Journal: Journal of Hematology & Oncology

Article Title: T cells are functionally not impaired in AML: increased PD-1 expression is only seen at time of relapse and correlates with a shift towards the memory T cell compartment

doi: 10.1186/s13045-015-0189-2

Figure Lengend Snippet: Similar expression of inhibitory molecules on peripheral blood T cells of AML patients at diagnosis compared to healthy controls. Expression of CD244 ( a , e ), PD-1 ( b , f ), CD160 ( c , g ), and TIM-3 ( d , h ) was measured on peripheral blood CD8 + ( a – d ) and CD4 + ( e – h ) T cells of 23 AML patients at diagnosis ( AML_diag ) in comparison to 30 healthy controls ( HC ) and 10 HIV patients ( HIV ), and percentages of positive cells were depicted. Samples were categorized according to age (≤40 vs. >40 years) within each group. Statistical differences were calculated to HC of the respective age cohort. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: Immunofluorescent staining of cell surface antigens was performed using the following fluorescence-conjugated monoclonal antibodies: CD244 (PE or APC, C1.7), PD-1 (APC or Brilliant Violet 421, EH12.7H7), CD3 (AlexaFluor 488, UCHT1), CD45RA (Brilliant Violet 421, HI100), CCR7 (PE, G043H7), CD27 (APC, O323; all BioLegend, San Diego, CA, USA), CD160 (APC, 688327), TIM-3 (PE, 344823; both R&D Systems, Minneapolis, MN, USA), CD8 (PerCP-eFluor 710, SK1; eBioscience, San Diego, CA, USA), and CD4 (APC-H7, RPA-T4; BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Biomarker Discovery, Comparison

Journal: Journal of Hematology & Oncology

Article Title: T cells are functionally not impaired in AML: increased PD-1 expression is only seen at time of relapse and correlates with a shift towards the memory T cell compartment

doi: 10.1186/s13045-015-0189-2

Figure Lengend Snippet: Increased PD-1 expression on peripheral blood T cells of AML patients at relapse after allogeneic SCT compared to diagnosis. Expression of CD244 ( a , e ), PD-1 ( b , f ), CD160 ( c , g ), and TIM-3 ( d , j ) was measured on peripheral blood CD8 + ( a – d ) and CD4 + ( e – h ) T cells of 8 patients with an AML relapse after intensive chemotherapy ( AML_rel ) and 6 patients with an AML relapse after allogeneic SCT ( AML_rel_allo ) in comparison to 16 AML patients at diagnosis ( AML_diag ), and percentages of positive cells were depicted. Patient selection was limited to the age cohort >40 years. Statistical differences were calculated to AML_diag. * p ≤ 0.05; **** p ≤ 0.0001

Article Snippet: Immunofluorescent staining of cell surface antigens was performed using the following fluorescence-conjugated monoclonal antibodies: CD244 (PE or APC, C1.7), PD-1 (APC or Brilliant Violet 421, EH12.7H7), CD3 (AlexaFluor 488, UCHT1), CD45RA (Brilliant Violet 421, HI100), CCR7 (PE, G043H7), CD27 (APC, O323; all BioLegend, San Diego, CA, USA), CD160 (APC, 688327), TIM-3 (PE, 344823; both R&D Systems, Minneapolis, MN, USA), CD8 (PerCP-eFluor 710, SK1; eBioscience, San Diego, CA, USA), and CD4 (APC-H7, RPA-T4; BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Biomarker Discovery, Comparison, Selection

Journal: Journal of Hematology & Oncology

Article Title: T cells are functionally not impaired in AML: increased PD-1 expression is only seen at time of relapse and correlates with a shift towards the memory T cell compartment

doi: 10.1186/s13045-015-0189-2

Figure Lengend Snippet: Upregulation of PD-1 expression on bone marrow T cells of AML patients at relapse after allogeneic SCT compared to diagnosis. Expression of CD244 ( a , e ), PD-1 ( b , f ), CD160 ( c , g ), and TIM-3 ( d , h ) was measured on bone marrow CD8 + ( a – d ) and CD4 + ( e – h ) T cells of 31 AML patients at diagnosis ( AML_diag ), 7 patients with an AML relapse after intensive chemotherapy ( AML_rel ), and 6 patients with an AML relapse after allogeneic SCT ( AML_rel_allo ) in comparison to 5 healthy controls ( HC ), and percentages of positive cells were depicted. Patient selection was limited to the age cohort >40 years. Statistical differences were calculated to HC and AML_diag. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Immunofluorescent staining of cell surface antigens was performed using the following fluorescence-conjugated monoclonal antibodies: CD244 (PE or APC, C1.7), PD-1 (APC or Brilliant Violet 421, EH12.7H7), CD3 (AlexaFluor 488, UCHT1), CD45RA (Brilliant Violet 421, HI100), CCR7 (PE, G043H7), CD27 (APC, O323; all BioLegend, San Diego, CA, USA), CD160 (APC, 688327), TIM-3 (PE, 344823; both R&D Systems, Minneapolis, MN, USA), CD8 (PerCP-eFluor 710, SK1; eBioscience, San Diego, CA, USA), and CD4 (APC-H7, RPA-T4; BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Biomarker Discovery, Comparison, Selection

Journal: The Journal of Experimental Medicine

Article Title: A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor

doi: 10.1084/jem.20100810

Figure Lengend Snippet: Expression of CD160 in tumor blood vessels but not in the blood vessels of healthy tissues. (A) Representative sections of B16 melanoma at day 16 after their subcutaneous injection (top) and healthy mouse heart (bottom), both stained with either CL1-R2 (CD160) or isolectin B4, an endothelial cell marker. Bar, 50 µm. Images are representative of four independent experiments (five mice/group). (B) Sections of human colon tumor (top) and healthy colon (bottom) stained with CL1-R2 (CD160) or a mAb against CD31, a human endothelial cell marker. Images are representative of two patient biopsies. Control IgG1 did not stain and is not shown. Bar, 50 µm.

Article Snippet: The cross-reactivity of CL1-R2 mAb with CD160 mouse proteins has been demonstrated by different means (Fig. S1): (1) using flow cytometry, we found that CL1-R2 stained mouse SVR endothelial cells as well as the BCL-1 mouse B cell chronic lymphocytic leukemia cell line (Fig. S1 A); (2) using immunoprecipitation and Western blotting, we found that the CNX46-3 anti–mouse CD160 mAb (Cedarlane laboratories) immunoprecipitated protein bands from mouse spleen or BCL-1 cells that were blotted by the CL1-R2 mAb (Fig. S1 B); and (3) five CD160 amino acids were identified by mass spectrometry in the CNX43-6–immunoprecipitated 37-kD band blotted with CL1-R2 mAb with a minimum coverage of 37.5% (Fig. S1 C).

Techniques: Expressing, Injection, Staining, Marker, Control

Journal: Alzheimer's & Dementia

Article Title: Early blood immune molecular alterations in cynomolgus monkeys with a PSEN1 mutation causing familial Alzheimer's disease

doi: 10.1002/alz.14046

Figure Lengend Snippet: Top 10 significant differentially expressed plasma proteins between PSEN1 ‐ΔE9 and control cynomolgus monkeys.

Article Snippet: For validation of plasma proteins, we used the enzyme‐linked immunosorbent assay (ELISA) to detect CD160 (CUSABIO, CSB‐EL004881HU) and C1QA (CUSABIO, CSB‐EL003637HU) in the plasma of 1.5‐year‐old monkeys.

Techniques: Clinical Proteomics, Control

Journal: Frontiers in immunology

Article Title: BTLA inhibition has a dominant role in the cis -complex of BTLA and HVEM.

doi: 10.3389/fimmu.2022.956694

Figure Lengend Snippet: FIGURE 3 Regulation of HVEM co-stimulation in trans and in cis. (A) Upper panels: cell surface expression of CD86, CD160 and LIGHT on TCS analysed via flow cytometry (open histograms: control TCS; grey histograms: TCS expressing the indicated molecules). Lower panel: membrane-bound anti-CD3-fragment (detected via its CD14 stem) on TCS analysed via flow cytometry. Parental BW5147 cells were used as a control. (B) Control reporter cells and HVEM expressing reporter cells were stimulated with control TCS and TCS expressing CD86, BTLA, LIGHT and CD160 for 24 h and NFkB::eGFP expression was measured by flow cytometry. Data is shown from 4 independent experiments performed in duplicates. For statistical evaluation, one-way ANOVA with Dunn’s multiple-comparisons test was performed (***p ≤0.001; **p ≤0.01; *p < 0.05; ns, p > 0.05). (C) Jurkat NFkB::eGFP reporter cells expressing HVEM, HVEM/BTLA, HVEM/LIGHT and HVEM/CD160 were analysed for the expression of the indicated molecules; open histogram show control reporter cells. (D) The indicated reporter cells were left unstimulated or were stimulated with control TCS or TCS CD86 and eGFP expression was measured via flow cytometry. Results are shown from 4 independent experiments performed in duplicate. For statistical evaluation, two-way ANOVA followed by Bonferroni’s test was performed (***p ≤0.001; ns, p > 0.05). (E) Expression levels of HVEM and BTLADcyt on reporter cells. Open histograms represent control reporter cells. (F) The indicated reporter cells were left unstimulated or were co-cultured with control TCS and TCS CD86. eGFP expression was assessed via flow cytometry. Data is shown for 4 independent experiments performed in duplicate. For statistical evaluation, two-way ANOVA followed by Bonferroni’s test was performed (***p ≤0.001; **p ≤0.01; ns, p > 0.05). (B, D, F) ± SD is shown.

Article Snippet: HVEM antibody AF356 (polyclonal goat IgG) and CD160 antibody (clone 688327) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Cytometry, Control, Membrane, Cell Culture

Journal: Frontiers in immunology

Article Title: BTLA inhibition has a dominant role in the cis -complex of BTLA and HVEM.

doi: 10.3389/fimmu.2022.956694

Figure Lengend Snippet: FIGURE 4 Assessment of co-expression of HVEM/BTLA signaling. (A) Control reporter cells and reporter cells expressing BTLA, HVEM or BTLA/HVEM were left unstimulated or stimulated with TCS ctrl, TCS BTLA and TCS HVEM. NF-kB::eGFP activation was measured after 24 h. Left: one representative experiment performed in duplicate is shown. Right: reporter gene expression is shown normalized to reporter gene expression induced by control-TCS (gMFI of reporter cells stimulated with the indicated TCS/gMFI of TCS ctrl stimulated cells). Data is depicted of at least eight independent experiments performed in duplicates. One-way analysis of variance followed by a Dunnett’s multiple comparison test were used for comparison to control reporter cells (***p ≤0.001; ns, p > 0.05). (B) Jurkat NFkB::eGFP ctrl and Jurkat NFkB::eGFP expressing HVEM, HVEM/BTLA or HVEM/BTLADcyt cells were stimulated with TCS ctrl, TCS CD86, TCS CD160 and TCS LIGHT. Reporter gene expression is shown normalized to control-TCS. Results are shown from three independent experiments performed in duplicates. For statistical evaluation, two-way ANOVA followed by Bonferroni’s test was performed (***p ≤0.001). (C) Left panel: Schematic representation of receptors on reporter cells and ligands on stimulator that were evaluated in absence or presence of a blocking BTLA antibody. Right panel: Reporter cells expressing BTLA/ HVEM were stimulated with TCS CD86, TCS CD160 or TCS LIGHT in the presence or absence of a blocking BTLA antibody (5 mg/ml; clone 6F4). Reporter activation induced by the indicated TCS is shown normalized to control-TCS. Data is depicted from three independent experiments performed in duplicate. For statistical evaluation, two-way ANOVA followed by Bonferroni’s test was performed (***p ≤0.001; ns, p > 0.05). (D) Flow cytometric analysis of cell surface molecules BTLA and HVEM on the indicated reporter cells. (E) Left panel: Reporter cells expressing HVEM, HVEM/BTLAhigh or HVEM/BTLAlow were left unstimulated or stimulated with TCS ctrl, TCS BTLA and TCS LIGHT. Right panel: Reporter cells expressing HVEM, HVEM/BTLAhigh or HVEM/BTLAlow were left unstimulated or stimulated with TCS ctrl or TCS HVEM. gMFI of NFkB::eGFP activation is shown. Results are depicted from four independent experiments performed in duplicate. (F) Cell surface expression of mHVEM and membrane-bound anti-CD3 on TCS analysed via flow cytometry (open histograms: control TCS; grey histograms: expression level of the indicated molecules). (G) Left panel: Schematic representation of stimulation experiments of reporter cells co-expressing BTLA and HVEM with TCS expressing human HVEM or mouse HVEM (mHVEM) with and without HVEM antibody SL030717 (10 µg/ml) that blocks human but not mouse HVEM. Right panel: Control reporter cells and reporter cells expressing HVEM/BTLA or BTLA were stimulated with TCS HVEM or TCS mHVEM in the presence or absence of a blocking HVEM antibody. Reporter gene expression is shown normalized to control-TCS. Results are depicted from four independent experiments performed in duplicate. (A, B, C, G) ±DSD is shown. Dotted line depicts control stimulation.

Article Snippet: HVEM antibody AF356 (polyclonal goat IgG) and CD160 antibody (clone 688327) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Control, Activation Assay, Gene Expression, Comparison, Blocking Assay, Membrane, Cytometry

Journal: Frontiers in immunology

Article Title: BTLA inhibition has a dominant role in the cis -complex of BTLA and HVEM.

doi: 10.3389/fimmu.2022.956694

Figure Lengend Snippet: FIGURE 5 Evaluation of CD160 receptor function in a triple parameter reporter cell system. (A) Left panel: HVEM expressing triple parameter reporter cells (TPR; grey histogram) and control-TPR (open histogram) were analysed for HVEM expression. Right panel: Control TPR and TPR expressing HVEM were stimulated with TCS control or TCS expressing BTLA, CD160 or LIGHT. Reporter activation (NFAT::eGFP, NFkB::eCFP and AP-1::mCherry) was assessed via flow cytometry. Data of three independent experiments in duplicate is shown. Normalized reporter activation is shown (gMFI reporter gene expression induced by the indicated TCS/gMFI reporter gene expression induced by TCS ctrl stimulated cells). (B) Left panel: BTLA expressing triple parameter reporter cells (TPR; grey histogram) and control-TPR (open histogram) were analysed for BTLA expression. Right panel: Control TPR and TPR expressing BTLA were stimulated with control TCS and TCS HVEM. Results are shown from three independent performed experiments in duplicate. Normalized reporter activation is shown (gMFI of TCS HVEM of stimulated cells/gMFI of TCS ctrl stimulated cells). (A, B) For statistical evaluation, two-way ANOVA followed by Bonferroni’s test was performed (***p ≤0.001; **p ≤0.01; *p < 0.05). (C) Left panel: Schematic representation of two CD160 isoforms: the glycosylphosphatidylinositol-anchored-CD160 (CD160-GPI) and the transmembrane isoform of CD160 (CD160-TM). Right panel: Cell surface expression of CD160-GPI and CD160-TM on Jurkat TPR cells assessed by using two different antibodies (clone BY55 and clone 688327) (open histogram: control cells; grey histograms: expression of the indicated CD160 molecules). (D) Control TPR and TPR expressing the two isoforms CD160-GPI and CD160-TM, respectively, were left unstimulated or stimulated with the indicated TCS. (E) Left panel: Schematic representation of a mICOS-CD160-TM chimera. Middle panel: Expression of mICOS chimera on TPR. Right panel: TCS mICOSL (grey histogram) and control TCS (open histogram) were stained with a mICOSL antibody (F) mICOS-chimera expressing reporter cells were stimulated with TCS control and TCS mICOSL. Reporter gene expression induced by TCS mICOSL normalized to TCS control. For statistical evaluation, one-way ANOVA with Dunn’s multiple-comparisons test was performed (***p ≤0.001; **p ≤0.01; *p < 0.05). (A, B, D, F) ±DSD or mean is shown. Dotted line depicts control stimulation.

Article Snippet: HVEM antibody AF356 (polyclonal goat IgG) and CD160 antibody (clone 688327) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Control, Activation Assay, Cytometry, Gene Expression, Staining

Journal: Frontiers in immunology

Article Title: BTLA inhibition has a dominant role in the cis -complex of BTLA and HVEM.

doi: 10.3389/fimmu.2022.956694

Figure Lengend Snippet: FIGURE 6 HVEM engagement in primary human T cells. (A) Representative contour plots of CFSE-labelled PBMCs stimulated with TCS ctrl, TCS CD86, TCS 4-1BBL, TCS BTLA, TCS HVEM, TCS CD160 and TCS LIGHT for 5 days. Percentages of proliferated (CFSElow) of CD4+ and CD8+ T cells is shown. (B, C) CFSE-labelled PBMCs from healthy donors were stimulated for 5 days with the indicated TCS. Proliferation (CFSElow) and CD25 upregulation of CD4+ and CD8+ T cells was measured at day 5. Data is normalized for each donor to stimulation with TCS ctrl. Each data point represents the mean of triplicate measurement of one donor (n = 15; TCS 4-1BBL n = 7). (D) Cell culture supernatants of PBMCs stimulated with TCS cells were harvested at day 5 and cytokine expression profile (IFN-g, GM-CSF and TNF-a) was measured via Luminex multiplex cytokine analysis. Each data point represents the mean of triplicate measurement of one donor (IFN-g: n = 8, TCS 4-1BBL n = 5; GM-CSF and TNF-a: n = 9, TCS 4-1BBL n = 6). (B-D) For statistical evaluation, a one-way ANOVA with Dunn’s multiple-comparisons test was performed (***p ≤0.001; **p ≤0.01). Median is shown (red line). Dotted line depicts control stimulation.

Article Snippet: HVEM antibody AF356 (polyclonal goat IgG) and CD160 antibody (clone 688327) were purchased from R&D systems (Minneapolis, MN).

Techniques: Cell Culture, Expressing, Luminex, Multiplex Assay, Control

Journal: Structure (London, England : 1993)

Article Title: Structural Basis of CD160:HVEM Recognition

doi: 10.1016/j.str.2019.05.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-6xHis lag antibody APC-conjugated Abeam Cat#ab72467; RRID: AB_1267596 Anti-6xHis tag antibody PE-conjugated Abeam Cat#ab72579: RRID: AB_1267597 Goat anti-human IgG FC (FITC) Abeam Cat#ab97224: RRID: AB_10680849 Bacterial and Virus Strains E.coli BL21(DE3) pLysS Promega N/A E.coli methionine auxotroph B834(DE3) Novagen N/A Chemicals, Peptides, and Recombinant Proteins PEI, MW 25000 Polysciences Inc. Cat#23966–1 Recombinant human HVEM Fc R&D systems Cat#356-HV/CF Recombinant human BTLA His-lag R&D systems Cat#9235-BT-050 Recombinant human CP160His-tag R&D systems Cat6177-CD-0S0 Critical Commercial Assays MCSG crystallization suite Anatrace MCSG-1 MCSG crystallization suite Anatrace MCSG-2 MCSG crystallization suits Anatrace MCSG-3 MCSG crystallization suite Anatrace MCSG-4 Deposited Data Crystal structure of gD:HVEM complex ( Carfi et al., 2001 ) PDB: 1JMA Crystal structure of BLA:HVEM complex ( Compaan et al., 2005 ) PDB:2AW2 Crystal structure oi TNF:TNFR2 complex ( Mukai et al., 2010 ) PDB:3ALQ Crystal structure of equine lentivirus recpetor 1 ( Qian et al. 2015 ) PDB:3WVT Crystal structure of HVEM ( Liu et al., 2015 ) PDB:4FHQ Crystal structure of Fab:LTα1β2:LTβR complex ( Sudhamsu et al., 2013 ) PDB:4MXW Crystal structure of Fab:CD40 complex ( Yu et al., 2018 ) PDB:6FAX Crystal structure of sc_CD160:HVEM This paper PDB:6NG3 Crystal structure of CD160 This paper PDB:6NG9 Crystal structure of SeMet V58M CD160 This paper PDB:6NGG Experimental Models: Cell Lima Drosophila S2 Thermo Fisher N/A HEK293 Thermo Fisher N/A Recombinant UNA Human CD160-pET3a This paper N/A Human BTLA-pET3a This paper N/A Human single chain CD16D-HVEM-pMT-Bip-V5-His This paper N/A Human CD160-pMT-Blp-V5-His This paper N/A Human CD160-pmCherry-N1 This paper N/A Human HVEM pGFP-N1 This paper N/A HSV1-gD-plRES-acGFP This paper N/A Software and Algorithms HHL2000 ( Otwinowski and Minor, 1997 ) http://www.hkl-xray.com/ SHELX ( Sheldrick, 2010 ) http://shelx.uni-goettingen.de/ CCP4 ( Winn et al., 2011 ) https://www.ccp4.ac.uk/ COOT ( Emsley et al., 2010 ) https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ ARP/wARP ( Winn et al., 2011 ) http://www.embt-hamburg.de/ARP/ REFMAC5 ( Winn et al., 2011 ) http://www.ccp4.ac.uk/html/refmac5.html Pymol Molecular Graphics System, Schrodinger, LLC https://pyimol.org/2/ CLUSTALW ( Thompson et al., 1994 ) https://www.genome.jp/tools-bin/clustalw ESRript 3.0 ( Robert and Gouet, 2014 ) https://espript.ibcp.fr/ESPript/ESPript/ PDBePISA ( Krissinel and Henrick, 2007 ) http://wwiw.ebi.ac.uk/pdbe/pisa/ Prism 5 Graphpad https://www.graphpad.com/scientific-software/prism/ Open in a separate window KEY RESOURCES TABLE.

Techniques: Virus, Recombinant, Crystallization Assay, Software

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: CD244 and CD160 on CD8 + T cells from healthy individuals were upregulated with age. Flow cytometry analysis of CD244 and CD160 expression was performed on PBMCs collected from healthy individuals of different ages. (A) Representative flow data show the expression of CD244 (up) and CD160 (down) gated on CD8 + T cells from five healthy individuals in different age groups. (B, C) Box plots of the percentage of CD244 + and CD160 + cells on CD8 + T cells from healthy individuals in different age groups (n = 52-84 each group). P values were obtained by one-way ANOVA followed by Tukey’s multiple comparisons test [CD244 (left)] or Kruskal-Wallis test followed by Dunn’s multiple comparisons test [CD160 (right)]. (D, E) Correlation analysis of age and CD244 (D) , CD160 (E) expression on CD8 + T cells from all healthy individuals. Spearman’s non-parametric test was used to test for correlations. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: The frequencies of CD244 + CD160 + and CD244 + CD160 - CD8 + T cells in healthy individuals from different age groups. Flow cytometry analysis of frequencies of CD244 + CD160 + and CD244 + CD160 - CD8 + T cells from healthy donors of different ages. (A) Representative flow data show the frequencies of CD244 + CD160 + and CD244 + CD160 - cells gated on CD8 + T cells from five healthy donors in different age groups. (B, C) Box plots of the percentage of CD244 + CD160 + (B) and CD244 + CD160 - (C) cells on CD8 + T cells from healthy donors in different age groups (n = 27-50 each group). P values were obtained by one-way ANOVA followed by Tukey’s multiple comparisons test. (D, E) Correlation analysis of age and the percentage of CD244 + CD160 + (D) , CD244 + CD160 - (E) CD8 + T cells from all healthy individuals. Spearman’s non-parametric test were used to test for correlations. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: Flow cytometry analysis of senescent features on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells from the elderly. Flow cytometry analysis of senescence-associated markers on CD244 - CD160 - , CD244 + CD160 - and CD244 - CD160 + CD8 + T-cell subsets. (A) Representative histograms (left) and box plots (right) display the β-Gal activity measured by the mean fluorescence intensity (MFI) on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells (n = 10). P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test. (B–E) Representative histograms (left) and box plots (right) display the expression of KLRG-1 (B) , CD57 (C) , CD28 (D) , and CD27 (E) on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells from the elderly (61-90 years old, n = 15-17). P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test (KLRG-1, CD57, and CD27) or Friedman’s test followed by Dunn’s multiple comparisons test (CD28). (F–H) Intracellular staining for TNF-α, IFN-γ, and IL-2 on CD244 - CD160 - , CD244 + CD160 - and CD244 - CD160 + cells (gated with CD8 + T cells) from the elderly (61-90 years old, n = 17) after in vitro anti-CD3/anti-CD28 stimulation. Representative histograms (left) and box plots (right) for TNF-α (F) , INF-γ (G) , and IL-2 (H) , respectively. P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test (TNF-α and INF-γ) or Friedman’s test followed by Dunn’s multiple comparisons test (IL-2). (I–K) Expression of CD107a, Granzyme B, and perforin on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells from the elderly (61-90 years old, n = 17). Representative histograms (left) and box plots (right) of CD107a (I) , Granzyme B (J) , and perforin (K) expression. P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test (CD107a) or Friedman’s test followed by Dunn’s multiple comparisons test (Granzyme B and perforin). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Flow Cytometry, Activity Assay, Fluorescence, Expressing, Staining, In Vitro

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: CD244 + CD160 - and CD244 + CD160 + CD8 + T cells from the elderly partially displayed exhaustion. (A–E) Flow cytometry analysis of the expression of PD-1 (A) , TIGIT (B) , LAG-3 (C) , TIM-3 (D) , and percentage of HLA-DR + CD38 hi cells (E) on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells (n = 11-17). Representative histograms or flow data (left), and box plots (right) display the expression of the above receptors on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + cells (gated with CD8 + T cells). P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test (PD-1, TIGIT, and LAG-3) or Friedman’s test followed by Dunn’s multiple comparisons test (TIM-3 and HLA-DR + CD38 hi ). (F–H) Percentage of apoptotic cells (Annexin V + 7AAD - ) (F) and expression of CD95 (G) and Ki67 (H) on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells from the elderly (61-90 years old, n = 15-17). Representative histograms (left) and box plots (right) display the percentage of apoptotic cells (Annexin V + 7AAD - ) (F) and expression of CD95 (G) and Ki67 (H) on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + cells (gated with CD8 + T cells). P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test (Annexin V + 7AAD - and CD95) or Friedman’s test followed by Dunn’s multiple comparisons test (Ki67). ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: Enhanced glucose uptake and reduced expression of metabolism-associated genes in CD244 + CD160 - CD8 + T cells. (A) Representative histograms (left) and box plots (right) of 2-NBDG uptake or CD71 and CD98 expression in CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells (n = 7-10). P values were obtained by repeated-measures ANOVA followed by Tukey’s multiple comparisons test. (B, C) Differential effects of CD244 and CD160 on markers of the glycolysis and oxidative phosphorylation in CD8 + T cells from older subjects (n = 5-7). Real-time quantitative PCR analysis of transcript levels of (B) glycolytic enzymes (GLUT1, HK2, ENO1, and PDK1), and (C) oxidative phosphorylation (ATP5G1 and mtNd1) in CD8 + T cells after 4 h of culture in the presence of plate-bound anti-CD3 and anti-CD28 (10 µg/mL). P values were obtained by Friedman’s test followed by Dunn’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Expressing, Phospho-proteomics, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: CD244 + CD160 - CD8 + T cells from the elderly exhibited elevated T-bet hi Eomes dim cells while CD244 + CD160 + CD8 + T cells comprised a high number of T-bet dim Eomes hi cells. (A, B) Representative flow data (A) and box plots (B) of the percentage of T-bet dim Eomes hi and T-bet hi Eomes dim cells on CD244 - CD160 - , CD244 + CD160 - and CD244 + CD160 + CD8 + T cells in the elderly (61-90 years old, n = 17). P values were obtained by Friedman’s test followed by Dunn’s multiple comparisons test. (C, D) Correlation analysis of the percentage of CD244 + CD160 - and CD244 + CD160 + cells and the frequencies of T-bet hi Eomes dim and T-bet dim Eomes hi cells on CD8 + T cells of all ages. Spearman’s non-parametric test was used to test for correlations. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques:

Journal: Frontiers in Immunology

Article Title: High Levels of CD244 Rather Than CD160 Associate With CD8 + T-Cell Aging

doi: 10.3389/fimmu.2022.853522

Figure Lengend Snippet: The senescent status and cytokine production of CD8 + T cells could be reversed by CD244 but not CD160 blockade. Purified CD8 + T cells from healthy donors (n = 7) were cultured with antagonist anti-CD244, anti-CD160 antibody, or isotype IgG at a concentration of 10 µg/mL. After culturing in vitro for 24h, the activity of β-Gal (A) , KLRG-1 (B) , CD57 (C) , TNF-α (D) , IFN-γ (E) , and IL-2 (F) expression on CD8 + T cells was measured by flow cytometry. Representative plots of the above markers in CD8 + T cells. P values were obtained by paired t-test.

Article Snippet: Purified cells were cultured at a concentration of 2 × 10 6 cells/mL in a 96 well tissue culture plate and 10 μg/ml anti-human CD244 antibody (clone 999602; R&D systems), anti-human CD160 antibody (clone 688327; R&D systems) or isotype control was added to the culture medium.

Techniques: Purification, Cell Culture, Concentration Assay, In Vitro, Activity Assay, Expressing, Flow Cytometry

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: Percentage of CD160 + CD8 + T cells in patients with CHB with different natural history is negatively associated with the progress of CHB. (A) The percentage of CD160 + CD8 + T cells in patients with CHB was detected using a FACSCalibur flow cytometer, and statistical analysis was performed. (B) The percentage of CD160 + CD8 + T cells in patients with different stages of CHB was detected. (C) Analysis of the percentage of CD160 + CD8 + T cells in patients with different stages of CHB. (D) The expression of CD160 was inhibited by CD160-siRNA. (E) CD160 + CD8 + T cells were transfected with CD160-siRNA, and the CD160-siRNA significantly inhibited the expression of CD160. Following inhibition of CD160, (F) the expression of SAP was reduced and (G) the percentage of SAP + CD160 + cells in total CD8 + T cells was inhibited. To further clarify the role of CD160 in the CD8 + T cell immune response, the concentrations of (H) IFN-γ and (I) TNF-α were detected, which are produced by CD8 + T cells. IFN-γ and TNF-α were significantly decreased following CD160-knockdown in CD8 + T cells. **P<0.01, ***P<0.005. HBV, hepatitis B virus; CHB, chronic HBV; siRNA, small interfering RNA; SAP, (SLAM)-associated protein con, control; IT, immune tolerance; LR, low-replicate; IC, immunological clearance.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Flow Cytometry, Expressing, Transfection, Inhibition, Produced, Knockdown, Virus, Small Interfering RNA, Control

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: CD160 inhibits HDAC11 expression via epigenetic regulation in CD8 + T cells. (A) A gene microarray assay was conducted with CD160 + CD8 + T cells and CD160 − CD8 + T cells, which were isolated from patients with chronic hepatitis B virus, with unsupervised clustering analysis. Green indicates decreased expression and red indicates increased expression. (B) Gene microarray assay with supervised clustering analysis was performed with CD160 + CD8 + T cells and CD160 − CD8 + T cells for epigenetic factor detection. (C) The expression of HDAC11 in CD160 + CD8 + T cells and CD160 − CD8 + T cells was detected by RT-qPCR assay. (D) An immunofluorescence assay for CD8, CD160 and HDAC11 detection was performed with CD160 + CD8 + T cells to obtain confocal microscopic images; magnification, ×1,000. (E) CD8 + T cells were transfected with CD160-siRNA and the expression of HDAC11 was detected by RT-qPCR, and the expression level of HDAC11 was negatively associated with the expression of CD160. (F) The protein level of HDAC11 was measured by western blotting following transfection of CD8 + T cells with CD160-siRNA. (G) The expression of HDAC11 following transfection with HDAC11 siRNA. CD8 + T cells were transfected with HDAC11-siRNA and the expression levels of (H) IFN-γ and (I) TNF-α were detected by RT-qPCR assay. (J) Flow cytometry was performed to For detect the percentage of HDAC11 +/− CD160 +/− CD8 + T cells in the total CD8 + T cell population. *P<0.05, ***P<0.005. HDAC11, histone-modification enzyme gene histone deacetylases 11; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; con, control.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Expressing, Microarray, Isolation, Virus, Quantitative RT-PCR, Immunofluorescence, Transfection, Western Blot, Flow Cytometry, Modification, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA, Control

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 expression is positively associated with CD160 expression in CD8 + T cells. (A) lncRNA gene microarray assay was conducted with CD160 + CD8 + T cells and CD160 − CD8 + T cells, which were isolated from patients with chronic HBV, with unsupervised clustering analysis. Green indicates decreased expression and red indicates increased expression. (B) lncRNA gene microarray assay with supervised clustering analysis was performed with CD160 + CD8 + T cells and CD160 − CD8 + T cells. (C) Reverse transcription-qPCR assay was performed to detect the lncRNA-CD160 expression level in CD160 +/− CD8 + T cells. (D) Chromosome analysis indicated that both CD160 and lncRNA-CD160 were located at Chr1q42.3, and lncRNA-CD160 was partly located at the region of CD160, which was between the B and C region; therefore, lncRNA-CD160 could also be termed lncRNA-CD160. Chromatin immunoprecipitation-qPCR was performed to investigate the relationship between (E) lncRNA-CD160 and H3K9Me1, (F) the relationship between lncRNA-CD160 and HDAC11 also was detected. HDAC11 and H3K9Me1 trimethylation levels were promoted in the lncRNA-CD160 loci. *P<0.05, **P<0.01, ***P<0.005. qPCR, quantitative PCR; lncRNA, long non-coding RNA; HBV, hepatitis B virus; HDAC11, histone-modification enzyme gene histone deacetylases 11.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Expressing, Microarray, Isolation, Reverse Transcription, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Virus, Modification

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 inhibits IFN-γ and TNF-α secretion in CD8 + T cells via epigenetic regulation. In order to demonstrate the role of lncRNA-CD160 on IFN-γ and TNF-α secretion, siRNA targeting lncRNA-CD160 was transfected into the CD8 + T cells. (A) The efficiency of lncRNA-CD160 siRNA was detected, and the concentrations of (B) IFN-γ and (C) TNF-α were detected by ELISA assay. A CHIP-qPCR assay was performed to demonstrate the mechanism of the IFN-γ and TNF-α secretion inhibition. When lncRNA-CD160 was knocked down, the H3K9Me1 expression levels, which could be mediated by HDAC11 at the (D) IFN-γ and (E) TNF-α promoters loci, were significant inhibited. (F) Immunoprecipitation and western blot assays were performed to detect the expression of HDAC11 in the immunoprecipitate using an anti-HDAC11-specific antibody. (G) Gel electrophoresis and (H) an image of biotinylated lncRNA-CD160. (I) Reverse transcription-qPCR analysis of lncRNA-CD160 retrieved by IgG or anti-HDAC11 from CD8 + T-cell lysates of patients with HBV. (J) FISH following lncRNA-CD160 siRNA transfection, magnification, ×1,000. (K) RNA pull-down and western blot assays were conducted to investigate the association between lncRNA-CD160 and HDAC11, and the data indicated that lncRNA-CD160 and HDAC11 could bind to each other. (L) Further RNA FISH and immunofluorescence analyses were performed to investigate the locations of lncRNA-CD160 and HDAC11, and the results demonstrated that both were located in the nucleus of CD8 + T cells, magnification, ×1,000. A CHIP-qPCR assay was also performed to reveal the location of the lncRNA-CD160 and HDAC11 complex, and the results revealed that lncRNA-CD160-siRNA could significantly inhibit the expression of HDAC11 at (M) IFN-γ and (N) TNF-α promoter regions. **P<0.01, ***P<0.005. FISH, fluorescent in situ hybridization; lncRNA, long non-coding RNA; con, control; siRNA, small interfering RNA; qPCR, quantitative PCR; HDAC11, histone-modification enzyme gene histone deacetylases 11.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Inhibition, Expressing, Immunoprecipitation, Western Blot, Nucleic Acid Electrophoresis, Reverse Transcription, Immunofluorescence, In Situ Hybridization, Control, Small Interfering RNA, Real-time Polymerase Chain Reaction, Modification

Journal: Oncology Letters

Article Title: lncRNA-CD160 decreases the immunity of CD8 + T cells through epigenetic mechanisms in hepatitis B virus infection

doi: 10.3892/ol.2020.11534

Figure Lengend Snippet: lncRNA-CD160 suppresses HBV replication during infection in vivo . (A) To investigate the effect of lncRNA-CD160 on HBV replication, an adoptive transfer model was established. (B) Following adoptive transfer, the serum HBsAg levels were detected at different time points using a Roche Cobas 6000 immuno-chemiluminescence analyzer. *P<0.05, ***P<0.005 vs. LV-lncRNA-CD160. (C) The HBV DNA load was detected by reverse transcription--quantitative PCR assay at different time points following adoptive transfer. *P<0.05, **P<0.01 vs. LV-lncRNA-CD160. (D) An immunohistochemistry assay was performed for HBcAg detection in the liver tissues, which were harvested from the adoptive transfer model mice, magnification, ×1,000. (E) The percentages of HBcAg-positive hepatocytes were quantified. **P<0.01 and ***P<0.005. (F) An overview of the role of lncRNA-CD160 in the mediation of IFN-γ and TNF-α. lncRNA, long non-coding RNA; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; LV, lentivirus; HBcAg, hepatitis B virus c antibody; SAP, (SLAM)-associated protein; siRNA, small interfering RNA.

Article Snippet: The sections were then incubated at 4°C overnight with CD160 monoclonal mouse anti-human IgG (1:100; cat. no. AF3899; R&D Systems, Inc.) in a 1:50 dilution with 5% skimmed milk PBS buffer, followed by incubation with the corresponding secondary goat anti-mouse IgG-HRP antibody at room temperature (1:300; sc-2005; Santa Cruz Biotechnology, Inc.) for 45 min.

Techniques: Infection, In Vivo, Adoptive Transfer Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemistry, Virus, Small Interfering RNA