International Immunology, Vol. 12, No. 5, pp. 585–596 © 2000 The Japanese Society for Immunology

γδ T cells of human early pregnancydecidua: evidence for cytotoxic potency

Lucia Mincheva-Nilsson, Olga Nagaeva, Karl-Gosta Sundqvist,Marie-Louise Hammarstrom1, Sten Hammarstrom1 and Vladimir Baranov1

Departments of Clinical Immunology and 1Immunology, Umeå University, 90185 Umeå, Sweden

Keywords: cytotoxic granules, Fas ligand, granulysin, granzyme A, granzyme B, immunoelectronmicroscopy, lipids, perforin

Abstract

The immune compromise in decidua allows a semiallogeneic fetus to survive without impairing theability of the maternal immune system to fight infections. Cytotoxic mechanisms are likely to beimportant in this compromise. Using RT-PCR, immunoflow cytometry and immunoelectronmicroscopy, the cytotoxic potential of isolated human decidual γδ T cells was studied. mRNA forperforin (Pf), granzymes A and B, granulysin and Fas ligand (FasL) was simultaneously expressedin decidual γδ T cells. Pf and FasL were not expressed on the cell surface. However, the cellsconstitutively synthesized Pf and stored it in cytolytic granules. Within the granules Pf mainlyresided in the granule core formed by Pf-containing microvesicles. Ultrastructurally, three groupsof Pf-containing granules were distinguished. They probably represent different stages of granulematuration in a process where Pf-containing microvesicles first attach to the core cortex and thenare translocated across the cortex into the core. Presynthesized FasL was also stored in the coreand microvesicles of the cytolytic granules. Upon degranulation by ionomycin/Ca2� treatment,FasL was rapidly translocated to the cell surface, demonstrating that its surface expression wasnot controlled by de novo biosynthesis. Thus decidual γδ T cells appear to perform Pf- andFasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granuleexocytosis. 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 cytotoxicproteins as well as in the killing process. An ultrastructural model of a cytolytic granule ispresented.

Introduction

Cytolytic granule exocytosis and Fas ligand (FasL)–Fas cross- binding of FasL (CD95L) on the activated effector cells to itscell-death transducing receptor, Fas (CD95), on the mem-linking are two major mechanisms utilized by cytotoxic T

lymphocytes (CTL) and NK cells to kill targets via apoptosis brane of the target cells (6,7). Membrane-bound FasL canbe actively cleaved by a metalloproteinase-like enzyme (8).(reviewed in 1). The simplified granule exocytosis model

(2) of lymphocyte-mediated cytotoxicity postulates that after Many cell types express Fas, whereas FasL expression istransient and rapid, and is mainly restricted to activatedeffector activation the lethal hit is initiated by target-directed

secretion of cytoplasmic granules. These granules contain immune cells (for review, see 9). It has recently been shownthat FasL is also constitutively expressed in some non-key cytolytic molecules—perforin (Pf) and several serine

proteases known as granzymes (Gr) (3). Upon exocytosis, Pf lymphoid tissues which are associated with immune privilegestatus (10,11).and Gr function in synergy: Pf undergoes polymerization and

forms pores on the target-cell membrane allowing the entry Accumulated evidence from in vitro and in vivo studies(12–15) shows that both cytotoxic mechanisms play a crucialof Gr, such as GrA and GrB, which trigger nuclear damage

and cell death. Another recently described constituent of the role in the clearance of viral and bacterial infections, tumorsurveillance, transplant rejection, homeostatic regulation ofcytolytic granules is the human protein granulysin (4) and its

porcine homologue NK-lysin (5). The second mechanism immune responses, and peripheral tolerance (16,17). Thesecytotoxic mechanisms may play an important role in theof lymphocyte-mediated cytotoxicity is believed to require

Correspondence to: L. Mincheva-Nilsson

Transmitting editor: H. Wigzell Received 1 September 1999, accepted 6 January 2000

586 Cytotoxic potential of decidual γδ T cells

pregnant uterine mucosa, the decidua of mammals, e.g. by Immunofluorescence staining and flow cytometric analysis ofPf expression in γδ T cellsprotecting the maternal–fetal unit against pathogens, control-

ling invasion of fetal extravillous trophoblast and creating a DMC, preincubated with normal goat serum for 15 min onlocal transient immunotolerance toward the semiallogeneic ice, were stained with FITC-conjugated anti-TCRγδ mAb (aconceptus. Indeed, recent studies in Pf- and FasL-deficient mixture of clones TCRδ1, δTCS1 and Vδ1). After washing, themice have shown that although functional deletion of Pf or cells were fixed in 1.5% paraformaldehyde (Merck, Darmstadt,FasL alone does not appear to affect fertility, the combined Germany), permeabilized with 0.1% saponin and stained withabsence of these two effector molecules induces infertility phycoerythrin (PE)-conjugated anti-Pf mAb. Appropriately(17,18). Most of the knowledge about lymphocyte cytotoxicity labeled, isotype-matched irrelevant mAb were used as nega-in decidua has been limited to the human NK-like CD56�bright

tive controls. Flow cytometric analyses were performed onlarge granular lymphocytes (19,20). In a previous study FACScan flow cytometer equipped with a single argon ionwe presented evidence indicating that CD56�bright cells are laser (Becton Dickinson), calibrated with CaliBRITE Flowprogenitors of decidual γδ T cell (21). γδ T cells in decidua Cytometer Beads (Becton Dickinson). The data were analyzedwere shown to specifically colonize non-pregnant murine and displayed as dot-plots using Lysys II software.endometrium, and to constitute a major cell population inmurine, sheep, horse and pig decidua (22–26). Human γδ T Immunofluorescence staining and flow cytometric analysis ofcells comprise about half of all decidual T cells in early cell surface and intracellular FasL expression in γδ T cellspregnancy (21, 27). The vast majority of them are Vδ1�, CD4–

The DMC were preincubated with normal goat serum con-CD8–, and express CD56 (21,27). Ultrastructurally, humantaining 1 mM of the metalloproteinase inhibitor 1,10-phen-decidual γδ T cells have the distinctive morphology of largeanthroline monohydrate (Sigma, St Louis, MO) for 1 h on ice.granular lymphocytes (27,28). It has been shown that murineThe cells were stained with a mixture of FITC-conjugated anti-decidual γδ T cells specifically recognize a conserved tropho-TCRγδ mAb, washed and incubated with biotinylated anti-blast antigen (29) and suppress the maternal anti-fetalFasL mAb (NOK-1 or H11) followed by streptavidin–PE (Dako).response probably by transforming growth factor-β production1,10-Phenanthroline was present throughout. For intracellular(30). Recently, several reports have suggested a regulatorystaining of FasL, the cells were incubated with anti-TCRγδrole for the murine decidual γδ T cells in abortions via themAb, fixed in 1.5% paraformaldehyde and permeabilized withrelease of abortogenic or anti-abortive cytokines (31,32). In0.1% saponin for 15 min. The cells were then stained for FasLhumans, γδ T cell clones from decidua have been shown toand analyzed by flow cytometry.mediate strong non-MHC restricted cytotoxicity (33).

The aim of the present study is to evaluate the cytotoxic Flow cytometric analysis of FasL release by degranulated γδpotency of the γδ T cells using the expression of Pf, GrA, T cellsGrB, granulysin and FasL as markers of cytotoxicity.

DMC were pretreated for 1 h at room temperature with 1 mM1,10-phenanthroline and stained for TCR γδ with FITC-labeledmAb in the presence of 0.02% NaN3. After washing, the cellsMethodswere incubated with a mixture of 5 µg/ml ionomycin, 0.2%HSA, 1 mM 1,10-phenanthroline and 1 mM CaCl2 in PBS,Decidual samples and mAbpH 7.2 (34), and biotin-conjugated anti-FasL mAb for 15 min,

Vacuum-extracted decidual specimens were donated by followed by a fixation in 1.5% paraformaldehyde for 10 min.healthy women undergoing elective termination of pregnancy Thereafter, the cells were stained with PE–streptavidin. Appro-at 8–14 weeks of gestation. The specificity and sources of priately labeled, isotype-matched irrelevant mAb were usedthe mAb used in this study are listed in Table I. as controls for non-specific fluorescence.

Isolation of decidual mononuclear cells (DMC) Total RNA extraction and RT-PCR

Lysates from immunomagnetic-bead selected γδ T cells wereDMC were prepared as described previously (21). Contamin-ating epithelial cells were removed using mAb BerEP4- used to extract total RNA by the acid guanidinum thiocyanate–

phenol–chloroform method (27). The isolated RNA samplescoupled immunomagnetic beads (Dynabeads M-450; Dynal,Oslo, Norway). The isolated cell population contained on were analyzed by RT-PCR with specific primers for Pf (35),

GrA (35), GrB (forward primer 5�-CAT GCT ACT GCA GCTaverage �1% BerEp4� cells (21) as judged by flow cytometry.DMC were further depleted of B cells and monocytes using GGA G-3� and reverse primer 5�-TCC AGA GCT CCC CTT

AAA G-3�), FasL (36), granulysin (forward primer: 5�-TAAimmunomagnetic beads charged with anti-CD19 and anti-CD14 mAb. For RT-PCR, decidual γδ T cells were obtained GCC CAC CCA GAG AAG TGT-3� and reverse primer 5�-TAA

GCC CAC CCA GAG AAG TGT-3�) and β-actin (27). Theby positive selection using immunomagnetic beads labeledwith mAb specific for TCRγδ. The cells bound to the beads primer sequences were located in different exons so that

amplification of mRNA could be distinguished by the size ofwere washed 5 times in 10–20 volumes of ice-cold PBS. Aminimum of 1000–1500 cells/sample was counted in a light the amplified product. Single-strand cDNA copies were made

from 1 µg of total RNA using random hexamers and murinemicroscope to ascertain that only cells bound to beads werepresent in the selected population. The cells were lysed while leukemia virus reverse transcriptase (Perkin-Elmer, Roche

Molecular Systems, Nutley, NJ). Reverse transcription wasattached to the beads and subsequently total RNA wasextracted. performed at 42°C for 15 min followed by denaturation at

Cytotoxic potential of decidual γδ T cells 587

Table 1. Specificities of mAb used in this study

Surface marker mAb/clone Isotype Main reactivity Source

CD45 2B11 � PD7/26 IgG1 leukocytes Dako, Glostrup, DenmarkTCR γδ TCRδ1 IgG1 monomorphic determinant of the δ-chain T Cell Diagnostics, Cambridge, MA

of TCRγδTCR γδ Vδ1 IgG1 Vδ1-encoded determinant T Cell DiagnosticsTCR γδ δTCS1 IgG1 Vδ1-encoded determinant T Cell DiagnosticsCD14 TUK4 IgG2a monocytes/macrophages DakoCD19 HD37 IgG1 B cells DakoCD56 MY31 IgG1 isoform of NCAM, NK cells Becton Dickinson, Mountain View,

CAFasL (CD95L) H11 IgG2a human FasL Holzel Diagnostika, Koln, GermanyFasL (CD95L) NOK-1 IgG1 human FasL PharMingen, Becton DickinsonFasL (CD95L) G 247-4 IgG1 human FasL PharMingenPerforin δ G9 IgG2b human perforin molecule of 70 kDa Ancell, Laufelfingen, SwitzerlandHuman epithelial antigen BerEP4 IgG1 all human epithelial tissues except Dako

squamous epitheliaNegative control DAK-G01 IgG1 Aspergillus niger glucose oxidase DakoNegative control DAK-G05 IgG2a A. niger glucose oxidase DakoNegative control DAK-G09 IgG2b A. niger glucose oxidase Dako

99°C for 5 min. Subsequent PCR was performed in a total Electron microscopic localization of lipids in cytolytic granulesof γδ T cellsvolume of 100 µl in a reaction mixture containing 1.0 U of

Taq DNA polymerase (Promega) in 2.5 mM MgCl2, 10 mM The lipid staining was carried out with malachite green (37).Tris (pH 8.3), 50 mM KCl, 0.01% gelatin (w/v) supplemented A γδ T cell fraction of DMC was obtained by a negativewith 0.25 µM each of dATP, dGTP, dCTP and dTTP, and 0.20 selection procedure using immunomagnetic beads chargedµM of each primer. The following thermocycle programs were with anti-CD14, anti-CD19, anti TCR αβ and anti-CD56 mAb.used: for Pf and GrA: 35 cycles of 95°C for 1 min, 62°C for The fractionated cells were fixed in a mixture of 3% glutaral-30 s and 72°C for 2 min; for GrB: 35 cycles of 94°C for 20 s, dehyde (Sigma) and 0.1% malachite green in 0.1 M sodium55°C for 20 s and 72°C for 30 s; for FasL: 40 cycles of 94°C cacodylate buffer, pH 6.8, for 30 min at room temperature.for 15 s, 55°C for 30 s and 72°C for 1 min; for granulysin: 38 After washing in the same buffer, the cells were fixed in 1%cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s. OsO4 for 30 min and embedded in Epon–Araldite. Ultrathin

sections were examined without an additional staining.Immunoelectron microscopy (IEM) procedure for simultaneousdetection of TCR γδ and Pf in isolated DMC Ultrastructural analysis of Pf-containing cytolytic granules in

γδ T cells on serial sectionsThe pre-embedding immunoperoxidase technique was usedShort ribbons of ultrathin sections (65–70 nm of thickness)to co-localize Pf and TCR γδ in the same cell. This immuno-were collected on formvar/carbon-coated single-hole grids.staining procedure provides reliable results when the antigensThe diameter of the hole (0.8 mm) allows us to analyze aare known a priori to be separately located. DMC wereseries of four to five consecutive sections of one cell, repres-incubated with a mixture of anti-TCR γδ mAb (TCRδ1, Vδ1enting 260–350 nm of total thickness. Since the estimatedand δTCS1) in 0.02 M PBS, containing 0.2% BSA for 30 minaverage diameter of the granule cores was 400–600 nm (seeon ice, washed and fixed in 1.5% paraformaldehyde forResults), four to five serial ultrathin sections of 70 nm thickness15 min. After washing with 0.1 M glycine the cells wereshould give images covering at least half the core volume.permeabilized with 0.05% saponin for 30 min and incubatedCells exhibiting a variety of granule core types were selectedwith anti-Pf mAb for 2 h. After washing the cells wereand photographed in consecutive sections.consecutively incubated with biotinylated sheep anti-mouse

IgG F(ab�)2 (Amersham, Little Chalfont, UK) and peroxidase-IEM procedure for double labeling of TCR γδ and FasLconjugated streptavidin (Jackson ImmunoResearch, Westmolecules in isolated DMC using a combination of immuno-Groove, PA). The peroxidase activity was revealed with agold/silver and immunoperoxidase methodssubstrate containing 0.05% 3,3�-diaminobenzidine tetrahydro-

chloride (DAB; Sigma) and 0.03% H2O2. Finally, the cells Our modification of a double-labeling technique, employingwere fixed in 1.33% OsO4 for 30 min, dehydrated in acetone, two murine mAb in the same sample, has been describedand embedded in a mixture of Epon and Araldite (Fluka, previously (27). In brief, DMC were incubated with anti-TCRBuchs, Switzerland). Ultrathin sections were examined in a γδ mAb for 30 min on ice and fixed in 1.5% paraformaldehydeZeiss EM 900 electron microscope (Carl Zeiss, Oberkochen, for 15 min. Washed cells were incubated with goat anti-mouseGermany). The specificity of IEM staining was confirmed by IgG coupled to ultra-small gold particles (AuroProbe Onereplacing primary mAb with isotype-matched normal mouse GAM; Amersham) and fixed again in the same fixative for

10 min, followed by incubations with 5% normal mouseIgG.

588 Cytotoxic potential of decidual γδ T cells

surface while Pf was present exclusively inside the cells(data not shown). After double immunoperoxidase staining ofisolated DMC, γδ T cells were clearly identified by the peroxid-ase reaction product distributed over the entire cell surface,indicating the localization of TCR γδ (Fig. 3A). In the majorityof γδ T cells the reaction product simultaneously stained theperinuclear space, cisternae of rough endoplasmic reticulumand Golgi complex, reflecting the Pf synthesis pathway (Fig.3A and B). However, the most prominent morphologic featureof γδ T cells was the frequent presence of cytoplasmicgranules, which were also labeled by the reaction product(Fig. 3A). The granules were membrane delimited and showedtwo ultrastructurally distinct compartments (Fig. 3B): a poly-morphic core, which was surrounded by an electron-translu-cent area with microvesicles of 20–40 nm in size. Within the

Fig. 1. Agarose gel electrophoresis of RT-PCR products from Pf, GrA, granules, the reaction product selectively stained the coreGrB, granulysin, FasL and β-actin mRNA of decidual γδ T cells from

and microvesicles (Fig. 3B). The number of the Pf-containingfour individual samples: �, total RNA from activated peripheralgranules varied among γδ T cells. Some cells displayed onlyblood mononuclear cells; –, total RNA from human muscle; St, mol.

wt markers. single small granules, although these cells exhibited generallypositive labeling of protein-synthesizing organelles (Fig. 3C).A small proportion of γδ T cells showed weak Pf expressionand did not contain any cytoplasmic granules (Fig. 3D). The

serum and unconjugated goat anti-mouse IgG Fab (Jackson latter was confirmed by serial sections of this cell type (dataImmunoResearch). After washing, the second primary mAb, not shown). Thus, the vast majority of isolated γδ T cellsanti-FasL (clone G247-4, PharMingen/Becton Dickinson), was actively synthesize and store Pf in the granule cores andapplied, followed by an incubation with biotinylated sheep microvesicles.anti-mouse IgG F(ab�)2 (Amersham) and peroxidase-conjug-ated streptavidin (Jackson ImmunoResearch). The peroxid- Microarchitecture of the cytolytic granules in γδ T cellsase activity (i.e. FasL) was detected with DAB–H2O2 and the

In random ultrathin sections there were obvious variations incells were fixed with 0.5% OsO4 for 25 min. Finally, afterthe images of the Pf-containing granules both within individualwashing in distilled water, the gold particles (showing TCRγδ T cells and between cells (Fig. 3B and C). To determine ifγδ location) were silver-enhanced (IntenSE-M; Amersham)the various images of the cores were truly different or if theyaccording to the instructions of the manufacturer.were the result of sectioning non-homogeneous cores indifferent planes, a serial sectioning technique was used. We

Results screened 60 Pf-containing granules. Based on the appear-ance of the core in consecutive sections the granules couldDecidual γδ T cells contain mRNA for five key cytolyticbe divided into three main groups. The first, most abundantmoleculesgroup (31 granules) displayed cores consisting of two mor-

RT-PCR for Pf, GrA, GrB, granulysin and FasL mRNA was phologically distinct regions: a central, electron-translucentperformed on total RNA extracted from positively selected γδ

space, surrounded by a cortical rim, or cortex, of fine granularT cells (Fig. 1). mRNA for all five cytotoxic molecules were

material. Serial sections of one such granule are shown insimultaneously identified in almost all individual samples (PfFig. 4(A–D). The thickness of the cortex was somewhat19 of 22; GrA 18 of 20; GrB 22 of 26; granulysin 20 of 20;variable, from 70 to 90 nm. Numerous tightly packed labeledFasL 18 of 20).microvesicles seemed to fuse with the outer surface ofthe cortex forming an electron-dense scalloped layer. SomePf is expressed intracellularly in decidual γδ T cellselectron micrographs (Fig. 4C and D) showed the presenceA representative flow cytometric experiment using fixed andof stained microvesicles at the internal surface of the cortex,permeabilized DMC stained with anti-TCRγδ and anti-Pf mAbsuggesting a passage of the microvesicles through it. Twentyis shown in Fig. 2(A). The vast majority of the γδ T cellsof the granules were attributed to the second group (Fig. 4E–(constituting 14 � 6% of DMC, n � 15) expressed PfH). These granules also exhibited ‘hollow’ cores and a labeledintracellularly as did almost all of other DMC including thecortex. However, the central translucent space of the coresCD56�bright cells (constituting 55 � 13% of DMC, n � 15)usually displayed an electron-dense material consisting ofand the αβ T cells (data not shown). Monocytes/macrophagesconfluent Pf-containing microvesicles (Fig. 4F and G). Lastly,were not present in the gated DMC (Fig. 2B). Unpermeabilizednine of the granules were classified in a third group (Fig. 4I–DMC did not stain for Pf (data not shown).L). These granules showed only a uniform core which was

IEM analysis of Pf expression in γδ T cells intensely stained by the reaction product and never containeda discernible ‘hole’. The grainy textured core material seemedSingle-staining control experiments with anti-TCRγδ mAb or

anti-Pf mAb showed that TCR γδ was located only on the cell to consist of tightly packed Pf-containing microvesicles.

Cytotoxic potential of decidual γδ T cells 589

Fig. 2. Representative immunoflow cytometric experiment showing expression of Pf in decidual γδ T cells from one donor (A). Fixed andpermeabilized DMC, were dually labeled with PE-conjugated anti-Pf mAb (clone δG9) and a FITC-conjugated cocktail of anti-TCR γδ mAb(clones TCRδ1, δTCS1 and Vδ1). The analysis gate was set on CD45�CD14– cells shown in (B). Irrelevant, isotype-matched FITC- and PE-labeled mAb were used as a negative control (C).

Fig. 3. Immunoelectron micrographs of DMC stained for co-localization of TCR γδ and Pf by the immunoperoxidase method. No additionalcounterstaining was performed. (A) TCR γδ� cell showing complete surface immunostaining (arrowheads). Thin arrows point at four polymorphiccytolytic granules stained by the peroxidase reaction product. Note the positive staining of the perinuclear envelope (open arrow), the roughendoplasmic reticulum (thick arrow) and the Golgi complex (star). Magnification �9000. (B) High-power micrograph of a portion of the γδ Tcell demonstrated in (A). Thin arrows indicate labeled polymorphic cores in four cytolytic granules. Three granules contain positively stainedmicrovesicles, which associate with the outer surface of the cores (arrowheads). The perinuclear envelope (open arrows), the roughendoplasmic reticulum (thick arrows) and the Golgi complex (star) are stained by the reaction product. Magnification �22,000. (C) Area ofthe cytoplasm of a γδ T cell. Arrowheads show its surface immunostaining. The cytoplasm contains a single small cytolytic granule (arrow).Note the positive staining of the perinuclear envelope (open arrows) and the Golgi complex (star). Magnification �18,000. (D) γδ T lymphocyteshowing an intense cell surface staining (arrowheads) and a weak staining of the perinuclear envelope (open arrows). Magnification �8000.

590 Cytotoxic potential of decidual γδ T cells

Fig. 4. Representative immunoelectron micrographs showing selected serial sections through three main groups of cytolytic granules, stainedwith Pf-specific mAb. (A–D) 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 (B–D). The core cortex is positively stained (B–D; arrows). Pf-containingmicrovesicles are present at the outer (A and B; arrowheads) and internal (C and D; arrowheads) surface of the cortex. Magnification �40,000.(E–H) Granule of group 2. This granule also exhibits a hollow core (asterisk) and shows a labeled cortex (arrows). A central transparent spaceof the core contains clusters of tightly packed Pf-containing microvesicles (F–H; arrowheads). Magnification �40,000. (I–L) Granule of group3. 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 �40,000.

The cytolytic granules of the γδ T cells contain lipids 6A). Incubation with 1,10-phenanthroline before and duringcell staining did not significantly change the level of FasLIt was reasonable to assume that the ‘hollow’ core structuresurface expression (Table 2). Upon permeabilization, theresulted from lipid extraction during acetone treatment in theentire γδ T cell fraction as well as other DMC were positivelyprocess of sample embedding. We therefore undertook astained for FasL in all experiments (n � 28), showing ancytochemical staining of lipids by malachite green. Indeed,intracellular localization of FasL (Fig. 6E). Identical resultswe found lipids inside the majority of cytoplasmic granules,regarding surface and intracellular FasL expression werebut surprisingly the lipids were located outside of the coreobtained with both mAb.(Fig. 5A). In random sections, single electron-dense lipid

To define the precise intracellular localization of FasL,drops were located between the granule-limiting membraneisolated DMC were double labeled and analyzed by IEM.and the core. Further, images of lipid-containing granules inTCR γδ was detected by immunogold/silver staining and FasLserial sections (Fig. 5B–E) suggested that intragranular lipidsby immunoperoxidase staining. The results of IEM showedwrap up the core.that the surface of the γδ T cells was labeled by clusters of

Freshly isolated decidual γδ T cells express FasL intracellularly gold/silver particles and was not stained by the peroxidasebut not on the cell surface reaction product (Fig. 7A). The only intracellular compartments

containing deposits of the peroxidase reaction product wereDMC were double labeled with anti-TCR γδ mAb and anti-the cytolytic granules (Fig. 7A). Inside the granules theFasL mAb (clones H11 or NOK-1) and analyzed by flowreaction product stained a pleomorphic core material andcytometry. The cells were either non-permeabilized (FasLintragranular microvesicles. Several images of the granulessurface expression, Fig. 6A–D) or fixed and permeabilizedin random sections showed binding and possible merging of(FasL, total cell expression, Fig. 6E–H). Only a minority of γδ

T cells expressed FasL on their cell surface (Table 2 and Fig. the FasL-containing microvesicles with the core material (Fig.

Cytotoxic potential of decidual γδ T cells 591

Fig. 5. Electron microscopic visualization of lipid inclusions in cytolytic granules of γδ T cells, stained by malachite green. (A) Densely stainedlipid droplets (arrows) are seen inside cytolytic granules between the granule-limiting membrane and the core (arrowheads). The latter isrelatively solid and electron-lucent. Magnification �31,000. (B–E) Serial sections through a cytolytic granule. In the first section (B), a lipiddroplet is seen between the granule-limiting membrane and the granule core (arrowhead). In the next section (C), the core (arrowhead) ispartly covered by electron-dense lipids (arrow). In the sequential sections (D and E) lipids (arrows) completely mask the underlying core.Magnification (B–E) �27,000.

7B–D). It is, therefore, reasonable to assume that FasL is Discussiontransported to the granule core by these microvesicles. Thus,

In this study we report a series of observations, whichfreshly isolated γδ T cells contain presynthesized FasL intaken together, provide evidence for the cytotoxic potency ofcytolytic granules and do not express it on the cell surface.decidual γδ T cells.

From the RT-PCR experiments we conclude that these cellsFasL is rapidly mobilized to the cell surface of the γδ T cellsexpress mRNA for five marker proteins of cytotoxicity: Pf,upon degranulationGrA, GrB, granulysin and FasL. In general, these results

The presence of preformed FasL within cytolytic granules confirm few previous studies of human γδ T cells (38–41).suggested that it might be possible to stimulate rapid mobiliza- However, our results concern mucosa-associated Vδ1� γδTtion of the protein to the cell surface. To test this, DMC were cells (27) in a physiological situation. Furthermore, this is thetreated with ionomycin, which induces an intracellular Ca2� first demonstration of granulysin mRNA in γδ T cells. Theinflux and triggers cell degranulation within minutes (34). recent reports of antimicrobial and antitumor activity of granu-During degranulation, cells were double labeled for TCR γδ lysin (42) and its porcine analogue NK-lysin (5) are in agree-and FasL and analyzed by flow cytometry. To prevent shed- ment with studies which suggest protective responses of γδding of FasL from the cell surface, the experiments were T cells in a variety of pathological conditions (43,44).performed in the presence of 1,10-phenanthroline. Identical We have focused on the expression of Pf and FasL asresults with both mAb were obtained and a typical degranula- marker proteins of the two major pathways of cytotoxicity. Astion experiment with mAb H11 is illustrated in Fig. 8. Freshly judged by immunoflow cytometric analyses, intracellular Pf isisolated γδ T cells (Fig. 8A) or DMC (Fig. 8B) do not express present in the vast majority of decidual γδ T cells, confirmingFasL on the cell surface. However, upon ionomycin treatment the existing idea that Pf expression by γδ T cells is a generalfor 15 min, the fraction of γδ T cells (Fig. 8E) and DMC (Fig. phenomenon (38,39,44). Our ultrastructural findings support8F) expressing FasL on the surface was increased by nearly this proposal and for the first time provide definitive IEM proof100%. In fact, the intracellular staining of FasL demonstrated that the majority of these cells not only store preformed Pf inin Fig. 6(E and F) strongly resembles the cell surface staining cytolytic granules, but actively synthesize it. Pf was found inof FasL after degranulation (Fig. 8E and F), suggesting a shift the nuclear envelope, rough endoplasmic reticulum and thein FasL expression from the intracellular compartment to the Golgi complex, demonstrating that newly synthesized Pfcell surface. Thus, following degranulation, intracellular FasL molecules are targeted via the usual protein biosynthesis

pathway.is rapidly translocated to the cell surface.

592 Cytotoxic potential of decidual γδ T cells

Fig. 7. Immunoelectron micrographs of DMC stained for co-localization of TCR γδ by the gold/silver method and of FasL by theimmunoperoxidase method. (A) The plasma membrane of a γδ Tcell is labeled by numerous gold/silver grains (arrowheads). Theperoxidase reaction product selectively stains a core of a cytolyticgranule (arrow). N, nucleus. Magnification �12,000. (B–D) Randomsections of FasL-positive cytolytic granules showing: (B) a core (arrow)which consists of tightly packed FasL-containing microvesicles, (C)Fig. 6. A representative immunoflow cytometric experiment of FasL FasL-containing intragranular microvesicles (arrowheads) and theand TCR γδ expression showing surface staining of non-permeabilized positively stained core cortex (arrow), and (D) a granule core withDMC (A–D), and surface and intracellular staining of fixed and labeled core cortex (arrow) and a hollow structure. Magnification (B–permeabilized DMC (E–H). The DMC were stained with a FITC- D) �31,000.conjugated cocktail of anti-TCR γδ mAb (clones TCRδ1, δTCS1 and

Vδ1) and a biotin-conjugated anti-FasL mAb (clone H11), followedby PE–streptavidin. Irrelevant, isotype-matched FITC- and PE-labeledmAb were used as negative controls (B, D, F and H). The analysisgate was set as in Fig. 2(B). Our IEM results demonstrate that decidual γδ T cells

resemble other cytotoxic cells (reviewed in 45) in the domainorganization of the cytolytic granules, and confirm the pres-ence of Pf in the granule core and in the intragranularmicrovesicles. The cytolytic granules are mainly membranedelimited and contain two structural domains: an electron-Table 2. Surface expression of FasL on isolated DMCdense core surrounded by an electron-lucent area with smallinternal vesicles. Pf and Gr were localized preferentially inmAb 1,10- % FasL� cells No. ofthe core but also in the region of the small internal vesicles.clone Phenanthroline (mean � 2 SD) of experimentsFurthermore, using the serial section technique in IEM, we

Total DMC γδ T cells were able for the first time to show that the granule core itselfhas a complex organization. The core displays a thin cortex,NOK-1 –a 7 � 4 2 � 1 10formed by a homogeneous material, which surrounds a centralNOK-1 �b 7 � 6 5 � 4 10

H11 – 6 � 4 3 � 1 7 electron-translucent space. These findings indicate that Pf-H11 � 5 4 1 containing microvesicles first attach to the core cortex and

then are translocated across the cortex into the core. Thus,aExperiments done in the absence of 1,10-phenanthroline.the appearance of any granule core seems to depend on thebExperiments done in the presence of 1,10-phenanthroline.degree of its ‘filling’ with the microvesicles. Peters et al. (46)have proposed an association and a possible fusion of the

Cytotoxic potential of decidual γδ T cells 593

appear to fill the core completely. Second, cores of thecytolytic granules might be used as major ‘reservoirs’ for Pf-containing microvesicles. Thirdly, the current classification ofcytolytic granules, based only on routine electron microscopyand the relative proportion of the core and the translucentspace in each granule (45), should be reconsidered.

Our studies provide the first demonstration and preciselocalization of lipids in the cytolytic granules at the ultrastruc-tural level. Using the malachite green/aldehyde technique,we found that all cytolytic granules contained lipids located inthe translucent space between the granule-limiting membraneand the core, wrapping the core. Unfortunately, the nature ofthe lipids could not be determined, as there are no currentelectron microscopic methods which discriminate betweendifferent types of lipids (37). Our finding is supported by anearlier Raman microspectroscopic study of human LAK cellswhich demonstrated that cytolytic granules contain highlyunsaturated lipids, in contrast to the moderately unsaturatedlipids found in plasma membranes (47). It seems that lipidsmay function at different levels of granule-mediated cell lysis.Inside the cytolytic granules, the lipids wrapping the coremay function as an ‘isolation layer’, protecting the CTL fromits own harmful effector molecules (47). Upon exocytosis ofcytolytic granules into the intercellular cleft between the CTLand the target cell, the lipids may play a dual role. It hasbeen shown that unsaturated fatty acids inhibit degranulationof CTL (48). This effect is explained by fatty acid-inducedalteration of the plasma membrane’s lipid phase, which mostlikely perturbs the function of the signaling membrane pro-tein(s). Thus, released granule lipids may serve as autocrinesignal molecules ending the current degranulation and pre-venting a subsequent secondary degranulation. In addition,it is highly probable that such lipid-induced plasma membranealteration may occur also in the target cell plasma membrane,increasing its susceptibility to attack by cytotoxic proteins (47).Fig. 8. Representative immunoflow cytometric experiment of FasLHowever, the intriguing function(s) of granule lipid moleculesand TCR γδ expression on non-permeabilized DMC, preincubated

for 1 h with 1,10-phenanthroline, and stained before and after remains to be elucidated.degranulation induced by the calcium ionophore ionomycin. (A–D) A novel ultrastructural model of a cytolytic granule isSurface staining of DMC before and (E–H) surface staining of the

presented in Fig. 9. We propose that Pf-carrying microvesicles,same cells after degranulation. The DMC were stained with a FITC-formed by budding off the granule-limiting membrane (46),conjugated cocktail of anti-TCR γδ mAb (clones TCRδ1, δTCS1 and

Vδ1) and a biotin-conjugated anti-FasL mAb (clone H11), followed pass through the inner lipid layer and first accumulate aroundby PE–streptavidin. Irrelevant, isotype-matched FITC- and PE-labeled the outer surface of the core cortex. The microvesicles thenmAb were used as negative controls (B, D, F and H). The analysis penetrate the cortex and invade the internal space of thegate was set as in Fig. 2(B).

core to varying degrees. Another possibility is that the micro-vesicles are translocated into the core by invagination of thecore cortex. As a result, in some sections the microvesiclesappear to invade the core only partially, while in others theygranule internal vesicles with the cores, and hypothesized

that the effector molecules are delivered to the granule core occlude the core completely. According to this model Pf isreleased from killer cells in its microvesicle-bound form andas a result of this fusion. However, further work will be needed

to answer the question of the origin of the intragranular Pf- not as a soluble protein. The precise role of this form ofrelease remains to be determined but it seems that it has thecarrying microvesicles.

It is difficult, on the basis of IEM alone, to interpret the advantage of creating a high local Pf concentration, due tothe lower diffusion capacity of the microvesicles.relationship between the three groups of cytolytic granules

we have distinguished in this study. Nonetheless, three conclu- We performed our studies of FasL using mAb NOK-1, H-11 and G247-4, since the specificity of some of the poly-sions can be drawn from our analysis. First, it is likely that

the characteristic changes in the content of Pf-containing and monoclonal anti-FasL antibodies used earlier has beenquestioned recently (49,50). According to the existing opinionmicrovesicles within the cores reflect a progressive maturation

of the cytolytic granules. The ‘immature’ cytolytic granules of the cell surface expression of FasL in CTL is not constitutive,and activation via TCR–CD3 complex or by phorbol myristategroup 1 have relatively few core-invading microvesicles. By

contrast, in the ‘mature’ granules of group 3, the microvesicles acetate/ionomycin treatment is needed to induce its transient

594 Cytotoxic potential of decidual γδ T cells

Fig. 9. Schematic interpretation of the ultrastructure of a Pf-containing cytolytic granule based on the findings in this study. The cytolyticgranule contains a core that has a core cortex (arrows), surrounding an internal translucent space of unknown nature (question mark). Thelimiting membrane of the granule gives rise to the Pf-containing intragranular microvesicles (arrowheads) by budding off (46). The latter passthrough the inner lipid layer (open arrows, only shown in the drawing) and accumulate around the cortex. Then the microvesicles penetratethrough the cortex and gradually fill the internal core space. Newly arrived microvesicles continue to be condensed into the core and eventuallyoccupy it completely. (A) Section cut through a layer of Pf-containing microvesicles associated with the core surface. (B) Section cut throughthe outer microvesicle layer and the core cortex, seen in gray. (C) Section cut through an ‘empty’ core showing stained microvesicles, corecortex and an internal transparent space. (D) Section cut through a core cortex, invaded by stained microvesicles. The latter also occupy theinternal space of the core.

expression (6,7,51). Decidual γδ T cells are already activated and/or DNA transcription. This observation is consistent withour IEM results which demonstrate a lack of FasL synthesisin vivo (28,44,52); therefore, it is reasonable to expect that

FasL is present on the cell surface. However, almost none of in the γδ T cells despite its presence in cytolytic granules. Itis, thus, becoming increasingly clear that the execution phasethe decidual γδ T cells displayed FasL on the cell surface

when tested by flow cytometry, even in the presence of a of FasL-mediated killing may occur in the absence of proteinor RNA synthesis. The transcriptional control of FasL synthesismetalloproteinase inhibitor (8). In contrast, after fixation and

permeabilization of the cells, abundant intracellular expres- (16) seems to be required at the first activation of naive CTL,i.e. the initial formation of cytolytic granules, and for a renewalsion of FasL was revealed. These results support several

previous reports (53–57), demonstrating that only intracellular of the latter after degranulation. Thus the lag phase usuallyobserved in cytotoxicity assays (58) is probably due to theexpression of FasL can be revealed in monocytes, granulo-

cytes, mast cells, NK cells and CTL. Recently, Bossi and rapid release of preformed FasL from the effector cells duringthe experimental set-up and the subsequent restoration ofGriffiths (57) were able to co-localize FasL and Pf in cytolytic

granules in a human NK cell line and in CTL clones using intracellular FasL.We conclude that, at the level of the effector cell, FasLconfocal microscopy. We have confirmed and further

extended their results by demonstrating for the first time at the exploits the same granule-mediated pathway for the storageof the pre-formed molecules and their translocation to the cellIEM level the presence of FasL in the core and microvesicles of

the cytolytic granules of freshly isolated human decidual surface as Pf. Although this study has helped to clarify theintracellular localization of FasL in decidual γδ T cells, someγδT cells.

Earlier in vitro observations have suggested that the transi- subjects such as co-expression of Pf and FasL in the samecytolytic granules, the adequacy of granule-stored FasL forent cell surface FasL expression is regulated at the transcrip-

tional level (16). In functional cytotoxicity experiments there efficient target cell death, and the fate of FasL on the cellsurface after degranulation remain to be elucidated.is always a lag phase of 3–4 h, which is interpreted as the

time needed for de novo synthesis of FasL (58). Recently, Recent studies in mice and horses have provided evidencethat the systemic, MHC-restricted, maternal T cell-mediatedhowever, Li et al. (56) have demonstrated that FasL-based

cytotoxicity is unaffected by inhibition of protein synthesis response to paternal alloantigens is down-regulated in preg-

Cytotoxic potential of decidual γδ T cells 595

and. Dietrich, P. Y. 1997. Fas ligand expression by astrocytomanancy by different, but overlapping, mechanisms (59–61).in vivo: maintaining immune privilege in the brain? J. Clin.The local alloantigen-specific response in human decidua isInvest. 99:1173.

probably down-regulated as well (21,28). The suppression of 11 Griffith, T. S. and Ferguson, T. A. 1997. The role of FasL-inducedthe specific T cell response may be compensated by the apoptosis in immune privilege. Immunol. Today. 18:240.

12 Kagi, D., Vignaux, F., Lederman, B., Burki, K., Depraetere, V.,non-specific innate immune system in order to meet theNagata, S., Hengartner, H. and Golstein, P. 1994. Fas and perforinparticular requirements at the feto-maternal border, i.e. protec-pathways as major mechanisms of T cell-mediated cytotoxicity.tion against microbial infections, control of trophoblast inva- Science 265:528.

sion and creation of local transient allotolerance (62). The γδ 13 Lowin, B., Beermann, F., Schmidt, A. and Tschopp, J. 1994. Anull mutation in the perforin gene impairs cytolytic T lymphocyte-T cells, as a plausible cellular component of innate immunityand natural killer cell-mediated cytotoxicity. Proc. Natl Acad. Sci.possessing the two major mechanisms of cytotoxicity asUSA. 91:11571.shown here, may perform one or more of these functions.

14 Kojima, H., Shinohara, N., Hanaoka, S., Someya-Shirota, Y.,Takagaki, Y., Ohno, H., Saito, T., Katayama, T., Yagita, H. andOkumura, K. 1994. Two distinct pathways of specific killing

Acknowledgements revealed by perforin mutant cytotoxic T lymphocytes. Immunity.1:357.We wish to express our sincere gratitude to Professor Thorbjorn

15 Walsh, C. M., Matloubian, M., Liu, C-C., Ueda, R., Kurahara,Backman, Associate Professors Mats-Goran Damber and OthonC. G., Christensen, J. L., Huang, M. T. F., Young, J. D.-E.,Lalos, the colleagues at the Department of Gynecology, UmeåAhmed, R. and Clark, W. R. 1994. Immune function in miceUniversity Hospital, for kindly providing decidual samples. This worklacking the perforin gene. Proc. Natl Acad. Sci. USA 91:10854.was supported by grants from the Swedish Medical Society (98020555

16 Nagata, S. and Golstein, P. 1995. The Fas death factor. Scienceto L. M.-N.), the Cancer Research Foundation, Umeå university (AMP267:1449.99-213, L. M.-N.), Stiftelsen Lars Hiertas Minne (L. M.-N.), Swedish

17 Spielman, J., Lee, R. K. and Podack, E. R. 1998. Perforin/Natural Science Research Council (M.-L. H.) and a research stipendFas ligand double deficiency is associated with macrophagefrom the Swedish Institute (V. B.).expansion and severe pancreatitis. J. Immunol. 161:7063.

18 Stallmach, T., Ehrenstein, T, Isenmann, S., Muller, C., Hengartner,H. and Kagi, D. 1995. The role of perforin-expression by granular

Abbreviations metrial gland cells in pregnancy. Eur. J. Immunol. 25:3342.19 King, A., Wooding, P., Gardner, L. and Loke Y. W. 1993. ExpressionCTL cytotoxic lymphocytes

of perforin, granzyme A and TIA-1 by human uterine CD56� NKDAB 3,3�-diaminobenzidine tetrahydrochloridecells implies they are activated and capable of effector functions.DMC decidual mononuclear cellsHum. Reprod. 8:2061.FasL Fas ligand

20 Rukavina, D., Rubesa, G., Gudelj, L., Haller, H. and Podack, E. R.Gr granzyme1995. Characteristics of perforin expressing lymphocytes withinIEM immunoelectron microscopythe first trimester decidua of human pregnancy. Am. J. Reprod.PE phycoerythrinImmunol. 33:394.Pf perforin

21 Mincheva-Nilsson, L., Kling, M., Hammarstrom, S., Nagaeva, O.,Sundqvist, K.-G., Hammarstrom, M.-L. and Baranov, V. 1997. γδTcells of human early pregnancy decidua: evidence for localReferences proliferation, phenotypic heterogeneity, and extrathymicdifferentiation. J. Immunol. 159:3266.1 Shresta S., Pham C. T., Thomas D. A., Graubert T. A. and Ley

22 Itohara, S., Farr, A. G., Lafaille, J. J., Bonneville, M., Takagaki, Y.,T. J. 1998. How do cytotoxic lymphocytes kill their targets? Curr.Haas, W. and Tonegawa, S. 1990. Homing of a γδ thymocyteOpin. Immunol. 10:581.subset with homogeneous T-cell receptors to mucosal epithelia.2 Henkart, P. A. 1985. Mechanism of lymphocyte-mediatedNature 343:754.cytotoxicity. Annu. Rev. Immunol. 3:31.

23 Hansen, P. J. 1995. Interaction between the immune system and3 Masson, D. and Tschopp, J. 1987. A family of serine esterasesthe ruminant conceptus. J. Reprod. Fertil. Suppl. 49:69.in lytic granules of cytolytic T lymphocytes. Cell 49:679.

24 Heyborne, K.D., Cranfil, R. L., Carding, S. R., Born, W. K. and4 Pena, S. V. and Krensky, A. M. 1997. Granulysin, a new humanO’Brien, R. L. 1992. Characterization of γδ T lymphocytes at thecytolytic granule-associated protein with possible involvement inmaternal–fetal interface. J. Immunol. 149:2872.cell-mediated cytotoxicity. Semin. Immunol. 9:117.

25 Fox, A., Lee, C. S., Brandon, M. R. and Meeusen, E. N. 1998.5 Andersson, M., Gunne, H., Ageberth, B., Boman, A., Bergman, T.,Effects of pregnancy on lymphocytes within sheep uterineSillard, R., Jornvall, H., Mutt, V., Olsson, B., Wigzell, H.,interplacentomal epithelium. Am. J. Reprod. Immunol. 40:295.Dagerlind, A., Boman, H. and Gudmundsson, G. 1995. NK-lysin,

26 Engelhardt, H., Croy, B. A. and King, G. J. 1997. Role of uterinea novel effector peptide of cytotoxic T and NK-cells. Structureimmune cells in early pregnancy in pigs. J. Reprod. Fertil.and cDNA cloning of the porcine form, induction by interleukinSuppl. 52:115.2, antibacterial and antitumour activity. EMBO J. 14:1615.

27 Mincheva-Nilsson, L., Hammarstrom, S. and Hammarstrom, M.-L.6 Rouvier, E., Luciani, M. F. and Golstein, P. 1993. Fas involvement1992. Human decidual leukocytes from early pregnancy containin Ca(2�)-independent T cell-mediated cytotoxicity. J. Exp.high numbers of γδ� cells and show selective down-regulation ofMed. 177:195.alloreactivity. J. Immunol. 149:2203.7 Vignaux, F., Vivier, E., Malissen, B., Depraetere, V., Nagata, S.

28 Mincheva-Nilsson, L., Baranov, V., Yeung, M. M.-Y.,and Golstein, P. 1995. TCR/CD3 coupling to Fas-basedHammarstrom, S. and Hammarstrom, M.-L. 1994. Immuno-cytotoxicity. J. Exp. Med. 181:781.morphologic studies of human decidua-associated lymphoid cells8 Kayagaki, N., Kawasaki, A., Ebata, T., Ohmoto, H., Ikeda, S.,in normal early pregnancy. J. Immunol. 152:2020.Inoue, S., Yoshino, K., Okumura, K. and Yagita, H. 1995.

29 Heyborne, K., Fu, Y.-X., Nelson, A., Farr, A., O’Brien, R. and Born,Metalloproteinase-mediated release of human Fas ligand. J. Exp.W. 1994. Recognition of trophoblasts by γδ T cells. J. Immunol.Med. 182:1777.153:2918.9 Walker, P. R., Saas, P. and Dietrich, P.-Y. 1997. Role of Fas ligand

30 Suzuki, T., Hiromatsu, K., Ando, Y., Okamoto, T., Tomoda, Y.(CD95L) in immune escape. The tumor cell strikes back. J.and Yoshikai, Y. 1995. Regulatory role of γδ T cells in uterineImmunol. 158:4521.intraepithelial lymphocytes in maternal antifetal immune response.10 Saas, P., Walker, P. R., Hahne, M., Quiquerez, A. L., Shnuriger, V.,

Perrin, G., French, L., Van Meir, I. G., De Tribolet, N., Tschopp J. J. Immunol. 154:4476.

596 Cytotoxic potential of decidual γδ T cells

31 Arck, P. C., Ferrick, D. A., Steele-Norwood, D., Croitoru, K. and 46 Peters, P. G., Borst, J., Oorschot, V., Fukuda, M., Krahenbuhl, O.,Tschopp, J., Slot, J. W. and Geuze, H. J. 1991. Cytotoxic TClark, D. A. 1997. Regulation of abortion by γδ T cells. Am. J.

Reprod. Immunol. 37:87. lymphocyte granules are secretory lysosomes, containing bothperforin and granzymes. J. Exp. Med. 173:1099.32 Arck, P. C., Ferrick, D. A., Steele-Norwood, D., Croitoru, K. and

Clark D. A. 1997. Murine T cell determination of pregnancy 47 Takai,Y., Masuko, T. and Takeuchi, H. 1997. Lipid structure ofcytotoxic granules in living human killer T lymphocytes studiedoutcome: I. Effects of strain, αβ TCR, γδ TCR, and γδ T cell

subsets. Am. J. Reprod. Immunol. 37:492. by Raman microspectroscopy. Biochim. Biophys. Acta 1335:199.48 Anel, A., Richieri, G. V. and Kleinfeld, A. M. 1994. A tyrosine33 Christmas, S. E. 1991. T-cell receptor gene expression by human

γδ T-cell clones from peripheral blood and reproductive tissues phosphorylation requirement for cytotoxic T lymphocytedegranulation. J. Biol. Chem. 269:9506.in relation to non-MHC-restricted cytotoxic function. Scand.

J. Immunol. 33:627. 49 Smith, D., Sieg, S., and Kaplan, D. 1998. Technical note: aberrantdetection of cell surface Fas ligand with anti-peptide antibodies.34 Rodriguez, A., Webster, P., Ortego, J. and Andrews, N. W.

1997. Lysosymes behave as Ca2�-regulated exocytic vesicles in J. Immunol. 160:4159.50 Stokes, T. A., Rymaszewski, M., Arscott, P. L., Wang, S. H.,fibroblasts and epithelial cells. J. Cell Biol. 137:93.

35 Chen, R. H. and Fuggle, S. V. 1993. In situ cDNA polymerase Bartron, J., Baker, J. R., Jr, Fiedler, P., Schae, C. E., Papoff, G.,Stassi, G., De Maria, R., Giordano, C., Bagnasco, A., Ruberti, G.reaction. A novel technique for detecting mRNA expression. Am.

J. Pathol. 143:1527. and Testi, R. 1998. Constitutive expression of FasL in thyrocytes.Science 279:2015a.36 Chappell, D. B., Zaks, T. Z., Rosenberg, S. A. and Restifo, N. P.

1999. Human melanoma cells do not express Fas (Apo-1/CD95) 51 Suda, T., Takahashi, T., Golstein, P. and Nagata, S. 1993. Molecularcloning and expression of the Fas ligand, a novel member of theligand. Cancer Res. 59:59.

37 Vermelin,L., Septier, D. and Goldberg, M. 1994. Iodoplatinate tumor necrosis factor family. Cell 75:1169.52 Szekeres-Bartho, J., Barakonyi, A., Polgar, B., Par, G., Faust, Z.,visualization of phospholipids in rat incisor predentine and

dentine, compared with malachite green aldehyde. Palkovics, T. and Szereday, L. 1999. The role of gamma/delta Tcells in progesterone-mediated immunomodulation duringHistochemistry 101:63.

38 Koizumi, H., Liu, C.-C., Zheng, L. M., Joag, S. V., Bayne, N. K., pregnancy: a review. Am. J. Reprod. Immunol. 42:44.53 Kiener, P. A., Davis, P. M., Rankin, B. M., Klebanoff, S. J.,Holoshitz, J. and Young J. D.-E. 1991. Expression of perforin and

serine esterases by human γδ T cells. J. Exp. Med. 173:499. Ledbetter, J. A., Starling, G. C. and Liles, W. C. 1997. Humanmonocytic cells contain high levels of intracellular FasL: rapid39 Nakata, M., Kawasaki, A., Azuma, M., Tsuji, K., Matsuda, H.,

Shinkai, Y., Yagita, H. and Okumura, K. 1992. Expression of release following cellular activation. J. Immunol. 159:1594.54 Mincheff, M., Loukinov, D., Zoubak, S., Hammett, M. andperforin and cytolytic potential of human peripheral blood

lymphocyte subpopulations. Int. Immunol. 4:1049. Meryman, H. 1998. Fas and Fas ligand expression on humanperipheral blood leukocytes. Vox. Sang. 74:113.40 Mami-Chouaib, F., Flament, C., Asselin-Paturel, C., Gaudin, C.

and Chouaib S. 1996. TCR alpha/beta and TCR gamma/delta 55 Wagelie-Steffen, A. L., Hartmann, K., Vliagoftis, H. and Metcalfe,D. D. 1998. Fas ligand (FasL, CD95L, APO-1L) expression inCD4–/CD8– HLA-DR alloreactive CTL clones do not use Fas/

Fas ligand pathway to lyse their specific target cells. Hum. murine mast cells. Immunology 94:569.56 Li, J. H., Rosen, D., Ronen, D., Behrens, C. K., Krammer, P. H.,Immunol. 51:13.

41 Vincent, M.-S., Roessner, K., Lynch, D., Wilson, D., Cooper, S. M., Clark, W. R. and Berke, G. 1998. The regulation of CD95 ligandexpression and function in CTL. J. Immunol. 161:3943.Tschopp, J., Sigal, L. H. and Budd, R. C. 1996. Apoptosis of

Fashigh CD4� synovial T cells by Borrelia-reactive Fas-ligandhigh 57 Bossi, G. and Griffiths G. M. 1999. Degranulation plays anessential part in regulating cell surface expression of Fas ligandγδ T cells in Lyme arthritis. J. Exp. Med. 184:2109.

42 Stanger, S., Hanson, D. A., Teitelbaum, R., Dewan, P., Niazi, K. R., in T cells and natural killer cells. Nat. Med. 5:90.58 Lowin, B., Mattman, C., Hahne, M. and Tschopp, J. 1996.Froelich, C. J., Granz, T., Thoma-Uszynski, S., Melian, A., Bogdan,

C., Porcelli, S. A., Bloom, B. R. Krensky, A. M. and Modlin, R. L. Comparison of Fas (APO-1/CD95)- and perforin-mediatedcytotoxicity in primary T lymphocytes. Int. Immunol. 8:57.1998. An antimicrobial activity of cytolytic T cells mediated by

granulysin. Science 282:121. 59 Tafuri, A., Alferink, J., Moller, P., Hammerling, G. J. and Arnold, B.1995. T cell awareness of paternal alloantigens during pregnancy.43 Li, B., Bassiri, H., Rossman, M. D., Kramer, P., Eyuboglu, A. F.-O.,

Torres, M., Sada, E., Imir, T. and Carding S. R. 1998. Involvement Science 270: 630.60 Jiang, S. P. and Vacchio, M. S. 1998. Multiple mechanisms ofof the Fas/Fas ligand pathway in activation-induced cell death of

mycobacteria-reactive human γδ T cells: a mechanism for the peripheral T cell tolerance to the fetal ‘allograft’. J. Immunol.160:3086.loss of γδ T cells in patients with pulmonary tuberculosis.

J. Immunol. 161:1558. 61 Baker, J. M., Bamford, A. I., and Antczak, D. F. 1999. Modulationof allospecific CTL responses during pregnancy in equids: an44 Born, W., Cady, C., Jones-Carson, J., Mukasa, A., Lahn, M. and

O’Brien, R. 1999. Immunoregulatory functions of gamma delta T immunological barrier to interspecies matings? J. Immunol.162:4496.cells. Adv. Immunol. 71:77.

45 Griffiths, G. M. and Argon, Y. 1995. Structure and biogenesis of 62 Sacks, G., Sargent, I and Redman, C. 1999. An innate view ofhuman pregnancy. Immunol. Today 20:114.lytic granules. Curr. Top. Microbiol. Immunol. 198:39.