Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Panel (a) of this figure depicts the expression of the T cell activation marker GITR on CD25+, CD25 bright and CD25 verybright cells from healthy controls (white bars, n = 24) and SSc patients having limited cutaneous SSc (light gray bars, n = 18), late diffuse SSc (dark gray bars, n = 22) and early diffuse SSc (black bars, n = 22) patients. In panel (b) the expression of CD62L on Tregs is investigated. CD25+ and CD25 bright cells from SSc and healthy controls express similar levels of CD62L, whereas CD25 verybright from SSc patient subsets exhibit lower levels of CD62L compared to those from healthy controls. Panel (c) reflects the expression of CD69 on Tregs from healthy donors and SSc patients. CD25+, CD25 bright and CD25 verybright cells from SSc patients express significant lower levels of CD69 than those from healthy donors. CD69 expression on CD25 bright and CD25 verybright cells from edSSc patients was significantly lower then that from ldSSc patients, and ldSSc expressed CD69 significantly lower than those from lSSc. In panel (d) the expression on CD3+ cells is shown for all investigated groups. In contrast with that observed on Tregs from SSc patients, CD69 expression on CD4+ cells was significantly higher in all SSc patient groups. Panel (e) reflects the potential association between CD69 expression on Tregs and disease duration. CD69 expression on T regs from patients with lSSc correlated with disease duration, whereas this was not the case either with ldSSc nor edSSc. In all figures the white bars represent healthy controls, whereas lSSc, ldSSc and edSSc patients are represented by light gray, dark gray and black bars, respectively.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Expressing, Activation Assay, Marker

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Unsorted CD3+ (MACS bead isolated) were stimulated with PHA (5 µg/ml) and consecutively incubated with CD25 high CD127 - or CD25 low CD127 high cells for 5 days. Thereafter, CD3+ cells were incubated with 3 H -thymidine for 24 more hours after which 3 H -thymidine incorporation was measured. Panel (a) reflects the suppressive capacity of Tregs from healthy donors and SSc patients. Proliferation of CD3+ effector cells was effectively inhibited by T regulatory cells from healthy controls, whereas a clearly diminished suppressive activity was observed in the experiments with Tregs from SSc patients. Suppressive effect of Treg (CD25 high CD127 - ) and non-Tregs (CD25 low CD127 high ) is presented in black and white bars, respectively. Results are the mean and SEM of 6 separate experiments using cells from healthy donors (n = 9), lSSc (n = 7), ldSSc (n = 9) and edSSc (n = 7). Panel (b) represents the correlation of CD69 expression and Treg suppressive capacity in Tregs from the various groups under investigation. The percentage of CD69 positive regulatory T cells (CD25 high CD127 - ) correlates well with the percentage of inhibition of CD3+ cells in healthy controls (triangles), lSSc (diamonds), ldSSc (circles) and edSSc (squares). Panel (c) reflects the expression of intracellular TGFβ in Tregs from healthy controls and SSc patients as measured using intracellular flow cytometry. CD25 high CD127 - cells from all SSc patients express lower TGFβ levels compared to controls. Left panel reflects an representative individual from each group whereas the right panel displays the mean of each group comprising 6 individuals (per group) coming forth from 4 independent experiments.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Isolation, Incubation, Activity Assay, Expressing, Inhibition, Flow Cytometry

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: (a) During the co-cultures of unsorted CD3+ cells with either Tregs (CD25 high CD127 - ) or non-Tregs (CD25 low CD127 high ) 10 or 25% plasma from an edSSc patient or healthy control was added to the culture. The graph represents data from 3 independent experiments using 3 healthy control cells, and plasma derived from two edSSc patients and two control individuals. (b) The effect of SSc plasma was evaluated by adding 10% to CD3+ cells for 24 hrs stimulated with PHA or unstimulated. As a control, CD69 expression was measured on CD3+ cells stimulated with PHA only. CD4 and CD25 high /FoxP3 high cells were gated based on the expression of these markers using flow cytometry. (c) CD69 expression and induction upon PHA mediated stimulation of CD4+ and CD25 high /FoxP3 high obtained from healthy donors, lSSc, ldSSc and edSSc patients was investigated using flow cytometry. One representative patient from each group is shown.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Clinical Proteomics, Control, Derivative Assay, Expressing, Flow Cytometry

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: 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: PLoS ONE

Article Title: miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis

doi: 10.1371/journal.pone.0265192

Figure Lengend Snippet: Expression of FAS (A), FASL (B), CD69 (C), and CCR7 (D) in splenic leukocytes from CanL and healthy dogs after culture and respective representative histograms obtained from flow cytometry analysis. The red line represents the CanL group, and the blue line represents the control group. Cells were cultured for 48 h at 37°C and 5% CO 2 without treatment (medium) and then incubated with monoclonal antibodies. Data are presented as median ± min-max. Asterisks represent statistical significance (Mann–Whitney, *p < 0.05).

Article Snippet: Cells were centrifuged at 1800 rpm for 7 minutes and then incubated with phycoerythrin (PE)-conjugated anti-human CD95 (FAS) monoclonal antibody (BD Biosciences, USA), anti-human CD178 (FASL) monoclonal antibody (BD Biosciences, USA), and anti-human CD69 polyclonal antibody (Lifespan Biosciences, USA).

Techniques: Expressing, Flow Cytometry, Cell Culture, Incubation, MANN-WHITNEY

Journal: PLoS ONE

Article Title: miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis

doi: 10.1371/journal.pone.0265192

Figure Lengend Snippet: Expression of CD69 protein in splenic leukocytes of the control (A) and CanL group (B). Splenic leukocytes of dogs naturally infected by L . infantum and healthy dogs were transfected with scrambled, miR-21 mimic, and miR-21 inhibitor, all in the presence of Hiperfect, following 48 h in culture at 37°C and 5% CO 2 . Data are presented as median ± min-max. The asterisk indicates significant differences (Friedman’s multiple comparison test followed by Dunn’s multiple comparisons test (comparing the mean rank of each treatment with every other treatments), * p < 0.05).

Article Snippet: Cells were centrifuged at 1800 rpm for 7 minutes and then incubated with phycoerythrin (PE)-conjugated anti-human CD95 (FAS) monoclonal antibody (BD Biosciences, USA), anti-human CD178 (FASL) monoclonal antibody (BD Biosciences, USA), and anti-human CD69 polyclonal antibody (Lifespan Biosciences, USA).

Techniques: Expressing, Infection, Transfection

Journal: PLoS ONE

Article Title: miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis

doi: 10.1371/journal.pone.0265192

Figure Lengend Snippet: Expression of CD69 protein in (A) CD4+, (B) CD8+ and (C) B lymphocytes (CD21+ cells) in the CanL group. Splenic leukocytes of dogs naturally infected by L . infantum were transfected with scrambled, miR-21 mimic, and miR-21 inhibitor, all in the presence of Hiperfect, following 48 h in culture at 37°C and 5% CO 2 . Data are presented as median ± min-max, and the asterisk indicates significant differences (Friedman’s multiple comparison test, * p < 0.05).

Article Snippet: Cells were centrifuged at 1800 rpm for 7 minutes and then incubated with phycoerythrin (PE)-conjugated anti-human CD95 (FAS) monoclonal antibody (BD Biosciences, USA), anti-human CD178 (FASL) monoclonal antibody (BD Biosciences, USA), and anti-human CD69 polyclonal antibody (Lifespan Biosciences, USA).

Techniques: Expressing, Infection, Transfection