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NEOPLASIA
From the Department of Pathology, Free University
Medical Center, Amsterdam, The Netherlands; Department of
Pathophysiology of Plasma Proteins, CLB, Amsterdam, The Netherlands;
Department of Clinical Pathology, General Hospital Vienna, Austria; and
Department of Pathology, University of New Mexico, Albuquerque.
In tumor cells, the serine protease granzyme B is the primary
mediator of apoptosis induced by cytotoxic T lymphocytes (CTLs)/natural killer (NK) cells. The human intracellular serpin proteinase inhibitor 9 (PI9) is the only known human protein able to inhibit the proteolytic activity of granzyme B. When present in the cytoplasm of T lymphocytes, PI9 is thought to protect CTLs against apoptosis induced by their own
misdirected granzyme B. Based on the speculation that tumors may also
express PI9 to escape CTL/NK cell surveillance, immunohistochemical studies on the expression of PI9 in various lymphomas were performed. Ninety-two cases of T-cell non-Hodgkin lymphoma (NHL), 75 cases of
B-cell NHL, and 57 cases of Hodgkin lymphomas were stained with a
PI9-specific monoclonal antibody. In T-cell NHL, highest PI9 expression
was found in the extranodal T-cell NHL. In nearly 90% of
enteropathy-type T-cell NHLs and 80% of NK/T-cell, nasal-type lymphomas, the majority of the tumor cells expressed PI9. In nodal T-anaplastic large cell lymphomas and peripheral T-cell lymphomas (not
otherwise specified), PI9 expression occurred less frequently. In
B-cell NHL, PI9 expression was associated with high-grade malignancy; 43% of diffuse large B-cell lymphomas showed PI9+ tumor
cells. Finally, PI9 expression was also found in 10% of Hodgkin
lymphomas. This is the first report describing the expression of the
granzyme B inhibitor PI9 in human neoplastic cells in vivo. Expression
of this inhibitor is yet another mechanism used by tumor cells to
escape their elimination by cytotoxic lymphocytes.
(Blood. 2002;99:232-237) Natural killer (NK) cells and cytotoxic T
lymphocytes (CTLs) form an important line of defense against virally
infected cells and tumor cells. CTLs and NK cells kill target cells by
inducing apoptosis in the latter. This mainly occurs via 2 pathways,
one involving the death receptor induced-apoptosis (mainly the Fas ligand/Fas pathway), the other being dependent on the exocytosis of
cytotoxic granules from the effector cell. Cytotoxic granules contain perforin, which forms pores in the membrane of the target cell,
and several serine proteases, termed granzymes. Studies with purified
components,1 transfected cells,2 and knockout mice3,4 have established perforin to be essential
for membrane lysis and granzyme B to be necessary for rapid target cell
DNA fragmentation and apoptosis.
Currently, strategies used by cells of the immune system and tumor
cells to avoid unwarranted apoptosis by CTLs are the focal point of
considerable investigation. In this regard, CTLs must protect
themselves against their own cytolytic machinery to prevent unwanted
lysis and must use several mechanisms to accomplish this. For example,
resistance to Fas-mediated apoptosis is induced by the expression of
FLICE inhibitory protein (FLIP), which directly inhibits the
Fas-mediated signaling pathway.5 A number of reports have
indicated that tumor cells use comparable mechanisms to resist lymphocyte-mediated cytotoxicity such as expression of FLIP and Fas
ligand.5,6 In addition, they use other mechanisms such as
the down-regulation of major histocompatability complex (MHC) class I
molecules.7
Recently, a new defense mechanism was reported for T lymphocytes,
particularly CTLs, involving a novel human intracellular serine
proteinase inhibitor (serpin), proteinase inhibitor 9 (PI9). This
serpin efficiently inhibits granzyme B in vitro and in vivo and cells
transfected with a PI9 expression vector are protected against granzyme
B-mediated apoptosis.8,9 Therefore, it was proposed that
PI9 protects CTLs against death induced by their own, misdirected
granzyme B.8 Notably, as we showed recently, this
inhibitor is also expressed by dendritic cells and at immune-privileged sites (eg, placenta, testis, and the eye). At these sites, PI9 most
likely provides protection against degranulating CTLs or NK cells.
We hypothesized that tumor cells also use expression of PI9 as another
mechanism to escape immune surveillance by tumor-infiltrating CTLs or
NK cells. To verify this hypothesis, we investigated whether lymphomas
derived from T or B cells express the granzyme B inhibitor PI9.
Materials
Patient selection
Immunohistochemistry and cytochemistry Sections (3-µm thick) from the paraffin-embedded biopsies were mounted on slides coated with poly-L-lysine. Endogenous peroxidase activity was blocked by incubation for 30 minutes with 0.3% (v/v) H2O2 in methanol. Following antigen retrieval by boiling in 0.01 M sodium citrate, pH 6, for 10 minutes in a microwave oven, tissue sections were incubated with the primary antibody for 1 hour at room temperature. The following antibodies were used: either purified mAb PI9-17 (at 2.5 µg/mL) or mAb PI9-K (at 4.25 µg/mL) in case of staining for PI9 and anti-granzyme B mAb GB7 (1.2 µg/mL). The secondary antibody was biotinylated rabbit-antimouse Fab2 immunoglobulin diluted 1:500. Biotinylated secondary antibodies were detected with sABC. PI9-stained tissue sections were further incubated with biotinylated tyramine followed by a second incubation with sABC. Bound antibodies were visualized by incubation with diaminobenzidine/H2O2 (DAB) or 3-amino-9-ethylcarbazole (AEC). Slides were counterstained with hematoxylin and mounted. Negative control slides were stained with mouse IgG of the appropriate subclass.Double immunofluorescence staining for PI9 and granzyme B was performed on sections of T-ALCL as follows. After antigen retrieval sections were preincubated for 10 minutes with normal goat serum, diluted 1:50. Subsequently, sections were stained with mAb PI9-17 at 2.5 µg/mL as well as with GB7 at 6 µg/mL for 1 hour. The secondary antibodies used were biotinylated goat-antimouse IgG1, diluted 1:100, and horseradish peroxidase-labeled goat-antimouse IgG2a, diluted 1:100. Granzyme B was detected by incubation with DAB. After blocking the remaining peroxidase activity with 0.3% (v/v) H2O2 in methanol, sections were subsequently incubated with sABC and biotinylated tyramine. Following a second incubation with sABC, PI9 was detected by incubation with AEC. Sections were counterstained with hematoxylin and mounted. Cases were scored independently by 2 pathologists. A case was considered positive for PI9 when cytoplasmic staining of tumor cells for PI9 was observed unequivocally. According to the number of tumor cells staining positively, these cases were divided into 5 categories: less than 5%, 5% to 25%, 25% to 50%, 50% to 75%, and 75% to 100% of the tumor cell population staining positive for PI9. Staining of reactive lymphocytes and dendritic cells within the tumor cell population acted as a positive, internal control. Quantification of relative numbers of lymphocytes positive for granzyme B present in tumor sections was performed using a commercially available interactive video overlay-based measuring system (Q-Prodit, Leica, Cambridge, United Kingdom), as described previously.16,17 Statistical methods The relationship between PI9 expression and the presence of granzyme B+ CTLs was analyzed by Pearson 2
test. P values, based on 2-tailed statistical analysis,
below .05 were considered significant. All analyses were performed
using the SPSS statistical software (SPSS, Chicago, IL).
PI9 is expressed in neoplastic cells in Hodgkin lymphomas and NHLs Neoplastic cells expressing PI9 were detected in 36 of 92 cases (39%) of T-cell lymphoma, 20 of 75 cases (27%) of B-cell lymphoma, and 6 of 57 cases (10%) of Hodgkin lymphoma (Table 1). The percentage of PI9+ neoplastic cells in a given type of tumor varied from less than 5% to 100%. Figure 1A shows a case in which most neoplastic cells were PI9+; Figures 1C and 2B show an example in which tumor cells were completely negative for PI9. In these cases, dendritic cells present in the tumor area served as an internal positive control for PI9 detection (arrowhead). In PI9+ cases, a strong cytoplasmic staining of the tumor cells was observed (Figures 1A and 2A), consistent with the reported intracellular localization of PI9.
Enteropathy-type T-cell lymphomas show highest PI9 expression Of all T-cell NHLs, the highest PI9 expression was observed in extranodal enteropathy-type T-cell NHLs, regarding not only the number of positive cases but also the percentage of positive neoplastic cells (Table 1). In 17 of 19 (89%) of the enteropathy-type T-cell NHLs, PI9-expressing neoplastic cells were present. Figure 1E shows PI9+ tumor cells infiltrating in the epithelial layer. In addition, PI9 protein-expressing tumor cells were found in 4 of 5 (80%) NK/T-cell, nasal-type lymphomas. Figure 1D shows PI9+ tumor cells in the mitotic phase (arrow). Of the nodal T-cell NHLs, 12 of 57 (21%) cases of T-ALCL and 3 of 11 (27%) cases of PTCL NOS contained PI9-expressing cells. A PI9+ and PI9 case of ALCL is presented in Figure 1, panels A and
C, respectively. Whereas in most cases of the enteropathy-type
lymphomas nearly all tumor cells expressed the PI9 protein, the
percentage of PI9+ tumor cells in the T-ALCL and PTCL NOS
groups varied widely.
Although the mAb PI9-17 used in this study has been extensively tested
for its specificity,10 the immunohistochemical staining was confirmed with a second mAb against PI9, mAb PI9-K, which recognizes a different epitope than mAb PI9-17. Five cases of PI9 PI9 is expressed in B-cell NHLs and some cases of Hodgkin lymphoma Expression of PI9 was not restricted to T-cell lymphomas because it was also found in B-cell NHLs and Hodgkin lymphomas (Table 1). PI9 expression was observed specifically in B-cell NHLs of intermediate to high grade. In 43% of diffuse large B-cell lymphoma and 25% of Burkitt lymphoma tumor cells, PI9 was expressed, whereas only 1 of 27 (4%) low-grade B-cell NHLs was positive for PI9 staining. An example of PI9 expression in diffuse large B-cell lymphoma is shown in Figure 2A. In most cases of diffuse large B-cell lymphoma, 75% to 100% of the tumor cells expressed PI9 (Table 1 and Figure 2A). In PI9 low-grade B-cell NHLs, such as follicular lymphomas
(Figure 2B) and B-CLLs (Figure 2C), dendritic cells served as a
positive internal control for PI9 staining (arrowhead).
Expression of PI9 in tumor cells was also found in 5 of 53 (9%) cases of nodular sclerosing Hodgkin lymphoma and in 1 of 4 (25%) cases of nodular lymphocyte predominant Hodgkin lymphoma. A PI9-expressing Reed-Sternberg cell, the characteristic neoplastic cell in Hodgkin disease, is shown in Figure 2D. In addition to staining with mAb PI9-17, several cases of diffuse large B-cell lymphoma and Hodgkin lymphoma were stained with mAb PI9-K as well. In all cases, the staining patterns obtained with the 2 antibodies were completely identical (data not shown). PI9 expression is not always related to the cytotoxic phenotype of neoplastic cells in T-cell NHLs In many cases of T-cell NHL, particularly in extranodal lymphoma, the neoplastic cells co-expressed the serine proteinase target of PI9, granzyme B (Table 2 and Figure 1F). In T-cell NHLs of the gastrointestinal tract and the nose, all cases were positive for granzyme B, with the exception of one NK/T-cell, nasal-type lymphoma. In most of these cases, high levels of PI9 were observed, but in 3 cases no PI9 expression was detected. Furthermore, comparison between the expression of PI9 and granzyme B revealed that in T-ALCL and PTCL NOS the expression of these proteins was also not related. Only about half of the PI9+ T-ALCLs were also positive for granzyme B. On the other hand, 23 of 28 granzyme B+ T-ALCLs were negative for PI9. Nearly all cases of PTCL NOS were negative for granzyme B, but 3 of 10 cases did express PI9. Overall, these results showed that PI9 and granzyme B expression are not always related in T-cell NHL.
PI9 is expressed mainly in lymphomas harboring many activated CTLs Expression of PI9 in T-ALCLs tended to be related to a high percentage (ie, 15%) of activated CTLs (Table
3). Nine of 12 PI9+ cases
showed 15% or more granzyme B+ CTLs, but this was just not
significant (P = .08). Figure 1G shows an example of a
T-ALCL that was positive for PI9 and had a high number of granzyme
B-expressing CTLs surrounding the tumor cells. Most Hodgkin lymphoma
contained less than 5% granzyme B+ CTLs. However, 3 of 4 PI9+ cases of Hodgkin lymphoma analyzed for CTL infiltrates
contained a relative high percentage of 10% or greater granzyme
B+ CTLs (data not shown).
We have described, for the first time, that PI9 is expressed in vivo by human tumors, more specifically by various types of lymphomas, that is, a substantial number of T-cell and B-cell NHLs as well as some cases of Hodgkin lymphoma. In B-cell NHL, PI9 is particularly expressed by high-grade lymphomas. These results implicate a novel mechanism by which tumor cells may evade the attack of the immune system. Tumor cells use various defense mechanisms against CTL-induced apoptosis, such as down-regulation of MHC class I molecules, resulting in less efficient tumor cell recognition by CTLs, and expression of proteins that confer resistance to Fas-mediated apoptosis.6 Examples of the latter mechanism are expression of Fas ligand, which kills attacking cytotoxic lymphocytes, and FLIP, which directly inhibits the Fas-mediated signal transduction pathway.5 Expression of PI9 would be an important additional defense mechanism, because this protein confers resistance to the other main pathway for induction of apoptosis, that is, granzyme B-induced apoptosis. Except for the viral serpin CrmA, PI9 is the only protein known that efficiently inhibits granzyme B in vitro and in vivo.8,9 It was initially described to be expressed by T- and B-cell lines. The function of PI9 in these cells was presumed to provide protection against their own, misdirected granzyme B.8 PI9 may play a protective role against granzyme B-induced apoptosis not only in CTLs, but also in other cell types as was suggested by its specific expression in dendritic cells and at immune-privileged sites.10 Thus, the present study points to a novel mechanism for tumor cells to escape from CTL-induced apoptosis. This is particularly important because granzyme B is the predominant mediator of early induction of DNA fragmentation and CTL-induced apoptosis of tumor cells.2,3 Several other serpins than PI9, especially those belonging to the
subfamily of so-called ovalbumin serpins, have been implicated in
processes associated with tumorigenesis and apoptosis. But, in contrast
to PI9, these ovalbumin serpins cause resistance to death receptor
rather than granzyme B-mediated apoptosis. Both human
PAI-218 and PI1019 can protect cells from
apoptosis induced by tumor necrosis factor Cytolytic molecules like granzyme B and perforin are reportedly expressed not only in reactive lymphocytes, but also in neoplastic cells in Hodgkin lymphomas and NHLs as well.27,28 Granzyme B+ tumor cells were mainly found in extranodal T-cell lymphomas and NK cell lymphomas, localized in mucosal lymphoid tissue.29 It was suggested that these lymphomas are the neoplastic counterparts of activated CTLs or NK cells.29 Although the function of granzyme B in these tumor cells is still unknown, its presence provides suggestive evidence that these tumors could defend themselves against an immune response by release of granzyme B, and in that way kill tumor-infiltrating CTLs. In such a scenario, the tumor cells would need to protect themselves against endogenous granzyme B and one would anticipate concomitant expression of granzyme B and PI9 in these cells. In agreement herewith, a large number of PI9+ tumor cells was present in enteropathy-type T-cell lymphoma and NK/T-cell, nasal-type lymphoma, that is, tumors that also contain high levels of granzyme B. However, from Table 2 it is clear that there is no correlation between PI9 and granzyme B expression in the nodal T-ALCL. Thus, PI9 expression in tumor cells was not strictly associated to granzyme B expression in these cells. Although its regulation is unclear, PI9 expression in tumors can be postulated to reflect selection due to immunologic pressure. It can be speculated that in these cases, the tumors may have become resistant to CTL-mediated apoptosis by expressing high levels of PI9. Indeed, PI9 expression in T-ALCL and Hodgkin lymphoma tended to be related to a high percentage of tumor-infiltrating, granzyme B+ CTLs (Table 3). Notably, neoplastic cells may use different defense mechanisms against CTL-induced apoptosis. This may explain why PI9 expression and the presence of a high percentage of granzyme B+ CTLs are not absolutely correlated. PI9 not only inhibits granzyme B but also some caspases,30,31 which may render these cells less susceptible to therapy. This may explain why tumors expressing high levels of PI9, such as gastrointestinal tract T-NHLs, NK/T cell nasal types, and high-grade B-cell NHLs, have a particularly poor clinical outcome. It can be postulated that PI9 expression is an intrinsic feature of normal T and B cells and that tumors derived from these cells, therefore, also express PI9. However, we showed that in normal human tissues PI9 was expressed mainly by T cells but hardly by B cells.10 Therefore, we expected to find PI9 preferentially in neoplastic equivalents of CTLs and NK cells, that is, T-cell NHL in the gastrointestinal tract and NK/T-cell, nasal type. Surprisingly, a high percentage of B-cell NHL was also found to be positive for PI9. Even more, PI9 was also detected in epithelial tumor cell lines (B.A.B., unpublished results, February 1999). These results indicate that PI9 expression is not exclusively used by tumors originating from T lymphocytes, but may be a common mechanism used by tumor cells of different origin. It might be argued that the lack of staining of low-grade B-cell NHL might be due to the lack of cytoplasm of small neoplastic B cells. However, this is very unlikely because these tumors are diagnosed by staining for their marker bcl-2, which like PI9 is a cytoplasmic protein. In conclusion, PI9 is expressed by a significant proportion of tumors derived from both T and B cells. Because of the ability of PI9 to efficiently inhibit granzyme B activity, PI9 expression in tumors may be an important new mechanism for tumor cells to escape from apoptosis induced by tumor-infiltrating CTLs.
We thank Angelique Verlaan for her help with the immunohistochemical stainings.
Submitted February 15, 2001; accepted August 17, 2001.
Supported by the Dutch Cancer Foundation (grant VU-98-1718).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: J. Alain Kummer, Dept of Pathology, Free University Hospital, PO Box 7057, 1007 MB Amsterdam, The Netherlands; e-mail: ja.kummer{at}vumc.nl.
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© 2002 by The American Society of Hematology.
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  Y. Getachew, H. Stout-Delgado, B. C. Miller, and D. L. Thiele Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses J. Immunol., December 1, 2008; 181(11): 7810 - 7817. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
. Loss of the serpin
maspin correlates with increased malignancy and metastasis of
breast carcinomas.
from controversy to consensus: the REAL and WHO classification of lymphoid neoplasms.
Ann Oncol.
2000;11(suppl)1:3-10