The MICA-NKG2D axis in clear cell renal cell carcinoma bolsters MICA as target in immuno-oncology.

NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.

Leveraging NKG2D Ligands in Immuno-Oncology.

Immune checkpoint inhibitors (ICI) revolutionized the field of immuno-oncology and opened new avenues towards the development of novel assets to achieve durable immune control of cancer. Yet, the presence of tumor immune evasion mechanisms represents a challenge for the development of efficient treatment options. Therefore, combination therapies are taking the center of the stage in immuno-oncology. Such combination therapies should boost anti-tumor immune responses and/or target tumor immune escape mechanisms, especially those created by major players in the tumor microenvironment (TME) such as tumor-associated macrophages (TAM). Natural killer (NK) cells were recently positioned at the forefront of many immunotherapy strategies, and several new approaches are being designed to fully exploit NK cell antitumor potential. One of the most relevant NK cell-activating receptors is NKG2D, a receptor that recognizes 8 different NKG2D ligands (NKG2DL), including MICA and MICB. MICA and MICB are poorly expressed on normal cells but become upregulated on the surface of damaged, transformed or infected cells as a result of post-transcriptional or post-translational mechanisms and intracellular pathways. Their engagement of NKG2D triggers NK cell effector functions. Also, MICA/B are polymorphic and such polymorphism affects functional responses through regulation of their cell-surface expression, intracellular trafficking, shedding of soluble immunosuppressive isoforms, or the affinity of NKG2D interaction. Although immunotherapeutic approaches that target the NKG2D-NKG2DL axis are under investigation, several tumor immune escape mechanisms account for reduced cell surface expression of NKG2DL and contribute to tumor immune escape. Also, NKG2DL polymorphism determines functional NKG2D-dependent responses, thus representing an additional challenge for leveraging NKG2DL in immuno-oncology. In this review, we discuss strategies to boost MICA/B expression and/or inhibit their shedding and propose that combination strategies that target MICA/B with antibodies and strategies aimed at promoting their upregulation on tumor cells or at reprograming TAM into pro-inflammatory macrophages and remodeling of the TME, emerge as frontrunners in immuno-oncology because they may unleash the antitumor effector functions of NK cells and cytotoxic CD8 T cells (CTL). Pursuing several of these pipelines might lead to innovative modalities of immunotherapy for the treatment of a wide range of cancer patients.

Altered NK cell receptor repertoire and function of natural killer cells in patients with acute myocardial infarction: A three-month follow-up study.

NK cells are important in the onset of acute myocardial infarction (AMI) by their ability to secrete IFN-γ and other inflammatory cytokines. They also participate in regulating pathological cardiac remodeling after myocardial infarction. Mechanisms of regulation, however, are incompletely understood. Herein, the aim of this study is to explore the possible association between the expression pattern of different NK cell receptors (phenotype), as well as the cytotoxic function of NK cells from AMI patients with their myocardial function after three months follow-up. We analyzed the phenotype and function of both CD56dimCD16+ and CD56brightCD16- NK cells from twenty-one patients within the first 72 h after ST-elevation AMI and three-month follow-up, as well as fifteen healthy controls. Clinical characteristics and ventricular function determined by echocardiography were also evaluated. NK cells from AMI patients showed an activated phenotype, characterized by high TNF-α production and low percentages of the activating receptor NKG2D. Interestingly, AMI patients display higher levels of circulating IL-10+ NK cells. Three-month follow-up showed that NK cells exhibit a diminished cytotoxic function. These data show that NK cells may have a role mediating myocardial remodeling by regulating the inflammatory response, mainly by the production of IL-10. We also propose that NKG2D may have a role in the onset of the inflammatory response immediately after AMI. The precise regulation of NK cells function may represent an important step in recovery of myocardial function.

Cervical Cancer Cells Express Markers Associated with Immunosurveillance.

Cervical cancer is the second most frequent cancer in women in Mexico, and its development depends on the presence of human papillomaviruses in the uterine cervix. These oncogenic viruses transform cells where the control over cell cycle disappears, and the capacity to induce apoptosis is absent. On the other hand, some mutations confer to the transformed cells the ability to evade recognition by the immune system. The expression of markers of the immune system such as CD95, MICA/B, CD39, CD73, NKp30, NKp46, CD44, CD24, NKG2A, and CTLA-4 was analysed by flow cytometry on cervical cancer cells INBL (HPV 18, stage IVB), HeLa (HPV 18), CaSki (HPV 16), and C33A (HPV-). Our results showed the presence of atypical markers on cervical cancer cells; some of them are molecules involved in tumour cell recognition such as MICA/B and CD95. Other markers associated with immune system escape, such as CD39, CD73, and CTLA-4, were also present. Furthermore, we found that some cervical cancer cells expressed typical markers of NK cells like NKp30, NKp46, NKG2A, and KIR3DL1. It is not clear whether these molecules confer any gain to the tumour cells or if they represent a disadvantage, but we hypothesise that these molecules that are present in cervical cancer cells allow them to mimic in front of the immune system.

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin-like receptors.

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody-dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T-cell receptor-γδ (TCR-γδ) T cells and TCR-αß CD8+ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin-like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon-γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8+ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.

Comparative analysis of NK cell receptor repertoire in adults and very elderly subjects with cytomegalovirus infection.

Human cytomegalovirus (HCMV) infection in children and young adults has been associated with changes in the innate immune system. We herein analyzed the possible effect of very long term HCMV infection on the expression of several NK cell receptors. Ninety HCMV-seropositive individuals were included and classified as young adults (n=30), elderly (n=30) and very elderly subjects (n=30). A peripheral blood sample was obtained and the expression of NK cell receptors (NKG2A, NKG2C, ILT2, CD161, KIR2DL1, KIR3DL1, and KIR3DL2) by NK and other lymphocyte subsets was assessed by flow cytometry. In addition, the frequency of the sixteen KIR genes was analyzed by polymerase chain reaction. We found a significant increase in the number of NKG2C+ NK and T cells in elderly individuals compared to young adults accompanied by an opposite trend in the number of NKG2A+ lymphocytes, and ILT2+ cells were also increased in elderly individuals. A significant increase in the levels of CD3-CD56+NKG2C+CD57+ cells was also detected in the elderly groups. Finally, KIR gene analysis revealed that the KIR genotype 2 was significantly less frequent in the elderly individuals. Our results support that long-term infection by HCMV exerts a significant progressive effect on the innate immune system.

Natural killer cell cytotoxicity is not regulated by folic acid in vitro.

OBJECTIVES: Folate supplementation may be associated with an increased risk of developing several types of cancer and a derangement of immune function. Among the latter, Natural killer (NK) cells are involved in non-MHC-restricted natural immunity against malignant target cells. Abnormalities in NK cell number or function have been associated with a higher cancer risk. The aim of this study was to study in vitro the possible effect of different concentrations of 5-methyltetrahydrofolic acid (5-MTHF) or folic acid on NK cell cytotoxic function, and expression of the stimulatory and inhibitory receptors KIRDL4, KIRDL3, and NKG2D. METHODS: Volunteer-derived peripheral mononuclear cells (PBMC) and highly enriched NK cells (95% CD56+ CD16+) were grown in folic acid free-RPMI 1640, supplemented either with folic acid or 5-MTHF (15-100 nM) during 72 h to 96 h. RESULTS: No differences in the cytolytic activity of PBMC and enriched NK cells were observed. After 96 h of in vitro culture without folate or supplemented with FA or 5-MTHF (30 or 100 nM), there were no changes in the percentage of HPNK receptor-positive cells. CONCLUSIONS: Our data indicate that a high dose of 5-MTHF or folic acid does not influence NK cell function in vitro.

Altered phenotype in peripheral blood and tumor-associated NK cells from colorectal cancer patients.

Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients-the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.

Age-related changes in natural killer cell receptors from childhood through old age.

Most studies on natural killer (NK) cells and aging have focused on overall cell numbers and global cytotoxic activity. NK cell functions are controlled by surface receptors belonging to three major families: killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), and C-type lectins. The expression of these receptors was investigated from childhood through old age in T, NKT- and NK cells and also in the CD56(dim) (cytotoxic) and CD56(bright) (responsible for cytokine production) NK cell subsets. A decrease in the expression of activating receptors (NKp30 and NKp46) was observed in NK cells in elderly individuals. KIR expression was increased only in the CD56(bright) subset. Children presented similar results regarding expression of NKp30 and KIR, but not NKp46. NKG2D expression was decreased in T cells of elderly subjects. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56(dim), observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. This initial study provides the framework for more focused studies of this subject, which are necessary to determine whether the changing balance of NK receptor expression may influence susceptibility to infectious, inflammatory, and neoplastic diseases.

Natural killer cell receptor and HLA-C gene polymorphisms among patients with hepatitis C: a comparison between sustained virological responders and non-responders.

BACKGROUND/AIMS: Killer cell immunoglobulin-like receptors (KIR) are involved in the activation/inhibition of NK cells through an interaction with HLA class I molecules on target cells. Our study aimed to evaluate the association between KIR gene polymorphisms and the response of patients with CHC to antiviral therapy. METHODS: We compared the frequency of KIR genes, as well as that of compound KIR/HLA-C genotypes, between groups of patients with CHC who presented a sustained virological response (n=66) and who were non-responders to a combination of pegylated or standard interferon and ribavirin (n=101). KIR and HLA-C genotyping were performed using commercial kits. RESULTS: We detected a greater frequency of the KIR2DL5 gene among non-responders to antiviral therapy compared with sustained virological responders (68.3 vs. 40.9%) (P<0.001). We used multiple logistic regression analysis to determine the association between therapy response and the presence of KIR2DL5, after a control for potentially confounding variables (genotype, alcohol, fibrosis, gender, age, ethnic background and route of HCV infection). The results confirmed the strong association between the presence of KIR2DL5 and the non-response to antiviral treatment (P=0.001). CONCLUSIONS: Host genetic factors may be associated with a non-response to antiviral therapy. KIR2DL5 is a candidate gene involved in immunomodulation associated with non-response to antiviral therapy.

Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children.

Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin-like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56-CD3- NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.

Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions.

BACKGROUND: Persistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection. METHODS: NKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays. RESULTS: We report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients. CONCLUSION: Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.

Binding of excreted and/or secreted products of adult hookworms to human NK cells in Necator americanus-infected individuals from Brazil.

The impact of the interaction between excreted and/or secreted (ES) Necator americanus products and NK cells from Necator-infected individuals was analyzed. We investigated the binding of ES products to NK cells, the expression of NK cell receptors (CD56, CD159a/NKG2A, CD314/NKG2D, CD335/NKp46, and KLRF1/NKp80), the frequency of gamma interferon (IFN-gamma)-producing NK cells after whole-blood in vitro stimulation, and the capacity of N. americanus ES products to induce NK cell chemotaxis. In contrast to those from noninfected individuals, NK cells from Necator-infected individuals demonstrated no binding with N. americanus ES products. This phenomenon was not due to alterations in NK cell receptor expression in infected subjects and could not be reproduced with NK cells from uninfected individuals by incubation with immunoregulatory cytokines (interleukin-10/transforming growth factor beta). Further, we found that a significantly greater percentage of NK cells from infected subjects than NK cells from uninfected individuals spontaneously produced IFN-gamma upon ex vivo culture. Our findings support a model whereby NK cells from Necator-infected individuals may interact with ES products, making these cells refractory to binding with exogenous ES products. During N. americanus infection, human NK cells are attracted to the site of infection by chemotactic ES products produced by adult Necator worms in the gut mucosa. Binding of ES products causes the NK cells to become activated and secrete IFN-gamma locally, thereby contributing to the adult hookworm's ability to evade host immune responses.

Expression of NK cells activation receptors after occupational exposure to toxics: a preliminary study.

The expression of NK cells activation receptors was assessed by comparative study of two groups of women workers at a chemical reagents factory, located in Zapopan, Jalisco, Mexico. Twenty of them were exposed to environmental toxics identified and quantified by gas chromatography, and 20 women unexposed to toxic substances. The expression of the surface markers CD56+ and CD3+, and of the activation receptors and co-receptors on NK cells was quantified by flow cytometry. To assess the cellular damage produced by chronic exposure to the toxics, the thiobarbituric acid reacting substances (TBARS) generated and the total plasma antioxidizing capacity (TPAC) were quantified in both groups. The exposed women had been exposed at least to 12 volatile toxic compounds, benzene, benz(a)pyrene, ethylbenzene, dimethylbenz(a)anthracene, xylene, toluene, styrene, chloroform, formaldehyde, iodine, chlorine and fluorine. Significant difference between the two groups was in the proportion of CD3 lymphocytes, 72.7+/-10.3% in the unexposed women versus 66.8+/-7.9% in the exposed group (p<0.05). The density of expression of NKG2D and NKp30 receptors was significantly higher in the unexposed women compared to the exposed group: NKG2D were 31.3+/-6.3 and NKp30 were 9.5+/-5.2 in the unexposed women and 5.14+/-2.9 (p<0.01) and 4.6+/-1.9 (p<0.05), respectively in the exposed women. No statistically significant differences were found in the expression of NKp80, NKp46 and 2B4 receptors. The concentration of TBARS was lower in women from the unexposed group than the corresponding data from women of the exposed group. However, no significant difference was observed in TPAC between the two groups studied. The results of this preliminary study suggest that from the five activation receptors and co-receptors of NK cells evaluated (NKp30, NKp46, NKp80, NKG2D and 2B4), only NKp30 and NKG2D receptor expression was diminished in women exposed to toxics when compared with data from unexposed women. These results suggest that the occupational exposure to mixture of toxics is one of the important factors in the diminution of the NK cell receptor expression.

Isolation and characterization of CD8+ regulatory T cells in multiple sclerosis.

To investigate CD8+ regulatory T cell influence on multiple sclerosis development, peripheral blood and cerebrospinal fluid (CSF) CD8+ T cell clones (TCCs) recognizing MBP(83-102) and MOG(63-87)-specific CD4+ T cells were isolated from 20 patients during acute exacerbations, 15 in remission and 15 controls. Blood and CSF CD8+ regulatory TCC cloning frequency decreased more during exacerbations than remissions or controls. Target cell pre-activation significantly enhanced CD8+ T granule-mediated cell killing of CD4+ targets, and was restricted by HLA-E. During exacerbations, killer-inhibitory receptor CD94/NKG2A expression was significantly higher in CD8+ TCCs, limiting their cytotoxic activity. Moreover, IL-15 and IFN-gamma significantly increased CD94 and NKG2A expression. These data provide evidence that CD94/NKG2A receptors play an important role in regulating T cell activity during the course of MS.

Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.

NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.

Cytokine-driven regulation of NK cell functions in tumor immunity: role of the MICA-NKG2D system.

Natural killer (NK) cells are critical players during tumor growth control in immunocompetent hosts. These cells also establish a cross-talk with dendritic cells (DCs) and promote a Th1-mediated immunity. NKG2D is a pivotal receptor that directs the tumoricidal activity of NK cells through the recognition of a group of ligands such as MICA widely expressed on different tumors. Here we will review the most important tumor immune escape mechanisms that compromise the functionality of NKG2D and its cognate ligands, including TGF-beta secretion, tumor shedding of soluble MICA, and additional mechanisms that compromise the tumoricidal activity of NKG2D-expressing cells. Such mechanisms may also dampen the cross-talk between NK cells and DCs during the anti-tumor immune responses. Recent knowledge may lead to innovative approaches to promote efficient NK cell-mediated anti-tumor immune responses.