Proteinase Inhibitor 9 Expression Research Articles

Serine protease inhibitor-6 inhibits granzyme B-mediated injury of renal tubular cells and promotes renal allograft survival.

Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B-mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2) into nephrectomized recipients (H-2). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 μmol/L; n=5; P<0.01), consistent with observed tubular injury and extensive mononuclear cell infiltration. Loss of donor kidney SPI-6 shortened graft survival time (20±19 vs. 66±33 days; n=8-10; P<0.001). Our data show for the first time that resistance of kidney TEC to cytotoxic T-cell granzyme B-induced death in vitro and in vivo is mediated by the expression of SPI-6. We suggest that SPI-6 is an important endogenous mechanism to prevent rejection injury from perforin or granzyme B effectors and enhanced PI-9 or SPI-6 expressions by TEC may provide protection from diverse forms of inflammatory kidney injury and promote long-term allograft survival.

Role of protease inhibitor 9 in survival and replication of Mycobacterium tuberculosis in mononuclear phagocytes from HIV-1-infected patients

Predisposition to opportunistic infections by Mycobacterium tuberculosis (MTB) is a concomitant of HIV-1 infection and occurrence of tuberculosis is independent of circulating CD4(+) T-cell count in HIV-1-infected patients. Infection of mononuclear phagocytes from healthy individuals by virulent MTB is associated with expression of the antiapoptotic molecule protease inhibitor 9 (PI-9), and PI-9 contributes to successful parasitism of macrophages by MTB. Here we studied the contribution of PI-9 to successful MTB infection of monocytes from HIV-1-infected patients. Blood monocytes obtained from HAART-treated HIV-1-infected patients (HIV+) and healthy controls were assessed for support of MTB H37Rv growth by assessment of MTB 16S ribosomal (r)RNA in cell lysates on day 1 and day 7 by real-time reverse transcription-PCR. PI-9 expression in monocyte cell lysates was assessed by ELISA and by reverse transcription-PCR. Inhibition of intracellular PI-9 was achieved by siRNA to PI-9 and compared to control constructs. Monocytes from HIV-infected patients supported higher MTB growth [MTB 16S rRNA (d7/d1)] as compared with monocytes from healthy controls. Both PI-9 protein and mRNA were significantly higher in monocytes from HIV-infected patients as compared with healthy controls. PI-9 protein levels prior to MTB infection correlated with MTB replication on day 7, and with plasma soluble CD14 levels. Silencing of PI-9 by transfection of monocytes from HIV-1-infected patients with PI-9-specific siRNA prior to infection improved intracellular containment of MTB. Increased intracellular PI-9 activity in mononuclear phagocytes from HIV-infected patients contributes to successful intracellular infection by virulent MTB.

Anti-Inflammatory Properties of Exenatide in Human Pancreatic Islets

Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN-γ, IL-17, IL-1β, and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3, and Akt in vitro. Immunostaining showed expression of GLP-1R in β-cells but not in α-cells. IL-1β colocalized with GLP-1R in β-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 expression in vitro but to a lower extent than exenatide. This effect was partially blocked by the antagonist exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in β-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of exenatide in human islets. Exenatide-mediated PI-9 expression, the only known granzyme B inhibitor, unveils potential immunoregulatory properties.

Increased proteinase inhibitor-9 (PI-9) and reduced granzyme B in lung cancer: Mechanism for immune evasion?

Cytotoxic CD8+ T-cells mount immune responses to cancer via cytotoxic pathways including granzyme B. Cancer cells are also known to develop immune evasion mechanisms. We hypothesised that lung cancer cells would over-express the granzyme B-inhibitor, proteinase inhibitor-9 (PI-9) and down-regulate granzyme B expression by neighbouring CD8+ T-cells.We investigated PI-9 expression in lung cancer cell lines, and primary lung cancer cells obtained at curative lung resection from cancer patients with/without chronic obstructive pulmonary disease (COPD). Granzyme B and PI-9 expression was also determined in CD8+ T-cells from the cancer and non-cancer areas of resected lung tissue and from bronchoalveolar lavage (BAL). We then evaluated the effects of conditioned media from lung cancer cell lines on granzyme B expression and the cytotoxic activity of CD8+ T-cells.PI-9 was highly expressed in lung cancer cell lines. Increased PI-9 expression was also observed in primary cancer cells vs. epithelial cells from non-cancer tissue or bronchial brushing-derived normal primary large airway epithelial cells. Expression significantly correlated with cancer stage. Significantly reduced granzyme B was noted in CD8+ T-cells from cancer vs. non-cancer tissue. Granzyme B production by CD8+ T-cells was reduced in the presence of conditioned media from lung cancer cell lines.Our data suggest that lung cancer cells utilise their increased PI-9 expression to protect from granzyme B-mediated cytotoxicity as an immune evasion mechanism, a function that increases with lung cancer stage.

The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 1984The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

The Novel Role of SERPINB9 in Cytotoxic Protection of Human Mesenchymal Stem Cells

Clinical trials using allogeneic mesenchymal stem cells (MSCs) are ongoing for the purpose of providing therapeutic benefit for a variety of human disorders. Pertinent to their clinical use are the accessibility to sufficient quantities of these cells allowing for repetitive administration, as well as a better understanding of the specific mechanisms by which allogeneic MSCs evade host immune responses that in turn influence their life span following administration. In this report, we sought to characterize and compare human peripheral blood MSCs (hPB-MSCs) with bone marrow-derived MSCs. hPB-MSCs met the established criteria to characterize this cellular lineage, including capacity for self-renewal, differentiation into tissues of mesodermal origin, and expression of phenotypic surface markers. In addition, hPB-MSCs suppressed alloreactive proliferation as well as the production of proinflammatory cytokines. Examination of the mechanisms by which allogeneic MSCs evade the host immune response, which is crucial for their therapeutic use, demonstrated that constitutive expression of serine protease inhibitor 9 (PI-9) on hPB-MSCs and bone marrow-derived MSCs is a major defense mechanism against granzyme B-mediated destruction by NK cells. Similarly, MSCs treated with small interfering RNA for PI-9 increased MSC cellular death, whereas expression of transgenic PI-9 following retroviral transduction protected MSCs. These data significantly advance our understanding of the immunomodulatory role for hPB-MSCs as well as the mechanisms by which they evade host immune responses. These findings contribute to the development of MSC-based therapies for diseases.

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.

Expression of proteinase inhibitor-9/serpinB9 in non-small cell lung carcinoma cells and tissues

Human proteinase inhibitor-9 (PI-9)/serpinB9 is an intracellular ovalbumin-family serpin with nucleocytoplasmic distribution which is expressed in certain normal cell types and cancer cells of different origin. Due to binding and inactivating of granzyme B (GrB), PI-9 can protect the cells from GrB-mediated apoptosis. High levels of PI-9 expression in certain cancer cells may contribute to their resistance against the immune mediated killing. So far, it is not known whether non-small cell lung cardinomas (NSCLCs) express PI-9 mRNA and a functional PI-9 protein. Herein we report for the first time that NSCLC cells express both PI-9 mRNA and protein and that there is a subset of NSCLC cells with upregulated PI-9 mRNA and protein expression. Futhermore, our work revealed that the PI-9 protein expressed in NSCLC cells can inhibit the active GrB. Finally, analysis of PI-9 mRNA expression in NSCLC tumours from surgically treated patients showed that the expression of this transcript is upregulated in the less-differentiated lung adenocarcinomas. We suggest, that the upregulated expression of PI-9 in NSCLC cells may serve to protect them from apoptosis induced by GrB.

Proteinase inhibitor–9 expression is induced by maturation in dendritic cells via p38 MAP kinase

Dendritic cells (DC) are crucial for the induction of CD8(+) T cell responses. However, as DC present antigen, they may also be at risk for being killed by recent activated CD8+ T cells. Previously we have shown that lipopolysaccharide (LPS)- or CD40L-stimulated monocyte-derived DC express high levels of the granzyme B-inhibitory serpin proteinase inhibitor-9 (PI-9). Furthermore we found that its murine homolog serine protease inhibitor-6 (SPI-6) protected murine DC against cytotoxic T lymphocyte-induced apoptosis. These data suggest that PI-9/SPI-6 may regulate survival of DC when they are under attack by effector cells. We therefore analyzed how PI-9 is regulated upon stimulation with several Toll-like receptor ligands. We found that the expression of PI-9 correlated with maturation, as measured with CD86 levels, but not with the production of interleukin-12-p70. Using LPS as stimulus, we also showed that the induction of PI-9 is dependent on the p38 mitogen-activated protein kinase signaling pathway. The data suggest that PI-9 is tightly linked to maturation and may allow DC to exert their function in a potentially hostile environment.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

Lithium-induced upregulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) in monocytes: Role of glycogen synthase kinase-3? (89.13)

Abstract Proteinase inhibitor 9 (PI-9) - the only known natural antagonist of the lymphocyte protease granzyme B (GrB) - is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. By intracellular flow cytometry, we have previously shown that ex-vivo stimulation by lipopolysaccharides leads to upregulation of PI-9 within 24 hours in the monocyte, but not the lymphocyte fraction; this can be inhibited by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Glycogen synthase kinase 3 (GSK) is a lithium-inhibitable, constutitively active serine/threonine kinase that controls a number of intracellular functions, especially those involving NF-kappaB regulated genes. We now asked whether lithium-induced inhibition of GSK might influence expression of PI-9 in monocytes and lymphocytes. A 24 hours incubation assay was done using heparinized full blood of healthy volonteers, and PI-9 expression was analysed by intracellular flow cytometry. We found that lithium chloride leads to PI-9 upregulation in monocytes at a medium concentration (1.5 μM), which could not further be enhanced by higher concentrations (10 or 20 μM lithium chloride). In lymphocytes, no alteration of PI-9 expression was induced. When cells were stimulated by LPS in the presence of lithium, only marginal additional upregulation of PI-9 could be induced. Our data show that a lithium-regulated factor, probably GSK, is involved in PI-9 expression in monocytes, down-regulating PI-9 in resting cells. Since it has been shown that PI-9 over-expression in antigen presenting cells, e.g. dendritic cells, leads to an enhanced immune response, our finding may be relevant for specific immune regulation. Pharmacological manipulation of PI-9 expression may represent a useful tool to study immune regulatory mechanisms as well as for therapy .

Expression of the granzyme B inhibitor PI9 predicts outcome in nasal NK/T-cell lymphoma: results of a Western series of 48 patients treated with first-line polychemotherapy within the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials

AbstractNasal NK/T-cell lymphoma is a rare disease entity with a poor outcome. Expression of antiapoptotic proteins has not been extensively investigated in this entity. Forty-eight patients with nasal T/NK-cell lymphoma who received first-line polychemotherapy (n = 44) or chemoradiotherapy (n = 4) were analyzed for expression of active caspase-3 (aC3), granzyme B protease inhibitor 9 (PI9), and Bcl-2 proteins. Lymphomas were CD3+/CD5−/granzyme B+ and EBV-associated. Median age was 46 years. Stage I/II disease was present in 75% of the cases and an International Prognostic Index (IPI) score less than 1 in 65%. With a median follow-up of 6.3 years, 5-year event-free survival (EFS) and overall survival (OS) rates were 39% and 49%, respectively. Apoptotic index was scored as high in 32% of cases and PI9 expression as positive in 68%, whereas 35% disclosed a high number of aC3+ tumor cells. Univariate analysis showed that absence of PI9 and low apoptotic index were associated with poor outcome, but not aC3 expression nor IPI score. By multivariate analysis, both parameters affected independently EFS (P = .02 and .08, respectively) and OS (P = .009 and .04). In view of its constitutive expression by normal NK cells, it is suggested that loss of PI9 expression in tumor cells may reflect some mechanism associated with progression.

Expression of Cellular FLICE Inhibitory Protein, Caspase-8, and Protease Inhibitor-9 in Ewing Sarcoma and Implications for Susceptibility to Cytotoxic Pathways

Ewing sarcoma is a common pediatric bone tumor with an unfavorable prognosis for metastatic or recurrent disease. Cellular immunotherapy may provide new treatment options and depends on the cytolytic death receptor and perforin/granzyme pathways. Expression of death receptor pathway inhibitor cellular FLICE inhibitory protein (cFLIP), initiator caspase-8, and granzyme B inhibitor protease inhibitor-9 (PI-9) have been reported to determine susceptibility to cell- and chemotherapy-mediated killing in several tumor types. Here, we have studied their in vitro and in vivo expression in Ewing sarcoma and the implications for susceptibility to cytotoxicity. Ewing sarcoma cell lines (n = 8) were tested for cFLIP, PI-9, and caspase-8 expression. Functional significance was tested by anti-Fas antibody (death receptor pathway) or natural killer cell (perforin/granzyme pathway) treatment. Immunohistochemistry was done on 28 sections from 18 patients. In half of the cases, sequential material, including metastases, was available. Although all tested Ewing sarcoma cell lines expressed cFLIP, resistance to CD95/Fas-mediated apoptosis was only observed in two cell lines lacking caspase-8 expression. PI-9 was expressed at low levels in four of eight Ewing sarcoma cell lines, but positive cell lines remained susceptible to perforin/granzyme-mediated killing. In primary Ewing sarcoma, including metastases, cFLIP was abundantly expressed in 18 of 18 patients. Caspase-8 was expressed in all patients but showed more intertumoral and intratumoral variation in both intensity and heterogeneity of staining. PI-9, in contrast, was undetectable. The expression patterns of cFLIP, caspase-8, and the absence of PI-9 provide a rationale to preferentially exploit the perforin/granzyme pathway in cytotoxic therapies against Ewing sarcoma.

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.

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.

Expression of Proteinase Inhibitor-9 in Primary AML Blasts and Its Regulation by Interferon-γ: A Potential Immune Escape Mechanism after Allogeneic Stem Cell Transplantation.

It is well established, that the curative potential of allogeneic peripheral blood stem cell transplantation (allo PBSCT) is due to immunocompetent donor T cells inducing potent anti-neoplastic effects against host tumor cells. This reaction, which is termed graft-versus-leukemia (GVL) effect, is clinically effective against a number of different hematologic malignancies such as myeloid and lymphoid leukemias. Despite great efforts of allo PBSCT in treatment of CML, the 5-year survival rate of AML patients after allo PBSCT is only about 30% due to relapsing disease. The recurrent disease is inefficiently controlled by the immune system, due most likely to the various immune escape mechanisms described for AML blasts including upregulation of anti-apoptotic molecules. Since cytotoxic T lymphocytes (CTL) and natural killer cells are the cells responsible for eliminating leukemic blasts, the most important effector molecule is Granzyme B (GrB). Misdirected GrB is quenched by its specific physiological inhibitor Protease Inhibitor-9 (PI-9) leading to inactivation of GrB. PI-9 expression by tumour cells can be used to escape immune surveillance and its presence has been shown for different tumors e.g. melanoma, colon carcinoma and lymphoma. Despite other regulators, interferon-γ (IFN-γ) has been shown to upregulate PI-9 expression in hepatocytes. Here, we wanted to investigate the expression of PI-9 in primary AML blasts and its regulation by IFN-γ. Using CD34+ positive magnetic selection, we isolated primary blasts with a purity of >90% from 20 AML patients with different FAB subtypes. For detection of PI-9 expression by Western Blotting, whole cell lysates were made from freshly purified blasts and after 24 h +/− 200 IU/ml IFN-γ. In some patients, PI-9 expression was confirmed by FACS analysis with an anti- PI-9 specific monoclonal antibody. Here we describe for the first time, that PI-9 is constitutively expressed in 16/20 (80%) of AML blasts. Treatment of AML blasts with IFN-γ could upregulate PI-9 expression in a dose-dependent manner (2–2,000 IU/ml) and strong expression of PI-9 was detectable in 6/18 patients within 4–5 h after IFN-γ exposure. Of note, a mild upregulation of PI-9 upon 24 h incubation w/o IFN-γ could be detected in 4/18 (22%) patients. We conclude, that cytokines such as IFN-γ which are secreted during the cytokine storm of acute graft-versus-host disease can contribute to the development of immune escape mechanisms in AML blasts.

Lymphomas are sensitive to perforin-dependent cytotoxic pathways despite expression of PI-9 and overexpression of bcl-2

AbstractThere is considerable interest in immunotherapeutic approaches for lymphoma. The expression of proteinase inhibitor 9 (PI-9), a molecule that inactivates granzyme B, is considered an immune escape mechanism in lymphoma. Further, lymphomas frequently overexpress the antiapoptotic molecule bcl-2, which is able to inhibit perforin-dependent cytotoxic pathways. In this study, the impact of PI-9 and bcl-2 expression on the sensitivity of lymphomas to T- and natural killer (NK) cell–mediated cytotoxicity was analyzed. We found PI-9 expression in 10 of 18 lymphoma cell lines and in 9 of 14 primary lymphomas. Overexpression of bcl-2 was found in 8 of 18 cell lines and in 12 of 14 primary lymphomas. All lymphoma cells were sensitive to cytolysis by specific T cells and cytokine-activated NK cells, and no difference in sensitivity was observed with respect to PI-9 or bcl-2 expression. Cytolysis was mediated predominantly through perforin-dependent pathways despite expression of PI-9 and bcl-2. Interestingly, the majority of lymphoma cells were resistant to cytolysis by resting allogeneic NK cells. This was due to the failure of lymphomas to induce degranulation of resting NK cells. These results show that resistance to perforin-dependent pathways is not a relevant immune escape mechanism in lymphoma and therefore is unlikely to impair clinical outcome of immunotherapeutic approaches.

Modulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) by activation of lymphocytes and monocytes in vitro and by Epstein–Barr virus and bacterial infection

Proteinase inhibitor 9 (PI-9) is an intracellular serpin expressed in lymphocytes and monocyte-derived cells. It is the only known endogenous natural antagonist of granzyme B (GrB), and its proposed function is protection of cells from misdirected GrB. We have studied the regulation of PI-9 in primary peripheral blood mononuclear cells (PBMCs) following ex-vivo stimulation, and in PBMCs from patients suffering from viral or bacterial infections. By intracellular flow cytometry, we found identical PI-9 expression in all lymphocyte subsets, lower levels in monocytes and none in granulocytes. PI-9 was stable for 48 h in the presence of cycloheximide, indicating slow protein turnover. Incubation of PBMCs with several stimuli including lipopolysaccharide (LPS) led to up-regulation in the monocyte, but not the lymphocyte fraction, within 48 h, inhibitable by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Up-regulation of PI-9 was observed in lymphocytes and monocytes of patients with acute Epstein-Barr virus (EBV), but not bacterial infection. Preterm infants had similar PI-9 expression as adults in monocytes, but lower in lymphocytes, decreasing during bacterial infection. Taken together, our data indicate that PI-9 is rapidly up-regulated upon stimulation of monocytes, but not lymphocytes. By protecting monocytes and macrophages from misdirected GrB in the inflammatory process, PI-9 might be involved in the regulation of antigen presentation.

Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T Lymphocytes and Natural Killer Cells

Exposure to estrogens is associated with an increased risk of developing breast, cervical, and liver cancer. Estrogens strongly induce the human granzyme B inhibitor, proteinase inhibitor 9 (PI-9). Because cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the granzyme pathway to induce apoptosis of target cells, we tested the ability of activated CTLs and the human NK cell line, YT cells, to lyse human liver cells. Estrogen induction of PI-9 protected the liver cells against CTL and NK cell-mediated, granzyme-dependent, apoptosis. Knockdown of PI-9 by RNA interference blocked the protective effect of estrogen. This work demonstrates that estrogens can act on target cells to control their destruction by immune system cells and shows that induction of PI-9 expression can inhibit both CTL and NK cell-mediated apoptosis. Estrogen induction of PI-9 may reduce the ability of cytolytic lymphocytes-mediated immune surveillance to destroy newly transformed cells, possibly providing a novel mechanism for an estrogen-mediated increase in tumor incidence.

Constitutive Expression of the Granzyme B Inhibitor PI-9 Protects Leukemic Cells from Granzyme B Induced Apoptosis.

Constitutive expression of the granzyme B inhibitor PI-9 protects leukemic cells from granzyme B induced apoptosis Introduction Apoptosis induced in leukemic blasts by alloreactive Cytotoxic T-Lymphocytes (CTL) is the molecular mechanism of cell death involved in the Graft versus leukemia (GvL) reaction following allogeneic bone marrow (BMT) and peripheral stem cell transplantation (PBSCT). Apoptosis in alloreactivity is mediated mainly by the granule exocytosis pathway. Granules rich in serine proteases and perforin are released into the target cell upon recognition by CTL. Of these proteases granzyme B (GrB) has been shown to be the most important effector for apoptosis induction by activating Caspase 3 and subsequently the apoptotic effector stage. Thus, resistance to GvL might be mediated at least in part by resistance to granzyme B induced apoptosis. The human serine proteinase inhibitor 9 (PI-9) is the only known endogenous proteinase inhibitor capable to inhibit proteolytic activity of granzyme B. Therefore, PI-9 may constitute a mechanism of GvL resistance. We investigated a number of human leukemic cell of myeloid and lympoid recognition for their pattern of PI-9 expression and the functional relevance on apoptosis by granzyme B. Methods and Results Following cell lines were investigated: the lymphoblastic cell lines Daudi and Jurkat, the myeloblastic cell lines K562 and U937 and two EBV transformed primary B-Cells (LCL) from volunteer donors. Furthermore we established PI-9 high expressing clones from U937 (U937/PI-9+) that is normally low expressing. PI-9 expression in cell lines was measured by western blot. Daudi, U937/PI-9+ and LCL1 showed a high expression level of PI-9. K562, LCL2 and U937 showed a low expression level of PI-9. Jurkat showed no expression of PI-9.Cytotoxic granules rich in granzyme B were purified using the human NK cell line YT. Cells were incubated with granzyme B granules and intracellular granzyme B activity was measured using a colorimetric assay based on the substrate IEPD. Granzyme B activity inversely correlated to the levels of PI-9 expression in all cell types. Furthermore, Caspase 3 activity was assessed by intracytoplasmatic staining for active Caspase 3 using a fluorochrome coupled antibody against human active Caspase 3 fragment.Caspase 3 activity also was directly correlated to granzyme B activity, i.e. negatively correlated to the level of PI-9 expression, respectively.Discussion Our data show a clear correlation of PI-9 expression levels and inhibition of granzyme B activity in a panel of myeloblastic and lymphoblastic leukemia derived cell lines as well as EBV-transformed B-cells. Furthermore, granzyme B activity was correlated to downstream Caspase 3 activation.Thus, leukemic cells constitutively expressing PI-9 exhibit intrinsic resistance towards granzyme B dependent Caspase 3 activation and apoptosis induction, respectively.This mechanism may be involved in resistance to GvL following allogeneic transplantation. Further studies involving primary leukemic cells might reveal if leukemias with high PI-9 expression are at higher risk of relapse post hematopoetic SCT.

Expression of the Apoptosis Inhibitor Protease Inhibitor 9 Predicts Clinical Outcome in Vaccinated Patients with Stage III and IV Melanoma

There have been reports of successful treatment of metastatic melanoma patients with active specific immunotherapy (ASI) using irradiated autologous tumor cell vaccination. It is still unknown why some patients respond and others do not. Tumor cells can evade the immune system, for example through interference with antigen presentation by down-regulation of MHC molecules or expressing proteins interfering with cytotoxic lymphocyte-induced apoptosis like the granzyme B antagonist protease inhibitor 9 (PI-9). PI-9 expression was detected in melanoma cell lines. To investigated if PI-9 is important in the response to ASI, paraffin-embedded tissues from stage III or IV melanoma patients were stained. PI-9 is expressed in melanoma cells and expression in metastasized melanoma cells is, in this group of patients, an adverse prognostic marker with regard to overall and disease-free survival. Moreover, loss of MHC-1 expression frequently occurs during tumor progression but is not associated with poor clinical outcome. Interestingly, melanoma patients with a favorable clinical outcome after ASI therapy usually have high percentages of activated (granzyme B-positive) tumor-infiltrating lymphocytes at time of first diagnosis and low percentages of activated lymphocytes at time of recurrent tumor. Expression of PI-9 in metastatic melanoma cells is associated with unfavorable clinical outcome whereas MHC-1 down-regulation is not. Although it cannot be proven that PI-9 expression is directly responsible for failure of immunotherapy, these data suggest that expression of PI-9 could be an important immune escape mechanism and that modulation of this inhibitor may enhance the efficacy of immunotherapy.