Serine Protease Inhibitor 6 Protects Cytotoxic T Cells from Self-Inflicted Injury by Ensuring the Integrity of Cytotoxic Granules

Abstract

How cytotoxic T lymphocytes (CTLs) kill intracellular pathogens without killing themselves has been a recurring question ever since their discovery. By using mice deficient in Serine Protease Inhibitor 6 (Spi6), we show that by inhibiting granzyme B (GrB), Spi6 protects CTLs from self-inflicted injury. Infection with either Lymphocytic Choriomeningitis virus (LCMV) or Listeria monocytogenes (LM) revealed increased apoptosis and diminished survival of Spi6 knockout (KO) CTLs, which was cell autonomous and could be corrected by GrB deficiency. Spi6 KO mice in turn were impaired in their ability to clear LCMV infection. Spi6 KO CTLs revealed a breakdown in the integrity of cytotoxic granules, increased cytoplasmic GrB, and ensuing apoptosis. We conclude that Spi6 protects CTLs from suicide caused by GrB-mediated breakdown of cytotoxic granules.

Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell Death

“…However, somewhat paradoxically, GzmB-deficient mice do not have increased numbers of CTL after viral infection, raising questions about the interpretation of the SPI-6 transgenic study (103). However, CTL from mice genetically deficient in SPI-6 have increased cytosolic GzmB and reduced viability (106). One surprising finding is a breakdown of the integrity of the cytotoxic granules in SPI-6-deficient CTLs (106).…”

“…However, CTL from mice genetically deficient in SPI-6 have increased cytosolic GzmB and reduced viability (106). One surprising finding is a breakdown of the integrity of the cytotoxic granules in SPI-6-deficient CTLs (106). The exact mechanism behind this collapse of cytotoxic granules is unclear.…”

Control of granzymes by serpins

“…Serpinb9 À/À mice show a predictable defect in response to viral infection, because of impaired CTL numbers. 81 Specifically, the cytoprotection model predicts that the lack of Serpinb9 should predispose CTL to endogenous gzmB-mediated death during their effector functions, as any gzmB escaping into the killer cell cytoplasm will not be controlled. Indeed, Serpinb9 À/À mice infected with lymphocytic choriomeningitis virus produce fivefold less virus-specific CTL than do wild-type mice.…”

“…Indeed, Serpinb9 À/À mice infected with lymphocytic choriomeningitis virus produce fivefold less virus-specific CTL than do wild-type mice. 81 This is because of an increase in apoptosis within the CTL population and is caused by intrinsic factors, as Serpinb9 À/À CTL transplanted into wild-type mice show the same effect. 81 Importantly, CTL numbers are fully restored when Serpinb9 À/À mice are crossed with gzmB À/À mice, showing that Serpinb9 controls endogenous gzmB.…”

“…81 This is because of an increase in apoptosis within the CTL population and is caused by intrinsic factors, as Serpinb9 À/À CTL transplanted into wild-type mice show the same effect. 81 Importantly, CTL numbers are fully restored when Serpinb9 À/À mice are crossed with gzmB À/À mice, showing that Serpinb9 controls endogenous gzmB. 81 However, the presumption that the features of mouse gzmB control by Serpinb9 accurately reflect those of human gzmB by SERPINB9 is complicated by the in vitro kinetics of the Serpinb9-mouse gzmB interaction (k ass ¼ 5.6 ± 1.4 Â 10 4 M À1 s À1 and SI ¼ 5.8±0.5).…”