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<p>The biology and function of NKG2H receptor, unlike the better characterized members of the NKG2 family NKG2A, NKG2C, and NKG2D, remains largely unclear. Here, we show that NKG2H is able to associate with the signaling adapter molecules DAP12 and DAP10 suggesting that this receptor can signal for cell activation. Using a recently described NKG2H-specific monoclonal antibody (mAb), we have characterized the expression and function of lymphocytes that express this receptor. NKG2H is expressed at the cell surface of a small percentage of peripheral blood mononuclear cell (PBMC) and is found more frequently on T cells, rather than NK cells. Moreover, although NKG2H is likely to trigger activation, co-cross-linking of this receptor with an NKG2H-specific mAb led to decreased T cell activation and proliferation in polyclonal PBMC cultures stimulated by anti-CD3 mAbs. This negative regulatory activity was seen only after cross-linking with NKG2H, but not NKG2A- or NKG2C-specific monoclonal antibodies. The mechanism underlying this negative effect is as yet unclear, but did not depend on the release of soluble factors or recognition of MHC class I molecules. These observations raise the intriguing possibility that NKG2H may be a novel marker for T cells able to negatively regulate T cell responses.</p

DataSheet_5_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_3_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_4_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_7_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_1_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_8_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_6_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

DataSheet_2_Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.pdf

IntroductionIt is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2.MethodsTo address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease.ResultsAlthough NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies.DiscussionThese observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.</p

MOESM1 of Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma

Additional file 1: Figure S1. Optimization of LFIA for exosomal MICA detection. A. Antibodies combination. Different antibody combinations for capture and detection, as indicated, were tried. B. Running buffer composition. The addition of different concentrations of Tween 20 and ethanol with anti-MICA in the running buffer was tested. In A and B, the amount of antibody-coupled AuNP was 10 µL, thus, for comparison, the same set of strips was used in the no ethanol (0% ethanol) and 10 µL of detection antibody sets. C. Incubation time. Melanoma exosomes were incubated with the detection antibody anti-MICA conjugated to AuNP for different times prior to the run on the strip. D. Effect of including two steps for dispensing exosomes and antibody-coupled NP to the LFIA strip. Exosomes were incubated with detection anti-MICA antibody NP and dispensed to the dipstick in one step. Alternatively, exosomes were run first and, in a second step, the detection antibody-coupled NP. The test strips in triplicates are shown. Right graph: quantitation of the test line signal represented as arbitrary units (a.u.). Data are the mean and SEM of the triplicates. The capture antibodies were immobilized manually in the strips and exosomes were not pre-incubated with detection antibody for one hour in A and B. In B, C and D, anti-CD9 was used as capture antibody and anti-MICA-NP for detection. Melanoma exosomes derived from Ma-Mel-55 (55) or Ma-Mel-86c (86c) were run (E, EXO) or no exosomes as control (B, BLANK). The position of the test (T) and control lines (C) are depicted. Figure S2. Visual analysis of the sample pads colour. Pads from the experiments above were scanned to appreciate the material still embedded in the pad at the end of the running time. A. Effect of incubation time. Pads from the experiments shown in Additional Figure 1C. B. Titration of exosome concentration. Pads from the experiments shown in Figure 4B