Journal: Frontiers in Genetics

Article Title: Exosomes containing long non-coding RNA AGAP2-AS1 promote the differentiation of CD4 + T cells through the miR-424-5p/SGK1 axis in psoriasis

doi: 10.3389/fgene.2025.1521470

Figure Lengend Snippet: ASAP2-AS1 promotes the differentiation of Th1 and Th17 in CD4 + T cells and serves as a sponge for miR-424-5p. (A) Fluorescence microscopy was used to detect the subcellular localization of AGAP2-AS1 in CD4 + T cells. AGAP2-AS1 was labeled with Cy3, cell nuclei were counterstained with DAPI (blue). (B) The expression of AGAP2-AS1 in CD4 + T cells after co-incubation of TNF-α-treated HaCaTs-derived exosomes. (C) CD4 + T cells were transfected with LV-AGAP2-AS1 or LV-NC. The percentage of Th1 and Th17 cells was detected by flow cytometry. (D) The protein levels of IFN-γ and IL-17A are measured by ELISA. (E) Prediction of the binding sequence between AGAP2-AS1 and miR-424-5p and construction of the AGAP2-AS1 MUT sequence. (F) Dual-luciferase reporter gene assay confirmed the binding relationship between AGAP2-AS1 and miR-424-5p in HEK-293T cells. (G) AGAP2-AS1 expression level in CD4 + T cells transfected with miR-424-5p mimics or NC mimics and miR-424-5p inhibitor or NC inhibitor. WT wild type, MUT mutant type. Th, T helper. Experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001. ns: no significance.

Article Snippet: For intracellular cytokine staining, cells were fixed with fixation buffer (E-CK-A109, Elabscience) and then stained intracellularly with antibodies against human IFN-γ (E-AB-F1196C, Elabscience) and IL-17A (E-AB-F1173D, Elabscience).

Techniques: Fluorescence, Microscopy, Labeling, Expressing, Incubation, Derivative Assay, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Luciferase, Reporter Gene Assay, Mutagenesis

Journal: Frontiers in Genetics

Article Title: Exosomes containing long non-coding RNA AGAP2-AS1 promote the differentiation of CD4 + T cells through the miR-424-5p/SGK1 axis in psoriasis

doi: 10.3389/fgene.2025.1521470

Figure Lengend Snippet: AGAP2-AS1/miR-424-5p regulated CD4 + T cell differentiation via the SGK1 signaling pathway. (A) The relative expression levels of SGK1 in 293T cells that were transfected with siRNA targeting SGK1. (B) SGK1 expression level in CD4 + T cells transfected with LV-AGAP2-AS1 or LV-NC and si-AGAP2-AS1 or si-NC. (C) Western blot confirmed that transfection of AGAP2-AS1 in CD4 + T cells promoted the expression of SGK1, and the miR-424-5p mimics reversed this process. (D) Flow cytometry analysis of the percentages of Th1 and Th17 cells in CD4 + T cells transfected with LV-AGAP2-AS1 and miR-424-5p mimics or NC mimics. (E) The protein levels of IFN-γ and IL-17A are measured by ELISA. (F) Flow cytometry analysis of the percentages of Th1 and Th17 cells in CD4 + T cells transfected with LV-AGAP2-AS1 and SGK1 siRNA or NC siRNA. (G) The protein levels of IFN-γ and IL-17A are measured by ELISA. The experiments were repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For intracellular cytokine staining, cells were fixed with fixation buffer (E-CK-A109, Elabscience) and then stained intracellularly with antibodies against human IFN-γ (E-AB-F1196C, Elabscience) and IL-17A (E-AB-F1173D, Elabscience).

Techniques: Cell Differentiation, Expressing, Transfection, Western Blot, Flow Cytometry, 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: 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 IFNγ expression, the Th2 cell percentage via IL4 expression, and the Th17 cell percentage via IL17A expression. (B) ELISA was applied to analyze serum IFNγ (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 IFNγ (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 (PE-Cy5, 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 IFNγ (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: DON treatment suppressed T cells differentiation in vitro . Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5μM DON, and cells were analyzed 24h after ConA stimulation. (A) The differentiation markers of CD4+ T cells (IFNγ for Th1 cells, IL4 for Th2 cells and IL17 for Th17 cells) were detected using flowcytometry. (B) qRT-PCR was applied to analyze IFN-γ, IL4 and IL17 mRNA levels. (C) ELISA was applied to analyze supernatant IFN-γ and IL17 secretions. Flow cytometry was also applied to detect the production of activation markers of CTL, such as IFNγ (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 (PE-Cy5, 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 IFNγ (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: Frontiers in Immunology

Article Title: VISTA + follicular regulatory T cells modulate the function of effector immune cells: implications for ovarian cancer immune escape

doi: 10.3389/fimmu.2025.1704048

Figure Lengend Snippet: Effects of VISTA + /VISTA − Tfr cells on CD25 − CD4 + T cell differentiation. (A) Flow cytometry scatter plots showing the effects of VISTA + /VISTA − Tfr cells in the co-culture system on the expression of differentiation markers IFN-γ, IL-4, and IL-17A on CD25 − CD4 + T cells (n=3). (B–D) Quantitative bar graphs showing the expression proportions of Th1, Th2, and Th17 cells after co-culture of CD25 − CD4 + T cells with VISTA − Tfr cells or VISTA − Tfr cells overexpressing VISTA (LV-VISTA). (E–G) Quantitative bar graphs showing the expression proportions of Th1, Th2, and Th17 cells after co-culture of CD25 − CD4 + T cells with VISTA + Tfr cells or VISTA + Tfr cells with VISTA silenced (shRNA-VISTA). The statistical results are expressed as the mean ± SD, * p < 0.05, ** p < 0.01, ns, no significant difference.

Article Snippet: Then, 5 μL each of IL-4-APC (MultiSciences, F11IL403), IFN-γ-PE (MultiSciences, F11IFNG02), and IL-17A-FITC (Elabscience, E-AB-F1173C) antibodies were added, followed by incubation at 4°C in the dark for 30 min.

Techniques: Cell Differentiation, Flow Cytometry, Co-Culture Assay, Expressing, shRNA