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
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![( 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 ) <t>ELISA</t> 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 <t>E</t> <t>(SEE)–activated</t> 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7029/pmc12757029/pmc12757029__sciadv.ady2539-f7.jpg)