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...