Serine Protease Inhibitor 6 Plays a Critical Role in Protecting Murine Granzyme B–Producing Regulatory T Cells

Abstract

Regulatory T-cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. While Tregs express Granzyme-B (GrB) to suppress effector T-cells via direct-killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. We show, for the first time, that both iTregs and nTregs increase their intracellular expression of GrB and its endogenous inhibitor, Serine Protease Inhibitor-6 (Spi6) upon activation. Sub-cellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6−/− Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6−/− nTregs and impaired suppression of alloreactive T-cells in vitro. Spi6−/− Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6−/− nTregs were less effective than WT nTregs in suppressing Graft-versus-host-disease (GVHD) due to their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared to WT recipients. Our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.

A Novel Serpin Regulatory Mechanism

“…By contrast, mice lacking both Sb9 and GzmB have normal numbers of virus-specific CD8 ϩ T cells, implicating uncontrolled GzmB as a mediator of CD8 ϩ T cell disappearance (11). GzmB-mediated death has also been reported in responding invariant NKT cells, helper T cell, and regulatory T cells (12,13). The pathophysiological importance of GzmB in immune cell homeostasis is also exemplified by decreased death of GzmB-null Th2 CD4 ϩ T helper cells (14).…”

Human regulatory T cells undergo self-inflicted damage via granzyme pathways upon activation

“…Our group and others have reported a central role of murine Treg-secreted GrB in inducing effector cell suppression and allograft tolerance (11,13,14). However, the importance of direct killing in the immunosuppressive function of Tregs compared with other mechanisms, including depletion of IL-2, is unclear.…”

“…Stimulated Tregs undergo apoptosis during stimulation, despite the coinduction of the GrB inhibitor, the serpin PI9. The mouse homolog of PI9 (Spi6) was previously shown to protect both conventional CD8 + cytotoxic T cells and mouse Tregs from self-inflicted death by any stray Grs that leak out of cytotoxic granules (13,(41)(42)(43), but it is clearly not adequate to completely protect stimulated human Tregs from apoptosis. Our data suggest that GrB is not only induced by Treg activation during kidney transplant rejection, but also plays a role in the activation-induced cell death of Tregs, dampening the controls that suppress rejection.…”

“…Similarly, GrA levels increased from 9.8% ± 2.5% to 65.6% ± 3.1% (****P < 0.0001). Spi6 is an endogenous murine serpin inhibitor of GrB and is a key homeostatic factor, regulating GrB activity and preventing GrB vesicular leakage (13,27). The counterpart of Spi6 in human leukocytes is PI9.…”

“…Tregs suppress effector cells via multiple mechanisms, including secretion of soluble factors (IL-10, TGF-β); upregulation of checkpoint inhibitory molecules, such as CTLA-4; and through direct granzyme-mediated (Gr-mediated) cytolysis of effector cells (11,12). In mice, Treg-secreted GrB has been shown to improve allograft survival significantly (13,14).…”