
T cells of human early pregnancy decidua: evidence for cytotoxic potency
Lucia Mincheva-Nilsson,
Olga Nagaeva,
Karl-Gösta Sundqvist,
Marie-Louise Hammarström1,
Sten Hammarström1 and
Vladimir Baranov1
Departments of Clinical Immunology and
1 Immunology, Umeå University, 90185 Umeå, Sweden
Correspondence to:
L. Mincheva-Nilsson
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Abstract
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The immune compromise in decidua allows a semiallogeneic fetus to survive without impairing the ability of the maternal immune system to fight infections. Cytotoxic mechanisms are likely to be important in this compromise. Using RT-PCR, immunoflow cytometry and immunoelectron microscopy, the cytotoxic potential of isolated human decidual 
T cells was studied. mRNA for perforin (Pf), granzymes A and B, granulysin and Fas ligand (FasL) was simultaneously expressed in decidual 
T cells. Pf and FasL were not expressed on the cell surface. However, the cells constitutively synthesized Pf and stored it in cytolytic granules. Within the granules Pf mainly resided in the granule core formed by Pf-containing microvesicles. Ultrastructurally, three groups of Pf-containing granules were distinguished. They probably represent different stages of granule maturation in a process where Pf-containing microvesicles first attach to the core cortex and then are translocated across the cortex into the core. Presynthesized FasL was also stored in the core and microvesicles of the cytolytic granules. Upon degranulation by ionomycin/Ca2+ treatment, FasL was rapidly translocated to the cell surface, demonstrating that its surface expression was not controlled by de novo biosynthesis. Thus decidual 
T cells appear to perform Pf- and FasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granule exocytosis. The first cytochemical visualization of lipids in the cytolytic granules is provided. These intragranular lipids probably wrap up the core and participate in packaging of the cytotoxic proteins as well as in the killing process. An ultrastructural model of a cytolytic granule is presented.
Keywords: cytotoxic granules, Fas ligand, granulysin, granzyme A, granzyme B, immunoelectron microscopy, lipids, perforin
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Introduction
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Cytolytic granule exocytosis and Fas ligand (FasL)Fas cross-linking are two major mechanisms utilized by cytotoxic T lymphocytes (CTL) and NK cells to kill targets via apoptosis (reviewed in 1). The simplified granule exocytosis model (2) of lymphocyte-mediated cytotoxicity postulates that after effector activation the lethal hit is initiated by target-directed secretion of cytoplasmic granules. These granules contain key cytolytic moleculesperforin (Pf) and several serine proteases known as granzymes (Gr) (3). Upon exocytosis, Pf and Gr function in synergy: Pf undergoes polymerization and forms pores on the target-cell membrane allowing the entry of Gr, such as GrA and GrB, which trigger nuclear damage and cell death. Another recently described constituent of the cytolytic granules is the human protein granulysin (4) and its porcine homologue NK-lysin (5). The second mechanism of lymphocyte-mediated cytotoxicity is believed to require binding of FasL (CD95L) on the activated effector cells to its cell-death transducing receptor, Fas (CD95), on the membrane of the target cells (6,7). Membrane-bound FasL can be actively cleaved by a metalloproteinase-like enzyme (8). Many cell types express Fas, whereas FasL expression is transient and rapid, and is mainly restricted to activated immune cells (for review, see 9). It has recently been shown that FasL is also constitutively expressed in some non-lymphoid tissues which are associated with immune privilege status (10,11).
Accumulated evidence from in vitro and in vivo studies (1215) shows that both cytotoxic mechanisms play a crucial role in the clearance of viral and bacterial infections, tumor surveillance, transplant rejection, homeostatic regulation of immune responses, and peripheral tolerance (16,17). These cytotoxic mechanisms may play an important role in the pregnant uterine mucosa, the decidua of mammals, e.g. by protecting the maternalfetal unit against pathogens, controlling invasion of fetal extravillous trophoblast and creating a local transient immunotolerance toward the semiallogeneic conceptus. Indeed, recent studies in Pf- and FasL-deficient mice have shown that although functional deletion of Pf or FasL alone does not appear to affect fertility, the combined absence of these two effector molecules induces infertility (17,18). Most of the knowledge about lymphocyte cytotoxicity in decidua has been limited to the human NK-like CD56+bright large granular lymphocytes (19,20). In a previous study we presented evidence indicating that CD56+bright cells are progenitors of decidual 
T cell (21). 
T cells in decidua were shown to specifically colonize non-pregnant murine endometrium, and to constitute a major cell population in murine, sheep, horse and pig decidua (2226). Human 
T cells comprise about half of all decidual T cells in early pregnancy (21, 27). The vast majority of them are V
1+, CD4CD8, and express CD56 (21,27). Ultrastructurally, human decidual 
T cells have the distinctive morphology of large granular lymphocytes (27,28). It has been shown that murine decidual 
T cells specifically recognize a conserved trophoblast antigen (29) and suppress the maternal anti-fetal response probably by transforming growth factor-ß production (30). Recently, several reports have suggested a regulatory role for the murine decidual 
T cells in abortions via the release of abortogenic or anti-abortive cytokines (31,32). In humans, 
T cell clones from decidua have been shown to mediate strong non-MHC restricted cytotoxicity (33).
The aim of the present study is to evaluate the cytotoxic potency of the 
T cells using the expression of Pf, GrA, GrB, granulysin and FasL as markers of cytotoxicity.
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Methods
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Decidual samples and mAb
Vacuum-extracted decidual specimens were donated by healthy women undergoing elective termination of pregnancy at 814 weeks of gestation. The specificity and sources of the mAb used in this study are listed in Table I.
Isolation of decidual mononuclear cells (DMC)
DMC were prepared as described previously (21). Contaminating epithelial cells were removed using mAb BerEP4-coupled immunomagnetic beads (Dynabeads M-450; Dynal, Oslo, Norway). The isolated cell population contained on average <1% BerEp4+ cells (21) as judged by flow cytometry. DMC were further depleted of B cells and monocytes using immunomagnetic beads charged with anti-CD19 and anti-CD14 mAb. For RT-PCR, decidual 
T cells were obtained by positive selection using immunomagnetic beads labeled with mAb specific for TCR
. The cells bound to the beads were washed 5 times in 1020 volumes of ice-cold PBS. A minimum of 10001500 cells/sample was counted in a light microscope to ascertain that only cells bound to beads were present in the selected population. The cells were lysed while attached to the beads and subsequently total RNA was extracted.
Immunofluorescence staining and flow cytometric analysis of Pf expression in 
T cells
DMC, preincubated with normal goat serum for 15 min on ice, were stained with FITC-conjugated anti-TCR
mAb (a mixture of clones TCR
1,
TCS1 and V
1). After washing, the cells were fixed in 1.5% paraformaldehyde (Merck, Darmstadt, Germany), permeabilized with 0.1% saponin and stained with phycoerythrin (PE)-conjugated anti-Pf mAb. Appropriately labeled, isotype-matched irrelevant mAb were used as negative controls. Flow cytometric analyses were performed on FACScan flow cytometer equipped with a single argon ion laser (Becton Dickinson), calibrated with CaliBRITE Flow Cytometer Beads (Becton Dickinson). The data were analyzed and displayed as dot-plots using Lysys II software.
Immunofluorescence staining and flow cytometric analysis of cell surface and intracellular FasL expression in 
T cells
The DMC were preincubated with normal goat serum containing 1 mM of the metalloproteinase inhibitor 1,10-phenanthroline monohydrate (Sigma, St Louis, MO) for 1 h on ice. The cells were stained with a mixture of FITC-conjugated anti-TCR
mAb, washed and incubated with biotinylated anti-FasL mAb (NOK-1 or H11) followed by streptavidinPE (Dako). 1,10-Phenanthroline was present throughout. For intracellular staining of FasL, the cells were incubated with anti-TCR
mAb, fixed in 1.5% paraformaldehyde and permeabilized with 0.1% saponin for 15 min. The cells were then stained for FasL and analyzed by flow cytometry.
Flow cytometric analysis of FasL release by degranulated 
T cells
DMC were pretreated for 1 h at room temperature with 1 mM 1,10-phenanthroline and stained for TCR 
with FITC-labeled mAb in the presence of 0.02% NaN3. After washing, the cells were incubated with a mixture of 5 µg/ml ionomycin, 0.2% HSA, 1 mM 1,10-phenanthroline and 1 mM CaCl2 in PBS, pH 7.2 (34), and biotin-conjugated anti-FasL mAb for 15 min, followed by a fixation in 1.5% paraformaldehyde for 10 min. Thereafter, the cells were stained with PEstreptavidin. Appropriately labeled, isotype-matched irrelevant mAb were used as controls for non-specific fluorescence.
Total RNA extraction and RT-PCR
Lysates from immunomagnetic-bead selected 
T cells were used to extract total RNA by the acid guanidinum thiocyanatephenolchloroform method (27). The isolated RNA samples were analyzed by RT-PCR with specific primers for Pf (35), GrA (35), GrB (forward primer 5'-CAT GCT ACT GCA GCT GGA G-3' and reverse primer 5'-TCC AGA GCT CCC CTT AAA G-3'), FasL (36), granulysin (forward primer: 5'-TAA GCC CAC CCA GAG AAG TGT-3' and reverse primer 5'-TAA GCC CAC CCA GAG AAG TGT-3') and ß-actin (27). The primer sequences were located in different exons so that amplification of mRNA could be distinguished by the size of the amplified product. Single-strand cDNA copies were made from 1 µg of total RNA using random hexamers and murine leukemia virus reverse transcriptase (Perkin-Elmer, Roche Molecular Systems, Nutley, NJ). Reverse transcription was performed at 42°C for 15 min followed by denaturation at 99°C for 5 min. Subsequent PCR was performed in a total volume of 100 µl in a reaction mixture containing 1.0 U of Taq DNA polymerase (Promega) in 2.5 mM MgCl2, 10 mM Tris (pH 8.3), 50 mM KCl, 0.01% gelatin (w/v) supplemented with 0.25 µM each of dATP, dGTP, dCTP and dTTP, and 0.20 µM of each primer. The following thermocycle programs were used: for Pf and GrA: 35 cycles of 95°C for 1 min, 62°C for 30 s and 72°C for 2 min; for GrB: 35 cycles of 94°C for 20 s, 55°C for 20 s and 72°C for 30 s; for FasL: 40 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 1 min; for granulysin: 38 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s.
Immunoelectron microscopy (IEM) procedure for simultaneous detection of TCR 
and Pf in isolated DMC
The pre-embedding immunoperoxidase technique was used to co-localize Pf and TCR 
in the same cell. This immunostaining procedure provides reliable results when the antigens are known a priori to be separately located. DMC were incubated with a mixture of anti-TCR 
mAb (TCR
1, V
1 and
TCS1) in 0.02 M PBS, containing 0.2% BSA for 30 min on ice, washed and fixed in 1.5% paraformaldehyde for 15 min. After washing with 0.1 M glycine the cells were permeabilized with 0.05% saponin for 30 min and incubated with anti-Pf mAb for 2 h. After washing the cells were consecutively incubated with biotinylated sheep anti-mouse IgG F(ab')2 (Amersham, Little Chalfont, UK) and peroxidase-conjugated streptavidin (Jackson ImmunoResearch, West Groove, PA). The peroxidase activity was revealed with a substrate containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.03% H2O2. Finally, the cells were fixed in 1.33% OsO4 for 30 min, dehydrated in acetone, and embedded in a mixture of Epon and Araldite (Fluka, Buchs, Switzerland). Ultrathin sections were examined in a Zeiss EM 900 electron microscope (Carl Zeiss, Oberkochen, Germany). The specificity of IEM staining was confirmed by replacing primary mAb with isotype-matched normal mouse IgG.
Electron microscopic localization of lipids in cytolytic granules of 
T cells
The lipid staining was carried out with malachite green (37). A 
T cell fraction of DMC was obtained by a negative selection procedure using immunomagnetic beads charged with anti-CD14, anti-CD19, anti TCR
ß and anti-CD56 mAb. The fractionated cells were fixed in a mixture of 3% glutaraldehyde (Sigma) and 0.1% malachite green in 0.1 M sodium cacodylate buffer, pH 6.8, for 30 min at room temperature. After washing in the same buffer, the cells were fixed in 1% OsO4 for 30 min and embedded in EponAraldite. Ultrathin sections were examined without an additional staining.
Ultrastructural analysis of Pf-containing cytolytic granules in 
T cells on serial sections
Short ribbons of ultrathin sections (6570 nm of thickness) were collected on formvar/carbon-coated single-hole grids. The diameter of the hole (0.8 mm) allows us to analyze a series of four to five consecutive sections of one cell, representing 260350 nm of total thickness. Since the estimated average diameter of the granule cores was 400600 nm (see Results), four to five serial ultrathin sections of 70 nm thickness should give images covering at least half the core volume. Cells exhibiting a variety of granule core types were selected and photographed in consecutive sections.
IEM procedure for double labeling of TCR 
and FasL molecules in isolated DMC using a combination of immunogold/silver and immunoperoxidase methods
Our modification of a double-labeling technique, employing two murine mAb in the same sample, has been described previously (27). In brief, DMC were incubated with anti-TCR 
mAb for 30 min on ice and fixed in 1.5% paraformaldehyde for 15 min. Washed cells were incubated with goat anti-mouse IgG coupled to ultra-small gold particles (AuroProbe One GAM; Amersham) and fixed again in the same fixative for 10 min, followed by incubations with 5% normal mouse serum and unconjugated goat anti-mouse IgG Fab (Jackson ImmunoResearch). After washing, the second primary mAb, anti-FasL (clone G247-4, PharMingen/Becton Dickinson), was applied, followed by an incubation with biotinylated sheep anti-mouse IgG F(ab')2 (Amersham) and peroxidase-conjugated streptavidin (Jackson ImmunoResearch). The peroxidase activity (i.e. FasL) was detected with DABH2O2 and the cells were fixed with 0.5% OsO4 for 25 min. Finally, after washing in distilled water, the gold particles (showing TCR 
location) were silver-enhanced (IntenSE-M; Amersham) according to the instructions of the manufacturer.
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Results
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Decidual 
T cells contain mRNA for five key cytolytic molecules
RT-PCR for Pf, GrA, GrB, granulysin and FasL mRNA was performed on total RNA extracted from positively selected 
T cells (Fig. 1
). mRNA for all five cytotoxic molecules were simultaneously identified in almost all individual samples (Pf 19 of 22; GrA 18 of 20; GrB 22 of 26; granulysin 20 of 20; FasL 18 of 20).
Pf is expressed intracellularly in decidual 
T cells
A representative flow cytometric experiment using fixed and permeabilized DMC stained with anti-TCR
and anti-Pf mAb is shown in Fig. 2
(A). The vast majority of the 
T cells (constituting 14 ± 6% of DMC, n = 15) expressed Pf intracellularly as did almost all of other DMC including the CD56+bright cells (constituting 55 ± 13% of DMC, n = 15) and the
ß T cells (data not shown). Monocytes/macrophages were not present in the gated DMC (Fig. 2B
). Unpermeabilized DMC did not stain for Pf (data not shown).
IEM analysis of Pf expression in 
T cells
Single-staining control experiments with anti-TCR
mAb or anti-Pf mAb showed that TCR 
was located only on the cell surface while Pf was present exclusively inside the cells (data not shown). After double immunoperoxidase staining of isolated DMC, 
T cells were clearly identified by the peroxidase reaction product distributed over the entire cell surface, indicating the localization of TCR 
(Fig. 3A
). In the majority of 
T cells the reaction product simultaneously stained the perinuclear space, cisternae of rough endoplasmic reticulum and Golgi complex, reflecting the Pf synthesis pathway (Fig. 3A and B
). However, the most prominent morphologic feature of 
T cells was the frequent presence of cytoplasmic granules, which were also labeled by the reaction product (Fig. 3A
). The granules were membrane delimited and showed two ultrastructurally distinct compartments (Fig. 3B
): a polymorphic core, which was surrounded by an electron-translucent area with microvesicles of 2040 nm in size. Within the granules, the reaction product selectively stained the core and microvesicles (Fig. 3B
). The number of the Pf-containing granules varied among 
T cells. Some cells displayed only single small granules, although these cells exhibited generally positive labeling of protein-synthesizing organelles (Fig. 3C
). A small proportion of 
T cells showed weak Pf expression and did not contain any cytoplasmic granules (Fig. 3D
). The latter was confirmed by serial sections of this cell type (data not shown). Thus, the vast majority of isolated 
T cells actively synthesize and store Pf in the granule cores and microvesicles.
Microarchitecture of the cytolytic ranules in 
T cells
In random ultrathin sections there were obvious variations in the images of the Pf-containing granules both within individual 
T cells and between cells (Fig. 3B and C
). To determine if the various images of the cores were truly different or if they were the result of sectioning non-homogeneous cores in different planes, a serial sectioning technique was used. We screened 60 Pf-containing granules. Based on the appearance of the core in consecutive sections the granules could be divided into three main groups. The first, most abundant group (31 granules) displayed cores consisting of two morphologically distinct regions: a central, electron-translucent space, surrounded by a cortical rim, or cortex, of fine granular material. Serial sections of one such granule are shown in Fig. 4
(AD). The thickness of the cortex was somewhat variable, from 70 to 90 nm. Numerous tightly packed labeled microvesicles seemed to fuse with the outer surface of the cortex forming an electron-dense scalloped layer. Some electron micrographs (Fig. 4C and D
) showed the presence of stained microvesicles at the internal surface of the cortex, suggesting a passage of the microvesicles through it. Twenty of the granules were attributed to the second group (Fig. 4E
H). These granules also exhibited `hollow' cores and a labeled cortex. However, the central translucent space of the cores usually displayed an electron-dense material consisting of confluent Pf-containing microvesicles (Fig. 4F and G
). Lastly, nine of the granules were classified in a third group (Fig. 4I
L). These granules showed only a uniform core which was intensely stained by the reaction product and never contained a discernible `hole'. The grainy textured core material seemed to consist of tightly packed Pf-containing microvesicles.

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Fig. 4. Representative immunoelectron micrographs showing selected serial sections through three main groups of cytolytic granules, stained with Pf-specific mAb. (AD) Granule of group 1. Note the solid structure of the core (arrow) in the first section (A) and a central electron-translucent space (asterisk) of the core in sequential sections (BD). The core cortex is positively stained (BD; arrows). Pf-containing microvesicles are present at the outer (A and B; arrowheads) and internal (C and D; arrowheads) surface of the cortex. Magnification x40,000. (EH) Granule of group 2. This granule also exhibits a hollow core (asterisk) and shows a labeled cortex (arrows). A central transparent space of the core contains clusters of tightly packed Pf-containing microvesicles (FH; arrowheads). Magnification x40,000. (IL) Granule of group 3. In all sections this granule shows a solid core (arrows), which is stained by the reaction product and seems to be formed by confluent Pf-containing microvesicles (arrowheads). Magnification x40,000.
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The cytolytic granules of the 
T cells contain lipids
It was reasonable to assume that the `hollow' core structure resulted from lipid extraction during acetone treatment in the process of sample embedding. We therefore undertook a cytochemical staining of lipids by malachite green. Indeed, we found lipids inside the majority of cytoplasmic granules, but surprisingly the lipids were located outside of the core (Fig. 5A
). In random sections, single electron-dense lipid drops were located between the granule-limiting membrane and the core. Further, images of lipid-containing granules in serial sections (Fig. 5B
E) suggested that intragranular lipids wrap up the core.
Freshly isolated decidual 
T cells express FasL intracellularly but not on the cell surface
DMC were double labeled with anti-TCR 
mAb and anti-FasL mAb (clones H11 or NOK-1) and analyzed by flow cytometry. The cells were either non-permeabilized (FasL surface expression, Fig. 6A
D) or fixed and permeabilized (FasL, total cell expression, Fig. 6E
H). Only a minority of 
T cells expressed FasL on their cell surface (Table 2
and Fig. 6A
). Incubation with 1,10-phenanthroline before and during cell staining did not significantly change the level of FasL surface expression (Table 2
). Upon permeabilization, the entire 
T cell fraction as well as other DMC were positively stained for FasL in all experiments (n = 28), showing an intracellular localization of FasL (Fig. 6E
). Identical results regarding surface and intracellular FasL expression were obtained with both mAb.
To define the precise intracellular localization of FasL, isolated DMC were double labeled and analyzed by IEM. TCR 
was detected by immunogold/silver staining and FasL by immunoperoxidase staining. The results of IEM showed that the surface of the 
T cells was labeled by clusters of gold/silver particles and was not stained by the peroxidase reaction product (Fig. 7A
). The only intracellular compartments containing deposits of the peroxidase reaction product were the cytolytic granules (Fig. 7A
). Inside the granules the reaction product stained a pleomorphic core material and intragranular microvesicles. Several images of the granules in random sections showed binding and possible merging of the FasL-containing microvesicles with the core material (Fig. 7B
D). It is, therefore, reasonable to assume that FasL is transported to the granule core by these microvesicles. Thus, freshly isolated 
T cells contain presynthesized FasL in cytolytic granules and do not express it on the cell surface.
FasL is rapidly mobilized to the cell surface of the 
T cells upon degranulation
The presence of preformed FasL within cytolytic granules suggested that it might be possible to stimulate rapid mobilization of the protein to the cell surface. To test this, DMC were treated with ionomycin, which induces an intracellular Ca2+ influx and triggers cell degranulation within minutes (34). During degranulation, cells were double labeled for TCR 
and FasL and analyzed by flow cytometry. To prevent shedding of FasL from the cell surface, the experiments were performed in the presence of 1,10-phenanthroline. Identical results with both mAb were obtained and a typical degranulation experiment with mAb H11 is illustrated in Fig. 8
. Freshly isolated 
T cells (Fig. 8A
) or DMC (Fig. 8B
) do not express FasL on the cell surface. However, upon ionomycin treatment for 15 min, the fraction of 
T cells (Fig. 8E
) and DMC (Fig. 8F
) expressing FasL on the surface was increased by nearly 100%. In fact, the intracellular staining of FasL demonstrated in Fig. 6
(E and F) strongly resembles the cell surface staining of FasL after degranulation (Fig. 8E and F
), suggesting a shift in FasL expression from the intracellular compartment to the cell surface. Thus, following degranulation, intracellular FasL is rapidly translocated to the cell surface.
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Discussion
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In this study we report a series of observations, which taken together, provide evidence for the cytotoxic potency of decidual 
T cells.
From the RT-PCR experiments we conclude that these cells express mRNA for five marker proteins of cytotoxicity: Pf, GrA, GrB, granulysin and FasL. In general, these results confirm few previous studies of human 
T cells (3841). However, our results concern mucosa-associated V
1+ 
T cells (27) in a physiological situation. Furthermore, this is the first demonstration of granulysin mRNA in 
T cells. The recent reports of antimicrobial and antitumor activity of granulysin (42) and its porcine analogue NK-lysin (5) are in agreement with studies which suggest protective responses of 
T cells in a variety of pathological conditions (43,44).
We have focused on the expression of Pf and FasL as marker proteins of the two major pathways of cytotoxicity. As judged by immunoflow cytometric analyses, intracellular Pf is present in the vast majority of decidual 
T cells, confirming the existing idea that Pf expression by 
T cells is a general phenomenon (38,39,44). Our ultrastructural findings support this proposal and for the first time provide definitive IEM proof that the majority of these cells not only store preformed Pf in cytolytic granules, but actively synthesize it. Pf was found in the nuclear envelope, rough endoplasmic reticulum and the Golgi complex, demonstrating that newly synthesized Pf molecules are targeted via the usual protein biosynthesis pathway.
Our IEM results demonstrate that decidual 
T cells resemble other cytotoxic cells (reviewed in 45) in the domain organization of the cytolytic granules, and confirm the presence of Pf in the granule core and in the intragranular microvesicles. The cytolytic granules are mainly membrane delimited and contain two structural domains: an electron-dense core surrounded by an electron-lucent area with small internal vesicles. Pf and Gr were localized preferentially in the core but also in the region of the small internal vesicles. Furthermore, using the serial section technique in IEM, we were able for the first time to show that the granule core itself has a complex organization. The core displays a thin cortex, formed by a homogeneous material, which surrounds a central electron-translucent space. These findings indicate that Pf-containing microvesicles first attach to the core cortex and then are translocated across the cortex into the core. Thus, the appearance of any granule core seems to depend on the degree of its `filling' with the microvesicles. Peters et al. (46) have proposed an association and a possible fusion of the granule internal vesicles with the cores, and hypothesized that the effector molecules are delivered to the granule core as a result of this fusion. However, further work will be needed to answer the question of the origin of the intragranular Pf-carrying microvesicles.
It is difficult, on the basis of IEM alone, to interpret the relationship between the three groups of cytolytic granules we have distinguished in this study. Nonetheless, three conclusions can be drawn from our analysis. First, it is likely that the characteristic changes in the content of Pf-containing microvesicles within the cores reflect a progressive maturation of the cytolytic granules. The `immature' cytolytic granules of group 1 have relatively few core-invading microvesicles. By contrast, in the `mature' granules of group 3, the microvesicles appear to fill the core completely. Second, cores of the cytolytic granules might be used as major `reservoirs' for Pf-containing microvesicles. Thirdly, the current classification of cytolytic granules, based only on routine electron microscopy and the relative proportion of the core and the translucent space in each granule (45), should be reconsidered.
Our studies provide the first demonstration and precise localization of lipids in the cytolytic granules at the ultrastructural level. Using the malachite green/aldehyde technique, we found that all cytolytic granules contained lipids located in the translucent space between the granule-limiting membrane and the core, wrapping the core. Unfortunately, the nature of the lipids could not be determined, as there are no current electron microscopic methods which discriminate between different types of lipids (37). Our finding is supported by an earlier Raman microspectroscopic study of human LAK cells which demonstrated that cytolytic granules contain highly unsaturated lipids, in contrast to the moderately unsaturated lipids found in plasma membranes (47). It seems that lipids may function at different levels of granule-mediated cell lysis. Inside the cytolytic granules, the lipids wrapping the core may function as an `isolation layer', protecting the CTL from its own harmful effector molecules (47). Upon exocytosis of cytolytic granules into the intercellular cleft between the CTL and the target cell, the lipids may play a dual role. It has been shown that unsaturated fatty acids inhibit degranulation of CTL (48). This effect is explained by fatty acid-induced alteration of the plasma membrane's lipid phase, which most likely perturbs the function of the signaling membrane protein(s). Thus, released granule lipids may serve as autocrine signal molecules ending the current degranulation and preventing a subsequent secondary degranulation. In addition, it is highly probable that such lipid-induced plasma membrane alteration may occur also in the target cell plasma membrane, increasing its susceptibility to attack by cytotoxic proteins (47). However, the intriguing function(s) of granule lipid molecules remains to be elucidated.
A novel ultrastructural model of a cytolytic granule is presented in Fig. 9
. We propose that Pf-carrying microvesicles, formed by budding off the granule-limiting membrane (46), pass through the inner lipid layer and first accumulate around the outer surface of the core cortex. The microvesicles then penetrate the cortex and invade the internal space of the core to varying degrees. Another possibility is that the microvesicles are translocated into the core by invagination of the core cortex. As a result, in some sections the microvesicles appear to invade the core only partially, while in others they occlude the core completely. According to this model Pf is released from killer cells in its microvesicle-bound form and not as a soluble protein. The precise role of this form of release remains to be determined but it seems that it has the advantage of creating a high local Pf concentration, due to the lower diffusion capacity of the microvesicles.

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Fig. 9 . Schematic interpretation of the ultrastructure of a Pf-containing cytolytic granule based on the findings in this study. The cytolytic granule contains a core that has a core cortex (arrows), surrounding an internal translucent space of unknown nature (question mark). The limiting membrane of the granule gives rise to the Pf-containing intragranular microvesicles (arrowheads) by budding off (46). The latter pass through the inner lipid layer (open arrows, only shown in the drawing) and accumulate around the cortex. Then the microvesicles penetrate through the cortex and gradually fill the internal core space. Newly arrived microvesicles continue to be condensed into the core and eventually occupy it completely. (A) Section cut through a layer of Pf-containing microvesicles associated with the core surface. (B) Section cut through the outer microvesicle layer and the core cortex, seen in gray. (C) Section cut through an `empty' core showing stained microvesicles, core cortex and an internal transparent space. (D) Section cut through a core cortex, invaded by stained microvesicles. The latter also occupy the internal space of the core.
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We performed our studies of FasL using mAb NOK-1, H-11 and G247-4, since the specificity of some of the poly- and monoclonal anti-FasL antibodies used earlier has been questioned recently (49,50). According to the existing opinion the cell surface expression of FasL in CTL is not constitutive, and activation via TCRCD3 complex or by phorbol myristate acetate/ionomycin treatment is needed to induce its transient expression (6,7,51). Decidual 
T cells are already activated in vivo (28,44,52); therefore, it is reasonable to expect that FasL is present on the cell surface. However, almost none of the decidual 
T cells displayed FasL on the cell surface when tested by flow cytometry, even in the presence of a metalloproteinase inhibitor (8). In contrast, after fixation and permeabilization of the cells, abundant intracellular expression of FasL was revealed. These results support several previous reports (5357), demonstrating that only intracellular expression of FasL can be revealed in monocytes, granulocytes, mast cells, NK cells and CTL. Recently, Bossi and Griffiths (57) were able to co-localize FasL and Pf in cytolytic granules in a human NK cell line and in CTL clones using confocal microscopy. We have confirmed and further extended their results by demonstrating for the first time at the IEM level the presence of FasL in the core and microvesicles of the cytolytic granules of freshly isolated human decidual 
T cells.
Earlier in vitro observations have suggested that the transient cell surface FasL expression is regulated at the transcriptional level (16). In functional cytotoxicity experiments there is always a lag phase of 34 h, which is interpreted as the time needed for de novo synthesis of FasL (58). Recently, however, Li et al. (56) have demonstrated that FasL-based cytotoxicity is unaffected by inhibition of protein synthesis and/or DNA transcription. This observation is consistent with our IEM results which demonstrate a lack of FasL synthesis in the 
T cells despite its presence in cytolytic granules. It is, thus, becoming increasingly clear that the execution phase of FasL-mediated killing may occur in the absence of protein or RNA synthesis. The transcriptional control of FasL synthesis (16) seems to be required at the first activation of naive CTL, i.e. the initial formation of cytolytic granules, and for a renewal of the latter after degranulation. Thus the lag phase usually observed in cytotoxicity assays (58) is probably due to the rapid release of preformed FasL from the effector cells during the experimental set-up and the subsequent restoration of intracellular FasL.
We conclude that, at the level of the effector cell, FasL exploits the same granule-mediated pathway for the storage of the pre-formed molecules and their translocation to the cell surface as Pf. Although this study has helped to clarify the intracellular localization of FasL in decidual 
T cells, some subjects such as co-expression of Pf and FasL in the same cytolytic granules, the adequacy of granule-stored FasL for efficient target cell death, and the fate of FasL on the cell surface after degranulation remain to be elucidated.
Recent studies in mice and horses have provided evidence that the systemic, MHC-restricted, maternal T cell-mediated response to paternal alloantigens is down-regulated in pregnancy by different, but overlapping, mechanisms (5961). The local alloantigen-specific response in human decidua is probably down-regulated as well (21,28). The suppression of the specific T cell response may be compensated by the non-specific innate immune system in order to meet the particular requirements at the feto-maternal border, i.e. protection against microbial infections, control of trophoblast invasion and creation of local transient allotolerance (62). The 
T cells, as a plausible cellular component of innate immunity possessing the two major mechanisms of cytotoxicity as shown here, may perform one or more of these functions.
 |
Acknowledgments
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We wish to express our sincere gratitude to Professor Thorbjörn Bäckman, Associate Professors Mats-Göran Damber and Othon Lalos, the colleagues at the Department of Gynecology, Umeå University Hospital, for kindly providing decidual samples. This work was supported by grants from the Swedish Medical Society (98020555 to L. M.-N.), the Cancer Research Foundation, Umeå university (AMP 99-213, L. M.-N.), Stiftelsen Lars Hiertas Minne (L. M.-N.), Swedish Natural Science Research Council (M.-L. H.) and a research stipend from the Swedish Institute (V. B.).
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Abbreviations
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CTL cytotoxic lymphocytes |
DAB 3,3'-diaminobenzidine tetrahydrochloride |
DMC decidual mononuclear cells |
FasL Fas ligand |
Gr granzyme |
IEM immunoelectron microscopy |
PE phycoerythrin |
Pf perforin |
 |
Notes
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Transmitting editor: H. Wigzell
Received 1 September 1999,
accepted 6 January 2000.
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