Journal: Science Advances

Article Title: Multidimensional mapping of stimulation-responsive regulatory elements and candidate causal variants in T cell activation

doi: 10.1126/sciadv.ady2539

Figure Lengend Snippet: ( A to D ) Western blot of phosphorylated ZAP70 (Y493) and Lck (Y394) in stimulated rs5837875-AT/AT versus -A/A Jurkat cells [(A) and (B)] and ZNF384-OE versus empty vector control CD4 + T cells [(C) and (D)]. ( E to G ) ELISA quantification of IL-2 in culture supernatants from resting and stimulated rs5837875-AT/AT versus -A/A Jurkat cells (E), ZNF384-OE versus empty vector control CD4 + T cells (F), and ZNF384-SI versus scrambled vector control CD4 + T cells (G). ( H ) Schematic of T–B cell coculture model. Superantigen staphylococcal enterotoxin E (SEE)–activated Raji cells were combined with Jurkat cells (1:1 ratio), synchronized on ice, centrifuged to promote contact, and incubated for 24 hours. ( I and J ) ELISA quantification of IL-2 following Raji/SEE stimulation in rs5837875-AT/AT versus -A/A Jurkat cells (I) and rs5837875-KO versus control Jurkat-Cas9 cells (J). ( K and L ) For in vivo validation, 4 × 10 6 resting or stimulated rs5837875-A/A and -AT/AT Jurkat cells were intravenously injected into 2-month-old female C-NKG mice ( n = 6 random mice). Serum IL-2 was quantified by ELISA at the indicated time points (K), with days 3 and 7 results detailed (L). ( M ) Proposed model for rs5837875 function in autoimmune diseases. In resting conditions, chromatin surrounding rs5837875 remains relatively inactive. Upon stimulation, the rs5837875-AT allele specifically engages the CD28 promoter through ZNF384-mediated chromatin looping, enhancing CD28 expression and contributing to CD4 + T cell dysfunction and autoimmune susceptibility. Data represent means ± SD; n = 3, 4, or 6 biologically independent samples. Unpaired two-tailed Student’s t test is used to calculate P values in (A) to (G), (I), (J), and (L): * P < 0.05; ** P < 0.01; ns, not significant.

Article Snippet: IL-2 concentrations were quantified under both resting and SEE-stimulated conditions using a commercially available ELISA kit (CUSABIO, CSB-E04626h) according to the manufacturer’s instructions.

Techniques: Western Blot, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Incubation, In Vivo, Biomarker Discovery, Injection, Expressing, Two Tailed Test

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 CD8 + T cell proliferation and cytokine secretion. After co-culture of VISTA + or VISTA − Tfr cells with CD8 + T cells (CD8 + T cells: Tfr cells = 4:1): (A) Representative flow cytometry histogram of CFSE proliferation assay for co-cultured cells. The x-axis represents CFSE fluorescence intensity, and the y-axis represents cell count (n=3). (B) Quantitative analysis of CFSE fluorescence intensity in VISTA − Tfr + CD8 + T cell group versus VISTA + Tfr + CD8 + T cell group. (C) Quantitative analysis of CFSE fluorescence intensity after co-culture of CD8 + T cells with VISTA − Tfr cells overexpressing VISTA (LV-VISTA). (D) Quantitative analysis of CFSE fluorescence intensity after co-culture of CD8 + T cells with VISTA + Tfr cells with VISTA silenced (VISTA-shRNA). (E–L) Levels of IL-2, IFN-γ, perforin (PFP), and granzyme B in culture supernatants of VISTA − Tfr + CD8 + T cell group (E–H) and VISTA + Tfr + CD8 + T cell group (I–L) as detected by ELISA (n=3). The statistical results are expressed as the mean ± SD, * p < 0.05, ** p < 0.01.

Article Snippet: For co-culture groups with CD8 + T lymphocytes, levels of IL-2, Interferon-γ (IFN-γ), granzyme B, and perforin were detected (MultiSciences, EK102, EK180, EK158; CUSABIO, CSB-E09313h).

Techniques: Co-Culture Assay, Flow Cytometry, Proliferation Assay, Cell Culture, Fluorescence, Cell Counting, shRNA, Enzyme-linked Immunosorbent Assay