Natural Killer Group 2D Receptor Research Articles

NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vβ chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D)

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein–protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Amygdalin-folic acid-nanoparticles inhibit the proliferation of breast cancer and enhance the effect of radiotherapy through the modulation of tumor-promoting factors/ immunosuppressive modulators in vitro

IntroductionBreast cancer (BC) cells often develop multiple mechanisms of chemo- and radio-resistance during tumor progression, which is the major reason for the failure of breast cancer therapy. Targeted nanomedicines have tremendous therapeutic potential in BC treatment over their free drug counterparts. Searching for chemo- and radio-sensitizers to overcome such resistance is therefore urgently required. The goal of this study is to evaluate and compare the radio-sensitizer efficacy of amygdalin-folic acid nanoparticles (Amy-F) on MCF-7 and MDA-MB-231 cells.Materials and methodsThe effects of Amy-F on MCF-7 and MDA-MB-231 cell proliferation and IC50 were assessed using MTT assay. The expression of proteins involved in several mechanisms induced by Amy-F in MCF-7 and MDA-MB-231 cells, including growth inhibition, apoptosis, tumor growth regulators, immuno-modulators, and radio-sensitizing activities were evaluated via flow cytometry and ELISA assay.ResultsNanoparticles demonstrated sustained Amy-F release properties and apparent selectivity towards BC cells. Cell-based assays revealed that Amy-F markedly suppresses cancer cell growth and improves radiotherapy (RT) through inducing cell cycle arrest (G1 and sub-G1), and increases apoptosis as well as reduces the proliferation of BC by down-regulating mitogen-activated protein kinases (MAPK/P38), iron level (Fe), nitric oxide (NO), and up-regulating the reactive oxygen species level (ROS). Amy-F has also been shown to suppress the expression of the cluster of differentiation (CD4 and CD80), and interfere with the Transforming growth factor beta (TGF- β)/Interferon-gamma (INF-g)/Interleukin-2 (IL-2)/Interleukin-6 (IL-6)/Vascular endothelial growth factor (VEGF) induced suppression in its signaling hub, while up-regulating natural killer group 2D receptor (NKG2D) and CD8 expression.ConclusionsCollectively, the novel Amy-F either alone or in combination with RT abrogated BC proliferation.

Ciclopirox olamine sensitizes leukemia cells to natural killer cell-mediated cytolysis by upregulating NKG2DLs via the Akt signaling pathway

The activating receptor natural killer group 2D (NKG2D) expressed by Natural killer (NK) cells functions as a “master-switch” in governing the awakening status of NK cells. The NKG2D-mediated cytotoxicity has been declared to be related with the expression levels of NKG2D ligands (NKG2DLs) expressed on tumor cells. Therefore, selective induction of NKG2DLs could be a reliable approach to enhance the efficacy of NK cell-mediated immunotherapy. Our existing study demonstrated that Ciclopirox Olamine (CPX), an off-patent antifungal agent, effectively elevated the expression of NKG2DLs on leukemia cells and sensitized leukemia cells to NK-cell mediated cytolysis. Induction of ROS production and AKT phosphorylation by CPX is essential for the up-regulation of NKG2DLs expressions. Inhibition of AKT by using AKT inhibitor MK2206 decreased both NKG2DLs expressions and NK cell cytotoxicity. These data indicated that increased sensitivity of CPX-treated leukemia cells to NK cell cytolysis was attributed to higher NKG2DLs expressions, resulting from activated AKT signaling pathway. Our findings support the ongoing development of CPX as an anti-tumor agent and suggest its promising immunotherapeutic value in the medication of leukemia.

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear.Method In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo.ResultsCombined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors.ConclusionRadiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

Natural Killer Group 2D Receptor and B1a Cells Crosstalk in Post-Hepatitis C Virus Infection Hepatocellular Carcinoma and Cirrhosis.

BackgroundNatural killer (NK) and B1a cells are implicated in innate immune surveillance against chronic hepatitis C virus (CHCV). NK group 2D (NKG2D) receptor is important for B cell differentiation. This study was designed to assess whether B1a cells and NK Cells expressing NKG2D are implicated in post-hepatitis C infection hepatocellular carcinoma (post-HCV HCC) and cirrhosis using flow cytometry and investigate the association between NK-expressing NKG2D and B1a in complications of CHCV infection.MethodsIn this cross-sectional study, 111 participants were included and divided into the post-HCV HCC (n = 50), post-HCV liver cirrhosis (n = 31), and CHCV (n = 30) groups.ResultsThe percentage of B1a cells (B1a%) and the mean fluorescence intensity (MFI) of NKG2D (NKG2D MFI) showed a significant increase in the CHCV group compared with those in the post-HCV liver cirrhosis and post-HCV HCC groups (P < 0.05). A positive correlation was observed between NKG2D MFI and B1a% (r = 0.6, P < 0.001). The receiver operating characteristic (ROC) curve revealed that NKG2D MFI and B1a% differentiated between patients with CHCV infection and those with HCC with a sensitivity of 92% and 98%, respectively, and differentiated between patients with CHCV infection and those with liver cirrhosis with a sensitivity of 94% and 90%, respectively.ConclusionDownregulation of B1a frequency and NKG2D intensity is implicated in the progression of CHCV infection to cirrhosis and HCC. NKG2D receptor is associated with the frequency of circulating B1a cells. NKG2D intensity and B1a% can be used as indicators of CHCV progression.

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.

Study of the association between a MICA gene polymorphism and cholangiocarcinoma in Egyptian patients.

An inflammatory environment is the common pathway for the development of cholangiocarcinoma (CCA). The natural killer group 2D receptor (NKG2D), an activating receptor for NK cells, is a potent immune axis in the antitumor and antimicrobial immune response through its binding to NKG2D ligands (NKG2DLs). NKG2DLs are normally absent or poorly expressed in most cells; conversely, they are upregulated in stressed cells. We studied the rs2596542 polymorphism located upstream of the MICA gene, which encodes an NKG2DL, in patients with CCA as a marker for early disease detection and a possible therapeutic target. A case-control study was conducted on 40 patients with CCA and 45 healthy individuals (as controls). After routine examination, the rs2596542 polymorphism of the MICA gene was investigated using real-time PCR. We found that a TT homozygous genotype was significantly predominant in patients with CCA (p = 0.039), with the T allele being dominantly distributed in CCA (p = 0.007). High levels of CA19-9 were significantly associated with the TT genotype in the patients. However, we did not detect significant differences in rs2596542C/T genotype and allele distribution between patients with CCA with cirrhosis and those without cirrhosis (p > 0.05). The MICA rs2596542 polymorphism may affect the susceptibility to CCA, but not its progression. The TT genotype could be used as a potential diagnostic marker for CCA and triggering the MICA pathway could be a promising therapeutic target.

Co-Expression of an shRNA Targeting MICA/Micb Improves the Clinical Activity of a NKG2D-Based CAR T in Patients with Relapsed / Refractory AML/MDS

While CAR T cell therapy has delivered impressive clinical efficacy in B cell malignancies, similar levels of clinical activity have not been demonstrated in acute myeloid leukemia (AML). One underlying reason for this lack of translation is the relative paucity of validated CAR T targets. Major Histocompatibility Complex Class 1 related proteins MICA and MICB along with the UL16 binding proteins 1-6 (ULBP1-6) are frequently over-expressed on AML and myelodysplastic syndrome (MDS) blasts. The Natural Killer Group 2D (NKG2D) is a single receptor able to bind MICA, MICB and ULBP1-6, thus providing a potentially powerful approach that could be exploited to target this family of targets thereby providing a novel CAR T approach for AML/MDS.Expressing the NKG2D receptor fused to the human CD3z cytoplasmic domain generates a CAR that, when expressed in primary T cells, generates a CAR T product (termed CYAD-01) that showed good anti-tumor activity in preclinical models. In clinical trials, CYAD-01 showed a good tolerability profile but with a disappointing level of clinical activity when combined with cyclophosphamide / fludarabine preconditioning (DEPLETHINK trial, NCT03466320).We hypothesised that the transient expression of MICA/MICB on the CAR T itself may inhibit the activity of the NKG2D CAR through self-targeting and consequent fratricide. We developed a single shRNA able to knockdown both MICA and MICB. Co-expression of this shRNA with the NKG2D CAR generated a second generation CAR T product termed CYAD-02 which was examined in a highly similar AML/MDS patient population and preconditioning regimen as employed in the DEPLETHINK trial.CYAD-02 was evaluated in the the first-in-human CYCLE-1 trial (NCT04167696) in patients with r/r AML/MDS. The dose-escalation phase evaluated three dose-levels (DL) (1x10 8, 3x10 8 and 1x10 9 total cells per infusion), administered as a single infusion after standard preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classic 3+3 design.As of July 2021, 11 patients have been treated with CYAD-02 at the different dose-levels (3 at each DL1 and 2 and 5 at DL3). In terms of safety and tolerability, there was no evidence of a differential safety profile of CYAD-02 within the CYCLE-1 trial as compared to that observed in the CYAD-01 clinical trial. Of note, five patients enrolled in all 3 DLs experienced Grade ≥ 3 adverse events (AE) at least possibly related to CYAD-02 (cytokine release syndrome, febrile neutropenia, white blood cell count decreased, infusion related reaction).With respect to clinical activity, seven patients in total achieved stable disease (2 at DL1, 3 at DL2 and 2 at DL3) with 4 demonstrating evidence of transient blast reduction or anti-leukemic activity (decrease of at least 50% of the bone marrow blasts). In addition, two MDS patients at DL3 achieved a marrow complete response. Overall, this suggests dose dependent response to CYAD-02. Furthermore, patients receiving CYAD-02 cells seem to display a greater duration of response and stronger blast reduction as compared to patients who received CYAD-01 in DEPLETHINK.Interestingly, peak engraftment levels were higher for CYAD-02 compared to CYAD-01 at the same dose (DEPLETHINK trial). At DL3, CYAD-02 cells could be readily detected in peripheral blood of 4/5 patients through month 2. In addition, and unlike what is reported in B cell CAR T-cells trials, there was very limited evidence of elevated homeostatic cytokines (IL-7/IL-15) following CyFlu administration with IL-15 not detected in any of the patients and IL-7 showing only a minor increase in 2 patients.The knockdown of MICA/MICB appears to have a positive contribution to the initial clinical activity of CYAD-02 as compared to that achieved with the first generation CYAD-01 CAR T, together with good safety and tolerability. The lack of homeostatic cytokines after preconditioning, likely limiting the engraftment and activity of CAR T cells, may be related to the biology of myeloid malignancies. One approach to further drive the potency of NKG2D-based CAR T cells would likely be armoring the CAR T through using the T cell as a vehicle to secrete cytokines alongside the CAR. Overall, shRNA knockdown technology provides a means to modify CAR T function and here shows that single shRNA can target two independent genes to enhance the phenotype of the CAR Ts. DisclosuresDeeren: Alexion: Consultancy; BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Sanofi: Consultancy, Research Funding; Sobi: Consultancy; Takeda: Consultancy. Lin: AbbVie, Aptevo Therapeutics, Astellas Pharma, Bio-Path Holdings, Celgene, Celyad, Genentech-Roche, Gilead Sciences, Incyte, Jazz Pharmaceuticals, Novartis, Ono Pharmaceutical, Pfizer, Prescient Therapeutics, Seattle Genetics, Tolero, Trovagene: Research Funding. Alcantar-Orozco: Celyad Oncology: Current Employment. Dheur: Celyad Oncology: Current Employment. Breman: Celyad Oncology: Current Employment. Braun: Celyad Oncology: Current Employment. Lonez: Celyad Oncology: Current Employment. Gilham: Celyad Oncology: Current Employment. Flament: Celyad Oncology: Current Employment. Lehmann: Celyad Oncology: Current Employment.

The natural killer cell activating receptor, NKG2D, is critical to antibody-dependent chronic rejection in heart transplantation

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor-recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag-/- recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.

Expression profiles of Natural Killer Group 2D Ligands (NGK2DLs) in colorectal carcinoma and changes in response to chemotherapeutic agents.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Natural Killer Group 2D Receptor (NKG2D) and their ligands (NKG2DLs) play crucial roles in natural killer (NK) cell-mediated cytotoxicity. Tumorigeneses cause increased NKG2DLs expression on tumor cell surfaces, thereby these cells individually eliminated by NK cells. However, CRC cells can reduce their NKG2DL expression to escape from NK-mediated immune surveillance which is associated with poor prognosis. Therefore, previous studies suggest that up-regulation of NKG2DLs can contribute to promising NK cell-mediated immunotherapy strategies. We aimed to analyze NKG2DLs expression profiles in response to chemotherapeutic drugs and increased MHC class I polypeptide-related sequence A (MICA) expression, which is related to favorable prognosis in CRC, using low doses of bortezomib and epirubicin combination without causing direct cytotoxicity. Results showed that MICA expression sligthlyincreased following drug treatment in the CRC cells but not for the normal cells. Also, we enriched our study with Gene Expression Omnibus (GEO) datasets including expression profiles of various NKG2DLs using in silico analyses. Accordingly, NKG2DL expression in CRC was screened in proportion to other cancers, histologic subtypes, TNM stages and metastatic samples to compare with our data. Overall, the analyzed data showed that NKG2DLs demonstrate different expression profiles in response to chemotherapeutic agents and a combination of low-dose bortezomib and epirubicin slightly increased MICA mRNA expression in CRC cell lines. However, performing further analysis of the combination therapy for MICA protein expression and studying its interaction with NK cells will make the results more meaningful.

Natural Killer Cell Phenotype and Functionality Affected by Exposure to Extracellular Survivin and Lymphoma-Derived Exosomes.

The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Clinical Impact of Natural Killer Group 2D Receptor Expression and That of Its Ligand in Ovarian Carcinomas: A Retrospective Study.

PurposeNatural killer (NK) cells are innate immune cells with antitumor activity. NKG2D is the most important activating receptor expressed on the NK cell surface; this receptor binds to the ligands MICA/B and ULBPs to activate NK cells. The current study aimed to evaluate the expression of NKG2D by NK cells, and to the evaluate expression of its ligands in ovarian carcinomas; it also examined the clinical relevance of NK receptor/ligand expression by analyzing the relationship between expression, clinicopathological parameters, and prognosis.Materials and MethodsFormalin-fixed paraffin-embedded archival ovarian high-grade serous carcinoma (HGSC, n=79) tissue samples were used for tissue microarray analysis. The expressions of NK cell markers (CD56 and NKG2D) and NKG2D ligands (MICA/B, ULBP1, ULBP3, and ULBP2/5/6) in carcinoma tissues were evaluated by immunohistochemical staining, and the association between these results and clinical prognostic parameters was analyzed statistically.ResultsULBP1 was highly expressed in 51 cases (64.6%), and ULBP2/5/6 was highly expressed in 56 cases (70.9%) of HGSC. High expression of ULBP1 and ULBP2/5/6 was significantly associated with lower recurrence of HGSC, whereas high expression of ULBP3 was significantly associated with higher recurrence. Multivariate Cox regression analysis revealed that high expression of ULBP1 was associated with increased overall survival and a decreased hazard ratio (0.150, p=0.044), suggesting that it is an independent predictor of better survival.ConclusionHigh expression of ULBP1 predicts a better prognosis for HGSC, suggesting that ULBP1 expression could be a novel prognostic indicator in this subset of carcinomas.

MICA ∗012:01 Allele Facilitates the Metastasis of KRAS-Mutant Colorectal Cancer.

Major histocompatibility complex (HLA) class I chain-related protein A (MICA) regulates immune surveillance through activation of NKG2D (natural killer group 2D) receptor. However, the genetic association, potential function, and predictive ability of MICA alleles with colorectal cancer (CRC) prognosis remain undefined. In this study, we characterized MICA alleles in tissue samples from 104 patients with CRC and 536 healthy controls and carried out genetic association studies by molecular and clinical CRC phenotypes. Preliminary sequence analysis revealed that MICA ∗009:01 or ∗049 alleles were significantly decreased in patients with CRC (p = 0.0049), and further stratification analysis indicated that MICA ∗012:01 allele was associated with patients with CRC and carrying KRAS codon 12 mutation (p = 0.027). The functional consequences of MICA alleles were examined via transfected CRC cell lines which showed that overexpression of MICA ∗012:01 enhanced the proliferation, invasion, and metastatic phenotype of CRC. Preliminary analysis of disease-free survival time in patients with and without MICA ∗012:01 suggest this allele may be predictive for poor prognosis of patients with KRAS codon 12 mutated CRC, as no somatic mutation of MICA gene was detected in CRC tumors compared to paracancerous tissues. Our study indicates that MICA ∗012:01 allele is associated with KRAS-mutated CRC, facilitates CRC invasion and metastasis, and possibly reduces the survival of patients with KRAS-mutated CRC.

Tumor-Derived Soluble MICA Obstructs the NKG2D Pathway to Restrain NK Cytotoxicity.

The natural killer group 2D (NKG2D) receptor and its ligands play important roles in immune surveillance. In this study, we observed that the average serum soluble MICA (sMICA) concentration of 174 hepatocellular carcinoma (HCC) patients was significantly higher than that in 80 healthy subjects (602.17 ± 338.15 vs. 72.26 ± 87.88 pg/ml, t = 3.107, P=0.002). The levels of serum sMICA in 44 HCC patients with initial levels above 400 pg/ml declined significantly after surgical removal of the liver cancer tissue (P<0.001). Moreover, the mean survival time of HCC patients who had sMICA above 400 pg/ml was significantly shorter than that HCC patients with lower sMICA levels (P<0.001). Using the reporter cell line (NKG2D-2B4) in which activation of the NKG2D receptor pathway results in GFP expression based on the stimulation of immobilized rMICA, we showed that the number of GFP-expressing cells decreased sharply in presence of sMICA. Upon adding sMICA, the release of cytokines IFN-γ, TNF-α, and IL-8 by NK cell line (NKL) under stimulation of immobilized rMICA was blocked. Using MICA-expressing cells as the target cells, we observed that about 80% of target cells were killed by NKL at E:T of 10:1, but in presence of sMICAhigh serum of HCC patients, the dead target cells were reduced to 30.8%. Compared in presence of sMICAlow serum from HCC patients, there were 63.7% of target cells dead (p=0.043). Thus, our data suggested that sMICA obstructs the activation of NKG2D pathway to protect tumor cells from NK cell-mediated cytotoxicity.

Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

Results from the Completed Dose-Escalation of the Hematological Arm of the Phase I Think Study Evaluating Multiple Infusions of NKG2D-Based CAR T-Cells As Standalone Therapy in Relapse/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Results from the Completed Dose-Escalation of the Hematological Arm of the Phase I Think Study Evaluating Multiple Infusions of NKG2D-Based CAR T-Cells As Standalone Therapy in Relapse/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

P1625A pivotal role for cytotoxic CD8+ T cells in development of cardiac fibrosis

Abstract Background/Introduction Pressure overload-induced cardiac fibrosis increases myocardial stiffness leading to reductions in cardiac performances and cardiac failure. CD8+ T cells have been shown to accumulate during development of fibrosis but their role has not been defined. Purpose We examined the role and significance of CD8+ T cells in development of cardiac fibrosis. Methods Trans-aortic constriction (TAC) or 2-kidney-1-clip (2K1C) procedures were used to generate pressure overload-induced cardiac fibrosis in mice. Rat anti-mouse CD8β (lyt-3) monoclonal antibody (clone YTS 156.7) was used to deplete CD8+ T cells. A mixed bone marrow chimera strategy was used to specifically delete innate receptor Natural Killer Group 2D (NKG2D) or cytotoxin perforin from CD8+ T cells. Results Depleting CD8+ T cells in mice subjected to TAC or 2K1C-renal hypertension attenuated left ventricular fibrosis by 93% and 84% without affecting blood pressure. In TAC mice this was associated with a 68% reduction in apoptotic cardiomyocytes, a 74% reduction in macrophage accumulation, a 65% reduction in TGF-beta positive cells and a 95% reduction in TGF-beta positive macrophages, whilst CD4+ T cells were unaffected. Cardiomyocytes in regions of developing cardiac fibrosis contained cytoplasmic DNA and expressed the NKG2D ligand, Rae-1, indicative of activation of a DNA damage response; CD8+ T cells expressed the NKG2D receptor. Deletion of the NKG2D receptor from CD8+ T cells attenuated cardiac fibrosis by 82%; deletion of cytotoxin perforin has similar effects. Conclusion(s) We conclude that CD8+ T cells contribute to development of cardiac fibrosis by targeting stressed/damaged cardiomyocytes via an NKG2D-Rae-1 cytotoxic mechanism inducing their apoptosis. Macrophages then accumulate in the heart in response to increased numbers of apoptotic cardiomyocytes, clearing the apoptotic cells through engulfment and increasing their expression of the pro-fibrotic factor TGF-beta1. Acknowledgement/Funding The National Health and Medical Research Council of Australia

Soluble NKG2D Ligands Are Potential Biomarkers and Sentinels of Immune-Mediated Bone Marrow Injury in Bone Marrow Failure Syndromes

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitroanalyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.

Higher Frequencies of Lymphocytes Expressing the Natural Killer Group 2D Receptor in Patients With Behçet Disease.

Behçet disease (BD) is an inflammatory systemic disease with a fluctuating course, which can affect the skin, eyes, central nervous system, musculoskeletal, gastrointestinal, and vascular systems. No laboratory tests are currently available for the diagnosis of BD and monitoring disease activity. Moreover there is a lack of knowledge on BD pathogenesis. This study focused on circulating Natural Killer (NK), NKT and T cells evaluated as CD3neg CD56pos, CD3pos CD56pos, and CD3pos CD56neg. Peripheral blood mononuclear cells (PBMCs) were collected from 38 BD patients and 20 healthy controls (HC). The frequencies of NK, NKT, and T cells expressing CD16, CD69, NKG2D, Nkp30, Nkp46, and NKG2A were assessed by flow cytometry. Cytotoxic potential of NK cells was evaluated by flow cytometry as the percentage of cells expressing the degranulation marker CD107a after incubation with K562 cells. The levels of 27 cytokines were determined in plasma with a multiplex bead-based assay. Higher percentages of NK, NKT, and T cells expressing NKG2D were detected in PBMCs of BD patients than HC. ROC curve analysis showed that the evaluation of NKG2Dpos NK, NKT, and T cell percentages discriminated between BD patients and HC. Moreover, there was a positive correlation between the BD Current Activity Form (BDCAF) scores and the frequencies of NKG2Dpos NK and NKT cells. A higher frequency of NK cells expressing CD107a was induced in PBMCs from BD patients than HC after incubation with K562 cells. Concentrations of IL-5, IL-6, IL-10, IL-13, IP-10, and MIP-1β were higher in plasma of BD patients than HC. Monitoring the frequencies of NKG2Dpos lymphocytes could help the clinicians in BD patients management. In addition, the increased expression of NKG2D in BD patients is likely involved in disease pathogenesis.