Journal: International Journal of Molecular Sciences

Article Title: Role of ADAM10 and ADAM17 in Regulating CD137 Function

doi: 10.3390/ijms22052730

Figure Lengend Snippet: ADAM10 is the major sheddase of endogenously expressed CD137 in T cells. ( A/C ) Primary CD4 + T cells or ( B/D ) CD8 + T cells were treated with ADAM10 inhibitor GI (3 µM), mixed ADAM10/ADAM17 inhibitor GW (3 µM) or broad-spectrum metalloprotease inhibitor marimastat (MM, 10 µM) for 6, 24, 72 or 96 h. ( A/B ) Supernatants were analyzed for soluble shedding products of CD137 with ELISA. Treatment with GI, GW and MM resulted in a strikingly reduced amount of sCD137. One representative experiment out of five experiments is shown with mean ± SD. ( C/D ) Expression of cell surface CD137 on CD4 + and CD8 + T cells was analyzed using FITC labeled anti-CD137 mAb 72 h after activation. Incubation with MM (blue), GW (orange) and GI (green) increased the amount of cell bound CD137 compared to untreated cells (red). Black line: mock-stained control. One representative out of three experiments is shown.

Article Snippet: Cells were stained in different combinations with the following antibodies according to manufacturer’s recommendations: CD4-APC-Vio-770 (M-T466), CD8-VioBlue (REA734) and CD137-VioBright-FITC (REA765), all Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Labeling, Activation Assay, Incubation, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Role of ADAM10 and ADAM17 in Regulating CD137 Function

doi: 10.3390/ijms22052730

Figure Lengend Snippet: Shed sCD137 augments proliferation of CD4+ and CD8+ T cells . Proliferation assay of primary ( A ) CD4 + T cells or ( B ) CD8 + T cells. WT HEK293T cells were either co-transfected with CD137 and mock vector, CD137 and hyperactive ANO6 (ANO6H), or ANO6H and mock vector. Then, 24 h after transfection, medium was changed and supernatants were harvested after further 24 h. Pre-activated T cells were cultured in the presence of the precleared conditioned HEK cell medium for 72 h and proliferation was analyzed. Untreated control cells were standardized to baseline. * indicates significant increase compared to mock-treated cells, # indicates significant decrease (*/# p < 0.05 ( A , B ) n = 4; ± s.e.m.). Data were analyzed by one-way analysis of variance and Sidak multiple comparison post hoc test.

Article Snippet: Cells were stained in different combinations with the following antibodies according to manufacturer’s recommendations: CD4-APC-Vio-770 (M-T466), CD8-VioBlue (REA734) and CD137-VioBright-FITC (REA765), all Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Proliferation Assay, Transfection, Plasmid Preparation, Cell Culture, Control, Comparison