16 Apr 2020

Abstract

Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, though the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)–positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon gamma up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia including derived tumors. Introducing GZMA-cleavable GSDMB into murine cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte killing mechanism, which may enhance antitumor immunity.

[Image]

Fig. 2 GZMA cleaves GSDMB to induce pyroptosis in GSDMB-reconstituted target cells.

(A) In vitro cleavage assay of GSDMB by five human granzymes. Shown is a representative Coomassie Blue-stained gel.

(B) Cartoon diagram of GSDMB structure and the GZMA cleavage sites (Lys244 and Lys229).

(C) Equal amounts of full-length (FL) GSDMB or GSDMB-(N+C) proteins were electroporated into or added directly to 293T cells. ATP-based cell viability is shown.

(D to F) GZMA was delivered into 293T cells expressing GSDMB WT or K229A/K244A (KK/AA) mutant perforin. Representative cell images (D) (arrowheads mark pyroptotic cells; scale bar, 25 μm), lactate dehydrogenase (LDH) release-based cell death (E), and immunoblotting of GSDMB cleavage (F) are shown.

(G) 293T cells expressing GSDMB WT or KK/AA double mutant were preloaded with calcein AM and then co-cultured with NK-92MI cells. Quantification of calcein AM release and immunoblotting of GSDMB cleavage are shown.

(H to J) Effect of siRNA knockdown of GZMA on pyroptotic killing of GSDMB-expressing 293T cells by NK-92MI cells. Anti-GZMA immunoblot in NK-92MI cells (H), immunoblotting of GSDMB cleavage (I), and quantification of calcein AM release (J) are shown. Data are means ± SD from three replicates [(C), (E), (G), (J)]. ***P < 0.001 (two-tailed unpaired Student’s t-test). Data are representative of three [(A), (C to F)] or two (G to J) independent experiments.