Figure 5. OX40 activates caspase 1 through TRAF6-dependent recruitment of MALT1.

(A) FACS-sorted iNKT cells from WT B6 mice were stimulated with OX86, followed by immunoprecipitation using anti-TRAF6 antibody. The immunoprecipitates were further immunoblotted with anti-TRAF6, anti-BCL10, and anti-MALT1 antibodies. The blot shows the detection of BCL10 and MALT1 in the TRAF6 immunoprecipitates. One of 6 representative experiments is shown. (B) Schematic rendering of the MALT1 molecule showing different domains and corresponding mutations made in the present study. ΔIKBS, IKBS deletion; ΔCaspD, CaspD deletion; ΔB10BS, B10BS deletion. (C) HEK293T cells were transfected with plasmids encoding MALT1, TRAF6, and caspase 1, and cell lysates were immunoprecipitated by anti-TRAF6 antibody. The immunoprecipitates were further immunoblotted using the indicated antibodies for the presence of MALT1 and caspase 1. One of 6 representative experiments is shown. (D) HEK293T cells were transfected with plasmids encoding the full-length (FL) and various mutations of MALT1. Cells were cultured for 36 hours, and cell lysates were immunoprecipitated by anti-TRAF6 antibody. The immunoprecipitates were then immunoblotted using the indicated antibodies for the presence of caspase 1. Data shown are one of 6 independent experiments. (E) FACS-sorted iNKT cells from WT B6 mice were transduced with retroviral vectors expressing MALT1 shRNA (shMALT1) or scrambled RNA for 24 hours, followed by treatment with OX86 for another 24 hours. Induction of caspase 1 cleavage was determined by FACS. The plot shown represents one of 6 experiments. (F) FACS-sorted iNKT cells from WT B6 mice were stimulated with OX86 (or a control IgG) with or without mepazine acetate (Mep; 0, 0.5, 1, 2 μg/ml). After 24 hours, caspase 1 cleavage was determined by immunoblotting. β-Actin was used as a loading control. Data shown are from one of 6 independent experiments.