Cd4 and cd8

Poly(I:C) was obtained from InvivoGen (San Diego, CA). Recombinant murine IFNγ was obtained from Peprotech (315-05, Rocky Hill, NJ). For immunohistochemical analysis and western blotting, we used the following specific antibodies: CD4 and CD8 (Biolegend, San Diego, CA), CXCL10 (R&D systems, Minneapolis, MN), IL-1β (Abcam, Cambridge, MA), caspase-1 (Cell Signaling Technology, Danvers, MA), NLRP3 and ASC (Adipogen, San Diego, CA), NKG2D (Abcam, Cambridge, MA) and actin (Santa Cruz Biotechnologies, Santa Cruz, CA).

Tumor cells (3 × 10⁵) and PBMCs or T cells (effector-to-target ratio of 5) were seeded in 96-well plates in 100 μl of medium. For cytokine production assays, 100 μl of the supernatants were added to the wells. Supernatants were collected after 24 h of incubation and assessed for human IFN-ɣ, TNF-α, and IL-2 using the ELISA MAX Deluxe set (Biolegend), following the manufacturer’s protocol. For PBMCs or T-cell proliferation assays, PBMCs or T cells were labeled with 1 μmol/L Carboxyfluorescein succinimidyl ester (CFSE) (Sigma Aldrich) and co-cultured as described above for 6 days (PBMCs) or 3 days (T cells). Cells were then stained for cell viability with LIVE/DEAD (Thermo Fisher Scientific) and for CD4 and CD8 (Biolegend). Flow cytometry analysis was performed by acquiring a total of 20,000 events.