Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells

Granzyme A lights a fire Cytotoxic T cells and natural killer cells use several strategies to kill infected or transformed cells. One such pathway entails the delivery of a family of serine proteases called granzymes to target cells through perforin-mediated pores to induce a form of programmed cell death called apoptosis. Zhou et al. show that granzyme A cleaves and activates gasdermin B (GSDMB), a central player in the highly inflammatory cell death process known as pyroptosis (see the Perspective by Nicolai and Raulet). GSDMB expression was highly expressed in some tissues and could be up-regulated by interferon-γ. Enforced expression of GSDMB in cancer cells enhanced tumor clearance in a mouse model, suggesting that this pathway may be a target for future cancer immunotherapies. Science, this issue p. eaaz7548; see also p. 943 Cytotoxic lymphocytes use granzyme A to cleave gasdermin B and induce pyroptosis in target cells. INTRODUCTION In cellular immunity, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use perforin to deliver serine protease granzymes into target cells to kill them. Gasdermins are pore-forming proteins that execute pyroptosis, a form of proinflammatory cell death. Gasdermin D (GSDMD) is cleaved by caspase-1/4/5/11 upon inflammasome activation, releasing the pore-forming domain for plasma membrane disruption. Gasdermin E (GSDME) is similarly cleaved by caspase-3, converting apoptosis to pyroptosis. The functional mechanism for other gasdermins is unknown. RATIONALE The view that granzymes induce target-cell apoptosis was proposed two decades ago, when apoptosis was thought to be the dominant form of programmed cell death and assays to ascertain apoptosis were insufficiently accurate. Furthermore, granzyme cytotoxicity was only assessed in a few cell types. Discovery of the gasdermin family, which are true cell death executors, has altered our understanding of programmed cell death. In this work, we explored whether members of the gasdermin family might respond to granzymes and induce pyroptosis. RESULTS The expression of gasdermin B (GSDMB) but no other gasdermins in human embryonic kidney (HEK) 293T cells induced pyroptotic killing by NK cells, accompanied by an interdomain cleavage of GSDMB. These processes were blocked by inhibiting the perforin–granzyme pathway. In vitro profiling of all five human granzymes identified granzyme A (GZMA), which readily cleaved GSDMB, predominantly at Lys244 within the interdomain linker. This cleavage unmasked the pore-forming activity of GSDMB. GZMA, delivered into GSDMB-reconstituted cells by electroporation or perforin, induced extensive pyroptosis with interdomain cleavage of GSDMB. These effects were diminished by a K229A/K244A (KK/A) mutation of GSDMB [in which lysine (K) was replaced by alanine (A) at positions 229 and 224, respectively]. In cells normally undergoing apoptosis upon GZMA delivery, the additional expression of GZMA-cleavable GSDMB converted apoptosis into pyroptosis. Pyroptotic killing by NK cells was blocked by both the KK/AA mutation and a knockdown of GZMA expression. Among 39 cell lines, three, including the esophageal carcinoma (OE19 cells), expressed GSDMB and underwent pyroptosis upon GZMA delivery. Knockout experiments revealed that pyroptosis in OE19 cells required the interdomain cleavage of GSDMB. Furthermore, GSDMB expression was up-regulated by interferon-γ (IFN-γ). Approximately one-third of GSDMB-negative cell lines showed IFN-γ–induced GSDMB expression. IFN-γ promoted GZMA- or NK cell–induced pyroptosis in several target cells. Primary T cells, including anti-CD19 chimeric-antigen receptor (CAR) T cells and NY-ESO-1–specific T cell receptor (TCR)–engineered T cells (TCR T cells), also induced pyroptosis in GSDMB-reconstituted cells through cleavage of GSDMB by GZMA. Introducing GZMA-cleavable GSDMB into mouse cancer cells promoted tumor clearance in mice. GSDMB was highly expressed in certain tissues, particularly digestive tract epithelia, including the derived tumors. GSDMB appeared to be silenced in gastric and esophageal cancers. The Cancer Genome Atlas database recorded a strong positive correlation between GSDMB expression and patient survival for bladder carcinoma and skin cutaneous melanoma. CONCLUSION GZMA from cytotoxic lymphocytes cleaves and activates GSDMB to induce target cell pyroptosis. This immune effector mechanism promotes CTL-mediated tumor clearance in mice. High GSDMB expression in the digestive system suggests the importance of GSDMB-mediated immunity in these tissues and will guide immunotherapy for related cancers. Our findings suggest that substrates such as gasdermins, rather than their upstream proteases, determine the nature of cell death. GZMA from cytotoxic lymphocytes cleaves GSDMB in target cells, predominantly at Lys244 within the interdomain linker. The cleavage allows GSDMB pore-forming domain (GSDMB-N) to perforate plasma membrane and induce pyroptosis. Expression of GSDMB wild type (WT) but not its GZMA-resistant K/A mutant in mouse cancer cells promotes cytotoxic T lymphocyte–mediated tumor clearance when the inhibitory checkpoint is blocked by antibody to programmed cell death 1 (PD-1). IFNGR, IFN-γ receptor. Cytotoxic lymphocyte–mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although 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-γ (IFN-γ) 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 mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte–killing mechanism, which may enhance antitumor immunity.