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.

中文翻译：

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来自细胞毒性淋巴细胞的颗粒酶 A 裂解 GSDMB 以触发靶细胞的焦亡

颗粒酶 A 点燃火焰 细胞毒性 T 细胞和自然杀伤细胞使用多种策略来杀死受感染或转化的细胞。一种这样的途径需要将称为颗粒酶的丝氨酸蛋白酶家族通过穿孔素介导的孔递送至靶细胞，以诱导一种称为细胞凋亡的程序性细胞死亡形式。周等人。表明颗粒酶 A 裂解并激活 Gasdermin B (GSDMB)，这是被称为细胞焦亡的高度炎症性细胞死亡过程的核心参与者（参见 Nicolai 和 Raulet 的观点）。GSDMB 表达在一些组织中高表达，并且可以被干扰素-γ 上调。GSDMB 在癌细胞中的强制表达增强了小鼠模型中的肿瘤清除率，表明该途径可能是未来癌症免疫疗法的目标。科学，这个问题 p。eaaz7548; 另见第 943 细胞毒性淋巴细胞使用颗粒酶 A 切割 gasdermin B 并诱导靶细胞焦亡。引言 在细胞免疫中，细胞毒性 T 淋巴细胞 (CTL) 和自然杀伤 (NK) 细胞使用穿孔素将丝氨酸蛋白酶颗粒酶递送到靶细胞中以杀死它们。Gasdermins 是一种成孔蛋白，可以执行细胞焦亡，这是一种促炎细胞死亡的形式。Gasdermin D (GSDMD) 在炎症小体激活时被 caspase-1/4/5/11 裂解，释放用于质膜破裂的成孔结构域。Gasdermin E (GSDME) 同样被 caspase-3 裂解，将细胞凋亡转化为细胞焦亡。其他 gasdermins 的功能机制尚不清楚。原理 颗粒酶诱导靶细胞凋亡的观点是在 20 年前提出的，当细胞凋亡被认为是程序性细胞死亡的主要形式时，确定细胞凋亡的分析不够准确。此外，仅在少数细胞类型中评估了颗粒酶的细胞毒性。gasdermin 家族是真正的细胞死亡执行者，其发现改变了我们对程序性细胞死亡的理解。在这项工作中，我们探讨了 gasdermin 家族的成员是否可能对颗粒酶作出反应并诱导细胞焦亡。结果 在人胚胎肾 (HEK) 293T 细胞中，gasdermin B (GSDMB) 的表达诱导了 NK 细胞的细胞焦亡，伴随着 GSDMB 的域间裂解，但没有其他 gasdermin 的表达。这些过程通过抑制穿孔素-颗粒酶途径而被阻断。所有五种人类颗粒酶的体外分析鉴定了颗粒酶 A (GZMA)，它很容易切割 GSDMB，主要位于域间链接器内的 Lys244。这种切割揭示了 GSDMB 的成孔活性。GZMA 通过电穿孔或穿孔素递送到 GSDMB 重组细胞中，诱导广泛的细胞焦亡，GSDMB 域间裂解。GSDMB 的 K229A/K244A (KK/A) 突变 [其中赖氨酸 (K) 分别在位置 229 和 224 被丙氨酸 (A) 取代] 减弱了这些影响。在 GZMA 递送后正常经历细胞凋亡的细胞中，GZMA 可裂解的 GSDMB 的额外表达将细胞凋亡转化为细胞焦亡。KK/AA 突变和 GZMA 表达的敲低都阻止了 NK 细胞的焦亡杀伤。在 39 个细胞系中，包括食管癌（OE19 细胞）在内的三个细胞系表达 GSDMB 并在 GZMA 递送后发生细胞焦亡。敲除实验表明 OE19 细胞中的细胞焦亡需要 GSDMB 的域间裂解。此外，GSDMB 表达被干扰素-γ (IFN-γ) 上调。大约三分之一的 GSDMB 阴性细胞系显示出 IFN-γ 诱导的 GSDMB 表达。IFN-γ 在几个靶细胞中促进 GZMA 或 NK 细胞诱导的细胞焦亡。原代 T 细胞，包括抗 CD19 嵌合抗原受体 (CAR) T 细胞和 NY-ESO-1 特异性 T 细胞受体 (TCR) 工程化 T 细胞 (TCR T 细胞)，也通过GZMA 对 GSDMB 的裂解。将 GZMA 可切割的 GSDMB 引入小鼠癌细胞中，可促进小鼠的肿瘤清除。GSDMB 在某些组织中高度表达，特别是消化道上皮细胞，包括衍生肿瘤。GSDMB 似乎在胃癌和食道癌中被沉默。癌症基因组图谱数据库记录了 GSDMB 表达与膀胱癌和皮肤皮肤黑色素瘤患者存活率之间的强正相关。结论来自细胞毒性淋巴细胞的 GZMA 裂解并激活 GSDMB 以诱导靶细胞焦亡。这种免疫效应机制促进了 CTL 介导的小鼠肿瘤清除。GSDMB 在消化系统中的高表达表明 GSDMB 介导的免疫在这些组织中的重要性，并将指导相关癌症的免疫治疗。我们的研究结果表明，底物如gasdermin，而不是它们的上游蛋白酶，决定了细胞死亡的性质。来自细胞毒性淋巴细胞的 GZMA 切割靶细胞中的 GSDMB，主要在域间接头内的 Lys244。裂解允许 GSDMB 成孔结构域 (GSDMB-N) 穿透质膜并诱导细胞焦亡。当抑制性检查点被程序性细胞死亡 1 (PD-1) 抗体阻断时，GSDMB 野生型 (WT) 而非 GZMA 抗性 K/A 突变体在小鼠癌细胞中的表达促进细胞毒性 T 淋巴细胞介导的肿瘤清除。IFNGR，IFN-γ受体。细胞毒性淋巴细胞介导的免疫依赖于颗粒酶。颗粒酶被认为通过诱导细胞凋亡来杀死靶细胞，尽管其潜在机制尚不完全清楚。在这里，我们报告自然杀伤细胞和细胞毒性 T 淋巴细胞通过细胞焦亡杀死 Gasdermin B (GSDMB) 阳性细胞，细胞焦亡是由 Gasdermin 成孔蛋白家族执行的一种促炎细胞死亡形式。GSDMB 被淋巴细胞衍生的颗粒酶 A (GZMA) 裂解，从而释放其成孔活性，从而导致杀伤。干扰素-γ (IFN-γ) 上调 GSDMB 表达并促进细胞焦亡。GSDMB 在某些组织中高度表达，特别是消化道上皮细胞，包括衍生肿瘤。将 GZMA 可裂解的 GSDMB 引入小鼠癌细胞中可促进小鼠的肿瘤清除。该研究将 gasdermin 介导的细胞焦亡确立为一种细胞毒性淋巴细胞杀伤机制，可增强抗肿瘤免疫。将 GZMA 可裂解的 GSDMB 引入小鼠癌细胞中可促进小鼠的肿瘤清除。该研究将 gasdermin 介导的细胞焦亡确立为一种细胞毒性淋巴细胞杀伤机制，可增强抗肿瘤免疫。将 GZMA 可裂解的 GSDMB 引入小鼠癌细胞中可促进小鼠的肿瘤清除。该研究将 gasdermin 介导的细胞焦亡确立为一种细胞毒性淋巴细胞杀伤机制，可增强抗肿瘤免疫。