Journal: Frontiers in immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: FIGURE 3 | JHU083 inhibited T cells differentiation in vivo. Mice of Vehicle-AIH and JHU083-AIH group were intravenously injected with 8ml/kg ConA, and Vehicle- WT group was injected with normal saline of equal volume. JHU083-AIH group was gavaged with 0.3mg/kg JHU083 24h and 1h before ConA injection, and Vehicle- WT and Vehicle-AIH group were treated with equal vehicle at the same time points. (A) Flow cytometry was applied to analyze the Th1 cell percentage via IFNg expression, the Th2 cell percentage via IL4 expression, and the Th17 cell percentage via IL17A expression. (B) ELISA was applied to analyze serum IFNg (Th1 cells) and IL17 (Th17 cells) secretions. Flow cytometry was also applied to analyze the differentiation of CD8(+) T cells into cytotoxic T lymphocytes, evidenced by secreting IFNg (C) and Granzyme (D). Data were expressed as means ± SEM. *P < 0.05, **P <0.01, and ***P <0.001. ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PECy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNg (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vivo, Injection, Saline, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: FIGURE 5 | DON treatment suppressed T cells differentiation in vitro. Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5mM DON, and cells were analyzed 24h after ConA stimulation. (A) The differentiation markers of CD4+ T cells (IFNg for Th1 cells, IL4 for Th2 cells and IL17 for Th17 cells) were detected using flowcytometry. (B) qRT-PCR was applied to analyze IFN-g, IL4 and IL17 mRNA levels. (C) ELISA was applied to analyze supernatant IFN-g and IL17 secretions. Flow cytometry was also applied to detect the production of activation markers of CTL, such as IFNg (D) and Granzyme B (E). Data were expressed as means ± SEM. **P <0.01 and ***P <0.001, ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PECy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNg (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Activation Assay

Journal: Immunity

Article Title: Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity

doi: 10.1016/j.immuni.2018.12.020

Figure Lengend Snippet:

Article Snippet: The cells obtained were fixed and permeabilized and were stained with PE conjugated anti-IL-12p40 antibody (Tonbo) or APC anti-mouse IL-6 antibody or its isotype control antibody APC Rat IgG1, κ (Biolegend).

Techniques: Control, Blocking Assay, Negative Control, Virus, Recombinant, Staining, Bacteria, Reverse Transcription, Amplification, Enzyme-linked Immunosorbent Assay, PCR Cloning, DNA Library Preparation, Software

Journal: The Journal of allergy and clinical immunology

Article Title: Anti-apoptotic Serine Protease Inhibitors contribute towards the survival of allergenic Th2 cells

doi: 10.1016/j.jaci.2017.07.055

Figure Lengend Snippet: (A) Experimental protocol. Arrows indicate i.p. injections of OVA + alum on d-5 and d0 and the times at which mice were analyzed (B) Flow cytometry plots after staining with OVA329–337/I-Ab tetramer (OVA tet) and anti-CD4 before (d0) after 40d after the final OVA + alum immunization. Percentage OVA tet+ CD4+ cells is indicated. (C) Histograms (filled WT, line Spi2A KO) after staining for ST2 on gated OVA tet+ CD4+ cells. Histograms after ICS for IL-4 or IL-5 on gated OVA tet+ CD4+ cells. Percentage positive population (line gate) is indicated for WT/Spi2A KO, respectively. (D) Histograms for mean percentage OVA tet+ CD4+, mean percentage OVA tet+ CD4+ IL-4+, mean percentage OVA tet+ CD4+ ST2+ and mean percentage OVA tet+ CD4+ IL-5+ of total splenocytes (± SEM, n=6–8). P <0.005 **; P <0.0005 ***; P>0.05 Not Significant (n.s.). Broken horizontal lines show the limit of detection.

Article Snippet: Each week, resting cells were stimulation of PMA, Ionomycin for 1 hr and Brifeldin A for 4h prior surface immunostaining with antihuman CD4-Percp (clone: L200) and intracellular immunostaining with anti human IL-5-APC (clone: TRFK), anti human IL-13-Percp cy5.5 (clone: JES-5AZ), antihuman GATA-3-PE-Cy7 (clone: L50823), anti human IFN-γ-PE Cy5.5, clone: B27) and antihuman T-bet V450 (clone: 04–46) antibodies (BD Biosciences, San Jose, CA) according the manufacturer’s protocol (Cytofix-Cytoperm, BD Biosciences).

Techniques: Flow Cytometry, Staining

Journal: The Journal of allergy and clinical immunology

Article Title: Anti-apoptotic Serine Protease Inhibitors contribute towards the survival of allergenic Th2 cells

doi: 10.1016/j.jaci.2017.07.055

Figure Lengend Snippet: (A). Representative flow-cytometry data illustrating expression of Th2/Th1 cytokines in non-polarized (NP) and polarized Th2 cells at week 3. (B–G). Mean (± SEM n= 8 patient samples) level of target mRNA (fold-change compared to EFA-2 housekeeping gene) in Th2 polarized and non-polarized (NP) cells. (H) Relationship between Th2 master regulator GATA-3 and SERPINB3 (filled symbols) and SERPINB4 (open symbols) mRNA levels in polarized Th2 cells from individuals after 3 weeks of culture. (I) Percentage of ILC2 and ILC1 cells of CD45+Lin− PBMC from pollen allergy patients (n=6) (horizontal line indicates mean percentage). Mean levels of (J) IL-5, (K) GATA-3, (L) T-BET, (M) SERPINB3 and (N) SERPINB4 mRNA in ICL2 cells relative to the ICL1 cells. P< 0.05*; P <0.005**; P<0.0005***.

Article Snippet: Each week, resting cells were stimulation of PMA, Ionomycin for 1 hr and Brifeldin A for 4h prior surface immunostaining with antihuman CD4-Percp (clone: L200) and intracellular immunostaining with anti human IL-5-APC (clone: TRFK), anti human IL-13-Percp cy5.5 (clone: JES-5AZ), antihuman GATA-3-PE-Cy7 (clone: L50823), anti human IFN-γ-PE Cy5.5, clone: B27) and antihuman T-bet V450 (clone: 04–46) antibodies (BD Biosciences, San Jose, CA) according the manufacturer’s protocol (Cytofix-Cytoperm, BD Biosciences).

Techniques: Flow Cytometry, Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: Anti-apoptotic Serine Protease Inhibitors contribute towards the survival of allergenic Th2 cells

doi: 10.1016/j.jaci.2017.07.055

Figure Lengend Snippet: (A) Representative flow-cytometry data for percentage GFP+ of lentivirus transduced CD27− CD4 T cells. (B). Mean (± SEM n= 8 patient samples) percentage GFP+ of lentivirus transduced CD27− CD4 T cells. (C) Mean (± SEM n= 8 patient samples) level of SERPINB3 mRNA (fold-change in compared to EFA-2 housekeeping gene). (D) Mean (± SEM n= 8 patient samples) level of SERPINB4 mRNA (fold-change in compared to EFA-2 housekeeping gene). (E and F) Mean percentage viability (± SEM n= 8 patient samples) of CD27− CD4+ Th2 cells and concentrations of IL-5 and IL-13 in culture supernatant after transduction with lentiviruses. P< 0.05*; P <0.005**; P<0.0005***.

Article Snippet: Each week, resting cells were stimulation of PMA, Ionomycin for 1 hr and Brifeldin A for 4h prior surface immunostaining with antihuman CD4-Percp (clone: L200) and intracellular immunostaining with anti human IL-5-APC (clone: TRFK), anti human IL-13-Percp cy5.5 (clone: JES-5AZ), antihuman GATA-3-PE-Cy7 (clone: L50823), anti human IFN-γ-PE Cy5.5, clone: B27) and antihuman T-bet V450 (clone: 04–46) antibodies (BD Biosciences, San Jose, CA) according the manufacturer’s protocol (Cytofix-Cytoperm, BD Biosciences).

Techniques: Flow Cytometry, Transduction