Expression Of Natural Killer Group 2D Research Articles

Natural killer group2D-major histocompatibility complex classI polypeptide-related sequenceA activation enhances natural killer cell-mediated immunity against hepatocellular carcinoma: A review.

The recent clinical introduction of immune checkpoint inhibitors has improved therapeutic outcomes in patients with advanced hepatocellular carcinoma. However, these therapies targeting CD8+ T lymphocytes have a response rate of approximately 30%. In addition to CD8+ T lymphocytes, natural killer (NK) cells represent promising therapeutic targets for hepatocellular carcinoma, because they comprise 30%-50% of all lymphocytes in the liver and contribute to antitumor immunity. A recent meta-analysis revealed that the percentage of infiltrating NK cells in hepatocellular carcinoma correlates with a better patient outcome. Similarly, our previous genome-wide association study on chronic viral hepatitis showed that a single-nucleotide polymorphism of major histocompatibility complex classI polypeptide-related sequenceA (MICA), a ligand to the NK activating receptor, plays a critical role in hepatocarcinogenesis. In this review, we summarize the mechanisms underlying the regulation of MICA and NK group 2D expression in chronic hepatitis. Furthermore, we describe recent reports on MICA single-nucleotide polymorphism-driven hepatocarcinogenesis. The suppression of MICA shedding could represent a promising approach for immunosurveillance, as increased expression of membrane-bound MICA achieved through the use of a MICA shedding inhibitor also enhances NK cell-mediated cytotoxicity.

Clearing soluble MIC reverses the impaired function of natural killer cells from patients with multiple myeloma

BackgroundMajor histocompatibility complex (MHC) class I chain-related protein (MIC) is a stress-induced ligand released from multiple myeloma (MM) cells during progression, and soluble MIC impairs natural killer group 2D (NKG2D)...

Highly Efficient & Specific Repair of MAGT1 Mutation in Xmen Patient T Cells and Hematopoietic Stem Cells

Introduction: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is a congenital glycosylation disorder with combined immune deficiency due to Magnesium Transporter 1 (MAGT1) mutations. Defective glycosylation of Natural Killer Group 2D (NKG2D), an activating receptor expressed on CD8 and NK cells critical for cytotoxicity, results in EBV lymphoproliferative disease and an inverted CD4:CD8 ratio in many XMEN patients. Oral magnesium supplementation for XMEN patients was not helpful, and hematopoietic stem cell transplants (HSCT) are complicated by high mortality. CRISPR-Cas9-AAV inserts MAGT1 specifically at endogenous locus but relies on double strand DNA breaks and adeno-associated virus donor that triggers DNA damage response. To address this, we evaluated base editing approach to repair a MAGT1 mutation that affects multiple patients with XMEN disease. We observed highly efficient mutation repair of XMEN T cells (75.5%) and hematopoietic stem cells (71.9%) to restore MAGT1 function and persistence of gene correction following transplant into immunodeficient mice. Materials and methods: Peripheral blood hematopoietic stem cells (HSPCs, CD34+) and mononuclear cells (PBMC) were collected from XMEN patients and healthy donors (HD) after G-CSF and plerixafor mobilization. PBMCs were stimulated (anti-CD3/anti-CD28, IL-2 (100 IU/mL), in ImmunoCult™-XF T cell expansion medium for 5-7 days prior to editing. HSPCs were cultured in StemSpan™ SFEM II Stem Cell Factor, Thrombopoietin, Fms-like tyrosine kinase 3 ligand (all 100ng/mL) and UM171 for two days prior to editing. Base editing is performed by electroporation (MaxCyte Inc.) of cells with ABE8e SpRY (CELLSCRIPT LLC.) and synthetic guide (Synthego) RNAs. For in vitro killing activity, T cells were co-cultured with EBV-B cells at a ratio of 1:1 for 48 hours and analyzed for apoptosis (Apoptracker Green+ 7AAD-), necrosis (Apoptracker Green+ 7AAD+) and degranulation (CD8+ CD107a+). HSPCs were transplanted into mice two days after EP and harvested 17-20 weeks after transplant. Results:Base editing of XMEN T cells: Flow cytometric analysis 2 days post BE show restored expression of biomarker NKG2D in XMEN CD8+ T cells (75.5%) compared to HD (93.2%) and naïve XMEN (2.39%). CD8+ cytotoxic activity against EBV-B cells was restored in BE XMEN patient cells; as indicated by increased apoptosis (BE: 6.78%, naïve: 4.82%, HD: 8.17%), necrosis (BE: 9.35%, naïve: 4.54%, HD: 7.19%), and degranulation (BE: 22.80%, naïve: 15.50%, HD: 21.40%), compared to naïve MAGT1 CD8+ T cells.In an in vivo tumor model, BE XMEN T cells injected into mice with induced luminescent EBV-B cell tumors lysed tumors and reduced average bioluminescence flux (photons/sec) (7 weeks) (8.23 × 107 ± 1.06 × 108) compared to naïve T cells (3.39 × 109 ± 2.22 × 109 and HD T cells (1.45 × 109 ± 2.22 × 109). Base editing of HSPCs: Immunodeficient NSGS pups transplanted with BE XMEN HSPCs engrafted and differentiated normally into lymphocytes with a normalized CD4:CD8 ratio in spleen (BE: 0.79 ± 0.14, naïve: 3.45 ± 1.1, HD: 1.3 ± 0.40) and peripheral blood (BE: 2.16 ± 0.84, naïve: 5.56 ± 2.32, HD: 1.79 ± 0.70) (Fig 1a). Importantly, restoration of biomarker NKG2D expression selectively on CD8+ T cells in spleen (BE: 80.5% ± 8.61%, naïve: 3.63% ± 1.45%, HD: 68.9% ± 10.32%) and peripheral blood (BE: 86.1% ± 2.88%, naïve: 2.25% ± 1.45%, HD: 62.6% ± 12.44%) (Fig 1b) confirmed correction of MAGT1 function in the glycosylation of critical NKG2D protein. High throughput targeted sequencing of on-target locus shows minimal bystander edits and additional off-target analysis is ongoing. Conclusion: Our data show highly efficient and specific base editing repair of MAGT1 mutation in XMEN T lymphocytes and CD34+ HSPCs. BE XMEN T cells increased cytotoxic activity in vitro and killing of tumor cells in mice. In vivo studies with mice transplanted with BE CD34+ HSPCs also confirm long term engraftment and differentiation into CD8+ T cells with restored NKG2D necessary for activation and cytolytic activity of CD8+ T and NK cells. The highly efficient and specific correction with base editing holds great promise for gene therapy for XMEN disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Differential modulation of natural killer cell cytotoxicity by 17β‑estradiol and prolactin through the NKG2D/NKG2DL axis in cervical cancer cells

Natural killer (NK) cells play a crucial role in cervical cancer (CC). As estrogens and prolactin (PRL) have been reported to be involved in CC, the present study attempted to elucidate the effects of both hormones on NK cells in CC. For this purpose, NKL cells, as well as CC-derived cell lines (HeLa, SiHa and C33A) and non-tumorigenic keratinocytes (HaCaT cells) were stimulated with 17β-estradiol (E2; 10 nM), PRL (200 ng/ml), or both (E2 and PRL) for 48 h. The expression of hormone receptors (estrogen receptor α and β, G protein-coupled estrogen receptor 1 and PRL receptor) and NK cell activating receptors [natural killer group 2D (NKG2D), natural cytotoxicity triggering receptor 3, natural cytotoxicity triggering receptor 2 and natural cytotoxicity triggering receptor 1] were measured using western blot analysis and flow cytometry, respectively. In the HeLa, SiHa, C33A and HaCaT cells stimulated with the hormones, the expression of NKG2D ligands [MHC class I polypeptide-related sequence A/B (MICA/B)] on the membrane and the soluble form of MICA was evaluated using flow cytometry and ELISA. Cytotoxicity assay was performed using GFP-transfected K562 cells as target cells. E2 reduced NKL cell-mediated cytotoxicity, while PRL exerted the opposite effect. NKL cells expressed different hormone receptor forms, of which PRL only induced a decrease in NKG2D expression compared to the untreated control NKL cells. PRL increased MICA/B expression in HeLa cells and E2 and PRL reversed this effect. However, in SiHa cells, the concurrent incubation with the two hormones decreased MICA/B expression. E2 and PRL, either alone or in combination, decreased soluble MICA secretion in all CC cell lines, while E2 solely increased soluble MICA secretion in SiHa cells. On the whole, the present study provides evidence that E2 and PRL mediate the mechanisms through which NK and CC cells mediate a cytotoxic response and these have an antagonistic effect on NK cell-mediated cytotoxicity.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells.

Chemotherapy-induced senescence promotes immunocyte aggregation in the tumor microenvironment by upregulating the surface expression of activating ligands in cancer cells. However, these senescent tumor cells cannot be completely cleared and can induce tumor recurrence. Previous studiesshowed that soluble natural killer (NK) group 2D (NKG2D) ligands impair the recognition of multiple immune cells. In this study, we established an in vitro senescence model using neuroblastoma cells subjected to low-dose Chemotherapeutic drug doxorubicin or the Aurora A inhibitor MLN8237. The results showed that different neuroblastoma cell lines showed increased secretion of the NKG2D ligand MHC class I polypeptide-related sequence A/B (MICA/B) following proteolysis after treatment, with MICA/B subsequently recruited to exosomes to downregulate NKG2D expression in NK cells. Interestingly, disintegrin and metalloproteinase domain-containing 10 (ADAM10) was upregulated in senescent tumor cells, and combined treatment with the ADAM10 inhibitor GI254023X and chemotherapeutic drugs inhibited MICA/B secretion and enhanced recognition and killing by NK cells. Additionally, we found that expression of the long noncoding RNA MALAT1 was significantly increased in senescent neuroblastoma cells, and that MALAT1 served as a sponge for microRNA (miR)-92a-3p to counteract miR-92a-3p-mediated repression of ADAM10 levels. Furthermore, administration of a MALAT1 inhibitor or an miR-92a-3p mimic reduced the MICA/B shedding and enhanced recognition and killing by NK cells. These results confirmed that low-dose chemotherapy induces senescence in neuroblastoma cells, and that senescent tumor cells promote the shedding of the NKG2D ligand MICA/B through the MALAT1/miR-92a/ADAM10 axis, thereby contributing to the formation of a suppressive immune microenvironment and promoting immune escape.

Liver-Resident Bystander CD8+ T Cells Contribute to Liver Disease Pathogenesis in Chronic Hepatitis D Virus Infection

The hepatitis D virus (HDV) causes the most severe form of chronic hepatitis, often progressing to cirrhosis within 5 to 10 years. There is no curative treatment, and the mechanisms underlying the accelerated liver disease progression are unknown. Innate and adaptive immune responses were studied in blood and liver of 24 patients infected with HDV and 30 uninfected controls by multiparameter flow cytometry in correlation with disease severity and stage. The 2 main intrahepatic innate immune-cell populations, mucosal-associated invariant T cells and natural killer (NK) cells, were reduced in the livers of patients infected with HDV compared with those of uninfected controls but were more frequently activated in the liver compared with the blood. Most intrahepatic cluster of differentiation (CD) 8-positive (CD8+) T cells were memory cells or terminal effector memory cells, and most of the activated and degranulating (CD107a+) HDV-specific and total CD8+ T cells were liver-resident (CD69+C-X-C motif chemokine receptor 6+). Unsupervised analysis of flow cytometry data identified an activated, memory-like, tissue-resident HDV-specific CD8+ T-cell cluster with expression of innate-like NK protein 30 (NKp30) and NK group 2D (NKG2D) receptors. The size of this population correlated with liver enzyme activity (r= 1.0). NKp30 and NKG2D expression extended beyond the HDV-specific to the total intrahepatic CD8+ T-cell population, suggesting global bystander activation. This was supported by the correlations between (i) NKG2D expression with degranulation of intrahepatic CD8+ T cells, (ii) frequency of degranulating CD8+ T cells with liver enzyme activity and the aspartate aminotransferase-to-platelet ratio index score, and by the invitro demonstration of cytokine-induced NKG2D-dependent cytotoxicity. Antigen-nonspecific activation of liver-resident CD8+ T cells may contribute to inflammation and disease stage in HDV infection.

Potential therapeutic effects of cyanidin-3-O-glucoside on rheumatoid arthritis by relieving inhibition of CD38+ NK cells on Treg cell differentiation

BackgroundCD38+ NK cells are overabundant in rheumatoid arthritis (RA). Cyanidin-3-O-glucoside (C3G) is an inhibitor of CD38. This study investigated the pathogenic role of CD38+ NK cells and the effect of C3G on RA.MethodsRats with bovine type II collagen-induced arthritis (CIA) were injected with C3G. RA synovial fibroblasts (RASFs) or mononuclear cells (MNCs) were cultured with C3G. MNCs were also cocultured with CD38+ NK cells following C3G pretreatment.ResultsC3G injection significantly alleviated CIA. C3G also significantly increased the level of interleukin (IL)-10 and the regulatory T (Treg) cell proportion, and it decreased the interleukin (IL)-6 and interferon (IFN)-γ levels and CD38+ NK cell proportion in rat peripheral blood and synovial fluid. Additionally, C3G significantly increased RASF apoptosis and decreased RASF proliferation and IL-6 production in the culture medium. Furthermore, C3G stimulated MNCs to increase IL-2 and IL-10 production and the Treg cell proportion, and it caused MNCs to decrease IL-6 and IFN-γ production and the CD38+ NK cell proportion. Although CD38+ NK cells significantly decreased the Treg cell proportion and IL-10 level in MNCs, CD38+ NK cells that had been pretreated with C3G increased the proportion of Treg cells and IL-10 levels and decreased the IL-6 and IFN-γ levels in the coculture. In CD38+ NK cells, C3G significantly increased Sirtuin 6 (Sirt6) expression and the tumor necrosis factor (TNF)-α level, and it decreased natural killer group 2D (NKG2D) expression and the IFN-γ level. However, when CD38+ NK cells were treated with Sirt6 siRNA, C3G did not change the NKG2D expression, the TNF-α level sharply decreased, and the IFN-γ level increased. When MNCs were cocultured with C3G-pretreated CD38+ NK cells in the presence of TNF-α and an anti-IFN-γ antibody, the IL-10+ Treg cell proportion significantly increased. When MNCs were cocultured with C3G-pretreated CD38+ NK cells in the presence of IFN-γ and an anti-TNF-α antibody, the IL-10+ Treg cell proportion sharply decreased. When CIA rats were injected with both C3G and the Sirt6 inhibitor OSS_128167, the rats exhibited joint inflammation and a low Treg cell proportion, but the CD38+ NK proportion was still low.ConclusionC3G has therapeutic effects on CIA and RA. C3G decreased the proportion of CD38+ cells, RASF proliferation, and proinflammatory cytokine secretion, and it increased the Treg cell proportion. C3G also elevated Sirt6 expression to suppress NKG2D expression, increase TNF-α secretion, and decrease IFN-γ secretion in CD38+ NK cells, which stimulates MNCs to differentiate into Treg cells. This study also demonstrates that the inhibition of Treg cell differentiation in MNCs by CD38+ NK cells is a potential cause of the immune imbalance in RA and CIA.

Ultrasound-guided radiofrequency ablation enhances natural killer-mediated antitumor immunity against liver cancer.

For patients with liver cancer who are not sufficiently fit for surgical resection, radiofrequency ablation (RFA) is an effective and low risk treatment modality; however, the mechanism underlying this procedure is not fully understood. In the present study, a series of experiments were conducted, which demonstrated that RFA therapy stimulates innate antitumor immunity via directly enhancing natural killer (NK) cell cytotoxicity, thus achieving a favorable outcome for patients with liver tumors. It was determined that the percentage of NK cells within the peripheral blood of the rabbits in the RFA treatment groups were significantly higher, compared with the control groups. The levels of interferon-γ and tumor necrosis factor-α in NK cells were also significantly upregulated following thermal coagulation induced via RFA. In addition, RFA enhanced the NK cell receptor, NK group 2D (NKG2D), expression and NK cell antitumor cytotoxicity in hepatic cancer cells. The results indicated that the RFA treatment could effectively eliminate liver tumors via enhancing NK-mediated antitumor activity and NKG2D expression.

Role of natural killer cells in liver transplantation treatment of liver cancer.

Liver cancer caused by diet or life style is a significant public health problem. Liver transplantation (LT) is a commonly used method of treatment for the liver cancer. The present study aimed to determine whether assessing the net state of natural killer (NK) cell function following LT distinguishes patients at risk for transplantation rejection. A total of 53 patients were involved; all underwent LT for hepatocellular carcinoma with (n=13) or without (n=40) transplantation rejection. The density of interferon-γ (IFN-γ) in blood serum was examined and patients were divided into two groups: Higher (H) and lower (L), on the basis of IFN-γ density. The percentage of NK cells and their producing cytokines was detected using fluorescence-activated cell sorting in peripheral blood and liver samples. As evaluation indexes of liver function, aspartate transaminase (AST) and alanine transaminase (ALT) were detected in blood serum. NK cell activation of the H-group was observed to be higher than the L-group, specifically the expression of NK group 2D, cluster of differentiation 69 and IFN-γ were higher than the L-group. The H-group exhibited a higher level of AST and ALT, which indicates the potential for acute transplantation rejection. The results of the present study indicate that NK cells and NK-derived IFN-γ serve an important function in regulating the rejection of LT and tumor metastasis in response to LT.

Natural Killer Group 2A Expressed on Both Peripheral CD3-CD56+NK Cells and CD3+CD8+T Cells Plays a Pivotal Negative Regulatory Role in the Progression of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

To explore the role of surface receptors natural killer group 2A (NKG2A) and natural killer group 2D (NKG2D) on CD3+CD8+T cells and CD3-CD56+NK cells in the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF), we measured the expression of NKG2A and NKG2D on the surface of these 2 types of circulating cells by flow cytometry in 3 groups. One group consists of 36 patients with chronic hepatitis B (CHB), another one consists of 22 patients with HBV-related ACLF, and the last one has 12 normal controls (NC). The experimental result indicated that there was no significant difference in the proportion of CD3+CD8+T cells in total lymphocytes between the 3 groups. However, the percentage of CD3-CD56+NK cells in ACLF group was evidently higher than that in the CHB group (P < 0.05). In addition, the expression of NKG2D on CD3+CD8+T cells in the ACLF group was significantly lower than that in the CHB group (P < 0.05), but there were no statistically significant differences in its percentages on CD3-CD56+NK cells between the 3 groups. The expression of NKG2A on CD3+CD8+T cells in the ACLF group was significantly higher than that in the NC group (P < 0.05), and on NK cells was significantly higher than that in the CHB group (P < 0.05) and NC group (P < 0.01). The increase in ratios of NKG2A to NKG2D on CD3+CD8+T cells and CD3-CD56+NK cells in the ACLF group was significantly more than that in the CHB group and NC group. The results indicate that the imbalance between NKG2A and NKG2D may contribute to the progression of HBV-related ACLF mediated by CD3-CD56+NK cells and CD3+CD8+T cells. Compared with NKG2D, NKG2A expressed on both peripheral CD3-CD56+NK cells and CD3+CD8+T cells plays a more pivotal negative regulatory role in the progression of HBV-related ACLF.

Regulation of NKG2D+CD8+ T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism

ABSTRACTThe natural killer (NK) group 2D (NKG2D) receptor, which displays on mouse and human NK cells, activates CD8+ T cells and small subsets of other T cells. NKG2D+CD8+ T cells play critical roles in both innate and adaptive immunity upon engagement with NKG2D ligands to eliminate tumor and infected cells. Despite the important role of NKG2D+CD8+ T cells in immune surveillance, the mechanisms of how NKG2D expression on CD8+ T cells is regulated remain poorly defined. We treated mouse and human CD8+ T cells with CD80 recombinant protein, plus a pharmacologic model with small molecular inhibitors to determine which signaling pathway leads to NKG2D regulation on CD8+T cells. This study revealed that CD28 activation gives rise to sustained NKG2D expression on both mouse and human CD8+ T cells in a signal transducer and activator of transcription 3 (STAT3) phosphorylation-dependent manner. Further, we found that CD28 activation stimulated sustained activation of the tyrosine kinase Lck, which recruits and triggers Janus kinase/STAT3 signaling to phosphorylate STAT3, and in turn increases NKG2D expression. Moreover, NKG2D induction on CD8+ T cells exerts cytolytic activity against target tumor cells in vitro, as well as significantly improves the antitumor therapeutic effects in vivo in an NKG2D-dependent manner. Taken together, these results elucidated a novel mechanism of NKG2D regulation by phosphorylated STAT3 (pSTAT3) on CD8+ T cells upon CD28 activation. This mechanism may shed light on the effectiveness of CD80-based, NKG2D-dependent antitumor immunotherapy.

71. Validation of Manufacturing NKG2D Chimeric Antigen Receptor T Cells for a First-In-Human Clinical Trial in AML/MDS and Multiple Myeloma

Canonical CAR-T cells are engineered using constructs encoding a single chain variable fragment derived from a murine or humanized antibody, a costimulatory domain connected by a hinge region, and the signaling domain of CD3 zeta. This restricts T cells transduced with these constructs to recognizing one tumor antigen, such as CD19, and a limited set of cancers. Prior work in murine models demonstrated preclinical efficacy of T cells transduced with a novel activating CAR construct that exploits the ability of NK cells to recognize a variety of tumor antigens. Specifically, a CAR was generated by fusion of native full-length human Natural Killer Group 2D (NKG2D) gene with the human CD3 zeta cytoplasmic signaling domain. The NKG2D receptor in association with natural costimulatory molecule DAP10 creates a unique 6 protein receptor complex enabling NK and CD8+ T cells to kill many cells types via recognition of ligands including MIC-A, MIC-B, and UL-16 binding proteins 1-6. NKG2D-ligands are upregulated in malignancy but protein expression is absent or minor in healthy tissues. Complete remissions and durable CD4+ and CD8+ T-cell memory have been demonstrated in murine models of lymphoma, myeloma and ovarian cancer following adoptive therapy with NKG2D CAR T cells. At the same time, NKG2D CAR T cells significantly alter the tumor microenvironment through cytokine secretion to further promote anti-tumor immunity. This technology is being evaluated clinically in a phase I dose-escalation study (ClinicalTrials. gov NCT02203825) arising through collaboration between academic investigators and commercial sponsors. GMP manufacturing procedures for NKG2D CAR T cells were developed to support this first-in-human trial of NKG2D CAR T cells to assess safety and feasibility in acute myeloid leukemia/myelodysplastic syndrome and multiple myeloma. Following isolation of mononuclear cells, T cells were activated with anti-CD3 mAb and IL-2, subjected to 2 rounds of transduction with SFG retroviral vector containing the NKG2D CAR construct (CM-CS1), and expanded in media containing IL-2. GMP manufacturing procedures were validated using T cells from healthy donors as well as patients with AML and myeloma. Validation studies and initial clinical manufacturing demonstrated consistent viability, robust cell expansion, vector-mediated surface expression of NKG2D on a median of 53.6% CD8+ and 90.3% of CD4+ T cells, consistent viral copy number/cell less than 5, and the absence of replication-competent retrovirus in CM-CS1 T cells. Functionally, CM-CS1 T cells exhibit potent IFN-γ production following exposure to NKG2D ligand-expressing tumor cells. We also demonstrated minimal carry-through of malignant myeloid or plasma cells after deriving autologous CM-CS1 T cells from patients with malignancies. The Phase I trial using CM-CS1 T cells is ongoing, with dose-escalation proceeding as planned. Future goals include application of this novel cell therapy to additional malignant diseases.

A gluten-free diet lowers NKG2D and ligand expression in BALB/c and non-obese diabetic (NOD) mice

The interplay between diet and immune parameters which could affect type 1 diabetes (T1D) pathogenesis is not sufficiently clarified. Intestinal up-regulation of the activating receptor natural killer group 2D (NKG2D) (CD314) and its ligands is a hallmark of coeliac disease. However, the direct effect of gluten on NKG2D expression is not known. We studied, by fluorescence activated cell sorter (lymphoid tissues) and reverse transcription–quantitative polymerase chain reaction (intestine and pancreatic islets), if a gluten-free diet (GF diet) from 4 weeks of age or a gluten-free diet introduced in breeding pairs (SGF diet), induced changes in NKG2D expression on DX5+(CD49b) natural killer (NK) cells, CD8+ T cells and in intestinal and islet levels of NKG2D and ligands in BALB/c and non-obese diabetic (NOD) mice. Gluten-free NOD mice had lower insulitis (P < 0·0001); reduced expression of NKG2D on DX5+ NK cells in spleen and auricular lymph nodes (P < 0·05); and on CD8+ T cells in pancreas-associated lymph nodes (P = 0·04). Moreover, the level of CD71 on DX5+ NK cells and CD8+ T cells (P < 0·005) was markedly reduced. GF and SGF mice had reduced expression of NKG2D and DX5 mRNA in intestine (P < 0·05). Differences in intestinal mRNA expression were found in mice at 8, 13 and 20 weeks. Intestinal expression of NKG2D ligands was reduced in SGF mice with lower expression of all ligands. In isolated islets, a SGF diet induced a higher expression of specific NKG2D ligands. Our data show that a gluten-free diet reduces the level of NKG2D and the expression of NKG2D ligands. These immunological changes may contribute to the lower T1D incidence associated with a gluten-free diet.

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

Background/AimsThis study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function.MethodsThe number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls.ResultsWhen compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D+ NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above.ConclusionsThe decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

Mature Cytotoxic CD56bright/CD16+Natural Killer Cells Can Infiltrate Lymph Nodes Adjacent to Metastatic Melanoma

Melanomas are characterized by high metastatic potential, with regional lymph node representing the most frequent site of early dissemination in this disease. These regional lymph nodes also represent the primary site for differentiation of natural killer (NK) cells. Although blood-derived NK cells can efficiently lyse melanoma cells isolated from metastatic lymph node (M-LN), there has been no study of the properties of the most disease-relevant NK cells isolated from M-LN in patients with melanoma. Here, we report that M-LN contains 0.5% to 11% of CD56(bright) NK cells among CD45(+) hematopoietic cells present and that this cell population surrounds tumor cell clusters in M-LN. This NK cell population was characterized by expression of CD62L, chemokine receptors, and high levels of natural cytotoxicity receptors (NCR), NK group 2 D (NKG2D), and DNAX accessory molecule 1 (DNAM-1). Expression of NCR-NKp30 and NKG2D correlated negatively with percentages of tumor cells in M-LN. Interestingly, M-LN contained a unique subset of mature CD56(bright)CD16(+) NK cells displaying coregulated expression of NCR and NKG2D activating receptors. Ex vivo analyses suggested that M-LN-derived NK cells were inactive but could be activated by appropriate cytokine signals [interleukin (IL)-2 or IL-15], and could lyse metastatic melanoma cells in a highly efficient manner compared with blood-derived NK cells. Taken together, the results offer evidence that adjuvant immunotherapy that targets NK cells in M-LN for activation may improve treatment of patients with sentinel lymph node-positive melanoma.

Interleukin‐15 mediates protection against experimental tuberculosis: A role for NKG2D‐dependent effector mechanisms of CD8+ T cells

CD8+ T cells are involved in protection against Mycobacterium tuberculosis infection and represent a promising target for new vaccine strategies. Because IL-15 is important for the homeostasis of CD8+ T cells, we studied the immune response in IL-15-deficient mice during tuberculosis. In the absence of IL-15, CD8+ T cells failed to efficiently accumulate in draining lymph nodes and at the site of infection. The expression of antigen-specific effector functions, such as the production of interferon-gamma and cytotoxicity, were impaired in CD8+ T cells, but not CD4+ T cells, from IL-15-deficient mice. This defect was associated with an increased mortality of IL-15-deficient mice during the chronic phase of infection. The lectin-like stimulatory receptor natural killer group 2D (NKG2D) was up-regulated on CD8+ T cells only from wild-type mice, but not from IL-15-deficient mice. Mechanistically, blocking NKG2D function with an mAb inhibited M. tuberculosis-directed CD8+ T cell responses in vitro. We conclude that in addition to regulating the expansion of CD8+ T cells, IL-15 is also necessary for inducing effector mechanisms in CD8+ T cells that depend on NKG2D expression. Hence, our results implicate IL-15 and NKG2D as promising targets for modulating CD8+ T cell-mediated protection against tuberculosis.