Activating Receptor NKG2D Research Articles

CD6 is a target for cancer immunotherapy.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with the aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung, and prostate cancer cells through direct effects on both CD8+ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme B production. The combined capability of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

24P miR-17-5p: A potential activator of natural killer cells through tuning STAT3/H19/ULBP2 axis in breast cancer

Natural killer (NK) cells are the first native invaders against malignancy. NK cells activity is regulated by an array of activating and inhibitory receptors present on their surface. UL-16 binding proteins (ULBP 1-6) are ligands expressed by malignant cells to be detected by NKG2D activating receptor and consequent eradication of transformed cells by NK cells. Breast cancer (BC) patients experience a shedding of ULBP2 ligand in an attempt to evade immune surveillance process exhibited by NK cells.

The Role of NKG2D in Vitiligo

Vitiligo is an acquired multifactorial disease that affects melanocytes and results in skin depigmentation. In this review, we examine the role of cells stress and self-reactive T cells responses. Given the canonical and non-canonical functions of NKG2D, such as authenticating stressed target and enhance TCR signaling, we examine how melanocyte stress leads to the expression of ligands that are recognized by the activating receptor NKG2D, and how its signaling results in the turning of T cells against self (melanocyte suicide by proxy). We also discuss how this initiation phase is followed by T cell perpetuation, as NKG2D signaling results in self-sustained long-lasting T cells, with improved cytolytic properties.

The Role of Tim-3 on dNK Cells Dysfunction During Abnormal Pregnancy With Toxoplasma gondii Infection.

Vertical transmission of Toxoplasma gondii (T. gondii) infection during gestation can result in severe complications such as abortion, congenital malformation, fetal teratogenesis, etc. Immune inhibitory molecule Tim-3 was discovered to be expressed on some decidual immune cells and participates in the maintenance of maternal-fetal tolerance. Dysregulation of Tim-3 expression on decidual NK (dNK) cells was observed in several cases of pregnancy complications, whereas the role of Tim-3 on dNK cells during T. gondii infection remains unclear. In the present study, T. gondii infected Tim-3-/- pregnant mice, and anti-Tim-3 neutralizing antibody treated and infected human dNK cells were successfully established to explore the role of Tim-3 in dysfunction of dNK cells during abnormal pregnancy. Our results illustrated that Tim-3-/- pregnant mice displayed more worse pregnancy outcomes with T. gondii infection compared to infected WT pregnant mice. Also, it demonstrated that Tim-3 expression on dNK cells was significantly down-regulated following T. gondii infection. Data suggested a remarkable activation of dNK cells in Tim-3-/- mice and anti-Tim-3 neutralizing antibody treated and infected groups, with higher ratios of activating receptor NKG2D to inhibitory receptor NKG2A or KIR2DL4, IFN-γ/IL-10, and increased granule production compared with that of the infected group. Mechanism analysis proved that T. gondii-induced Tim-3 down-regulation significantly activated the phosphatidylinositol-3-kinase (PI3K)-AKT and JAK-STAT signaling pathway, by which the GranzymeB, Perforin, IFN-γ, and IL-10 production were further up-regulated. Our research demonstrated that the decrease of Tim-3 on dNK cells caused by T. gondii infection further led to dNK cells function disorder, which finally contributed to the development of abnormal pregnancy outcomes.

Effector memory CD8 T cell response elicits Hepatitis E Virus genotype 3 pathogenesis in the elderly.

Genotype 3 Hepatitis E virus (HEV-3) is an emerging threat for aging population. More than one third of older infected patients develops clinical symptoms with severe liver damage, while others remain asymptomatic. The origin of this discrepancy is still elusive although HEV-3 pathogenesis appears to be immune-mediated. Therefore, we investigated the role of CD8 T cells in the outcome of the infection in immunocompetent elderly subjects. We enrolled twenty two HEV-3-infected patients displaying similar viral determinants and fifteen healthy donors. Among the infected group, sixteen patients experienced clinical symptoms related to liver disease while six remained asymptomatic. Here we report that symptomatic infection is characterized by an expansion of highly activated effector memory CD8 T (EM) cells, regardless of antigen specificity. This robust activation is associated with key features of early T cell exhaustion including a loss in polyfunctional type-1 cytokine production and partial commitment to type-2 cells. In addition, we show that bystander activation of EM cells seems to be dependent on the inflammatory cytokines IL-15 and IL-18, and is supported by an upregulation of the activating receptor NKG2D and an exuberant expression of T-Bet and T-Bet-regulated genes including granzyme B and CXCR3. We also show that the inflammatory chemokines CXCL9-10 are increased in symptomatic patients thereby fostering the recruitment of highly cytotoxic EM cells into the liver in a CXCR3-dependent manner. Finally, we find that the EM-biased immune response returns to homeostasis following viral clearance and disease resolution, further linking the EM cells response to viral burden. Conversely, asymptomatic patients are endowed with low-to-moderate EM cell response. In summary, our findings define immune correlates that contribute to HEV-3 pathogenesis and emphasize the central role of EM cells in governing the outcome of the infection.

Defective Natural Killer Cells in Melanoma: Role of NKG2D in Pathogenesis and Immunotherapy.

Melanoma is the most aggressive and deadliest form of skin cancer, and its prognosis is very poor. Although the early detection is responsive to many treatments, metastatic melanoma is refractory to most of them. In the United States, skin melanoma is the fifth most common type of cancer in men and the sixth in women. Current treatment modalities, depending on the cancer stage, consist primarily of surgical excision, chemotherapy, adjuvant therapy, targeted therapies, and immunotherapy. Despite the wide range of therapeutic options and the steadily increasing response rates, a large subset of the treated patients relapse and develop resistance to further treatments. One novel approach in preclinical and clinical trials in immunotherapy is the adaptation of natural killer (NK) cells against resistant cancer cells. NK cells can kill a variety of cancer cell types, as well as the cancer stem cells, while leaving normal cells intact. In skin melanoma, as in most cancers, NK cells in the tumor microenvironment (TME) are functionally impaired. Several factors underlie the defective cause of NK cells, one of which is the dysregulation of the activating receptor NKG2D. This is the dominant receptor in regulating the cytotoxic activity, cytokine production, and regulation of other receptors expressed on NK cells and other lymphocytes. The defective NK cells in cancer models were associated with tumor growth and metastasis. In this review, we discuss the role of NK cells and their phenotypic variants in skin melanoma. Using bioinformatics, we have further analyzed the expression of NKG2D, confirming its low transcript levels in patients with skin melanoma. Furthermore, we show that the CD133 subset of cancer stem cells expresses low levels of NKG2D. Based on these findings we discuss the potential therapeutic approaches that can be exploited to upregulate NKG2D in patients' NK cells and restore their anti-melanoma effects, resulting in tumor regression and prolonged survival.

PD-1 blockade improves the anti-tumor potency of exhausted CD3+CD56+ NKT-like cells in patients with primary hepatocellular carcinoma

ABSTRACT CD3+CD56+ NKT-like cells play pivotal roles in the anti-tumor immune defense response. However, little is known regarding circulating NKT-like cells in patients with primary hepatocellular carcinoma (HCC). In the present study, we demonstrate that circulating NKT-like cells in HCC patients are functionally impaired and anti-PD-1 blockade improves their anti-tumor potency. Circulating NKT cells were mainly comprised of CD8+ T cells. The frequencies and absolute counts of circulating NKT-like cells were comparable between HCC patents compared to healthy donors. NKT-like cells in HCC patients were impaired in their production of TNF-α and IFN-γ as well as cytotoxicity. The level of activating receptor NKG2D was significantly decreased on NKT-like cells in HCC patients. In contrast, the expression of inhibitory receptors PD-1, Tim-3, and CTLA-4 were markedly increased on NKT-like cells in HCC patients. Meanwhile, the expression of PD-L1 was also upregulated on NKT-like cells in HCC patients. In detail, PD-1+ NKT-like cells expressed lower levels of NKG2D, higher levels of Tim-3, and CTLA-4, and less IFN-γ when compared with PD-1− NKT-like cells. Importantly, PD-1 blocked with anti-PD-1 antibody effectively improved the effector function of NKT-like cells from HCC patients or healthy donors. Our findings unveil the functional characterization of NKT-like cells in HCC patients and provide the potential targets to improve their function, which might benefit the optimization of HCC immunotherapy.

Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition.

Natural killer (NK) cells, a type of cytotoxic lymphocytes, can infiltrate into ischemic brain and exacerbate neuronal cell death. Astragaloside IV (ASIV) is the major bioactive ingredient of Astragalus membranaceus, a Chinese herbal medicine, and possesses potent immunomodulatory and neuroprotective properties. This study investigated the effects of ASIV on post-ischemic brain infiltration and activation of NK cells. ASIV reduced brain infarction and alleviated functional deficits in MCAO rats, and these beneficial effects persisted for at least 7 days. Abundant NK cells infiltrated into the ischemic hemisphere on day 1 after brain ischemia, and this infiltration was suppressed by ASIV. Strikingly, ASIV reversed NK cell deficiency in the spleen and blood after brain ischemia. ASIV inhibited astrocyte-derived CCL2 upregulation and reduced CCR2+ NK cell levels in the ischemic brain. Meanwhile, ASIV attenuated NK cell activating receptor NKG2D levels and reduced interferon-γ production. ASIV restored acetylation of histone H3 and the p65 subunit of nuclear factor-κB in the ischemic brain, suggesting inhibition of histone deacetylase (HDAC). Simultaneously, ASIV prevented p65 nuclear translocation. The effects of ASIV on reducing CCL2 production, restoring acetylated p65 levels and preventing p65 nuclear translocation were mimicked by valproate, an HDAC inhibitor, in astrocytes subjected to oxygen-glucose deprivation. Our findings suggest that ASIV inhibits post-ischemic NK cell brain infiltration and activation and reverses NK cell deficiency in the periphery, which together contribute to the beneficial effects of ASIV against brain ischemia. Furthermore, ASIV's effects on suppressing NK cell brain infiltration and activation may involve HDAC inhibition.

NK Cell Interaction With Platelets and Myeloid Cells in the Tumor Milieu.

Natural killer (NK) cells recognize and kill tumor cells via germ-line encoded receptors and polarized degranulation of cytotoxic molecules, respectively. As such, NK cells help to inhibit the development of cancers. The activating receptor NKG2D induces NK cell-mediated killing of metastasizing tumor cells by recognition of the stress-induced ligands MICA, MICB, and ULBP1-6. However, platelets enable escape from this immune surveillance mechanism by obstructing the interactions between NK cells and tumor cells or by cleaving the stress-induced ligands. It is also being increasingly appreciated that NK cells play additional roles in cancer immunity, including chemokine-mediated recruitment of antigen presenting cells in the tumor microenvironment that is followed by generation of adaptive immunity. However, the NK cell interplays with dendritic cells, and macrophages are extremely complex and involve molecular interactions via NKG2D and cytokine receptors. Specifically, NKG2D-mediated chronic interaction between NK cells and tumor-infiltrating macrophages causes immune suppression by differentiating NK cells toward a dysfunctional state. Here we discuss the underlying mechanisms of NK cell control by platelets and myeloid cells with focus on NKG2D and its ligands, and provide a timely perspective on how to harness these pathways with novel immunotherapeutic approaches.

Development of a Novel MICA/B-Specific CAR As a Pan-Tumor Targeting Strategy for Off-the-Shelf, Cell-Based Cancer Immunotherapy

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p<0.0001) and 93% in the liver (p<0.006) for CAR-MICA/B iNK cells vs non-CAR controls (Figure 1F). Additionally, CAR-MICA/B iNK cells were effective at controlling Nalm6-MICA progression in a disseminated leukemia model, suggesting potential application against both hematopoietic and solid tumors. Ongoing work is focused on extending these studies into disease-specific models of endogenous MICA/B expression to further advance CAR-MICA/B iNK cells in both solid and hematologic cancers. In summary, these preclinical data support the development and translation of an off-the-shelf NK cell immunotherapy targeting the conserved α3 domain of MICA/B with potential therapeutic application to multiple hematopoietic and solid tumor types. Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Selenium nanoparticles regulates selenoprotein to boost cytokine-induced killer cells-based cancer immunotherapy

Abstract As one of the typical adoptive cell transfer modality, cytokine-induced killer cell (CIK)-mediated immunotherapy exhibits promising applications in cancer treatment. However, the short in vivo persistence of cytokine-induced killer (CIK) cells and complex tumour microenvironment are major challenges for CIK-based immunotherapy. Herein, we demonstrate a safe and effective strategy by combining selenium nanoparticles (SeNPs) with CIK cells for efficient cancer immmunotherapy. Intriguingly, SeNPs could effectively prolong the in vivo persistence of CIK cells in peripheral blood. Furthermore, SeNPs can significantly enhance the cytotoxicity of CIK cells from cancer patients to tumour cells through upregulating the expressions of activation receptor-NKG2D and its ligands, while exhibiting no toxicity to CIK and tumor cells. Series of evidences indicate that SeNPs metabolism into selenocystine mainly contributes to its unique advantages in facilitating CIK treatment. Importantly, this strategy can effectively induce natural killer cells infiltrating in tumours and shape tumour-associated macrophages polarizing into M1 phenotype to trigger robust immune responses for combating multiple tumours progression involving hepatic, breast and prostate ones. This study provides a novel strategy to promote the clinical applications of CIK therapy in cancer treatment.

Interleukin-4-induced natural killer cell antitumor activity in metastatic melanoma patients.

NK cells are important effectors of innate immunity that mount the first line of defense toward tumor growth. Interleukin-4 (IL-4) has recently been shown to regulate NK cell function, although its role in the regulation of NK cell function in cancer patients has not been clarified. The aim of this study was to investigate the effect of IL-4 on the function and the receptor characteristics of CD16-defined NK cells and their cytotoxic CD16bright and regulatory CD16dim subsets. Peripheral blood lymphocytes obtained from 36 metastatic melanoma (MM) patients treated for 18 h with 10 ng/mL IL-4 were evaluated for NK cell cytotoxicity using the radioactive 51chromium release assay. Expression of the activating receptors NKG2D and CD161, as well as the inhibitory receptors CD158a and CD158b, was analyzed on CD3-CD16+ NK cells and their subsets by flow cytometry. IL-4 induced significant in vitro enhancement of NK cell activity, as well as increased expression of the CD107a degranulation marker, by CD3-CD16dim NK cells. NKG2D expression was also increased on CD3-CD16+ cells by IL-4 with no alteration of the expression of CD161 and inhibitory KIR receptors. Although in vitro treatment with IL-4 increased both the expression of NKG2D and the cytotoxicity of NK cells, it had no detectable effect on the transcription of the TGF-β gene in NK cells of MM patients. The IL-4-induced NK cell cytotoxicity and increased activating NKG2D receptor expression may indicate an important antitumor effect of IL-4 with a potential application for immunotherapy of MM patients.

Abstract 3333: EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity

Abstract Purpose: Breg (B regulatory) cells have been implicated in suppression of anti-tumor response in murine and human solid tumors. Prior studies in our laboratory demonstrated that murine EMT-6 breast carcinoma is rejected in B cell deficient mice (BCDM), but not in wild type (WT) mice, due to an enhanced immune response. Tumor infiltrating B cells (TIL-B), and normal splenic B cells co-cultured with EMT-6 (EMT6-B), express LAP/TGF-β, PD-L1, and suppress CD8+ and NK cell proliferation in vitro. Increased CD8+ and NK cell infiltration into the tumor bed is seen in BCDM. BCDM tumor rejection is abrogated by NK depletion. We studied EMT6-B effects on NK cell phenotype, proliferation and cytokine expression in vitro and in vivo. Experimental design: Normal splenic B cells were co-cultured with mitomycin C treated EMT-6, and Breg phenotype, and effects on NK cell phenotype and proliferation and cytokine function were studied in vitro and in vivo. Results: EMT6-B but not WT splenic B cells inhibited CFSE labeled CD49b+ NK cell proliferation in response to IL-15 in dose dependent fashion. EMT-6-B suppressed expression of CD226 and NKG2D activating receptors on NK cells, and increased expression of PD-1 and PD-L1 respectively. EMT6-B suppressed the induction of IFNγ+/CD49b+ NK cells following PMA/ION stimulation as assayed by intracellular flow cytometry (10%±1.2 to 2%±0.5) and also suppressed IL-2 induced induction of IFNγ+/CD49b+ NK cells (70%±5 to 20%±7.8). EMT-6 and CT26 colon carcinoma conditioned B cells, but not C166 endothelial cell conditioned B cells suppressed NK degranulation (IFNγ+/CD107a+ NK cells). EMT6-B inhibited YAC-1 cell induced NK cell degranulation, and killing of YAC-1 targets, more effectively than naïve B cells (40%±6.2 to 18%±5.1) (P=0.05). NK suppression was inhibited by anti-PD-L1, and anti-TGF-β antibody. EMT6-B expressed increased FasL compared to splenic B cells, and induced NK apoptosis as measured by 7AAD/Annexin-V staining. Blocking antibody to FasL decreased NK apoptosis, and restored NK cell proliferation. Infiltrating NK cells in the EMT6 tumor bed demonstrated increased PD-1 compared to splenic and TDLN NK cells, and higher levels of IFN-γ, CD107a, and Granzyme B in BCDM vs. WT mice. EMT6 expressed high levels of CD155 NK inhibitory ligand compared to C166 endothelial cells. The EMT-6 tumor bed contained higher levels of CD19+CD155+ B cells (22% / tumor bed vs 6% / TDLN, vs 9%/ spleen). BCDM expressed higher levels of CD226+/Ki67+ NK cells in spleen compared to WT splenic NK cells. Antibodies to Fas-L, TGF-beta, and PD-L1 partially restored anti-tumor response in WT mice and decreased tumor growth. Conclusions: EMT-6 TIL-B or normal B cells conditioned by EMT-6 coculture acquire an NK suppressive phenotype which inhibits NK cell activation, proliferation, degranulation and cytotoxic function. Targeting of Breg-NK interactions may improve anti-tumor immune response. Citation Format: Yu Zhang, Muhammad Husnain, Zhifen Luo, Mahmood Al Bayati, Joseph D. Rosenblatt. EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3333.

Investigation of the PD-1 and PD-L1 Immune Checkpoint Molecules Throughout Healthy Human Pregnancy and in Nonpregnant Women

Background: A growing body of evidence supports the importance of PD-1 and PD-L1, especially in the materno-fetal interface, although limited information is available about the peripheral expression of these molecules during the trimesters of pregnancy. Methods: 13 healthy women were enrolled from the 1st, 10 from the 2nd and 12 from the 3rd trimester of pregnancy at the same time, 10 healthy, age-matched nonpregnant women formed the control group. From peripheral blood, mononuclear cells were separated and stored at –80 °C. From freshly thawed samples, surface and intracellular staining were performed for flow cytometric analyses. CD107a degranulation assay was used to evaluate the cytotoxicity. Results: significant alternation was detected in PD-1 expression by CD8+T cells and in PD-L1 expression by CD8+T, CD4+T and Treg cells. An interesting relationship was revealed between the PD-1 and PD-L1 expression by the investigated subpopulations in 2nd trimester of pregnancy. Different expression patterns of an activation receptor NKG2D by the PD-1+ CD8+T cells was observed during pregnancy. The notable relationship was further determined in cytotoxicity between PD-1+ and NKG2D+ CD8+T cells throughout pregnancy. Conclusions: the different PD-1 presence and the relationship with NKG2D could contribute to the dynamic changes of the Th1 and Th2 predominance throughout the three trimesters of a healthy pregnancy.

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

Joint study of the associations of HLA‐B and the transmembrane short tandem repeat polymorphism of MICA protein with alopecia areata shows independent associations of both with the disease

Alopecia areata (AA) is a skin disease that produces hair loss in patches of skin. The underlying mechanism of AA is a loss of immune privilege of hair follicles, which are then attacked by natural killer (NK) cells. A previous genome-wide association study linked single nucleotide polymorphisms of the protein MHC class I chain-related A (MICA) to this disease. MICA is the ligand for the activating receptor NKG2D, expressed mainly by NK cells and CD8+ cytotoxic T cells. As the aforementioned study did not include short tandem repeats (STRs) of MICA, we decided to study these in relation to AA. To study the association of STRs with AA, alongside that of human leucocyte antigen (HLA) locus B, which is closely linked to MICA. DNA amplicon size analysis was carried out, and HLA-B locus genomic typing was performed by PCR-sequence-specific oligonucleotide analysis. We observed an association between AA and both MICA*009 and HLA-B14; associations were also observed between HLA-B alleles and MICA alleles, which have both been previously found to be connected with AA, but never studied together. We conclude that it is important to study HLA-B and MICA together to avoid the influence of their association in experiments in which they are investigated separately.

Inhibition of MICA and MICB Shedding Elicits NK-Cell-Mediated Immunity against Tumors Resistant to Cytotoxic T Cells.

Resistance to cytotoxic T cells is frequently mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations of B2M or JAK1 genes. Natural killer (NK) cells could potentially target such resistant tumors, but suitable NK-cell-based strategies remain to be developed. We hypothesized that such tumors could be targeted by NK cells if sufficient activating signals were provided. Human tumors frequently express the MICA and MICB ligands of the activating NKG2D receptor, but proteolytic shedding of MICA/B represents an important immune evasion mechanism in many human cancers. We showed that B2M- and JAK1-deficient metastases were targeted by NK cells following treatment with a mAb that blocks MICA/B shedding. We also demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA/B antibody acted synergistically to enhance MICA/B surface expression on tumor cells. The HDAC inhibitor enhanced MICA/B gene expression, whereas the MICA/B antibody stabilized the synthesized protein on the cell surface. The combination of panobinostat and the MICA/B antibody reduced the number of pulmonary metastases formed by a human melanoma cell line in NOD/SCID gamma mice reconstituted with human NK cells. NK-cell-mediated immunity induced by a mAb specific for MICA/B, therefore, provides an opportunity to target tumors with mutations that render them resistant to cytotoxic T cells.

Characterization of the immune cell landscape of patients with NAFLD

Multiple factors are involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), but the exact immunological mechanisms that cause inflammation and fibrosis of the liver remain enigmatic. In this current study, cellular samples of a cohort of NAFLD patients (peripheral blood mononuclear cells (PBMC): n = 27, liver samples: n = 15) and healthy individuals (PBMC: n = 26, liver samples: n = 3) were analyzed using 16-color flow cytometry, and the frequency and phenotype of 23 immune cell subtypes was assessed. PBMC of NAFLD patients showed decreased frequencies of total CD3+, CD8+ T cells, CD56dim NK cells and MAIT cells, but elevated frequencies of CD4+ T cells and Th2 cells compared to healthy controls. Intrahepatic lymphocytes (IHL) of NAFLD patients showed decreased frequencies of total T cells, total CD8+ T cells, Vd2+γδ T cells, and CD56bright NK cells, but elevated frequencies of Vδ2-γδ T cells and CD56dim NK cells compared to healthy controls. The activating receptor NKG2D was significantly less frequently expressed among iNKT cells, total NK cells and CD56dim NK cells of PBMC of NAFLD patients compared to healthy controls. More strikingly, hepatic fibrosis as measured by fibroscan elastography negatively correlated with the intrahepatic frequency of total NK cells (r2 = 0,3737, p = 0,02). Hepatic steatosis as measured by controlled attenuation parameter (CAP) value negatively correlated with the frequency of circulating NKG2D+ iNKT cells (r2 = 0,3365, p = 0,0047). Our data provide an overview of the circulating and intrahepatic immune cell composition of NAFLD patients, and point towards a potential role of NK cells and iNKT cells for the regulation of hepatic fibrosis and steatosis in NAFLD.

Recent Advances in Molecular Mechanisms of the NKG2D Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma is a common malignant tumor with high mortality. Its malignant proliferation, invasion, and metastasis are closely related to the cellular immune function of the patients. NKG2D is a key activated and type II membrane protein molecule expressed on the surface of almost all NK cells. The human NKG2D gene is 270 kb long, located at 12p12.3–p13.1, and contains 10 exons and 9 introns. The three-dimensional structure of the NKG2D monomeric protein contains two alpha-helices, two beta-lamellae, and four disulfide bonds, and its’ signal of activation is transmitted mainly by the adaptor protein (DAP). NKG2D ligands, including MICA, MICB, and ULBPs, can be widely expressed in hepatoma cells. After a combination of NKG2D and DAP10 in the form of homologous two polymers, the YxxM motif in the cytoplasm is phosphorylated and then signaling pathways are also gradually activated, such as PI3K, PLCγ2, JNK-cJunN, and others. Activated NK cells can enhance the sensitivity to hepatoma cells and specifically dissolve by releasing a variety of cytokines (TNF-α and IFN-γ), perforin, and high expression of FasL, CD16, and TRAIL. NK cells may specifically bind to the over-expressed MICA, MICB, and ULBPs of hepatocellular carcinoma cells through the surface activating receptor NKG2D, which can help to accurately identify hepatoma, play a critical role in anti-hepatoma via the pathway of cytotoxic effects, and obviously delay the poor progress of hepatocellular carcinoma.

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.