SPI-CI and SPI-6 cooperate in the protection from effector cell–mediated cytotoxicity

Abstract

Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrMinduced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cells. IntroductionCytotoxic T lymphocytes (CTLs) and natural killer (NK) cells induce cell death via 2 distinct mechanisms: the oligomerization of death receptors and secretion of cytolytic granules. These granules contain several important death effector molecules, such as the pore-forming protein perforin and a variety of serine proteases known as granzymes. 1,2 Granzyme B (GrB) is the key granzyme in rapid induction of apoptosis. 3,4 However, other proapoptotic enzymes, such as granzyme A (GrA) [5][6][7] and granzyme C (GrC), 8 can independently induce cell death with morphologic features reminiscent of apoptosis. Entry of these proteases into the target cells depends on the presence of perforin, but how this is achieved is debated. The original concept relied on the capacity of perforin to induce plasma membrane damage that provided access for entry of granzymes into target cells. It is clear, though, that GrB can enter target cells independent of perforin. 9 Upon such receptor-mediated endocytosis, perforin is suggested to release granzymes from the endosomes by disruption of the endosomal membrane. [9][10][11] Despite the multitude of granzymes, in vitro data using granzyme or perforin-deficient mice indicate that perforin is crucial in the induction of target cell lysis, whereas most granzymes are dispensable. 12 This hypothesis was corroborated by Trapani et al, showing that tumor eradication is unaffected in GrA/GrB double knock-out mice, 13,14 which are reportedly also diminished in the expression of granzymes C, D, F, and G. 15 This suggests that perforin, possibly aided by an as yet undefined granzyme, is sufficient for effector cells to exert their lytic function in vivo. Importantly, recent data indicate that granzyme M (GrM) with perforin can induce target cel...

A Novel Serpin Regulatory Mechanism

“…Sb9 is expressed in endothelial tumors, lymphomas, and leukemias and has been proposed to protect neoplastic cells from CLs (10,48). Sb9-positive cells resist killing by GzmB/perforin in vitro, as well killing by CLs (6,10,49). However, this appears to be cell type-dependent, because Sb9-positive lymphomas are sensitive to killing (50).…”

Interplay between the levels of estrogen and estrogen receptor controls the level of the granzyme inhibitor, proteinase inhibitor 9 and susceptibility to immune surveillance by natural killer cells

“…The mouse serpin SPI-6, which is related to PI-9, reportedly does not block CTL-induced killing of target cells without another serpin, SPI-CI, which has no known human homolog (Bots et al, 2005). The sensitivity of lymphomas to CTL-mediated cytotoxicy was unrelated to PI-9 level (Godal et al, 2006).…”

Control of granzymes by serpins

“…94 A variation in substrate specificity could lead to increased kinetics should mouse gzmM be used. Furthermore, it is interesting to note that Serpinb9b is completely absent from humans and exists only as a pseudogene in rats (encoding a premature stop codon within the RCL), suggesting distinct functions for mouse gzmM.…”

“…94 However, kinetic analyses were not performed to measure the efficiency of the interaction, which is especially relevant as the formation of a complex between recombinant Serpinb9b and gzmM seems to be inefficient, producing a prevalence of cleaved serpin, rather than an inhibitory complex. 94 It is possible that the high degree of overexpression in transfection studies may have been sufficient to overcome the poor kinetics and produce an inhibitory result.…”