human cd160  (R&D Systems)

Journal: Immunity & Ageing : I & A

Article Title: Phenotypic and functional alterations of monocyte subsets with aging

doi: 10.1186/s12979-022-00321-9

Figure Lengend Snippet: Percentage of monocyte subsets expressing co-inhibitory molecules decreased in older adults. A . Representative flow cytometry histograms display the expression of the co-inhibitory molecules 2B4, T-cell immunoglobulin domain and mucin domain 3 (TIM-3), CD200R, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), B and T lymphocyte attenuator (BTLA), CD160, programmed death-1 (PD-1), and lymphocyte-activation gene 3 (LAG-3) on monocyte subsets from young adults. B . The percentage of monocyte subsets expressing TIM-3, 2B4, CD200R, TIGIT, and BTLA from young, middle-aged, and older adults (young: 21–40 years, n = 42; middle-aged: 41–60 years, n = 34; older: > 60 years, n = 34). P-values were calculated using the non-parametric Kruskal–Wallis rank test, followed by post hoc analysis

Article Snippet: The gating strategy used is shown in Fig. S . Antibodies used included anti-human CD160-Alexa Fluor 488, CD4-APC-Fire750, CD8-BV510, HLA-DR-Alexa Fluor 700, CD14-APC, PD-1-PE, 2B4-PE-CF594, CD16-BV711, TIM-3-BV650, CD200R-PE, BTLA-BV650, CD45-BV786 (BD Biosciences, San Diego, CA, USA), CX3CR1-BV421, CD3-PerCP-Cy5.5, CD15-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD29-Alexa Fluor 488, CD62L-BV650, CD11b-BV605, CCR2-PE (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, and LAG-3-APC (eBioscience, San Diego, CA, USA), along with the corresponding isotype controls.

Techniques: Expressing, Flow Cytometry, Activation Assay

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: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet: Mucosal immune cells isolated from the colorectal tissue biopsies by enzymatic digestion were exposed to HIV-1 BaL (250 ng/mL), either free (F-HIV), complement-opsonized (C-HIV), or virions opsonized by a cocktail of complement and antibodies (CI-HIV) or mock-treated, by spinning the cultures. The effect HIV exposure had on mucosal T cells phenotype was assessed after 4 days. ( A–C ) The immune cells were stained with CD3, CD4, CD8, CD38, PD-1, LAG3, TIM3, and CD160 mAbs. ( A ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160+ CD4+ T cells, ( B ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160, and ( C ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed CD38+CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry (N = 12–15). Statistical significance was tested using repeated measures of ANOVA followed by Tukey's posttest. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM.

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Isolation, Staining, Expressing, Flow Cytometry

Journal: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet: Mucosal immune cells isolated from the colorectal tissue biopsies by enzymatic digestion were exposed to HIV-1 BaL (250 ng/mL), either free (F-HIV), complement-opsonized (C-HIV), or virions opsonized by a cocktail of complement and antibodies (CI-HIV) or mock-treated, by spinning the cultures. The effect HIV exposure had on mucosal T cells phenotype was assessed after 4 days. ( A–D ) The immune cells were stained with CD3, CD4, CD8, Tbet, EOMES, PD-1, LAG3, TIM3, and CD160 mAbs. ( A ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD4+ was constructed on CD4 T cells, data presented as dot plots with colored channels. Data from a donor shows CD4 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD4 following exposure to different conditions of viruses. ( B ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD4+ T cells expressing PD-1. ( C ) Percentage of Tbet+CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD8+ was constructed on CD8 T cells, and data are presented as dot plots with colored channels. Data from a donor shows CD8 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD8 following exposure to different conditions of viruses. ( D ) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells and percentage of Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD8+ T cells expressing PD-1 (N = 12–15). Statistical significance was tested using repeated measures of ANOVA followed by Tukey's posttest. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM.

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Isolation, Staining, Expressing, Construct, Flow Cytometry

Journal: eLife

Article Title: Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells

doi: 10.7554/eLife.57869

Figure Lengend Snippet:

Article Snippet: , anti-human CD160-PE(Mouse monoclonal) , BD Biosciences , Cat#: 562118 , FACS (1 ul per test).

Techniques: Sequencing, Software, FCAP Assay