Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: Interleukin-3 (IL-3) induces CD69 expression on human basophils and eosinophils. Flow cytometry analysis data are shown of CD69 expression in basophils and eosinophils after 18 hr of culture in medium alone or in medium containing 300 pm IL-3. The figure shows representative histograms of three independent experiments. The percentage of positive cells is indicated.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (LY) blocks interleukin-3 (IL-3)-induced cell survival and CD69 expression in human basophils. (a) LY inhibits the IL-3-induced basophil survival. Freshly isolated human basophils were cultured in medium alone or with 300 pm IL-3, or in the presence or absence of LY294002 (25 µm or 50 µm), or 50 µm PD98059. At 48 hr, basophils were collected and stained with annexin-V and propidium iodide (prop. iod.), and analysed by flow cytometry. Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide negativity indicates cell viability (% cell survival). *P < 0·05 when compared with IL-3 alone. (b) LY inhibits CD69 expression on basophils. The experimental condition was the same as in (a), except that the cells were cultured for 18 hr and then stained with anti-CD69 or isotype-control antibody (IgG2b). A representative histogram is shown of human basophils stained with anti-CD69 or control antibody after 18 hr of culture with IL-3 alone and IL-3 plus 50 µm LY. Values in parenthesis indicate the mean fluorescence intensity. The figure is representative of three independent experiments.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry, Control, Fluorescence

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: Lactacystin (LC) inhibits interleukin-3 (IL-3)-induced cell survival and CD69 surface expression in human basophils. (a) LC inhibits IL-3-induced cell survival. Freshly isolated human basophils were cultured in medium alone, or in medium containing IL-3 or IL-3 + LC, for 48 hr. Basophils were then collected and stained with annexin-V and propidium iodide, and analysed by using flow cytometry. Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (prop. iod.) negativity indicates cell viability (% cell survival). These data presented here are representative of three experiments. (b) LC inhibits IL-3-induced cell-surface expression of CD69. A representative histogram is shown of human basophils stained with anti-CD69 or control antibody after 18 hr of culture with IL-3 alone or IL-3 + 10 µm LC. The experimental conditions were the same as in (a), except that the cells were cultured for 18 hr and then stained with anti-CD69 or isotype-control antibody (IgG2b). Values in parenthesis indicate the mean fluorescence intensity. The figure is representative of three independent experiments.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry, Control, Fluorescence

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: A schema presenting our hypothesis that interleukin-3 (IL-3) inactivates phosphatidylinositol 3-kinase (PI3-K), initiating signalling cascades that lead to the generation of survival genes through both an NF-κB pathway, as well as through an alternate mechanism. In summary, we demonstrated that the in vitro spontaneous rate of apoptosis of human basophils is higher than that of eosinophils, and that IL-3 inhibits basophil apoptosis as well as up-regulating basophil CD69 surface expression via a PI3-K-dependent mechanism(s) that entails new RNA and protein synthesis, partially via NF-κB signalling. As basophils are active participants in allergic reactions, and IL-3 is one of the most abundant proinflammatory cytokines in the secretions from allergic tissue, IL-3-mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: In Vitro, Expressing, Inhibition

Journal: Frontiers in Immunology

Article Title: Proline metabolism reprogramming of trained macrophages induced by early respiratory infection combined with allergen sensitization contributes to development of allergic asthma in childhood of mice

doi: 10.3389/fimmu.2022.977235

Figure Lengend Snippet: Lung CD11b + trained macrophages increased after PVM-OVA early sensitization and mediated asthma in childhood mice. (A) The lung CD11b + cells in the memory phase after sensitization by flow cytometry. (B) Lung CD11b + macrophages morphologic evaluation. (C) Timeline of mouse adoptive transfer experiment. (1) PBS transfer group, (2) PBS-OVA transfer group, (3) PVM transfer Group, (4) PVM-OVA transfer group. (D) RI and lung Cdyn in mice after adoptive transfer of the lung CD11b + macrophages following OVA challenge. PVM-OVA transfer group compared with PBS transfer group, *, with PBS-OVA transfer group, #, with PVM transfer group, <. (E) Relative expression of MCP-1 and IL-5 in lung tissue following OVA challenge by Real-time PCR. Each experimental group contained three biology repeats. Data are presented as means standard deviations of 5-6 mice per group. (F) H&E staining of lung tissue slices after adoptive transfer the lung CD11b + macrophages (100X) and inflammation scores following OVA challenge. (G) CD11b + CD69 + macrophages or CD11b + TLR4 + macrophages in lungs by flow cytometry. (H) TLR4 expression of the sorted CD11b+ macrophages after LPS or OVA stimulation by flow cytometry. (I) Relative mRNA expression of chemokines MCP-1 or GRO-α of the sorted CD11b + macrophages after LPS or OVA stimulation by real-time PCR. *P<0.05. ** P<0.01, ***P<0.001, **** P<0.001. ## means PVM-OVA transfer group compared with PBS-OVA transfer group (P<0.01). The symbol “■” but not “<” means PVM-OVA transfer group compared with PVM transfer group (P<0.05).

Article Snippet: Lung single cells were incubated with fluorescein labeled antibodies, anti-mouse CD11b-PE, CD69-FITC, TLR4-APC, CD49a-PE-vio770, Gr-1-FITC (MACS, Miltenyi, Germany).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: Frontiers in Immunology

Article Title: Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

doi: 10.3389/fimmu.2020.559810

Figure Lengend Snippet: T cell immune response after TBI progress differently in K14-VEGFR3-Ig and WT littermate mice. Panels (A, B) represent the number and frequency of TCRβ+ T cells (A) and the CD4/CD8 ratio (B) in the brain of WT and TG mice, as analyzed in the perilesional and contralateral cortices 3 days post injury (WT ipsi, n = 4; WT contra, n = 4; TG ipsi, n = 3; TG contra, n = 3). No differences between the genotypes have been observed. (C–F) Analysis of T cells infiltration in the brain of K14-VEGFR3-Ig and WT littermate mice 60 days post-injury (WT ipsi, n = 5; WT contra, n = 5; TG ipsi, n = 4; TG contra, n = 4). Box plot represents the number of infiltrating T cells, defined by expression of TCRβ (C) and stacked bargram represents the percentage of CD4+ and CD8+ T cells (D) in the perilesional areas (ipsi) and correspondent contralateral areas (contra) of WT and TG mice. Bargrams in (C, D) show respectively the frequencies of CD8+ and CD4+ T cell subpopulations, as analyzed in the perilesional cortices of WT and TG mice. In CD8+ subpopulation we observed a significant reduction in the frequency of the CD44 hi CD69+ subpopulation in K14-VEGFR3-Ig compared to WT mice, which corresponded to the increase in the frequency of CD44 neg CD69+ phenotype. In CD4+ subpopulation, instead, we did not observed differences in distribution between the two genotypes. Data are presented as median ± SD. A binomial negative regression or a linear mixed model was applied to assess statistical differences in the counts of TCRβ + T cells. The Kruskal Wallis test was used for the analysis of frequency distribution. **p < 0.01 vs. WT ipsi. #p < 0.05 vs. respective contra. In all tests, Bonferroni correction was used to adjust p-values in multiple comparisons.

Article Snippet: Antibodies used: TCRβ PE-Cy7 (1:100 or 1:200 clone H57-597), CD44 PE (1:300 clone IM7) (both BioLegend); CD8a APC-R700 (1:150 or 1:200, clone 53-6.7), CD69 BV421 (1:100, clone H1.2F3), CD25 BB515 (1:150, clone PC61) (BD Biosciences); CD4 FITC (1:500, clone RM4-5), CD4 eFluor506 (1:500, clone RM4-5), CD8 PerCP eFluor710 (1:300, clone 53-6.7), CD44 APC (1:300 or 1:400, clone IM7), FoxP3 (1:40, clone FJK-16s) (eBioscience Thermo Fisher Scientific, Waltham, MA, USA); CD69 APC (1:20, clone H1.2F3, Miltenyi Biotech).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

doi: 10.3389/fimmu.2020.559810

Figure Lengend Snippet: Analysis of CD69 and CD44 T cell activation and memory markers in CD4+ and CD8+ subpopulations. Pseudocolor dot plots (A, B) represent gated subpopulations CD69 vs. CD44 of CD8+ and CD4+, respectively. Bargrams in (C, D) show respectively the counts and frequencies of CD8+ T cell subpopulations, as analyzed in the perilesional cortices of WT and TG mice. No significant differences in CD8+ subpopulations were found between genotypes. In CD4+ subpopulation, instead, we observed a significant reduction in the counts of CD44 hi CD69+ and CD44 hi CD69- subpopulations (E) , in K14-VEGFR3-Ig compared to WT mice. However, no differences were observed in the different subpopulation frequencies (F) . Data are presented as median ± SD. A binomial negative regression was applied to assess statistical differences in the counts of total T cells between WT ipsi and TG ipsi. The Kruskal Wallis test was used for the analysis of frequency distribution. # p < 0.05; *p < 0.05 vs. WT ipsi.

Article Snippet: Antibodies used: TCRβ PE-Cy7 (1:100 or 1:200 clone H57-597), CD44 PE (1:300 clone IM7) (both BioLegend); CD8a APC-R700 (1:150 or 1:200, clone 53-6.7), CD69 BV421 (1:100, clone H1.2F3), CD25 BB515 (1:150, clone PC61) (BD Biosciences); CD4 FITC (1:500, clone RM4-5), CD4 eFluor506 (1:500, clone RM4-5), CD8 PerCP eFluor710 (1:300, clone 53-6.7), CD44 APC (1:300 or 1:400, clone IM7), FoxP3 (1:40, clone FJK-16s) (eBioscience Thermo Fisher Scientific, Waltham, MA, USA); CD69 APC (1:20, clone H1.2F3, Miltenyi Biotech).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: mTORC2 contributes to murine lupus

doi: 10.1101/2021.03.27.437347

Figure Lengend Snippet: (A) Immunoblot analysis of p-AKT 473 in B6 and Lpr CD4 + T cells isolated from peripheral lymph nodes (pLN). Right, summary of the relative p-AKT 473 expression (normalized to that in B6 CD4 + T cells). (B) Immunoblot analysis of p-AKT 473 , p-STAT1, and p-STAT2 in CD4 + T cells from B6 and CD4 cre Rictor fl/fl mice stimulated with or without anti-CD3/anti-CD28 in the presence or absence of IFNα for 1 and 6 hours. Right, summaries of the relative p-AKT 473 expression (normalized to that in B6 CD4 + T cells without any stimulation) for 1 and 6 hours, respectively. (C) Flow cytometry analysis of p-AKT 473 expression in CD4 + T cells treated with IFNα alone, or in combination with anti-CD3/anti-CD28 overnight. Right, summary of the relative pAKT 473 expression (normalized to that in B6 CD4 + T cells without any stimulation). (D) and (E) B6 and CD4 cre Rictor fl/fl mice were administered with poly(I:C) intraperitoneally. (D) Expression of CD69 in blood CD4 + T cells from B6 and CD4 cre Rictor fl/fl mice after poly(I:C) administration. Numbers indicate the percentages of CD69 + cells. Right, summary of CD69 + percentage in CD4 + T cells at baseline or treated with poly(I:C) for 16 h and 40 h. (E) Blood CD4 + T cell counts were determined before and after poly(I:C) treatment. (F) Expression of CD69 in CD4 + T cells from B6, Lpr and Lpr. Rictor −/− mice. Right, summary of CD69 + percentages among pLN CD4 + T cells. (G) Blood CD4 + T cell counts were determined in 4-6 months old B6, Lpr and Lpr. Rictor −/− mice. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (A-E, unpaired Student’s t test, F and G, one-way ANOVA). Results were presentative of 4 (A, B), or pooled from at least 3 (A-G) independent experiments. Error bars represent SEM.

Article Snippet: For analysis of surface markers, cells were stained in PBS containing 1% (w/v) BSA on ice for 30 min, with APC-labeled anti-ICOS (clone: 7E.17G9; BD), PE-Cy7-labeled anti PD-1 (clone: RMPI-30; BioLegend), Super Bright 600–labeled anti-CD4 (Clone: SK-3; eBioscience), BV510-labeld anti-CD8a (clone:53-6.7; BioLegend); APC-Cy7-labeled TCRβ (clone: H57-597; BioLegend), APC anti-CD162 (PSGL1; clone: 2PH1; BD), BV605-labeled anti-CD25 (clone: PC61; BioLegend), PE-Cy7–labeled anti-CD19 (clone: 6D5; BioLegend), BV605-labeled B220 (clone: RA3-6B2; BioLegend), PE-labeled anti-Fas (clone: Jo2; BD), BV785-labeled anti-CD138 (clone: 281-2; BioLegend), APC-labeled anti-IgD (clone: IA6-2; BioLegend), PerCP-Cyanine5.5-labeled anti-CD38 (clone: 90; BioLegend), FITC-labeled anti-IgG1 (clone: RMG1-1; BioLegend), APC-labeled anti-IL7Rα (clone: SB/199; BioLegend), FITC-labeled anti-CD69 (clone: H1.2F3; Tonbo Bio), PE-labeled anti-CD153 (clone: RM153; eBioscience), PE-labeled anti-CD73 (clone: TY/11.8; BioLegend) and APC-labeled anti-FR4 (clone: 12A5; BD).

Techniques: Western Blot, Isolation, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: mTORC2 contributes to murine lupus

doi: 10.1101/2021.03.27.437347

Figure Lengend Snippet: (A) Immunoblot analysis of pSTAT1, pSTAT2 and p-S6 expression in CD4 + T cells from B6 and CD4 cre Ricto fl/fl mice treated with or without IFNα in presence of anti-CD3/anti-CD28 for 6 hours. The relative expressions were normalized to them in B6 CD4 + T cells without any stimulation. (B) Blood CD19 + B cell counts were determined before and after poly(I:C) treatment in B6 and CD4 cre Rictor fl/fl mice. (C) Expression of CD69 was measured by flow cytometry in blood CD4 + T cells from 4-6 months old B6, Lpr and Lpr. Rictor fl/fl mice. Right, summary of CD69 + percentages among blood CD4 + T cells. (D) Percentages of CD19 − CD4 + T cells in blood from B6, Lpr and Lpr. Rictor−/− mice among total lymphocytes. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (A and B, unpaired Student’s t test, C and D, one-way ANOVA). Results were pooled from at least 3 independent experiments. Error bars represent SEM.

Article Snippet: For analysis of surface markers, cells were stained in PBS containing 1% (w/v) BSA on ice for 30 min, with APC-labeled anti-ICOS (clone: 7E.17G9; BD), PE-Cy7-labeled anti PD-1 (clone: RMPI-30; BioLegend), Super Bright 600–labeled anti-CD4 (Clone: SK-3; eBioscience), BV510-labeld anti-CD8a (clone:53-6.7; BioLegend); APC-Cy7-labeled TCRβ (clone: H57-597; BioLegend), APC anti-CD162 (PSGL1; clone: 2PH1; BD), BV605-labeled anti-CD25 (clone: PC61; BioLegend), PE-Cy7–labeled anti-CD19 (clone: 6D5; BioLegend), BV605-labeled B220 (clone: RA3-6B2; BioLegend), PE-labeled anti-Fas (clone: Jo2; BD), BV785-labeled anti-CD138 (clone: 281-2; BioLegend), APC-labeled anti-IgD (clone: IA6-2; BioLegend), PerCP-Cyanine5.5-labeled anti-CD38 (clone: 90; BioLegend), FITC-labeled anti-IgG1 (clone: RMG1-1; BioLegend), APC-labeled anti-IL7Rα (clone: SB/199; BioLegend), FITC-labeled anti-CD69 (clone: H1.2F3; Tonbo Bio), PE-labeled anti-CD153 (clone: RM153; eBioscience), PE-labeled anti-CD73 (clone: TY/11.8; BioLegend) and APC-labeled anti-FR4 (clone: 12A5; BD).

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Vaccines

Article Title: Influenza Vaccination Mediates SARS-CoV-2 Spike Protein Peptide-Induced Inflammatory Response via Modification of Histone Acetylation

doi: 10.3390/vaccines12070731

Figure Lengend Snippet: The activation of immune cell subtypes in PBMCs following IV and ex vivo stimulation. ( A ) Representative plots of the gating strategy of CD3 + T cells, CD3 - immune cells, and CD45 + immune cells from PBMCs. ( B ) Representative plots of the T-cell subset gated on total CD3 + T cells expressing CD4 and CD8 surface markers. ( C , D ) Dot plots of the frequencies of cells gated out of CD4 + and CD8 + T cells expressing the AIM marker (CD69 and CD137) and the percentage of the designated population (AIM + CD4 + and AIM + CD8 + ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( E , F ) Dot plots of the frequencies of cells gated out of CD3 - immune cells expressing the NK1.1 marker and the percentage of the designated population (NK1.1 + CD3 − ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( G , H ) Dot plots of the frequencies of cells gated out of CD3 − immune cells expressing the MHC-II marker and the percentage of the designated population (MHC-II + CD3 − ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( I , J ) Dot plots of the frequencies of monocytes gated out of CD45 + immune cells expressing CD11b and CD115 markers and the percentage of the designated population (CD11b + CD115 + CD45 + ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. n = 4/group. Data are presented as mean ± SEM and * p < 0.05, ** p < 0.01, and *** p < 0.001 (one-way ANOVA with Tukey’s test).

Article Snippet: Afterward, the cells were incubated with 200 μL of FACS buffer of antibody cocktail for surface marker staining, including CD3 (ThermoFisher Scientific, Waltham, MA, USA, cat# 12-0038; Biolegend, San Diego, CA, USA, cat# 100233), CD45 (ThermoFisher Scientific, cat# 11-0459), CD4 (ThermoFisher Scientific, cat# 11-0041), CD8 (ThermoFisher Scientific, cat# 25-0088), CD69 (Tonbo, Tucson, AZ, USA, cat# 20-0691), CD137 (ThermoFisher Scientific, cat# 12-1371), CD11b (Tonbo, cat# 20-0112), CD115 (Biolegend, cat# 135527), NK1.1 (ThermoFisher Scientific, cat# 45-5941), and MHC-II (ThermoFisher Scientific, cat# 17-5320).

Techniques: Activation Assay, Ex Vivo, Expressing, Marker, Control

Journal: iScience

Article Title: Temporal and spatial dynamics of immune cells in spontaneous liver transplant tolerance

doi: 10.1016/j.isci.2023.107691

Figure Lengend Snippet: List of metal-labeled antibodies used for mass cytometry

Article Snippet: Anti-CD69 , Fluidigm , 3143004C.

Techniques:

Journal: iScience

Article Title: Temporal and spatial dynamics of immune cells in spontaneous liver transplant tolerance

doi: 10.1016/j.isci.2023.107691

Figure Lengend Snippet:

Article Snippet: Anti-CD69 , Fluidigm , 3143004C.

Techniques: Transplantation Assay, Recombinant, Staining, Cell Isolation, Software, Cytometry, Flow Cytometry