The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor
9, Is Up-Regulated During Accessory Cell Maturation and
Effector Cell Degranulation, and Its Overexpression Enhances
Claire E. Hirst,* Marguerite S. Buzza,* Catherina H. Bird,* Hilary S. Warren,†
Paul U. Cameron,‡Manling Zhang,§Philip G. Ashton-Rickardt,§and Phillip I. Bird2*
Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic
pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The
nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis
in model systems. Here we show that PI-9 is present in CD4?cells, CD8?T cells, NK cells, and at lower levels in B cells and
myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules
in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9
enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because
dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8?T cells and therefore may also
require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3?, CD4?, CD8?,
CD45?), tonsillar DCs, and DC subsets purified from peripheral blood (CD16?monocytes and CD123?plasmacytoid DCs).
Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-?-induced maturation of monocyte-
derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective
role against ectopic or misdirected grB during an immune response. The Journal of Immunology, 2003, 170: 805–815.
viewed in Ref. 1). Granzyme B (grB)3is a key cytotoxic granule
proteinase that is endocytosed by the target cell following cyto-
toxic lymphocyte (CL) degranulation, probably by binding to the
mannose-6-phosphate receptor (M6PR) (2). The cytotoxic pore-
forming protein, perforin, is also internalized and mediates the
release of grB into the cytoplasm of the target cell. GrB then rap-
idly induces apoptosis by cleaving one or more cytoplasmic or
nuclear proteins. These include Bid (3–6), caspases (7, 8), com-
ponents of the DNA repair machinery (7, 9), and inhibitor of
caspase-activated DNase (10, 11).
atural killer cells and CTLs kill virus-infected and tumor
cells by inducing apoptosis through exocytosis of cyto-
toxic granule contents or ligation of death receptors (re-
Although CL granule contents are efficiently directed into the
target cell via the immunological synapse (12), some grB may
escape from granules or the synaptic zone into the cytoplasm of the
CL or into the extracellular milieu. For example, free grB is evi-
dent in the sera of patients with elevated CTL responses (13) or
severe Gram-negative bacterial infections (14). Given its cytotoxic
potency and ability to degrade extracellular proteins (13, 15–18), it
is likely that protective mechanisms exist to counter misdirected or
GrB is efficiently inhibited by the nucleocytoplasmic serpin,
proteinase inhibitor 9 (PI-9) (19, 20), with transfection studies
demonstrating that PI-9 protects cells from grB-mediated apopto-
sis (21). PI-9 is present in cells at immune-privileged sites such as
testis and placenta (22, 23), and the expression of PI-9 in endo-
thelial and mesothelial cells suggests that it protects bystander
cells from grB released during an immune response (24). Here we
show that PI-9 is up-regulated in CTLs in response to grB pro-
duction and degranulation, that it associates with grB-containing
granules in activated CTLs and NK cells, and that overexpression
increases CTL potency. We also show that PI-9 is present in sev-
eral dendritic cell (DC) types. The localization and regulated ex-
pression of PI-9 in these leukocyte subsets strongly support the
hypothesis that PI-9 protects effector, accessory, and bystander
cells from ectopic grB during an immune response.
Materials and Methods
PI-9 was detected with the specific mAb 7D8 (22) or with rabbit 12 or
rabbit 15 polyclonal antisera raised against recombinant PI-9 (19). PI-6 was
detected with mAb 3A (25). GrB was detected with 2C5 (26) or rabbit
anti-grB/grH polyclonal antiserum (27), both provided by J. Trapani (The
Peter MacCallum Cancer Institute, East Melbourne, Australia), or GrB-7
*Department of Biochemistry and Molecular Biology, Monash University, Clayton,
Australia;†Division of Immunology and Cell Biology, John Curtin School of Medical
Research, Australian National University, Canberra City, Australia;‡Department of
Microbiology and Immunology, University of Melbourne, Parkville, Australia; and
§Department of Pathology, Ben May Institute for Cancer Research and Gwen Knapp
Center for Lupus and Immunology Research, University of Chicago, Chicago, IL
Received for publication April 19, 2002. Accepted for publication November 5, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the National Health and Medical Research Council of
Australia, and the National Institutes of Health (Grant AI45108). H.S.W. is funded by
Senior Research Fellowship 148950.
2Address correspondence and reprint requests to Dr. Phillip Bird, Department of
Biochemistry and Molecular Biology, Building 13B, Room G09, Monash University,
Clayton 3800, Australia. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: grB, granzyme B; CL, cytotoxic lymphocyte; DC,
dendritic cell; GFP, green fluorescence protein; MDDC, monocyte-derived DC;
M6PR, mannose-6-phosphate receptor; NP40, Nonidet P-40; PI-9, proteinase inhib-
The Journal of Immunology
Copyright © 2003 by The American Association of Immunologists, Inc.0022-1767/03/$02.00
(Chemicon, Temecula, CA). CD markers were detected with hybridoma
supernatants from 3C10 (CD14; provided by R. Steinmann, Rockefeller
Institute, New York), FMC63 (CD19; provided by H. Zola, Child Health
Research Institute, Adelaide, Australia), and OKT3 (CD3) and OKT8
(CD8; both from American Type Culture Collection (Manassas, VA). Anti-
CD4 and anti-CD56 were obtained from BD Biosciences (Franklin Lakes,
NJ). Actin was detected with anti-actin (I-19) from Santa Cruz Biotech-
nology (Santa Cruz, CA).
Intracellular flow cytometry
Intracellular flow cytometry was performed using peripheral blood drawn
from healthy volunteers. Erythrocytes were removed from whole blood by
lysis in 167 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA for 5 min at
room temperature, centrifugation at 100 ? g for 5 min, then washing twice
in PBS containing 1% FCS and 0.02% sodium azide (repeated for all sub-
sequent washes). Approximately 2.5 ? 106PBLs were fixed in 4% form-
aldehyde in PBS for 10 min at room temperature, centrifuged, resuspended
in FACS Permeabilizing Solution (BD Biosciences) for 10 min, centri-
fuged, washed, and incubated with 7D8 or isotype control Ab (IgG1; BD
Biosciences) for 30 min. Cells were washed twice, then incubated with
FITC-conjugated anti-mouse Ig (Chemicon). After two more washes the
cells were analyzed using a FACSCalibur and CellQuest software (BD
Localization of ov-serpins in peripheral blood leukocytes
Highly purified B, CD4?, CD8?, monocyte, and NK cell populations were
obtained from whole blood using RosetteSep Enrichment cocktails (Stem-
Cell Technologies, Vancouver, Canada) according to the manufacturer’s
instructions. Cells were plated on poly-L-lysine-coated slides, fixed in 4%
formaldehyde for 20 min, then permeabilized in 0.5% Triton X-100 for 5
min. PI-9 and PI-6 were detected with 7D8 hybridoma supernatant or 3A
hybridoma supernatant, respectively. Bound Ab was detected with anti-
mouse Ig conjugated to FITC. Cells were counterstained with propidium
iodide (1 ?g/ml), and cross-sections through the nucleus were obtained
using laser scanning confocal microscopy (TCS-NT; Leica, Wetzlar, Ger-
many). The purity of each cell population and the presence of any con-
taminating cell types were assessed using Abs to CD3, CD4, CD8, CD14,
CD19, and CD56.
Retroviral transduction of human CTLs
A cDNA encoding PI-9 (19) was subcloned into the MIGR1 retroviral
vector in the forward or reverse orientation. PI-9 mRNA was transcribed as
a bicistronic message with green fluorescence protein (GFP) (28). Viral
supernatant for transduction was obtained by transient transfection of the
293GP packaging cell line (29) using Lipofectamine Plus (Invitrogen,
Carlsbad, CA). Supernatant was tested for transduction efficiency on Jurkat
cells. PBLs were obtained from normal healthy donors and were cultured
in for 2 days with PHA and human IL-2 (300 U/ml), then transduced with
retrovirus (29). After transduction, PBLs underwent one round of rapid
expansion with irradiated feeder cells (BRT Laboratories, Baltimore, MD),
and the brightest GFP-expressing PBLs were purified by FACS. Sorted
PBLs (72% GFP?, 75–80% CD8?) were then kept in culture by rapid
expansion every 3 days. PI-9 mRNA was quantitated by real-time PCR on
cDNA (30) using primers and probes specific for PI-9 and GAPDH, re-
spectively (MegaBases, Evanston, IL). Retrovirally transduced PBLs were
analyzed in51Cr release assays (in triplicate wells) with hybridoma targets
that express the anti-human CD3 mAb OKT3 (31). The percent specific
lysis of JY cells, a human B lymphoblastoid cell line, was determined in
parallel (?6% specific lysis) and subtracted from the lysis of the OKT3
hybridoma to report CTL killing only.
Expression of PI-9 in stimulated cytotoxic lymphocytes
YT cells (32) were cultured as previously described (20). PBMCs were
isolated from whole blood using Ficoll-Paque Plus. PBMCs (depleted of
monocytes by adherence to plastic) were cultured in IL-2 (Sigma-Aldrich,
St. Louis, MO) or a combination of Con A and PMA (both from Sigma-
Aldrich) as previously described (33). Culture-generated quiescent NK
cells were cultured and activated as described previously (34, 35). At the
indicated time points, activated cells were harvested, and cell lysates were
prepared by lysis in Nonidet P-40 (NP40) lysis buffer (1% NP-40 in 50 mM
Tris-HCl (pH 8.0), and 10 mM EDTA (pH 8.0)). To prevent postlysis
association of PI-9 and grB, cells were lysed on ice in the presence of
protease inhibitors (1 mM PMSF, 1 ?g/ml aprotinin, 1 ?g/ml leupeptin,
and 1 ?g/ml pepstatin). Lysates from 0.5 ? 106cells were resolved by
reducing 12.5% SDS-PAGE, transferred to nitrocellulose, immunoblotted
for PI-9 with 7D8, and detected with HRP-conjugated anti-mouse Ig
(Chemicon) using ECL (NEN, PerkinElmer, Boston, MA). The membranes
were stripped (62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 0.1 M 2-ME for
30 min at 50°C), then reprobed for grB (2C5) and actin (diluted 1/1000).
Densitometric analysis of PI-9 levels was determined using MCID Image
Analysis software (BD Biosciences).
Formation of PI-9/grB complexes
Lysate from COS-1 cells transfected with PI-9 (20) was incubated with or
without 100 ng of recombinant grB (36) at 37°C for 10 min. Samples were
resolved by reducing 12.5% SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with rabbit 12 and rabbit 15 antisera (PI-9) or 2C5 (grB).
YT cells (1 ? 106) were cultured for 20 h with or without 25 ?M calpain
inhibitor I (N-acetyl-Leu-Leu-Norleu; Sigma-Aldrich). Cells were lysed in
NP-40 lysis buffer or modified Laemmli sample buffer (60 mM Tris-HCl
(pH 6.8), 2% SDS, and 10% glycerol) (37) and passed through a 26-gauge
needle to shear the DNA. Twenty micrograms of cell lysate was resolved
by reducing 12.5% SDS-PAGE, transferred to nitrocellulose, immunoblot-
ted with rabbit 12 antiserum, then stripped and reprobed with rabbit 15
Colocalization of PI-9 and grB
Activated CTLs were attached to poly-L-lysine-treated slides, fixed and per-
for PI-9 (7D8), grB (rabbit anti-grB antiserum, diluted 1/200), or cathepsin A
(rabbit antiserum, diluted 1/500, obtained from A. Pshezhetsky, University
of Montreal, Montreal, Canada). Activated NK cells were cultured on
irradiated MM-170 cells for 4 h, fixed in 4% formaldehyde, then perme-
abilized in 100% methanol and stained for PI-9 (7D8) and grB (rabbit
anti-grB antiserum, diluted 1/200). YT cells were attached to poly-L-lysine-
treated slides, fixed and permeabilized in acetone/methanol, and stained for
PI-9 (rabbit anti-PI-9 antiserum, diluted 1/2000) and grB (2C5, diluted
1/200). BeWo cells were cultured and handled for microscopy as previ-
ously described (20). Primary Abs were detected with the appropriate sec-
ondary Ab conjugated to FITC or rhodamine B isothiocyanate (Chemicon).
usinglaser scanning confocal
Tissue localization of PI-9
Human tissues fixed in neutral buffered formalin were obtained from the
archive of the Pathology Department of Box Hill Hospital (Melbourne,
Australia). Immunohistochemistry was performed as previously described
(22). Cryopreserved normal human thymus was provided by R. Boyd (De-
partment of Pathology and Immunology, Monash University, Prahran,
Australia). Sections (6 ?m) of tissue were air-dried onto silanized slides.
Sections were washed and blocked in normal goat serum before incubation
with 7D8 or an isotype-matched control Ab (IgG1). Bound Ab was de-
tected with anti-mouse Ig conjugated to FITC. Sections were then stained
with anti-CD3, -CD4, or -CD45 mAbs directly conjugated to PE (Diatec,
Oslo, Norway). Two-color images were obtained using laser scanning con-
Dendritic cell purification
PBMCs were isolated from normal donors over Ficoll-Paque gradients
(Red Cross Blood Transfusion Service, Melbourne, Australia). Monocytes
were prepared by elutriation at 2100 rpm using a Beckman J6 M (Palo
Alto, CA) with a standard chamber. Monocytes were further purified by
sorting using a MoFlo CLS cell sorter (Cytomation, Fort Collins, CO)
based on forward and side light scatter and subpopulations sorting for
CD16?and CD14?expression. Lineage-negative DCs were purified from
the monocyte elutriation fractions by negative selection with Abs to CD3,
CD14, CD11b, CD19, and CD16. Lineage-negative DCs were sorted into
CD1b/c?(B-B5; BioSource International, Camarillo, CA) Langerhans cell
precursors (38) or CD123?(CD123, BD Bioscience) plasmacytoid DCs
(39). MDDCs were generated from CD14?monocytes positively selected
using MACS columns after labeling for CD14 and goat anti-mouse MACS
beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were selected
on a MACS column as recommended by the manufacturers. CD14?mono-
cytes were cultured in IL-4 (20 ng/ml) and GM-CSF (40 ng/ml) for 5–7
days as previously described (40). Immature DCs were matured in the
presence of TNF-? (10 ng/ml; R&D Systems, Minneapolis, MN) for
806 EXPRESSION AND REGULATION OF PI-9 IN CLs AND DCs
Distribution of PI-9 in peripheral blood leukocytes
To extend our previous observations that PI-9 is expressed in lym-
phoid (T, B, and NK-like) cell lines, but not myeloid cell lines
(19), we examined PI-9 in PBLs by intracellular flow cytometry
using a specific mAb (22). PI-9 was evident in the majority of
PBLs in both mononuclear cells (R1) and granulocytes (R2; Fig.
1A). Most mononuclear cells were clearly positive (?90%). Dual-
color analysis showed that ?95% of CD3?cells are PI-9 positive,
100% of CD4?and CD8?cells are PI-9 positive, and ?75% of
CD19?cells are PI-9 positive (data not shown). Many granulo-
cytes (?70%) were also positive, but appeared to have lower
amounts of PI-9, with only a slight positive shift in staining ap-
parent over the isotype control (Fig. 1A). Comparison of the mean
fluorescence intensities indicated that mononuclear cells express at
least 5-fold more PI-9 than granulocytes.
To further identify the populations of mononuclear cells that
produce PI-9, B cells, CD4?and CD8?T cells, NK cells, mono-
cytes, and granulocytes were enriched from PBLs using estab-
lished immunodepletion procedures. These were analyzed for PI-9
by indirect immunofluorescence so that its intracellular distribu-
tion could be simultaneously assessed (Fig. 1B). The efficiency of
immunodepletion was monitored by staining for CD3, CD4, CD8,
CD14, CD19, or CD56 (Fig. 1C). PI-9 was evident in all popula-
tions, with the highest levels in NK and T cells (CD4?and CD8?
T cells expressed equivalent levels). B cells, monocytes, and gran-
ulocytes had much lower levels. In all cell types PI-9 exhibited a
nucleocytoplasmic localization, consistent with previous reports
(19, 20). The expression pattern of PI-9 differed from that of the
closely related serpin, PI-6, which is a cathepsin G inhibitor
present in myeloid cells (41).
Intracellular localization of PI-9 in grB-expressing cells
The observation of high PI-9 expression in NK and CTLs suggests
that it protects grB-producing cells against endogenous grB. Using
indirect immunofluorescence and laser scanning confocal micros-
copy we examined the intracellular localization of PI-9 in primary
NK cells and the NK-like cell line, YT. This showed that in ad-
dition to its previously reported cytoplasmic and nuclear distribu-
tion (Fig. 2A) (20), PI-9 is associated with vesicles in the CL
cytoplasm that also contain grB (Fig. 2, B and C). These vesicles
were positive for the lysosomal/granule marker lysosome-associ-
ated membrane protein-1 and not with markers for the endoplas-
mic reticulum or Golgi apparatus (data not shown), indicating that
they are secretory lysosomes (cytotoxic granules). To determine
whether the association of PI-9 with lysosomes is unique to CLs,
the distribution of endogenous PI-9 in the epithelial cell line BeWo
was also examined (Fig. 2D). Here PI-9 was evident only in the
cytoplasm and nucleus, and no colocalization between PI-9 and
lysosomal markers was observed.
In a previous study we separated membrane and cytosolic frac-
tions of CLs and showed that PI-9 is present in the cytosol and
does not copurify with grB in the granule fractions (19). Given that
PI-9 is not present within granules, the results shown in Fig. 2
suggested that it is associated with the cytoplasmic surface of the
blood leukocytes. A, Flow cytometric analysis of
PI-9 in PBLs. PBLs were stained with a PI-9-spe-
cific mAb, 7D8 (filled histogram), compared with
an IgG1 isotype control (open histogram). This fig-
ure is representative of four different donors. B,
Distribution and subcellular localization of PI-9 in
PBL subsets. Leukocyte subsets were enriched and
assessed as described in Materials and Methods.
Indirect immunofluorescence was performed with
a mAb specific to PI-9 (7D8) or PI-6 (3A) and was
detected with FITC-conjugated anti-mouse Ig
(green). The nuclei were counterstained with pro-
pidium iodide (red), and images were obtained us-
ing laser scanning confocal microscopy. Relative
levels of PI-9 can be compared as all images were
captured using the same settings. Thisfigure is rep-
resentative of three separate experiments. C, Pro-
portion of PI-9- and PI-6-positive cells in enriched
leukocyte subsets. Subsets were assessed for
marker expression as described in Materials and
Distribution of PI-9 in peripheral
807 The Journal of Immunology
granule and is poised to rapidly inactivate grB entering the cyto-
plasm from leaking granules. To test this idea we examined YT
cells for evidence of extragranular grB in complex with PI-9. We
took advantage of the fact that grB-PI-9 complexes are stable un-
der SDS-PAGE and immunoblotting conditions (19, 21), and that
we have two high affinity PI-9 Abs that detect PI-9 in complex
with grB very efficiently. As shown in Fig. 3A, these Abs (rabbit
12 and 15) detect the grB-PI-9 complex better than they detect free
PI-9, and they also detect the degradation products commonly ob-
served following serpin-proteinase complex formation. Degrada-
tion of the complex occurs because distortion of the protease (in
particular) renders it highly susceptible to proteolysis (42, 43). The
Abs do not bind grB (Fig. 3A), and their corresponding preimmune
sera do not recognize PI-9, complex, or degradation products (data
not shown). None of the four grB Abs we have tested detected the
complex or degradation products with similar sensitivity to the
PI-9 Abs. An immunoblot using the most sensitive of these grB
Abs (2C5) is shown in Fig. 3A.
When YT cells are lysed in buffers containing NP-40, active
grB is released from granules and binds cytoplasmic PI-9
postlysis (20, 21). By contrast, lysis of CTLs in buffers con-
taining SDS prevents postlysis interactions, presumably by rap-
idly denaturing cellular proteins (37). Thus, lysis of YT cells in
SDS will denature free PI-9 and free grB, and a complex evi-
dent in SDS-treated YT cell extracts will have formed before
lysis. As shown in Fig. 3B, lysis of YT cells in NP40 generated
a postlysis complex of ?70 kDa as well as smaller products
resulting from complex degradation that were detected by both
PI-9 antisera, but more efficiently by rabbit 15. When cells were
lysed in SDS buffer, small, but reproducible, amounts of com-
plex and degradation products were detected by rabbit 15 (Fig.
3B). This suggests that there is a pool of extragranular grB in
the cytoplasm of YT cells that is bound to PI-9 and undergoing
Because serpin/proteinase complexes are irreversible and in-
volve distortion of both serpin and proteinase (42, 43), we pre-
dicted that cytoplasmic PI-9/grB complexes would be recognized
and degraded by the ubiquitin-proteosomal machinery. Cells cul-
tured in the presence of a proteosome inhibitor should therefore
accumulate complexes. Indeed, greater amounts of complexes
were detected by both PI-9 antisera when cells were incubated
with the proteosomal inhibitor, calpain inhibitor I, and lysed in
SDS (Fig. 3B). This also supports the idea that grB enters the
cytoplasm of CLs during normal cellular function and is rapidly
inactivated by PI-9.
grB in CLs. Activated CTLs (A) were
stained for PI-9 (green), and the nucleus
was counterstained with propidium iodide
(PI; red). Activated NK cells on targets (B)
or YT cells (C) were stained for PI-9
(green) and grB (red). Two-color images
were obtained using laser scanning confo-
cal microscopy. A and B, Single sections
through the cell; C, a projected view of
multiple scans through the depth of the cell
at 1-?m intervals to give a three-dimen-
sional representation of the entire cell; D,
representative single sections of BeWo
cells (choriocarcinoma) stained for PI-9
(green) and the lysosomal marker cathep-
sin A (red). No PI-9 association with lyso-
somes was evident in any of the cells in-
spected (n ? 15; sampling all 0.3-?m
sections generated through each cell).
Colocalization of PI-9 and
808 EXPRESSION AND REGULATION OF PI-9 IN CLs AND DCs
PI-9 enhances the potency of human CTLs
Since PI-9 potentially protects CLs from death induced by misdi-
rected grB, we wanted to determine the effect on CTL potency of
overexpressing PI-9 in activated PBLs. Normal donor PBLs were
activated with PHA and transduced with retroviral vectors con-
taining the PI-9 gene in either the forward (coding) or the reverse
(noncoding) orientation. These vectors allow transcription of the
inserted cDNA as a bicistronic mRNA that also encodes GFP (28,
44). Cultures transduced with either the coding or noncoding vec-
tors were enriched to equivalent amounts of GFP-expressing cells
(?72%) by FACS (data not shown). Those transduced with the
PI-9 gene in the coding orientation expressed 4-fold higher levels
of PI-9 mRNA (p ? 0.02) compared with those in which the PI-9
gene was in the noncoding orientation (Fig. 4A). Both cultures
contained an equal proportion of CD8 cells, which was 75–80% of
the total PBLs (data not shown).
The ability of uncloned populations of transduced CTLs to lyse
target cells expressing an anti-CD3 mAb (OKT3) (31) was eval-
uated. In parallel, we also determined the lysis of JY target cells,
which do not express anti-CD3 mAbs, so we were able to account
for NK cell activity in the cultures. In each experiment the specific
lysis of JY targets (NK cell killing) was subtracted from that of
OKT3 targets (total killing) to give the level of CTL activity. At
every E:T cell ratio tested we observed increased potency of CTLs
that expressed elevated levels of PI-9 (Fig. 4B). For example, at an
E:T cell ratio of 0.5:1 we observed a 3-fold increase in potency of
killing by CTLs that overexpressed PI-9 (p ? 0.02). Since PI-9
can protect cells from apoptosis caused by grB, we infer that over-
expression of PI-9 increases CTL potency by improving viability.
Regulation of PI-9 in grB-expressing cells
CL stimulation, which increases the level of grB, should also result
in a corresponding increase in PI-9. We therefore examined the
regulation of PI-9 in T and NK cells under conditions known to
induce grB synthesis and release. PBLs were stimulated with IL-2,
which expands and activates T cells, or a combination of Con A
and PMA, which results in T cell activation and granule exocyto-
sis. As shown by immunoblotting, both these stimuli induced grB
expression, peaking on days 6 and 3, respectively (Fig. 5, A and B).
No significant increase in PI-9 was observed in T cells stimulated
with IL-2 alone (Fig. 5A). However, stimulation of T cells with
Con A/PMA resulted in a 3-fold induction of PI-9 over endoge-
nous levels (Fig. 5B). This treatment also generated the higher Mr
form of grB (35 kDa) that is only observed following T cell de-
granulation (45). Thus, in vitro, PI-9 up-regulation is associated
with the release of grB from CLs.
The regulation of PI-9 was also investigated in NK cells. Cul-
ture-generated quiescent NK cells were activated by incubation
with targets (irradiated MM-170 cells) in the presence of IL-2.
These NK cells constitutively express both PI-9 and grB, as evi-
dent on day 1 of stimulation (Fig. 5C). While activated NK cells
killed MM-170 cells (data not shown) and up-regulated grB, which
peaked on day 3, no significant increase in PI-9 was detected over
time. Taken together, these results suggest that the level of PI-9 in
proliferating, nondegranulating T cells and activated NK cells is
sufficient to protect against grB. However, the increase in PI-9
expression during T cell degranulation suggests that granule exo-
cytosis is associated with increased entry of grB into the effector
cell cytoplasm, and that degranulating cells consequently require
higher levels of PI-9.
Since PI-9 is up-regulated in response to T cell degranulation in
vitro, its expression was also investigated in activated T cells in
vivo by immunohistochemical analysis of normal and inflamed
human tissues. When normal spleen was examined using the spe-
cific PI-9 mAb, 7D8, in standard immunohistochemical proce-
dures, no PI-9-positive lymphocytes were detected (Fig. 6, b and
c). However, when lymphocytes were extracted from freshly iso-
lated spleen and stained for PI-9 using the more sensitive tech-
nique of indirect immunofluorescence, PI-9-positive cells were ap-
parent (Fig. 6a). This indicated that the level of PI-9 expressed in
CLs. A, Rabbit anti PI-9 polyclonal antisera prefer-
entially recognize complexes of PI-9 and grB as well
as complex degradation products. Lysates of COS
cells expressing PI-9 were incubated in the presence
(?) or the absence (?) of excess grB, and samples
were detected by immunoblotting with rabbit anti-
PI-9 antisera R12 or R15, or the anti-grB mAb 2C5.
that the Ab can detect grB in complex. B, Detection of
preformed complexes in cytolytic cells. YT cells were
incubated in the presence (?) or the absence (?) of a
proteosome inhibitor (Prot. Inhib.), then lysed in NP40
or SDS lysis buffer. Cell lysates were immunoblotted
with rabbit 15 antiserum, then stripped and reprobed
with rabbit 12 antiserum. The position of uncomplexed
PI-9 is arrowed, and the positions of the complex
and primary degradation products are bracketed. Mi-
nor complex degradation products (?) are indicated
on the right.
Detection of preformed complexes in
809The Journal of Immunology
quiescent lymphocytes is below the level of immunohistochemical
detection using this Ab. By contrast, when we examined inflamed
tissue samples by immunohistochemistry, PI-9-positive cells were
clearly evident within lymphocytic infiltrates. For example, PI-9-
positive lymphocytes were observed in sections of a ductal carci-
noma of the breast (Fig. 6d) that were also positive for grB (Fig.
6e). Thus, PI-9 is up-regulated in grB-expressing effector cells
Expression of PI-9 in DC populations
In addition to protecting CLs from endogenous grB, PI-9 should
protect accessory and bystander cells from exogenous grB released
during the immune response. Supporting this idea, PI-9-positive
DCs were evident in tonsillar germinal centers using the immuno-
histochemical procedures described above (Fig. 6, g and h). PI-9-
positive DCs were also detected by two-color immunofluorescence
in the thymus. These thymic PI-9-positive cells were CD3?CD4?
CD45?(Fig. 6, j–l) and CD8?(data not shown) with DC mor-
phology and were located in the keratin-negative medulla. The
morphology and phenotype of these PI-9-positive DCs are consis-
tent with several populations of human thymic DCs recently de-
scribed (46). It is important to note that no PI-9-positive lympho-
cytes were detected in either tonsil or thymus, suggesting that DCs
constitutively express higher levels of PI-9 compared with resting
or immature lymphocytes.
Since immunohistochemistry is qualitative, highly purified DC
populations were isolated from whole blood to assess the relative
levels of PI-9. Consistent with the FACS data, low levels of PI-9
were present in monocytes purified from PBLs. IL-4- and GM-
CSF-induced differentiation of these cells into immature MDDCs
resulted in a significant increase in PI-9 expression (Fig. 7A). Mat-
uration of MDDCs by exposure to TNF-? resulted in a further
increase in the expression of PI-9 (Fig. 7B). By densitometric anal-
ysis, this increase in PI-9 in MDDCs was ?4-fold over monocytes
and increased a further 3.5-fold upon maturation with TNF-?.
To identify specific DC types that express PI-9, subpopulations
were purified from whole blood and examined for PI-9 by immu-
noblotting. The CD16?monocytic precursors of tissue DCs (47,
48) expressed higher levels of PI-9 than CD16?monocytes (Fig.
7B). Lineage-negative DCs purified from peripheral blood ex-
pressed PI-9 at an intermediate level between monocytes and
MDDCs (Fig. 7A). These lineage-negative DCs contain two major
populations: CD1b/c?Langerhans cell precursors (38) and
CD123?plasmacytoid DCs (39). The plasmacytoid DCs ex-
pressed an intermediate level of PI-9, while Langerhans cell pre-
cursors expressed virtually no PI-9 (Fig. 7B).
The differential expression of PI-9 in DC populations from both
the myeloid and lymphoid lineages is illustrated in Fig. 7C. The
distribution of PI-9 in DC subsets suggests that expression is not
limited by ancestry, and that differing requirements for protection
from grB during Ag presentation to naive CD8?T cells during
induction of effector functions determines PI-9 expression.
Cells have evolved a number of mechanisms to control apoptosis.
For example, inhibitor of apoptosis proteins inhibit caspases di-
rectly (49), cellular FLIP blocks Fas-mediated apoptosis (50), and
Bcl-2 family members regulate apoptosis at the mitochondria (re-
viewed in Refs. 51 and 52). We and others believe that PI-9 and
other intracellular serpins are part of the anti-apoptotic machinery
of cells involved in or exposed to the cellular immune response
(23, 41, 53, 54). In particular, we have proposed that PI-9 protects
cells against misdirected grB (19, 21, 22, 24).
Expression, regulation, and function of PI-9 in CLs
A role for PI-9 in protecting grB-expressing cells is supported by
its unusual intracellular distribution, described in the first part of
this study. In addition to its cytoplasmic and nuclear localization
(20), PI-9 in CLs is clearly associated with grB-containing cyto-
toxic granules. Fractionation studies indicate that PI-9 is not
present within granules (19). Other studies show that, like PI-6
(55), when PI-9 is provided with an efficient signal peptide, it is
retained in the endoplasmic reticulum and cannot move through
the secretory pathway (A. Gillard and P. Bird, unpublished obser-
vations). Therefore, PI-9 must associate with the external, cyto-
plasmic face of the granule through interaction with a specific
granule component. Our demonstration that PI-9 does not associate
with lysosomes in epithelial cells suggests that it interacts with a
protein or other component not found on nonsecretory lysosomes
or on other membrane-bound organelles. For example, it may bind
a specific lipid, as the lipid content of the granule membrane differs
from that of the plasma membrane and other organelles (56).
transduced with bicistronic retroviral vectors containing a PI-9 cDNA in
the forward (coding) or reverse (noncoding) direction in a transcriptional
unit also encoding GFP. A, Relative levels of PI-9 mRNA in GFP-positive
PBLs. Results are expressed as the concentration of PI-9 mRNA/the con-
centration of GAPDH mRNA, and are the mean ? SEM of four determi-
nations from two separate experiments. Cells transduced with PI-9-coding
virus expressed more PI-9 mRNA than those transduced with noncoding
virus (??, p ? 0.02). B, Specific CTL lysis by PBLs after transduction with
retrovirus. These values represent the specific lysis of OKT3 targets minus
the lysis of JY targets (?6%) and thus reflect killing by TCR-positive
CTLs. Results are the mean ? SEM of four wells. CTLs that overexpressed
coding PI-9 mRNA gave greater percent specific lysis than CTLs trans-
duced with noncoding control virus (??, p ? 0.02; ?, p ? 0.05). Similar
results were found in two separate experiments.
PI-9 enhances the potency of human CTLs. PBLs were
810 EXPRESSION AND REGULATION OF PI-9 IN CLs AND DCs
GrB is stored in granules in an active form (57); thus, leakage
from granules or from the synapse would allow it to access nu-
cleocytoplasmic substrates and kill the CL. The positioning of PI-9
at the granule surface and of the PI-9/grB complexes evident
within CLs suggests that granules do leak and that PI-9 is present
to rapidly inactivate extragranular grB. Once granule exocytosis is
triggered, the granules are refilled with newly synthesized grB;
however, some of the newly synthesized grB is constitutively se-
creted as a 35-kDa form (45). The 35-kDa form of grB is specif-
ically associated with degranulating cells, so the correlation we
report with a 3-fold increase in PI-9 strongly suggests that grB
misdirection occurs during T cell degranulation. While this in vitro
system probably does not fully recapitulate the situation in vivo,
our demonstration of an increase in CTL killing efficiency on 3- to
vated lymphocytes and DCs. a, Indirect im-
munofluorescence of PI-9 (green) in lympho-
cytes isolated from normal spleen; b, PI-9 is
undetectable by immunohistochemistry in
formalin-fixed, paraffin-embedded normal
spleen; c, isotype control of a serial section
of the same normal spleen; d, PI-9 in acti-
vated lymphocytes in ductal breast carcino-
ma; e, coexpression of grB in activated lym-
phocytes; f, isotype control; g, localization of
PI-9 to DCs within a tonsillar germinal cen-
ter; h, higher magnification illustrating the
dendritic morphology of the PI-9-positive
cells; i, isotype control; j–l, PI-9-positive
cells within the thymus. PI-9-positive cells
(green) are located within the medulla iden-
tified by the presence of Hassall corpuscles
(HC). PI-9 does not colocalize with CD3?
thymocytes (j, red), but slight colocalization
is noted where cytosolic PI-9 overlaps with
membrane CD45 (k, red) and CD4 (l, red)
expressed on DCs.
Expression of PI-9 in acti-
(10 ?g/ml and 10 ng/ml, respectively; B), or culture-generated quiescent NK cells (C) were activated in the presence of irradiated MM-170 cells and 200
U/ml IL-2. On the indicated days cells were harvested, and lysates were prepared. Cells (0.5 ? 106) loaded/lane) were resolved by reducing SDS-PAGE
and sequentially immunoblotted with mAbs to PI-9 (7D8, hybridoma supernatant diluted 1/10), grB (2C5, diluted 1/2000), and an antiserum to actin (diluted
1/1000). FACS analysis indicated that 40% of IL-2-stimulated T cells and 64% of Con A/PMA-stimulated T cells were CD8?. ?, Presence of the 35-kDa
form of grB. Densitometric analysis was performed on immunoblots from four separate stimulations, and the relative levels of PI-9 are plotted.
Regulation of PI-9 expression in CLs. PBMCs were activated in the presence of 100 U/ml IL-2 (A) or a combination of Con A and PMA
811 The Journal of Immunology
5-fold overexpression of PI-9 suggests that the similar fold up-
regulation seen in Con A/PMA-treated cells is physiologically rel-
evant. We therefore conclude that CTL viability is enhanced by a
PI-9-mediated reduction in suicide or fratricide induced by
What might cause granule leakage? Granules are secretory ly-
sosomes containing lysosomal hydrolases in addition to granzymes
and perforin (58–60). While there is no direct evidence that gran-
ules leak, it is clear that lysosomal rupture can be induced by
stressors such as oxidation and UV irradiation, leading to apoptosis
and necrosis (61, 62). Increased levels of reactive oxygen species
have been observed in CLs, which may lead to apoptosis (63). This
suggests that granule leakage occurs during effector cell activation
Release of grB into the effector cell cytoplasm need not only
occur from leaking granules. It is possible that secreted grB is
endocytosed by effectors or bystanders. The uptake of grB into
target cells is thought to be mediated by the 300-kDa M6PR (2).
The M6PR is expressed in all nucleated cells, with up to 20% of
the receptor present at the cell surface (64–68). Interestingly, the
M6PR is up-regulated on activated T cells (69), which might in-
crease their susceptibility to secreted grB.
Role of PI-9 in accessory cells
The cellular immune response involves a complex interplay be-
tween many cell types. APC or accessory cells (DCs, macro-
phages, and B cells) induce differentiation of naive T cells into
cytotoxic or Th lymphocytes by secretion of cytokines and expres-
sion of costimulatory molecules. These accessory cells are closely
associated with CLs and are likely to be exposed to collateral dam-
age mediated by grB and perforin during the immune response.
The presence of PI-9 would provide protection against inadvertent
killing of these cells.
How likely is such inadvertent killing? DCs have established
roles in presenting Ag to CD4?Th cells and eliciting Th1 (CTL)
or Th2 (B cell) responses. However, distinct DC subpopulations
also directly interact with B cells or CD8?CTLs. For example,
follicular DCs in the germinal center directly contribute to B cell
proliferation and differentiation (70), while virally infected DCs
and DCs purified from blood can directly present to CD8?T cells
in the absence of CD4?Th cells (71–74).
This close association of activated CTLs and DCs may result in
elimination of DCs by effector CTLs (33, 75). Elimination of DCs
in normal mice is unusual, presumably due to protective mecha-
nisms, but elimination of Ag-specific DCs by cognate CTLs has
been observed in transgenic mouse models (76, 77). Although this
demonstrates that DCs are potentially susceptible to CTLs, the
mechanism of CTL-dependent clearance of DCs is unclear. One
study found DC elimination to be independent of Fas and perforin
(77), while another reports that it is mediated partly by the perforin
pathway (78). Consistent with the latter results, we and others have
suggested that expression of PI-9 in DCs prevents grB-mediated
apoptosis during Ag presentation to CTLs (23, 53). This is further
supported by the recent report that SPI6, one of seven murine PI-9
homologues (79), protects murine DCs from CTL-induced apopto-
Our results show that PI-9 is expressed in specific DC subsets.
Thymic medullary DCs comprise three different subsets: a major
monocytes (sMono), lineage-negative DCs (lin?DC), monocyte-derived DCs (MDDC), and T cells; B, sorted monocytes were further divided into CD16?
and CD16?populations, lineage-negative DCs were divided into CD1b/c?(Langerhans cell precursors) and CD123?(plasmacytoid DCs), and MDDCs
were cultured in the presence (?) or the absence (?) of TNF-? (10 ng/ml) for 2 days. Samples were resolved by 12.5% reducing SDS-PAGE, transferred
to nitrocellulose, and immunoblotted for PI-9 with 7D8 hybridoma supernatant diluted 1/10, detected with HRP conjugated anti-mouse Ig, and ECL. Blots
were stripped and reprobed for actin (diluted 1/1000). C, Simplified diagram illustrating the differentiation of DCs from either a myeloid or a lymphoid
precursor. Boxed populations were assessed for PI-9 by immunoblotting and immunohistochemistry (shades of gray represent relative levels of PI-9).
Checkered boxes indicate that while PI-9-positive DCs were observed in the thymus, the phenotype of these DCs is not yet known. Dashed arrows indicate
ex vivo stimulation. Data in this figure are modified from Ref. 94 with additional information from Refs. 46, 48, 81, and 95.
Expression of PI-9 in primary DCs and MDDCs. A, Fifty micrograms of cell lysates were prepared from elutriated monocytes (Mono), sorted
812EXPRESSION AND REGULATION OF PI-9 IN CLs AND DCs
CD11b?subset of lymphoid origin, a minor CD11b?subset of
myeloid origin (46), and a population of plasmacytoid DCs (80).
The CD11b?thymic DCs resemble tonsillar germinal center DCs
(81) and are thought to be phenotypically and morphologically
related (46). Considering this relationship, it is likely that the PI-
9-positive DCs observed in the thymus are related to the PI-9-
positive DCs located in the tonsil. Both these DC populations are
probably derived from CD16?myeloid precursors (47, 48, 82).
PI-9 is highly expressed in CD16?monocytes, which is consistent
with PI-9 expression in the CD11b?thymic DCs and tonsillar
germinal center DCs.
The differential expression of PI-9 in DCs suggests differing
requirements for protection from grB-mediated apoptosis in DC
subsets. Thymic medullary DCs are essential in the positive and
negative selection of thymocytes. GrB-positive cells are present in
the thymic medulla, with grB transcripts detected in double-posi-
tive (CD4?CD8?) thymocytes (83) and in both CD4?or CD8?
single-positive thymocytes (84). This suggests that thymocytes un-
dergoing selection express grB and have cytotoxic potential. Thus,
the presence of PI-9 in thymic medulla DCs is consistent with a
role in protecting these DCs from grB-mediated apoptosis.
Some subsets of germinal center DCs are involved in the pre-
sentation and activation of T cells directly (81), while plasmacy-
toid DCs are IFN-producing cells (85–87) that initiate potent Th1
(CTL) responses (88). The expression of PI-9 in these cells sug-
gests that DC populations that present to and activate CD8?pre-
cursor T cells are at risk from the effector functions of the T cells
they activate. This is also supported by the up-regulation of PI-9 in
MDDCs upon TNF-?-induced maturation, suggesting that mature
APC require protection from inadvertent apoptosis.
Role of PI-9 in other cells
It would be advantageous for other cell types to express PI-9 to
protect against misdirected grB during the immune response. For
example, PI-9 is expressed in endothelial and mesothelial cells
likely to be exposed to CLs and is up-regulated by inflammatory
stimuli (24, 89). The expression of grB and perforin has also been
demonstrated in human CD4?CTLs (90, 91), suggesting that PI-9
has a cytoprotective role in some CD4?T cells. The level of PI-9
expressed in B cells, monocytes, and granulocytes is lower than in
CLs or DCs, and it is unlikely that they would be protected from
direct CTL attack. Hence, these cells have sufficient PI-9 to cope
with low levels of misdirected grB, but could still be cleared by
CTLs if they become infected or tumorigenic.
Two recent papers have indicated a role for PI-9 in tumor eva-
sion by providing neoplastic cells with an advantage against grB-
mediated cytotoxicity (92, 93). However, both studies failed to
establish the baseline expression of PI-9 in tissues and cells or to
compare the level of PI-9 expression in normal and tumor samples.
The expression of PI-9 in epithelial cells has been reported (24),
and PI-9 is in the ductal tissue of the breast, the columnar epithelia
of the colon, and the ciliated columnar epithelia lining the female
reproductive tract (M. Buzza, unpublished observations). The pres-
ence of PI-9 in carcinomas of breast, colon, and cervix (92) is
therefore not surprising.
Additionally, the work reported here shows that PI-9 is widely
distributed in normal leukocytes, which may account for the pres-
ence of PI-9 in T, B, and Hodgkin lymphomas (93). Analysis of the
latter data indicates that only 28% of 224 biopsies were positive
for PI-9. Furthermore, the number of PI-9-positive cells within
each biopsy varied, with only 17% containing a majority of PI-9-
positive tumor cells. The lack of PI-9 in most of these lymphomas
suggests it is unlikely that PI-9 up-regulation is a common mech-
anism by which lymphomas resist immune destruction.
We thank J. Sun for recombinant grB, J. Trapani (Peter MacCallum Cancer
Institute, Melbourne, Australia) for grB Abs, A. Pshezhetsky (University of
Montreal, Montreal, Canada) for cathepsin A Abs, P. Hosking (Department
of Anatomical Pathology, Box Hill Hospital, Melbourne, Australia) for
archival tissues, and R. Boyd and M. Malin (Department of Pathology and
Immunology, Monash University, Clayton, Melbourne, Australia) for thy-
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815 The Journal of Immunology