Journal: Stem Cell Research & Therapy

Article Title: HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats

doi: 10.1186/s13287-021-02647-5

Figure Lengend Snippet: HO-1/BMMSCs affect the level of BTLA and CD160 expression in NKT cells. The expression of co-stimulatory receptors of NKT cells had no significant difference among the groups ( A ). NKT cell co-inhibitory receptors, BTLA and CD160, differed significantly between each group ( B ). In the HMP and BMP groups, BTLA and CD160 expression in the NKT cells (proportion of positive cells and MFI) ( C and D ) were significantly higher than that in the NMP group. * P < 0.05

Article Snippet: The following antibodies were used for extracellular staining to identify lymphocytes: CD3 (BioLegend); NK1.1 (BioLegend); CD44 (Thermo Scientific); glucocorticoid-induced tumor necrosis factor receptor (GITR) (BioLegend); CD69 (BioLegend); CD28 (BioLegend); cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (BioLegend); CD160 (Biorbyt, Cambridgeshire, UK); B- and T-lymphocyte attenuator (BTLA) (Thermo Scientific).

Techniques: Expressing

Journal: Stem Cell Research & Therapy

Article Title: HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats

doi: 10.1186/s13287-021-02647-5

Figure Lengend Snippet: HO-1/BMMSCs had a weakened regulatory effect on NKT cells when the co-inhibitory receptors were blocked. A The cells in the control group were incubated with mIgG1 mAb 6 h in advance, and the other groups were incubated with BTLA or CD160 mAb, and co-cultured with HO-1/BMMSCs for 48 h. B Level of serum cytokines (IFN-γ, TNF-α, and IL-2). * P < 0.05

Article Snippet: The following antibodies were used for extracellular staining to identify lymphocytes: CD3 (BioLegend); NK1.1 (BioLegend); CD44 (Thermo Scientific); glucocorticoid-induced tumor necrosis factor receptor (GITR) (BioLegend); CD69 (BioLegend); CD28 (BioLegend); cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (BioLegend); CD160 (Biorbyt, Cambridgeshire, UK); B- and T-lymphocyte attenuator (BTLA) (Thermo Scientific).

Techniques: Control, Incubation, Cell Culture

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: Antibodies [CD8 (1:100; sc-1177; Santa Cruz Biotechnology, Inc.); CD160 antibody (1:1,000; AF3899; R&D Systems, Inc.)] specific for cell surface markers diluted in FACS buffer were added directly to this mixture and incubated for 30 min at 4°C.

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: Antibodies [CD8 (1:100; sc-1177; Santa Cruz Biotechnology, Inc.); CD160 antibody (1:1,000; AF3899; R&D Systems, Inc.)] specific for cell surface markers diluted in FACS buffer were added directly to this mixture and incubated for 30 min at 4°C.

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: Antibodies [CD8 (1:100; sc-1177; Santa Cruz Biotechnology, Inc.); CD160 antibody (1:1,000; AF3899; R&D Systems, Inc.)] specific for cell surface markers diluted in FACS buffer were added directly to this mixture and incubated for 30 min at 4°C.

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: Antibodies [CD8 (1:100; sc-1177; Santa Cruz Biotechnology, Inc.); CD160 antibody (1:1,000; AF3899; R&D Systems, Inc.)] specific for cell surface markers diluted in FACS buffer were added directly to this mixture and incubated for 30 min at 4°C.

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: Antibodies [CD8 (1:100; sc-1177; Santa Cruz Biotechnology, Inc.); CD160 antibody (1:1,000; AF3899; R&D Systems, Inc.)] specific for cell surface markers diluted in FACS buffer were added directly to this mixture and incubated for 30 min at 4°C.

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