k0353  (MedChemExpress)

Journal: Nature Communications

Article Title: TIGIT deficiency promotes autoreactive CD4 + T-cell responses through a metabolic‒epigenetic mechanism in autoimmune myositis

doi: 10.1038/s41467-025-59786-z

Figure Lengend Snippet: PBMCs were collected from polymyositis (PM), dermatomyositis (DM), systemic lupus erythematosus (SLE), or healthy controls (HC). TIGIT, CD226, and CD96 expression in cells in PBMCs was measured by flow cytometry. a Schematic employed to visualize TIGIT, CD226, and CD96 on T cells binding to CD155 on antigen-presenting cells (APC). b – g CD4 + T cells from HC or PM, DM or SLE patients were stimulated with anti-CD3/CD28 beads (αCD3/CD28) for 3 d. The expression of TIGIT, CD226, and CD96 in pre- and post-activation of CD4 + T cells was measured by flow cytometry of biological replicates (HC = 27, PM = 27, DM = 23, SLE = 23). h Naive CD4 + T cells isolated from PBMCs of HC or patients with PM, were stimulated with αCD3/CD28. TIGIT expression was measured by flow cytometry. Data from four biologically independent replicates. h – l Total CD4 + T cells from patients with PM or HC were transfected with pcDNA3.1-hTIGIT or pcDNA3.1-Vector by electroporation. Cells were then stimulated by αCD3/CD28 for 3 d. h Overexpression of TIGIT was confirmed by Western blot. The experiment was repeated three times independently with similar results. i , j Overexpression of TIGIT in CD4 + T cells from HC or PM was confirmed by flow cytometry. Data from five biologically independent replicates. k , l IFNγ and IL-17A production by CD4 + T cells from PM patients or HC was measured by flow cytometry. Data from five biologically independent replicates. m – o CD4 + T cells from PM patients or HC were stimulated with αCD3/CD28 in the presence of CD155-Fc (10 μg/ml) or control IgG1 for 3 d. IFNγ, IL-17A, and FoxP3 expression in CD4 + T cells from PM patients or HC was measured by flow cytometry. Data from five biologically independent replicates. All data are mean ± SEM. Statistics were done by one-way ANOVA with adjustments for multiple comparisons in c , e , g , and two-way ANOVA with adjustments for multiple comparisons in j , l , n , and o .

Article Snippet: Briefly, CD4 + T cells were activated with anti-CD3/CD28 beads in RPMI containing 2 g/L 12 C-glucose for 3 d. T cells were then collected and labeled in modified RPMI 1640 containing 2 g/L [ 13 C6]-glucose (MCE, Cat# HY-B0389A) for 24 h. For GC‒MS, cells were plated at 2 × 10 6 cells/well in 24-well plates.

Techniques: Expressing, Flow Cytometry, Binding Assay, Activation Assay, Isolation, Transfection, Plasmid Preparation, Electroporation, Over Expression, Western Blot, Control