer T (NKT) cells are implicated in the development and progression of colorectal cancer (CRC). Tumor cells express NK cell receptor ligands that modulate their function. This study aimed to investigate the expression of such ligands in CRC in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome. METHODS: Primary tumor tissues were analyzed for protein expression of NK cell ligands using immunohistochemistry with automated image analysis in a cohort of 78 CRC patients. For 24 of the 78 patients, RNA expression of NK cell ligands was analyzed in primary tumor tissue using RNA sequencing. Receptor expression on circulating NK- and NKT cells was previously measured by us in 71 of the 78 patients using flow cytometry. RESULTS: High Proliferating Cell Nuclear Antigen (PCNA) protein expression in the primary tumor associated with shorter disease-free survival (DFS) of CRC patients (P = 0.026). A trend was observed towards shorter DFS in CRC patients with above-median galectin-3 protein expression in the primary tumor (P = 0.055). High protein expression of galectin-3, CD1d, and human leukocyte antigen (HLA) class I, and high RNA expression of UL16-binding protein (ULBP)-1, -2, and -5, and HLA-E in the tumor tissue correlated with low expression of the corresponding receptors on circulating NK- or NKT cells (P < 0.05). CONCLUSIONS: Galectin-3 and PCNA expression in the primary tumor may be prognostic biomarkers in CRC patients. Furthermore, our results suggest that NK cell receptor ligands expressed by tumor cells may modulate the phenotype of circulating NK- and NKT cells, and facilitate immune escape of metastasizing cells.

Mass Cytometry Analysis of the NK Cell Receptor-Ligand Repertoire Reveals Unique Differences between Dengue-Infected Children and Adults.

Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56dimCD38bright NK cells and an increase in the percentage of CD56dimperforinbright NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

The Activating Receptors of Natural Killer Cells and Their Inter-Switching Potentials.

The global incidence of cancer is on the increase and researchers are prospecting for specific and non-selective therapies derived from the immune system. The killer activating receptors of NK cells are known to be involved in immunosurveillance against tumor and virally-infected cells. These receptors belong to two main categories, namely the immunoglobulin like and C-lectin like families. Though they have different signal pathways, all the killer activating receptors have similar effector functions which include direct cytotoxicity and the release of inflammatory cytokines such as IFN-gamma and TNF-alpha. To transduce signals that exceed the activation threshold for cytotoxicity, most of these receptors require synergistic effort. This review profiles 21 receptors: 13 immunoglobulin-like, 5 lectin-like, and 3 others. It critically explores their structural uniqueness, role in disease, respective transduction signal pathways and their status as current and prospective targets for cancer immunotherapy. While the native ligands of most of these receptors are known, much work is required to prospect for specific antibodies, peptides and multi-target small molecules with high binding affinities.

Cutting Edge: Inhibition of the Interaction of NK Inhibitory Receptors with MHC Class I Augments Antiviral and Antitumor Immunity.

NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.

Engineering of chimeric natural killer cell receptors to develop precision adoptive immunotherapies for cancer.

Natural killer (NK) cells are innate immune effectors which play a crucial role in recognizing and eliminating virally infected and cancerous cells. They effectively distinguish between healthy and distressed self through the integration of signals delivered by germline-encoded activating and inhibitory cell surface receptors. The frequent up-regulation of stress markers on genetically unstable cancer cells has prompted the development of novel immunotherapies that exploit such innate receptors. One prominent example entails the development of chimeric antigen receptors (CAR) that detect cell surface ligands bound by NK receptors, coupling this engagement to the delivery of tailored immune activating signals. Here, we review strategies to engineer CARs in which specificity is conferred by natural killer group 2D (NKG2D) or other NK receptor types. Multiple preclinical studies have demonstrated the remarkable ability of chimeric NK receptor-targeted T cells and NK cells to effectively and specifically eliminate cancer cells and to reject established tumour burdens. Importantly, such systems act not only acutely but, in some cases, they also incite immunological memory. Moreover, CARs targeted with the NKG2D ligand binding domain have also been shown to disrupt the tumour microenvironment, through the targeting of suppressive T regulatory cells, myeloid-derived suppressor cells and tumour vasculature. Collectively, these findings have led to the initiation of early-phase clinical trials evaluating both autologous and allogeneic NKG2D-targeted CAR T cells in the haematological and solid tumour settings.

Significance of KIR like natural killer cell receptors in autoimmune disorders.

Killer cell immunoglobulin-like receptors (KIRs), act as the regulators for the cytolytic activity of natural killer and certain T cells by interacting with the HLA class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases. However, their specific roles are still not very clear. Autoimmune diseases are multifactorial in nature, highlighting the influence of both genetic and environmental factors. The innate immune response plays an important role in autoimmunity as it alters the self-glycans that mimic molecular patterns found on different intracellular pathogens. Natural killer (NK) cells have an important position in the innate immune response. NK cell receptors are encoded by the leukocyte receptor complex located on the chromosome 19q13.4 and lectin-like receptors on chromosome 12p13. This review focuses on the role of KIRs and their relationship with different autoimmune diseases.