Gamma-Delta T Cell Killing of Human Oligodendrocytes is Perforin-Mediated

1. Journal of Neuroimmunology 87 Ž1998. 49–61
Mechanism of gd T cell-induced human oligodendrocyte cytotoxicity:
relevance to multiple sclerosis
a,b a,b
Rana Zeine , Robert Pon , Uma Ladiwala c , Jack P. Antel c , Lionel G. Filion b,
Mark S. Freedman a,b,)
a
Neurology DiÕision, Department of Medicine, UniÕersity of Ottawa, Ottawa, Ontario, Canada
b
Department of Microbiology and Immunology, UniÕersity of Ottawa, Ottawa, Ontario, Canada
c
Neuroimmunology Unit, Department of Neurology and Neurosurgery, McGill UniÕersity, Montreal, Quebec, Canada
Received 31 October 1997; revised 4 February 1998; accepted 5 February 1998
Abstract
gd T cells may contribute to the pathogenesis of Multiple Sclerosis ŽMS. via cytotoxicity directed at the myelin-oligodendrocyte unit.
We have previously demonstrated that peripheral blood-derived gd T cells lyse fresh human oligodendrocytes in vitro. The present work
extends these observations to gd T cells derived from both peripheral blood ŽPBL. and cerebrospinal fluid ŽCSF. of MS and non-MS
neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased
proportions of Vd 1q gd T cells in both CSF and PBL samples compared to other neurological disease ŽOND. controls. Although gd T
cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte
targets, OND-derived, Vd 2q rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines ŽDaudi, RPMI 8226.
known to express high levels of heat shock proteins Žhsp.. To clarify the mechanismŽs. of cytotoxicity used by gd T cells, we first
showed that cell-target contact was necessary by the use of physical barriers Žtranswells., which reduced target cell lysis by at least 75%.
The use of Ca2q-free media reduced lysis by up to 50%, but fully blocking gd T cell Perforin release and function by either Ca2q
chelation ŽMg 2 EGTA. or the Hq-ATPase inhibitor Concanamycin-A ŽCMA., completely abrogated the lysis of Fasyrhsp60 high
expressing targets ŽDaudi, U937.. However, additional treatment with Brefeldin A was required for the complete inhibition of gd T cell
mediated killing of Fasq expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an
isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly
affect lysis. These findings suggest that in MS, gd T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity
pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant. q 1998 Elsevier Science B.V. All rights
reserved.
Keywords: Multiple sclerosis; gd T cells; Cytotoxicity
1. Introduction al., 1996; Stinissen et al., 1997., these still require the
presence of intermediate antigen presenting cells ŽAPC.,
Immune-mediated damage to myelin or its cell of origin such as microglia, before they can be directed against
in the central nervous system ŽCNS., the oligodendrocyte, myelin or oligodendrocytes. Alternatively, gd T cells can
is probably orchestrated through a T cell interaction, though react directly with oligodendrocytes ŽFreedman et al.,
the exact mechanism of immune damage is still unclear. 1991., without the need for intermediary APC, possibly
Although antigen-directed responses Že.g., MBP. of ab T involving the recognition of heat shock proteins Žhsp.
cells may be involved, possibly initiated by viral infection ŽFreedman et al., 1992, 1997a; Birnbaum et al., 1993;
with ensuing autoimmune responses to cross reactive CNS Battistini et al., 1995., ab crystallin Žvan Noort et al.,
antigens Ž Hafler and Weiner, 1989; Sobel, 1995; Evans et 1995. or even non-peptide antigens ŽConstant et al., 1994;
Schoel et al., 1994; Bukowski et al., 1995.. This interac-
)
Corresponding author. Ottawa General Hospital, 501 Smyth Road,
tion can lead to cytotoxic damage to the oligodendrocyte
Ottawa, Ontario, Canada K1H 8L6. Tel.: q1 613 7378917; fax: q1 613 ŽFreedman et al., 1991.. gd T cells are concentrated in
7378857; e-mail: mfreedman@ogh.on.ca multiple sclerosis ŽMS. plaques ŽWucherpfennig et al.,
0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

2. 50 R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61
1992; Selmaj et al., 1991a., demonstrate limited hetero- study of lytic pathways, since it accelerates Perforin degra-
geneity of their TCRs suggesting a common antigen reac- dation by increasing the pH of lytic granules ŽKataoka et
tivity ŽBattistini et al., 1995; Nick et al., 1995; Stinissen et al., 1996.. CMA, thus, inhibits the Perforin-based compo-
al., 1995. and appear to be important in mediating early nent in a cytotoxicity interaction such that lysis occurring
damage in murine experimental allergic encephalomyelitis in the presence of CMA would be Fas-mediated and
ŽOlive, 1996; Rajan et al., 1996.. sensitive to Brefeldin A, which blocks Fas ligand surface
The gd T cells are still a poorly understood T cell expression on effector cells ŽKataoka et al., 1996.. A series
subset, constituting only a small proportion of peripheral T of isocoumarin compounds have been reported to interfere
cells. They have been demonstrated to secrete predomi- with cytotoxicity by biochemically inhibiting granzyme
nantly Th1-type cytokines, though under certain conditions activity ŽOdake et al., 1991.. In this paper, we decipher the
may also produce IL-4 ŽKaufmann, 1996.. Recently, CD1 cytotoxicity mechanisms utilized by CSF- or PBL-derived
has been shown to be a useful marker of immature human gd T cells from MS patients against targets that include
TCR gd thymocytes ŽOffner et al., 1997.. One of the most fresh human derived oligodendrocytes, by systematically
potent functions of mature activated gd T cells is that of disrupting key steps in the major cell death pathways.
MHC unrestricted cytotoxicity to a number of cell types
including oligodendrocytes ŽHaas, 1993; Tsukaguchi et al.,
1995; Freedman et al., 1997a.. 2. Materials and methods
Two major pathways have been described for the suc-
cessful killing of target cells by cytotoxic T cells ŽHenkart
2.1. gd T cell donors
et al., 1995; Atkinson and Bleackley, 1995; Kagi et al.,
1994; Lowin et al., 1994.. One leads to membrane frag-
mentation and lysis as a result of the pore-forming protein CSF and peripheral blood ŽPBL. samples were collected
Perforin ŽLowin et al., 1995., whereas the other leads to from consenting patients with clinically definite ŽPoser et
DNA fragmentation and apoptosis due to either granzyme al., 1983. MS and patients with other neurological diseases
ŽOND. that included: viral meningitis, benign intracranial
activity ŽHeusel et al., 1994; Nakajima and Henkart, 1994;
Irmler et al., 1995. or to the transduction of a death signal hypertension, brain stem tumor, cerebrovascular disease,
via membrane cross-linking of FasrCD95 molecules ŽJu et anxiety disorder, chronic fatigue syndrome, diabetes melli-
al., 1994; Golstein et al., 1995.. It is believed that these tus, systemic lupus erythematosus and Leber’s optic neu-
pathways may be related in a manner that allows switching ropathy. In some cases, PBL samples were obtained from
in the decision between apoptosis and lysis depending on normal controls including hospital personnel and patients’
the biochemical microenvironment ŽLeist et al., 1997.. spouses.
Furthermore, it has been possible to demonstrate that in
addition to pore formation, Perforin plays a critical role in 2.2. Expansion and isolation of human gd T cells
the initiation of the apoptotic process ŽShi et al., 1997..
Human gd T cells constitutively express Perforin Petri dishes ŽFalcon, VWR Canlab, London, ON. and
ŽNakata et al., 1990. and possess cytoplasmic granules 96 flat-bottomed wells ŽCostar, Fischer Scientific, Ottawa,
containing the cytolytic mediators Perforin and granzymes Ontario. were coated with a sheep anti-mouse IgG1 anti-
ŽKoizumi et al., 1991.. Fas-mediated death requires the body ŽT cell Sciences, Intermedico, Markham, ON. at 5
interaction between Fas ligand ŽSuda et al., 1993. on the m grml for 1 h at room temperature, then washed twice
killer cell and Fas on the target cells ŽYagita et al., 1995.. with PBS Žphosphate-buffered saline. and once with 5%
gd T cells have been shown to express Fas ligand mRNA FCS Žfetal calf serum. ŽGibco BRL, Burlington, ON..
ŽNagata and Golstein, 1995; Suda et al., 1995. and surface Monoclonal anti-TCRgd Žcourtesy of Dr. M.B. Brenner,
antigen ŽVincent et al., 1996.. This is significant in the Harvard University and titrated to yield maximal stimula-
study of MS pathogenesis, since oligodendrocytes have tion. in complete media ŽCM. ŽRPMI supplemented with
recently been shown to express Fas ŽCD95. by confocal 10% FCS, 2 mM glutamine, 100 Urml penicillin, 100
microscopy ŽD’Souza et al., 1996.. Urml streptomycin. was then added and the dishesrwells
A number of strategies have been useful in differentiat- were placed at 378C for 20 min. The cells were collected
ing between the Perforin-based and the Fas-based cytotoxi- from freshly obtained CSF samples by gentle centrifuga-
city mechanisms. The removal of calcium ions from the tion followed by resuspension in CM containing irradiated
media by either exclusion or chelation inhibits granule 30 Gy Ž3000 rads. autologous feeders Ž10 6 cellsrml., then
exocytosis preventing the release of Perforin and granzymes placed in the pre-coated flat-bottomed wells. We had
from cytotoxic T cells. Fas-mediated death does not appear previously determined that 10 ml of CSF contain roughly
to require the presence of extracellular Ca2q ŽTrenn et al., 5–10 = 10 4 cells; so, in the interest of maximizing yields,
1987; Rouvier et al., 1993; Rovere et al., 1996.. Recently, the samples were resuspended without direct counting into
an inhibitor of vacuolar type Hq-ATPase, Concanamycin a total volume of about 1 ml and then divided into
A ŽCMA., has been introduced as a powerful tool in the approximately 5 wells. Mononuclear cells were collected

3. R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61 51
from the PBL by Ficoll-Hypaque ŽPharmacia, Biotech, cells were maintained in the 24-well plate until the wells
´
Baie D’Urfe, PQ. centrifugation, as previously described became more confluent, prior to transferring to flasks. On
ŽFreedman et al., 1991.; some were then used as feeders approximately day 14, the cells were harvested, gd T cell
Ždescribed above. and the remainder was cultured at 10 6 purity was assessed by flow cytometry Žsee below. and the
cellsrml in CM in pre-coated petri dishes. Both CSF- and ab T cells were removed as described above for PBL
PBL-derived cells were expanded by the addition of 20 samples. In many cases, the CSF-derived cultures con-
Urml recombinant human IL-2 ŽCourtesy of Chiron, tained ) 90% pure gd T cells ŽFig. 1., but where purity
Emeryville, CA. and 20 Urml IL-4 Žcourtesy of was - 90%, ab T cells were depleted. Final cultures
Schering-Plough, Kennelworth, NJ. on day 2. On day 5, were estimated to contain - 2% NK cells by flow cytome-
PBL cells were collected, resuspended at 2.5 = 10 5 try Ždata not shown..
cellsrml in serum-free media ŽAim V; Gibco BRL. and The use of PHA was avoided in experiments designed
cultured for another 4 or 5 days with fresh IL-2 and IL-4. to study the mechanisms of cytotoxicity. Cells collected on
The ab T cells were then eliminated by complement lysis day 8 were re-cultured in the presence of irradiated he-
using anti-CD4 ŽOKT4, Ortho Diagnostics, Markham, ON. terologous feeders in either petri dishes or 24-well plates
and anti-CD8 ŽOKT8, Ortho Diagnostics. monoclonal anti- that had been freshly pre-coated with anti-TCRgd mAb.
bodies, as previously described ŽFreedman et al., 1991. IL-2 and IL-4 were added the following day and the cells
and used either immediately in cytotoxicity experiments, were harvested on day 12 and used immediately in cyto-
or maintained in culture by re-stimulation with phyto- toxicityrinhibition experiments.
hemagglutinin ŽPHA. ŽSigma, St. Louis, MO. at 1 m grml
and the addition of fresh 10% heterologous irradiated 2.3. Flow cytometry
feeder cells. CSF cells were given fresh media containing
IL-2 and IL-4 on day 5 and further cultured for 3 days. On
Cells were double stained with monoclonal antibodies
day 8, CSF cells were collected, counted and replated at
to CD3 ŽLeu4-PE, Becton Dickinson ŽBD., Mountain View,
3 = 10 5rwell of a 24-well plate ŽNunclon, Gibco BRL.
CA., TCRgd ŽTCR d 1-FITC, T cell Sciences, Inter-
containing 1 = 10 6 fresh irradiated heterologous feeders, 1
medico; TCRgd-PE, BD., Vd 1 ŽTCS-1-FITC, T cell Sci-
m grml PHA and CM, supplemented with 10% NHS Žnor-
ences., or Vg 9 Žrepresenting the majority of the Vd 2 gd T
mal human serum. ŽPel-freeze, Toronto, Ontario. instead
cell subset. ŽTg iA-FITC, T cell Sciences., as previously
of FCS. IL-2 and IL-4 were added the following day and
described ŽFreedman et al., 1991.. Cells were stained with
the cells were maintained until day 11, at which samples
a combination of 3 monoclonal antibodies to CD3rCD16
from each well were transferred to a 25-cm2 flask in 10 ml
q CD56 ŽSimultest, BD. for identification of NK cell
of fresh media containing IL-2 and IL-4. In some cases,
markers. Antibodies were conjugated to either phyco-
erythrin ŽPE. or fluorescein isothiocyanate ŽFITC.. Target
cells were stained with anti-human Fas FITC-labelled UB2
monoclonal ŽOncor, PDI-Joldon, Aurora, ON.. A COUL-
TER EPICS flow cytometer was used for data acquisition.
Statistical markers were set using isotype control antibo-
dies. Dead cells were identified by staining with propidium
iodide ŽPI., as described elsewhere ŽCoico, 1995. and
excluded by forward and side scatter gating and PI stain-
ing. The 5–10 = 10 3 events were acquired and the data
was analyzed using EPICS XL software.
2.4. Cytotoxicity assay
The 2 = 10 6 target cells were labelled in suspension
with 100 m Ci of 51 Cr Ž5 mCi, Amersham, Oakville, ON.
for 1 h at 378C. Adherent oligodendrocyte target cells were
incubated in 96-microwell plates ŽNunc. with 1 m Ci 51 Cr
per well overnight at 378C. Targets were then washed 3
times to remove free label. The gd T cells were titrated in
duplicates and triplicates onto 1.5 = 10 4 labelled targets
with effector:target ŽE:T. ratios of 1, 5, 10, 20, 40:1, and
Fig. 1. Flow cytometric analysis of typical CSF-derived gd T cells.
CD3q TCRgdq cells make up 90% of the population Župper right panel,
incubated in 96-microwell plates for 5 h at 378C. The 100
quadrant 2.. Of those, 11% expressed Vd 1 Žlower left panel, quadrant 2., m l of supernatant was transferred to monospot plates be-
and 89% expressed Vd 2 Žlower right panel, quadrant 2.. fore and after a 30-min treatment with 1% cetrimide

4. 52 R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61
Žmixed alkyltrimethylammonium bromide, Sigma, St. cubated for 30 min with titrations of 7-amino-4-chloro-3-
Louis, MO.. Radioactivity was measured by an indirect Ž3-isothioureidopropoxy. isocoumarin Žcourtesy of Dr. J.
Gamma Counter ŽMatrix 9600, Canaberra Packard, Missis- Powers, Georgia Institute of Technology, Atlanta, GA;
sauga, ON.. Percent specific lysis was calculated from the Odake et al., 1991. at final concentrations in the range of
ratios of induced and spontaneous chromium release as 5–49 m M. The stock solution of isocoumarin was prepared
follows: directly in CM. To disrupt Perforin activity, gd T cells
were pre-incubated for 2 h with CMA ŽSigma. at concen-
Induced Ž sample.
% Specific Lysis s trations of 1, 1000 and 2000 nM ŽKataoka et al., 1996.. To
Inducedq Residual interfere with Fas–Fas ligand interactions, target cells were
Spontaneous Ž control. pre-incubated for 2 h at various concentrations, up to 3
y = 100 m grml, of an anti-Fas mAb, clone ZB4 ŽImmunotech,
Spontaneousq Residual
Westbrook, ME., shown to neutralize Fas–thymocyte
apoptosis ŽYonehara et al., 1994. and to inhibit the cyto-
2.5. Isolation of fresh human brain-deriÕed oligodendro- toxic function of human autoreactive CD4q T cell clones
cyte targets ŽVergelli et al., 1997.. To interfere with Fas ligand expres-
sion on effector cells, gd T cells were pre-incubated for 2
For oligodendrocyte cultures, human brain tissue was h with Brefeldin A ŽSigma. at either 20 or 40 m M
ŽKataoka et al., 1996.. In some experiments, gd T cells
obtained from patients undergoing temporal lobe resection
or callostomy as part of a surgical therapeutic treatment for were pre-treated with combinations of the above men-
intractable epilepsy. Enriched cultures of oligodendrocytes, tioned agents. The reagents were not washed away prior to
approximately 60–90% galactocerebroside or 2X-3X-cyclic the addition of target cells except for the anti-Fas mono-
nucleotide phosphodiesterase ŽCNP. positive, were estab- clonal, which when used in the microgram range, was
lished, as detailed elsewhere ŽGrenier et al., 1989; Kim et reduced by 50% prior to the addition of the targets. Given
al., 1983.. above were the final concentrations for all reagents, fol-
lowing the addition of an equal volume containing the
targets. In control experiments, Mg 2 EGTA, Ca2q-free me-
2.6. Target cell lines
dia, CMA, Brefeldin A and anti-Fas did not cause lysis of
either the target cells or the gd T effectors in the concen-
Target cell lines included the Daudi B cell lymphoma trations used, as measured by a 51 Cr-release assay Ždata
ŽFasy, hsp60 high ., the U937 monocytic cell line ŽFasy,
not shown..
hsp60 low ., the Jurkat T cell line ŽFasq. , the RPMI 8226
Žhsp60 high ., Žthe latter 3 were obtained from ATCC,
Rockville, and maintained according to recommended con- 2.8. Statistical analysis
ditions. and an oligodendroglioma cell line called KG-1
ŽFasy. Ža generous gift of David H. Mattson, Rochester, Where indicated, groups were compared using an un-
NY; Miyake, 1979.. Hsp refers to the expression of the paired Student’s t-test and analyzed using a software
heat shock protein 60 protein Ž lowrhigh ., as determined package for IBM computers from GraphPade. The results
previously by staining using a human mAb to hsp60 depicted in the figures represent means " SD or SEM, as
ŽFreedman et al., 1997a.. Fas expression Žqry . was deter- indicated.
mined by flow cytometry Žresults not shown..
2.7. Disruption of cytotoxicity 3. Results
In transwell experiments, targets and effectors were 3.1. Increased proportions of Vd 1 expressing gd T cells in
separated by placing the gd T cells in a tissue culture MS
insert ŽNunc. designed for 96-microwell plates and fitted
with a 0.2 m m anopore membrane. To block granule The surface phenotype of gd T cells isolated from the
exocytosis, gd T effector cells were pre-incubated for 2 h blood and CSF of MS patients and OND was analysed by
in the presence of the Ca2q chelator Mg 2 EGTA ŽSigma. flow cytometry using monoclonal antibodies to CD3,
at various concentrations including 0.01, 0.1, 2, 2.5, 3.5 TCRgd , TCR Vd 1 and Vg 9 ŽVd 2. ŽFig. 1.. Despite
and 7.0 mM. Alternatively, effector and target cells were significant variability, there was a trend suggesting a shift
washed in Ca2q-free media DMEM 95-0302 ŽLife Tech- towards more Vd 1q cells vs. Vd 2q cells in MS, ŽFig. 2.,
nologies, Burlington, ON. and the assay was performed however, this was not statistically significant. In both the
using complete Ž10% FCS, 2 mM glutamine, 100 Urml MS and OND groups, the predominant subset was Vd 2q,
penicillin, 100 Urml streptomycin. Ca2q-free media. To however, there were significant proportions of Vd 1q cells,
inhibit granzyme activity, gd T effector cells were pre-in- indicating that the expansion process described here is not

5. R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61 53
appeared to be more resistant to lysis by PBL-derived gd
T cells as compared to the other cell lines. Similar results
were noted previously using cultured human oligodendro-
cytes in a comparative study of cell line lysis ŽFreedman et
al., 1997a.. These results suggest that human gd T cells
utilize an efficient mechanism for cell-mediated cytotoxi-
city which could operate independently of heat shock
protein or Fas recognition.
3.3. Cellular contact and soluble factor requirements for
gd T cell cytotoxicity
When gd T cells were physically unable to contact
target cells in transwell experiments, cytotoxicity was
greatly reduced 70–75% ŽFig. 3., but some cytotoxicity
remained. The data depicted ŽFig. 3. is from a representa-
tive experiment using Daudi cells, however, similar data
was seen using other cell types Žresults not shown.. These
data suggest that while physical contact is a definite
requirement for efficient killing by gd T cells, diffusible
soluble factors, such as those released from cytotoxic
granules, may also be involved.
Fig. 2. gd T cell subsets in PBL and CSF. Proportions of Vd 1 and Vd 2
expressing gd T cells from the PBL Ž ns14, MS; ns8, OND. and CSF 3.4. Calcium and granule exocytosis requirements for gd
Ž ns9, MS; ns6, OND. of MS and OND patients, as analysed by flow
cytometry. Depicted are the means"SEM.
T cell cytotoxicity
The release of soluble mediators from cytoplasmic
granules is mediated by Ca2q release and exocytosis can
selective for the Vd 2q subset, compared with the use of be blocked using the Ca2q chelator Mg 2 EGTA. Fig. 4
stimulator cell lines, such as Daudi ŽFisch et al., 1992. or demonstrates the effect of removing Ca2q with increasing
RPMI 8226 ŽSelin et al., 1992; Freedman et al., 1991.. concentrations of chelator, prior to the addition of 51 Cr-
labelled target cells. While the experiments shown in Fig.
3.2. gd T cell cytotoxicity of cell lines 4 were performed using gd T cells from MS patients,
similar findings were noted for gd T cells derived from
The gd T cells derived from both PBL and CSF of both OND patients Žresults not shown.. Ca2q chelation inter-
MS and OND patients adequately lysed all target cells, feres with granule exocytosis, thus blocking the release of
seemingly irrespective of hsp60 or Fas expression ŽTable Perforin and granzymes, and with the binding of Perforin
1.. Significant increases were noted in the degree of to target cell membranes. Mg 2 EGTA, at the highest con-
cytotoxicity exerted by OND CSF gd T cells against centrations used Ž7 mM., was not cytotoxic to the effector
hsp60 expressing target cells ŽDaudi and RPMI 8226. cells as measured by 51 Cr-release Ždata not shown.. In
compared to the MS-derived gd T cells. This possibly titration experiments, we determined that at concentrations
reflects the trend seen in the OND for higher proportions of 2 mM, Mg 2 EGTA blocked lysis only partially, and at
of Vd 2q cells, the subset of gd T cells known to respond 0.1 mM and 0.01 mM, it had no effect on lysis Ždata not
to hsp ŽHaas, 1993.. Overall, the oligodendroglioma cells shown.. It was very difficult to identify a concentration of
Table 1
Cytotoxicity of cell lines induced by gd T cells derived from blood and CSF of patients with MS and OND
Target cell Blood CSF
MS n OND n p MS n OND n p
Daudi 21.3 " 2.4 16 23.4 " 4.3 12 ns 15.5 " 3 15 31.4 " 3.6 11 0.0023
RPMI 8226 17.6 " 2.7 7 19.7 " 5.9 6 ns 9.4 " 2.2 5 25 " 5.2 5 0.0246
U937 22.7 " 2.9 16 18.2 " 4 11 ns 15.6 " 2.9 13 20.4 " 3.9 9 ns
Oligodendro-glioma ŽKG-1. 13.3 " 4 12 7.5 " 1.7 7 ns 15.3 " 2.7 10 21.1 " 6.7 6 ns
Mean cytotoxicity at a 20:1 E:T ratio " SEM; n s number of individuals; ns s not significant Ž p - 0.05., Student’s t-test.

6. 54 R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61
Mg 2 EGTA that could differentially influence the lysis of
Daudi and Jurkat targets in our system, emphasizing the
potency of the calcium-dependant killing mechanism for
the gd T cells. However, to further confirm the calcium
requirement for gd T cell-mediated cytotoxicity, the experi-
ments were repeated using Ca2q-free media Žsee Section
2.. As expected, Daudi and Jurkat target cell lysis was
significantly diminished with the use of Ca2q-free media,
although it was never completely abrogated ŽFig. 5.. The
reduction in achievable lysis was more pronounced when
effector and target cells were washed 3 = ŽFig. 5, bottom
panel. in Ca2q-free media prior to setting up the assay.
Taken collectively, these findings suggest a predominant
role for the Ca2q-dependentrPerforin-based mechanism in
gd T cell cytotoxicity.
Fig. 3. Disruption of gd T cell-mediated cytotoxicity by impeding 3.5. Intact granzyme actiÕity requirement for gd T cell
cell–cell contact. Using a filter-transwell system to physically separate gd cytotoxicity
T cells from Daudi target cells significantly reduced percent specific lysis
in a 5-h 51 Cr-release assay Žmean of triplicates"SD.. Depicted is a In order to biochemically inhibit the activity of
representative of 3 experiments. granzymes, gd T cells were pre-incubated for 30 min with
Fig. 4. Requirement for Ca2q in gd T cell-mediated cytotoxicity: effects of chelation. Complete abrogation by Ca2q chelation of cytotoxicity induced by
gd T cells from the PBL and CSF of MS patients against Daudi, oligodendroglioma, Jurkat, and U937 cells Žmean of triplicates" SD.. The gd T cells
were pre-incubated with Mg 2 EGTA at indicated concentrations prior to the addition of labelled target cells Žfinal concentrations: 5 mM for Daudi
experiments; 3.6 and 7.2 mM for other targets..

7. R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61 55
m M concentrations of an isocoumarin compound prior to
the addition of target cells. Fig. 6 provides data from
representative experiments using a titration of 7-amino-4-
chloro-3-Ž3-isothioureidopropoxy. isocoumarin with CSF-
and PBL-derived gd T cells from MS patients. We de-
tected little change or a slight decrease in the overall lysis
of Jurkat, U937 and oligodendroglioma cell targets. These
results indicate that although intact granzyme activity may
be important for efficient gd T cell cytotoxic function,
Fig. 5. Requirement for Ca2q in gd T cell-mediated cytotoxicity: effects
of Ca2q-free media. Reduced cytotoxic capacity in Ca2q-free media of Fig. 6. The role of granzymes in gd T cell-mediated cytotoxicity.
gd T cells from the PBL of a normal control Žtop. and the blood and CSF Biochemical inhibition of granzyme activity by pre-incubation of gd T
of MS patients Žmiddle and bottom. against Daudi and Jurkat targets cells from the PBL and CSF of MS patients with the indicated m M
Žmean of triplicates"SD.. The gd T cells and targets were pre-washed concentrations of isocoumarin. A marginal inhibitory effect on subse-
either once Žtop and middle. or 3 times Žbottom. with Ca2q-free media, quent gd T cell-mediated cytotoxicity against Jurkat, oligodendroglioma
and the 5-h assay was performed entirely in Ca2q-free media. and U937 target cells is depicted Žmean of triplicates"SD..

8. 56 R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61
granzymes probably do not play a major role in target cell
lysis within the 5-h time constraint of this assay.
3.6. gd T cells utilize both the Perforin-based and Fas-
based cytotoxicity mechanisms
Even though many Fasy targets are lysed by gd T
cells, it is still possible that Fas-mediated killing occurs,
but is overshadowed by other mechanisms. In order to
evaluate the importance of Fas–Fas ligand interactions, we
used the richly Fas expressing Jurkat target cell line,
previously shown to be susceptible to gd T cell-induced
cytotoxicity even in the absence of Ca2q ŽHaecker and
Wagner, 1994.. The top panel of Fig. 7 shows that pre-in-
cubation for 2 h at 378C with an anti-Fas mAb Žclone ZB4,
Immunotech. at concentrations Žup to 500 ngrml. that
were recommended by the manufacturer to neutralize Fas-
mediated apoptosis did little to interfere with Jurkat cell
lysis by gd T cells from an MS patient. Even at concentra-
tions in the microgram range Žup to 3 m grml., pre-treat-
Fig. 8. Contribution by both Fas and Perforin to gd T cell cytotoxicity.
Effects of disruption of Perforin function and Fas ligand expression on
the cytotoxic capacity of gd T cells from the PBL of a normal control
against Daudi and Jurkat targets Žmean of triplicates"SD.. Cytotoxicity
was assessed following the pre-incubation of gd T cells with the indi-
cated concentrations of either CMA ŽConcanamycin A., Brfd A ŽBrefel-
din A. or both. CMA abrogated Daudi cell lysis ŽPerforin-based pathway.
but only partially inhibited Jurkat cell lysis. Further addition of Brefeldin
A, however, inhibited the Fas-mediated component of Jurkat cell lysis at
a 20:1 E:T ratio.
ment with anti-Fas mAb did not inhibit lysis as shown in
the experiments depicted in the bottom panel of Fig. 7.
Similar results were seen with gd T cells from OND
Žresults not shown.. This is in agreement with others who
have experienced difficulties with blocking CTL and gd T
cell cytotoxicity by the direct addition of anti-Fas mono-
clonals into their assay systems ŽKataoka et al., 1996;
Vincent et al., 1996.. In order to estimate the contributions
of the Perforin-based and the Fas-based mechanisms, we
used Concanamycin A separately and in combination with
Brefeldin A. Whereas treatment with CMA completely
abrogated Daudi cell lysis by blood-derived gd T cells
Fig. 7. The role of Fas in mediating gd T cell cytotoxicity of Jurkat from a normal control ŽFig. 8, top. and an MS patient ŽFig.
targets. ZB4, an anti-Fas monoclonal antibody does not interfere signifi-
cantly at concentrations up to 500 ngrml Žtop. and 3 m grml Žbottom.
9, top., the lysis of Jurkat targets was only partially
with the lysis of Fasq Jurkat targets by gd T cells from human PBL and inhibited ŽFig. 8, bottom; Fig. 9.. These effects were noted
CSF derived from MS patients Žmean of triplicates"SD.. for each E:T ratio tested. When Brefeldin A was used

9. R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61 57
3.7. Inhibition of gd T cell-mediated lysis of fresh human
brain-deriÕed oligodendrocytes
Fig. 10 provides data from 5 experiments in which
blood-derived gd T cells from patients with MS ŽM.R.,
H.E., G.C. and W.N.. and 1 normal individual ŽR.B.. lysed
fresh human brain-derived oligodendrocytes in a CMA-
sensitive manner. The oligodendrocytes were used within 7
days of having been plated at 30,000 cells per microwell.
These experiments were performed in flat-bottomed plates
at a time when the oligodendrocytes were adherent and
had begun extending processes. The 5-h assays were per-
formed in triplicate and the percent specific lysis was
Fig. 9. Contributions of the Fas-based and the Perforin-based mechanisms
to gd T cell cytotoxicity in MS. Effects of disruption of Perforin function
and Fas ligand expression on the cytotoxic capacity of gd T cells from
the PBL and CSF of MS patients against Daudi and Jurkat targets Žmean
of triplicates"SD.. Cytotoxicity was assessed following the pre-incuba-
tion of gd T cells with the indicated concentrations of either CMA
ŽConcanamycin A., Brfd A ŽBrefeldin A., a combination of CMA and
Brfd A, or Mg 2 EGTA. CMA abrogated Daudi cell lysis ŽPerforin-based
pathway. but only partially inhibited Jurkat cell lysis. Addition of
Brefeldin A to the CMA further inhibited the Fas-mediated component of
Jurkat cell lysis at E:T ratios of 20:1 ŽPBL. and 10:1 ŽCSF..
alone, it had no effect on Jurkat cell lysis ŽFig. 8, bottom;
Fig. 9, top.. However, when a combination of CMA and
Brefeldin A was used, it was possible to further diminish
Jurkat cell lysis over and above the reduction caused by
CMA alone ŽFig. 8, bottom; Fig. 9.. These findings were
also true for CSF-derived gd T cells from MS patients
Fig. 10. Inhibition of gd T cell-mediated killing of fresh human brain-de-
ŽFig. 9, bottom.. CMA essentially abrogated the Perforin-
rived oligodendrocytes. Effects of disruption of Perforin function and Fas
dependent Daudi cell lysis but only partially inhibited ligand expression on the killing of human oligodendrocytes by blood-de-
Jurkat cell lysis, whereas, Brefeldin A inhibited the pre- rived gd T cells from 4 MS patients and 1 normal control ŽR.B...
sumed Fas-mediated component of Jurkat cell lysis. The Cytotoxicity was assessed before and after pre-incubation of gd T cells
differential pattern of lysis inhibition observed in these with the indicated concentrations of CMA ŽConcanamycin A. with or
without Brfd A ŽBrefeldin A.. CMA inhibited oligodendrocyte lysis
experiments demonstrates that human gd T cells can ŽPerforin-based pathway. significantly. Further addition of Brefeldin A
utilize both the Perforin-based as well as the Fas-based inhibited the Fas-mediated component. E:T ratios were 10:1 Žtop. and
cytotoxicity mechanisms. 20:1 Žbottom.. Those plotted are means of triplicates"SD.

10. 58 R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61
calculated on a per well basis, as indicated in Section 2. human oligodendrocytes in vitro, and therefore probably
CMA at concentrations of 1000 nM and 2000 nM inhibited have the greatest propensity for inflicting oligodendrocyte
lysis significantly but not completely Ž50–90%., suggest- damage in vivo. Using PBL-derived gd T cell lines, we
ing the predominance of the Perforin-based cytotoxicity reported that this cytotoxicity extended to other human
mechanism in gd T cell-mediated oligodendrocyte killing. glial cells with a typical profile of killing seen using
The addition of 40 m M Brefeldin A to the pre-treatment several tumour cell lines ŽFreedman et al., 1997a.. We now
conditions inhibited lysis further, suggesting a minor role extend these observations by examining the profile of
for a Fas-mediated mechanism in oligodendrocyte lysis killing seen with gd T cells that are derived from both
ŽFig. 10, bottom.. These results implicate Perforin in gd T CSF and PBL of both MS and OND patients. We com-
cell-mediated oligodendrocyte cell death and suggest that pared their cytotoxic potential against a series of target
interfering with this Perforin-based cytotoxicity mecha- cells, and similar to others ŽNick et al., 1995; Stinissen et
nism in vivo might have therapeutic potential in MS. al., 1995., found that they killed a variety of different cell
lines, with no perceived differences between MS and
OND. There was a slight increased killing of Daudi and
RPMI 8226 cells, both cell lines known to richly express
4. Discussion hsp and induce gd T cell expansion ŽFisch et al., 1992;
Selin et al., 1992; Freedman et al., 1997b., by OND CSF
Accumulated evidence supports a role of gd T cells in cells, that also contained higher proportions of Vd 2q cells
mediating the immune damage observed in acute and ŽTable 1; Fig. 1. that may be particularly responsive to hsp
chronically affected MS patients ŽFreedman et al., 1991; ŽHaas, 1993; Kaufmann, 1996.. Stinissen et al. Ž1995.
Selmaj et al., 1991a; Wucherpfennig et al., 1992; Shi- suggested that the higher proportions of Vd 1q cells they
menkovitz et al., 1993; Stinissen et al., 1995.. We have observed in MS CSF indicated that they were antigen-
focused on the cytotoxic properties of gd T cells, since driven, though the nature of the antigen is not known.
cell-mediated cytotoxicity could be a major cause of tissue We have not observed any differences in the profile of
damage regardless of the cytopathic effects of some pro-in- cytotoxicity against target cell lines induced by gd T cells,
flammatory cytokines. We chose to expand gd T cells whether or not they were generated from PBL using Daudi
directly from the PBL and CSF by stimulation via cross- or RPMI 8226 cells as stimulators Žthat generate mainly
linking the TCR and adding IL-2 and IL-4, as opposed to Vd 2q T cells. or the currently used anti-TCR cross-linking
either cloning or expanding cells with large amounts of method that yields both gd T cell subsets Žunpublished
IL-2 or IL-2 in conjunction with PHA ŽNick et al., 1995; results.. The latter is in contention with the notion that gd
Stinissen et al., 1995.. In the latter case, the predominant T cell cytotoxic activity appears to be influenced by the
response is still the proliferation of ab T cells, and there mode of primary activation ŽHaecker and Wagner, 1994..
is the need to subsequently clone out gd T cells in order to However, similar widespread and somewhat non-specific
perform functional assays. We found that the addition of killing was also demonstrated by others using gd T cell
IL-4 to the CSF samples was crucial to the consistent clones of various subtypes ŽNick et al., 1995; Stinissen et
success of generating gd T cell lines, without the need for al., 1995.. It is therefore likely that both gd T cell
cloning and often not even requiring weeding out of subtypes exert a similar profile of cytotoxicity, although
contaminating ab T cells. The derivation of pure Ž) 90%. some differences in antigen reactivity have been described
gd T cells by culturing CSF cells without any additional ŽKaufmann, 1996; Haas, 1993..
purification might indicate that CSF gd T cells either have In order to examine the cytotoxic mechanisms utilized
higher potency for survival or for killing ab T cells in by CSF- or PBL-derived gd T cells, we sequentially
vitro. Although both Vd 1q and Vd 2q gd T cell subsets disrupted a series of key steps along the major cell death
were represented, there was a trend towards an increased pathways. Since direct gd T cell–target cell contact would
proportion of Vd 1q cells, particularly in the CSF-derived be required for Fas ligand:Fas binding and physically
cultures from MS patients, as has been observed by others interfering with this contact using transwells reduced but
ŽShimenkovitz et al., 1993; Stinissen et al., 1995.. did not fully abolish cytotoxicity, we surmised that dif-
We have previously shown that human oligodendro- fusible factors were also likely involved. Such factors
cytes can be lysed by both ab and gd T cells in a 5-h might have either been triggered in vivo, reflecting perhaps
assay, the former involving class-I MHC-directed re- gd T cell activity in either CSF or PBL, or as a result of
sponses ŽFreedman et al., 1991; Ruijs et al., 1990.. Others the in vitro expansion process itself. These substances
have demonstrated oligodendrocyte cytotoxicity by could include the pore-forming protein, Perforin, or serine
promiscuous CD4q T cells ŽAntel et al., 1993., TNF esterases Žgranzymes., which are constrained by pH and
ŽSelmaj et al., 1991b; D’Souza et al., 1995., or anti-Fas Ca2q requirements for their integrity and release. Ca2q is
monoclonals ŽD’Souza et al., 1996., but this involved essential not only for Perforin granule exocytosis but also
culture periods of 18–24 h or even days. To our knowl- for binding of Perforin to target cell membranes ŽIshiura et
edge, gd T cells so far induce the most rapid cytolysis of al., 1990.. Two lines of evidence indicate that Fas-media-

11. R. Zeine et al.r Journal of Neuroimmunology 87 (1998) 49–61 59
ted lysis is independent of Ca2q. First, mutant cytotoxic T injury in vitro, however, this required at least 24 h of
cell clones have been described to be able to kill via the incubation with cross-linked anti-Fas monoclonal antibody.
Fas-based mechanism despite their failure to mobilize The fact that more than one cytotoxic mechanism may
intracellular Ca2q following TCR engagement ŽEsser et be used by gd T cells against oligodendrocytes is possibly
al., 1996.. Second, anti-Fas ŽCD95.-mediated death of typical of how these cells function, since others have
susceptible gd T cell clones has been shown to occur in shown that Borrelia-reactive human gd T cells utilize
Ca2q-free media ŽOdake et al., 1991.. In some systems, both the Ca2q-dependent and the Fas-based pathways
Fas-mediated killing can lead to cell death within hours ŽVincent et al., 1996.. To date, there have been no reports
ŽGolstein et al., 1995., however, when working with popu- concerning the expression of Perforin in the CNS of MS
lations of cytotoxic cells that are utilizing both the Per- patients, however, the expression of Perforin and
forin-based and the Fas-based mechanisms simultaneously, granzymes has been demonstrated in both synovial fluid
we and others have encountered difficulties in blocking lymphocytes and synovial tissue from patients with
cytotoxicity using anti-Fas agents in the absence of reagents rheumatoid arthritis ŽGriffiths et al., 1992; Young et al.,
that block the Perforin-based mechanism. Thus, we are in 1992; Kummer et al., 1994; Tak et al., 1994; Muller-
agreement with Kataoka et al. Ž1996. that although Ladner et al., 1995., another autoimmune disease in which
Brefeldin A in combination with CMA can significantly gd T cells have been postulated to play an important role.
affect cytotoxicity, Brefeldin A and anti-Fas monoclonals Though we used mature human adult oligodendrocytes in
used alone have minimal and variable effects in many such our experiments, similar gd T cell-mediated damage may
experimental systems ŽFigs. 7–9, and data not shown.. also be directed against oligodendrocyte precursor cells.
Furthermore, we demonstrated that an isocoumarin com- The gd T cells may thus be directly involved in damaging
pound, expected to specifically block the activity of mature myelin as well as perhaps interfering with re-
granzymes, interfered only minimally with gd T cell- myelination. Limiting gd T cell-mediated cytotoxicity in
mediated lysis ŽFig. 6., most likely for the same reason, MS might therefore not only prevent ongoing damage to
since granzymes activate apoptotic DNA fragmentation. the myelin-oligodendrocyte unit, but also indirectly pro-
Taken collectively, these observations suggest that, in this mote myelin repair.
type of fast cytotoxicity Žmeasured at 5 h., the Fas-based
component of the gd T cell-induced cytotoxicity is minor
and can only be seen when the Perforin-based component Acknowledgements
is eliminated.
In keeping with the above, the killing of the Fas high We are indebted to Vi Nguyen for her excellent techni-
Jurkat targets was exquisitely sensitive to even low chela- cal assistance. We acknowledge Dr. Sameer D. D’Souza,
tor concentrations ŽFig. 9, bottom., suggesting that gd T formerly of McGill University, for his input at the early
cells prefer a Perforin-based mechanism of killing, even stages of this project. We thank Dr. Rose Goldstein, of the
when given the opportunity to use a Fas-based system. University of Ottawa ŽRheumatology., for stimulating dis-
CMA, which blocks the Perforin pathway, completely cussions. This research was supported by a grant to MSF
protected Fasy targets ŽDaudi. from gd T cytotoxicity from the Multiple Sclerosis Society of Canada.
ŽFigs. 8 and 9., even with concentrations as low as 1 nM
Ždata not shown., whereas Fasq Jurkat cell lysis was only
partially inhibited, even with concentrations as high as References
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