The Role of Proteinase Inhibitor 9 (PI-9) in Granzyme B/Perforin-Mediated Cytotoxicity Induced by Cytotoxic T Cells in Allogeneic Hematopoietic Stem Cell Transfusion.

Abstract The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)/perforin-induced apoptosis and cytotoxicity, which is one of the important mechanisms in immunotherapy against tumors. However, the role of PI-9, which specifically inhibits GrB, in tumor immune escape is still under debate. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least partly through apoptotic molecules GrB and perforin produced by the donor-derived cytotoxic T cells (CTLs) and natural killer cells. However, the role of PI-9 in GVHD and GVL is unknown. In this study we analyzed the role of PI-9 in GVHD and GVL after HSCT through expression of PI-9 relative to GrB and perforin. First, we analyzed the expressions of PI-9 and three apoptosis-related molecules GrB, perforin and Fas ligand by quantitative RT-PCR in leukocytes of five patients with chronic GVHD and six patients without obvious GVHD, from whom a written informed consent was obtained. The level of GrB expression was significantly increased in three of the patients with GVHD and two of them exhibited elevation both in GrB/perforin and FasL. One of the cases with GVHD showed an isolated high expression of FasL. However, significantly, the expression of PI-9 was prominently decreased in all of the cases with GVHD relative to those without GVHD. None of the patients with GVHD who were positive for any of the apoptosis-related molecules relapsed. Two of the six patients without GVHD showed an elevated expression of GrB and perforin. The one of these two patients suffered from relapsed leukemic infiltration into various tissues and the other from herpes zoster reactivation. Thus, the expression of these molecules was thought to reflect the recipients’ protective reactions against the intrinsic diseases. We observed a tendency that the ratio of GrB and PI-9 expressions were higher in patients with GVHD. Next, human CTL and natural killer cell (YT-N10) lines were analyzed to check the time course of PI-9 and GrB expressions in vitro. The CTL line had been established by immortalization of normal T cells with herpesvirus saimiri. The stimulation of these cells with phorbol 12-myristate 13 acetate (PMA) resulted in the prior peak of expression of PI-9 within 3 hours followed by the late expression of GrB peaked after 8 hours. The co-stimulation of these cells with IL-2 and IL-12 showed similar results. These results have indicated that the earlier synthesis of PI-9 might be important for escaping autolysis of immunocompetent cells and that the expressions of PI-9 and the apoptosis-related molecules may be controlled in a different manner. The low expression of PI-9 relative to GrB/perforin in patients who received HSCT may indicate that the balance between the expressions of PI-9 and GrB/perforin could monitor the activity of CTLs in patients with post-transplantation GVHD (and possibly with various other immune disorders) and predict the prognosis of these diseases. It could also be correlated with the GVL activity and hence predict the probability of relapses.