Download gamma-delta T cells target the red blood cell

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Blood First Edition Paper, prepublished online November 1, 2011; DOI 10.1182/blood-2011-08-376111
Control of Plasmodium falciparum erythrocytic cycle: gamma-delta T cells
target the red blood cell-invasive merozoites
Giulia Costa1,2,4, Séverine Loizon1,2, Marianne Guenot1,2, Iulia Mocan1,2,
Franck Halary1,2, Geneviève de Saint-Basile6, Vincent Pitard1,2, Julie
Déchanet-Merville1,2, Jean-François Moreau1,2,3, Marita Troye-Blomberg5,
Odile Mercereau-Puijalon4, and Charlotte Behr1,2
1
CNRS, UMR 5164, F-33000 Bordeaux, France; 2Univ. Bordeaux, F-33000
Bordeaux, France; 3CHU Bordeaux, Service d’Immunologie et
Immunogénétique, F-33000 Bordeaux, France, 4Institut Pasteur, Immunologie
Moléculaire des Parasites, CNRS URA 2581, F-75015 Paris, France;
5
Department of Immunology, Stockholm University, Stockholm, Sweden;
6
INSERM, U768, F-75015 Paris, France
Address correspondence to: Charlotte BEHR, PhD, CNRS UMR 5164,
Université de Bordeaux Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex,
France. Phone: +33-5-5757-9233
E-mail: [email protected]
Running title: γδ T cells target extra-erythrocytic merozoites
This article contains supplemental data
1
Copyright © 2011 American Society of Hematology
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Abstract
The control of Plasmodium falciparum erythrocytic parasite density
is essential for protection against malaria, as it prevents pathogenesis and
progression towards severe disease. P.falciparum blood-stage parasite
cultures are inhibited by human Vγ9Vδ2 gamma-delta T cells, but the
underlying mechanism remains poorly understood. Here, we show that
both intra-erythrocytic parasites and the extracellular red blood cellinvasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell
receptor dependent manner and trigger their degranulation. In contrast,
the γδ T cell-mediated anti-parasitic activity only targets the extracellular
merozoites. Using perforin-deficient and granulysin-silenced T cell lines,
we demonstrate that granulysin is essential for the in vitro anti-plasmodial
process, whereas perforin is dispensable. Patients infected with
P.falciparum exhibited elevated granulysin plasma levels associated with
high levels of granulysin-expressing Vδ2+ T cells endowed with parasitespecific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2
T cells along with granulysin triggering and discharge during primary
acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells
as unconventional immune effectors targeting the red blood cell-invasive
extracellular P.falciparum merozoites and opens novel perspectives for
immune interventions harnessing the anti-parasitic activity of Vγ9Vδ2 T
cells to control parasite density in malaria patients.
2
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Introduction
Clinical malaria is associated with the intra-erythrocytic asexual
replication cycle of the Plasmodium sp. parasite. Whereas young intraerythrocytic-stage parasites circulate in the blood, mature intra-erythrocyticstage parasites (trophozoites and schizonts) are sequestered in the
microcirculation.
Upon
completion
of intra-erythrocytic
development,
extracellular invasive merozoites are released into the blood stream, where they
invade new red blood cells (RBCs), thus exponentially amplifying the density
of blood-stage parasites. Control of parasite density is essential for protection
against malaria, as it prevents pathogenesis and progression towards severe
disease.
Despite major research efforts, the immune mechanisms involved in the
control of parasite biomass remain poorly understood. This lack of
understanding
impedes
the
rational
development
of
immune-based
interventions to prevent or cure malaria. Analysis of immune effectors that
control blood-stage parasites has mainly focused on antibody-dependent
mechanisms, as passive transfer of immunoglobulins dramatically reduced
parasite density in children with malaria.1,2 Little attention has been paid to
early immune responses, which however, play a pivotal role in the race
between parasite development and the deployment of protective adaptive
immune mechanisms. Recent studies have highlighted the role of innate
immune effectors, including innate lymphocytes, in the early control of
parasitemia before significant levels of specific antibodies are produced;
however, the underlying effector mechanisms remain poorly understood.3
3
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Gamma-delta (γδ) T cells expressing the Vγ9 and Vδ2 chains of the T
cell receptor represent a non-conventional T lymphocyte subset found only in
primates that accounts for 0.5 to 5% of peripheral T cells. Gamma-delta T
cells circulate in the blood, patrolling vascular beds, readily responding to
tumor cells and blood-borne pathogens such as Plasmodium, and participating
in the early immune response and in the onset of adaptive immunity.4 They
react specifically to minute amounts of small non-peptidic metabolites, known
as phosphoantigens, independent of processing or presentation by professional
antigen presenting cells.5 The most active phosphoantigens are intermediate
products of the deoxy-xylulose-5-phosphate (DOXP) isoprenoid synthesis
pathway, which occurs in the Plasmodium apicoplast.6,7 During a primary
malaria infection, Vγ9Vδ2 T cells are rapidly activated and expanded, reaching
frequencies of up to 30% of circulating T cells.8,9
Previous studies have demonstrated that Vγ9Vδ2 T cell clones can
inhibit blood-stage P.falciparum parasites in vitro;10,11 however the
contribution of this effect to the overall antiparasitic activity of PBMC, the
actual parasite developmental stage(s) that activate and trigger γδ T cellinhibitory activity, the parasite stage(s) targeted by the γδ T cells, and the role
of cytotoxic effector molecules in this process remain unclear. Furthermore
little is known concerning the in vivo activation and antiparasitic status of
Vγ9Vδ2 T cells. Yet, this information is crucial to understand how γδ T cells
contribute to the early control of parasite density and to harness this process to
improve the control of parasite multiplication in primary infected patients.
In the present study, we elucidate the mechanism by which γδ T cells
inhibit in vitro P.falciparum blood-stage development. We report that both
4
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intracellular and extracellular stages specifically trigger Vγ9Vδ2 T cell antiparasitic activity through a TCR-dependent mechanism. Furthermore, we
demonstrate that the free RBC-invasive merozoite is the only vulnerable stage
in this process and that granulysin, but not perforin, is the crucial mediator of
parasite inhibition. We also documented the in vivo relevance of these data by
showing the presence of high levels of Plasmodium-reactive Vγ9Vδ2 T
lymphocytes expressing granulysin along with high concentrations of plasma
granulysin in P.falciparum-infected patients. These data identify the
Plasmodium merozoite as a novel activator and target of an unconventional
lymphocyte subset and highlight the RBC as a niche protecting the parasite
from cytotoxic mediators. Furthermore, our findings emphasize Vγ9Vδ2 T
cell-mediated production of granulysin as an underestimated immune effector
in malaria.
5
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Materials and Methods
Parasite culture
P.falciparum FCR3 parasites were grown in O+ RBC (EFS, Aquitaine),
cultured in RPMI 1640 supplemented with 10% human serum as described12
and regularly tested for absence of mycoplasma contamination. Parasites were
synchronized by repeated sorbitol treatments. Trophozoite or schizont stages
were purified by gel flotation.13
Free merozoites were isolated as previously described.14 Briefly, highly
synchronized mid-stage schizonts were purified by gel flotation, set to mature
until the late-schizont stage (4-16 nuclei) and incubated with 10 µM E64
(Sigma Aldrich, USA). After 8 hours, the culture was centrifuged for 5 minutes
at 500×g to remove contaminant schizonts. Supernatant containing
parasitophorous-enclosed-membrane-merozoites
(PEMS)
was
harvested,
centrifuged at 2,000×g for 10 minutes and enumerated. Subsequently, free
merozoites were released by mild mechanical treatment. Contamination by
intact schizonts was assessed by microscopic examination of Giemsa-stainedsmear and represented less than 5%.
Generation of Vγ9Vδ2 T cell lines
To generate Vγ9Vδ2 T cell lines, peripheral blood mononuclear cells
(PBMCs) from healthy donors (EFS, Aquitaine) were stimulated with
phosphoantigens [(50 μM isopentenyl pyrophosphate (IPP, Sigma Aldrich) ;
400 nM bromohydrin pyrophosphate (BrHPP) (IPH1101, Innate Pharma,
France); a P.falciparum phosphoantigen enriched fraction6] or with intact
purified P.falciparum trophozoites and expanded in bulk in complete medium
(CM = RPMI 1640 medium supplemented with 10% human serum, glutamine
6
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and antibiotics) in the presence of IL-2 (300 IU/mL, PeproTech, USA). Shortterm Vγ9Vδ2 T cell lines were functionally tested without further restimulation
when they reached at least 75% of purity. For long-term Vγ9Vδ2 T cell lines
(S1 and JT lines), Vδ2 cells were sorted after 2 weeks of stimulation by
positive magnetic separation (Miltenyi Biotec, Germany) and subsequently restimulated with PHA-L (Sigma Aldrich), IL-2 (1000 IU/mL) and irradiated
PBMCs as previously described.15 Long-term T cell lines were tested between
3 to 4 weeks after re-stimulation. The same procedure was followed to generate
the Vγ9Vδ2 T PFN-def cell from a genetically perforin-deficient familial
hemophagocytic lymphohistiocytosis patient (FHL) with heterozygous
missense mutations (G3A and G695A) (Hôpital Necker, Paris, France).16 The
Vγ4Vδ5 (4-29) T cell clone and the Vδ3VγI T cell line, both reactive against
the colon cancer cell line HT29 and the clone T4A.5 αβ CD4+ T cell clone
were derived as described elsewhere15,17 and re-stimulated under the same
conditions.
Lentiviral shRNA design and T lymphocyte transfection
The shRNA target-specific sequences previously described: GNLYsh1
5’-AAGCCCACCCAGAGAAGTGTT-3’
and
GNLYsh2
5’-
GGAGGTATCAGTCTAGAGTTT-3’ were used.18 The non-silencing (NS)
sequence 5’-AAGAACGACTAGGTGGAGAA-3’ was used as a control. To
produce granulysin-specific shRNA lentiviral vectors (LVs), 68 mers were
designed
as
follows:
5’-AGCTTCC-sense21mer-TTCAAGAGA-
antisense21mer-TTTTTGGAAG-3’
and
phosphorylated
as
described
(http://tronolab.epfl.ch/). Double-stranded oligonucleotides were cloned into a
7
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HindIII/Sal1-digested pIC20R (vector 30, Plateforme de Vectorologie,
Bordeaux University, France) downstream of the H1 RNA polymerase III
promoter. pIC20R-shRNA were subsequently digested by Cla1 and Sal1, and
the H1/shRNA inserts obtained were subcloned into pTRIPdsRED2 (vector
117, Plateforme de Vectorologie, Bordeaux University, France) . All the
constructs were confirmed by sequencing.
Lentiviral vectors (LV) were produced by transient transfection of
293T cells according to standard protocols.19 Subsequently, Vγ9Vδ2 T cell
lines were infected with LV (MOI=10) in the presence of protamine sulphate
(Sigma Aldrich). After 1 week, infected dsRED positive Vγ9Vδ2 T cells were
sorted by flow cytometry (FACSAria, BD-Biosciences, USA) and restimulated
as described above. The efficiency of silencing was assessed at the mRNA and
protein levels using RT-PCR and Western-Blotting respectively (see
Supplemental Methods).
CD107a assay
The degranulation assay was performed as previously described.20
Briefly, cells were incubated for 6 hours with schizonts, merozoites, or
uninfected RBC (uiRBC) at various Effector/Target (E/T = cells/parasites)
ratios in the presence of IL-2 (20 IU/mL), except in experiments using patients’
cells, for which no IL-2 was added. Anti-CD107a mAb or isotype control (BD
Biosciences) was added at the onset of the incubation. Cells were harvested,
stained with anti-Vδ2 or anti-CD3 mAb (Beckman Coulter, USA), and then
analyzed by flow cytometry using a FACSCanto (BD-Biosciences). For
blocking experiments, cells were pre-incubated for 1 hr with anti-Vδ2 (clone
8
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immu389, Beckman Coulter, USA) or anti-NKG2D (clone 149810, R&D
Systems) at varying concentrations.
Parasite growth inhibition assay
For assays using PBMC, PBMC magnetically-depleted or not of Vδ2+
T cells (Miltenyi Biotec) were stimulated with 400 nM BrHPP (Innate Pharma)
in CM without cytokines for 40 hours before being tested. For assays using
Vγ9Vδ2 T cell or control cell lines, cells were incubated in CM supplemented
with IL-2 20 IU/mL or IL-15 50 ng/mL (Peprotech) for 24 hours before being
tested. Cells were co-cultured in a 96-well microtiter plates at a specific
cell/parasite ratio with a trophozoite synchronized culture adjusted to 1%
parasitemia and 1% final hematocrit for 24 hours and ring parasitemia was
assessed
by
flow
cytometry
(FACSCanto,
BD-Biosciences)
using
hydroethidine staining21 (see Supplemental Methods and Figure S1 for details).
Results were confirmed by microscopic examination of Giemsa-stained
smears. Anti-parasitic activity (%) was calculated as follows: 100-[(average %
parasitemia of replicate test culture/average % parasitemia of replicate control
culture)x100].
To test whether schizonts were targeted by Vγ9Vδ2 cells, schizonts (34 to 40
hours after invasion) were incubated with the Vγ9Vδ2 T cells for 6 hours and
Vγ9Vδ2 T cells were removed before schizont rupture using anti-CD2Dynabeads (Dynal Biotech, Norway). Mock-treated parasites cultured without
cells were used as controls. Parasites were then cultured for an additional 18
hours and parasitemia assessed by hydroethidine staining.
9
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Merozoite reinvasion assay
T cells were incubated with purified merozoites and uiRBC for 28
hours and the parasitemia resulting from red blood cell re-invasion was
assessed by hydroethidine staining. Inhibition of merozoite reinvasion (%) was
calculated as follows: 100-[(average % parasitemia with cells/average %
parasitemia without cells)x100].
Clinical samples and flow cytometry
PBMCs were isolated from clinical blood sample leftovers of
P.falciparum-infected patients admitted to the Teaching University Hospital of
Bordeaux and from healthy donors from the blood bank (EFS, Aquitaine).
Patients were informed according to the rules of the Hospital ethical board, and
the research was approved by the University of Bordeaux IRB. PBMCs were
stained directly with mAb specific for CD3, CD27, and CD45RA (BDBiosciences), as well as Cδ and Vδ2 (Beckman-Coulter). For granulysin
staining, PBMCs were first stained with anti-CD3 and anti-Vδ2 mAbs, then
fixed in 2% formaldehyde, permeabilized in 0.1% Triton, and incubated with
granulysin mAb (BD-Biosciences). Cells were analyzed by six-color flow
cytometry using a FACSCanto and FACSDiva software (BD-Biosciences).
Granulysin ELISA
Granulysin ELISAs were performed on plasma and culture supernatants
as previously described.22
10
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Statistical analysis
Wilcoxon signed-rank test was used for paired comparison, Mann–
Whitney or the Kruskal-Wallis for unpaired comparison and Spearman's rank
for correlation analysis. P values ≤.05 were considered significant.
11
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Results
Vγ9Vδ2 T cells exhibit anti-parasitic activity in PBMCs or as purified T
cell lines
We first assessed the anti-parasitic activity of Vγ9Vδ2 T cells in PBMCs
using a parasite growth inhibition assay. In comparison to parasites alone, a
decrease of parasitemia was observed in parasite co-culture with resting
PBMCs for all but one donor. This decrease was slightly enhanced when the
PBMCs were activated before performing the parasite growth inhibition assay
with the synthetic phosphoantigen BrHPP, which selectively stimulates the
Vγ9Vδ2 T cells. Depletion of Vδ2+ T cells from PBMCs before activation
reduced the inhibitory effect of BrHPP-activated PBMCs (Figure 1A). Of note,
the effect of resting PBMCs on parasitemia was similar whether or not Vδ2+ T
cells were depleted (data not shown). Together, this suggests that although
Vγ9Vδ2 T cells represent a minor subset of PBMCs (ranging from 0.5 to 1.9%
of PBMCs for the donors analyzed), once activated, they contribute to PBMCs
parasite growth inhibition.
To directly assess the effect of Vγ9Vδ2 T cells, 10 short-term Vγ9Vδ2 T
cell lines (STLA to STLJ) derived from different donors by stimulation of
PBMCs with phosphoantigens (either BrHPP or P.falciparum-phosphoantigenenriched fraction) were tested for their ability to inhibit parasite growth.
Parasite growth was inhibited by γδ T cell lines in absence of added cytokines
and similarly when cells were exposed to low quantities of IL-2 (20IU/mL)
(Figure 1B). As IL-15 has been shown to favor the differentiation of cytotoxiceffector phenotype of γδ T cells23 and enhance their growth inhibitory
activity24, cells were tested after 24 hours of incubation with IL-15 in addition
12
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to IL-2. Parasite growth inhibition was most pronounced under this condition
(Figure 1B) and individual γδ T cell lines growth inhibitions ranged from 35 to
72% (Table S1). These data indicate that freshly expanded Vγ9Vδ2 T cell
lines inhibit parasite growth, with an optimal effect achieved by cells incubated
with IL-15.
To gain insight into the mechanisms underlying the γδ T cell-mediated
anti-parasitic activity, two stable long-term Vγ9Vδ2+ cells T cells lines (more
than 95% pure) were generated from two different donors following
stimulation with IPP (S1) or intact live trophozoites (JT). These T cell lines
were maintained through repeated PHA-restimulation and required IL-2
(20IU/mL) for their survival. For both T cell lines, reproducible parasite
growth inhibitions was dependent on the IL-15 dose with the maximum
inhibition reached (80%) with IL-15 doses greater than 25 ng/mL (Figure 1Ci)
and E/T ratios of 4/1 (Figure 1Cii). Of note, inhibition efficiency did not
depend on the antigen used to derive the cell line (i.e. phosphoantigen or intact
trophozoite). Vγ9Vδ2 T cells express various NK receptors that can modulate
their functions.25 The S1 and JT cell lines exhibited stable expression of NK
cell receptors, most of which were comparable in both cell lines, except
CD94/NKG2A and CD56, which were more highly expressed on JT cells
(Figure S2). These data indicated that the S1 and JT cell lines, although
generated differently, were phenotypically and functionally similar and
represented useful tools to further investigate the anti-parasitic activity of γδ T
cells. Based on these findings, a 4/1 E/T ratio after 24 hours of incubation in
the presence of IL-15(50 ng/mL) in addition to IL-2(20IU/mL) was used
13
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throughout the study to assess the anti-parasitic activity of the long-term γδ T
cell lines.
Granulysin, not perforin, mediates Vγ9Vδ2 T cell anti-parasitic activity
Separation of γδ T cells from the parasite culture in a Transwell insert
culture system abolished the parasite growth inhibition (Figure S3), confirming
that contact or close proximity between effector cells and parasites was
required.10,26 The cytotoxic activity of T lymhocytes, including γδ T cells,
operates via two major pathways: one involves the ligation of death receptors
on the target cells, whereas the other is dependent on the exocytosis-mediated
delivery of cytotoxic granule content, such as perforin, granzymes, or
granulysin, in the vicinity of the target cell.27 Neither infected or uninfected
RBCs express Fas or death receptors for TRAIL (Figure S4), ruling out a
possible involvement of this pathway in cytotoxicity against blood-stage
parsites. This is line with previous findings showing that the anti-parasitic
activity of γδ T cells relies on granule exocytosis.26 We thus investigated the
roles of perforin and granulysin, two cytotoxic molecules involved in the
killing of various microorganisms including bacteria28-30 and fungi18. To assess
the role of perforin, a long-term Vγ9Vδ2 T cell line (PFNdef) was generated
from a perforin-deficient patient. The PFNdef line expressed normal levels of
granulysin but no detectable perforin (Figure 2A). Parasite inhibition by
PFNdef was similar to the perforin-proficient cell lines S1 and JT (p>.05),
indicating that perforin was dispensable for anti-plasmodial activity (Figure
2B). Importantly, the irrelevant αβ T cell clone T4A5 did not display any
inhibitory activity under the same conditions.
14
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To assess the role of granulysin, we used RNAi to generate granulysin
knock-down T cell lines. The S1 line was infected with a lentiviral vector
expressing GNLYsh1 or GNLYsh2 granulysin-specific shRNA, resulting in
Gnly1 or Gnly2 cell lines, respectively, and a non-silencing shRNA (NS) was
used as a control. Granulysin mRNA expression was found to be reduced in the
Gnly1 and Gnly2 cell lines compared to the NS or non-infected S1 lines
(Figure 3A). Importantly, immunoblotting showed that the amount of
granulysin protein in both shRNA-expressing cell lines was much lower than
that in the NS or the non-infected S1 lines (Figure 3B). Perforin expression
remained unaltered, indicating specific shRNA targeting. Moreover, the three
lentivirus-infected Vγ9Vδ2 T cell lines produced similar levels of TNF-α upon
phosphoantigen stimulation, although slightly lower than the non-infected S1
cell line (Ctrl) (Figure S5). This supports the notion that, aside from decreased
granulysin expression, the functional responses of the lentivirus-infected lines
are maintained. Reduced granulysin expression was stable over time and
consistently more pronounced in the Gnly2 than in Gnly1 cell line (Figure 3C).
The inhibitory activity of the silenced cell lines was significantly reduced (p ≤
.05)(Figure 3D). Interestingly, the greatest loss of inhibitory activity was
observed in the Gnly2 cell line, which also displayed the lowest expression of
granulysin. These data show that granulysin, but not perforin, is essential for
the Vγ9Vδ2 T cell-mediated anti-parasitic activity.
Accordingly, the granulysin concentrations measured in the supernatants
harvested at the end of the parasite growth inhibition test in the 10 short-term
Vγ9Vδ2 T cell lines positively correlated with the anti-parasitic activities of
the individual lines (Figure S6).
15
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Schizonts-infected RBC and extracellular merozoites trigger Vγ9Vδ2 T
cell degranulation in a TCR-dependent manner
The release of granulysin and other cytotoxic lysosomal molecules via
exocytosis (degranulation) is associated with transient cell surface expression
of lysosomal membrane-associated glycoproteins such as CD107a(Lamp1).20
To identify the parasitic stages that trigger Vγ9Vδ2 T cell degranulation, we
performed a CD107a mobilization assay in the presence of schizonts or
merozoites. Both stages triggered dose-dependent CD107a surface expression
by the S1 and JT lines, whereas uiRBCs did not induce any significant CD107a
expression (Figure 4A and 4B). In contrast, no degranulation was observed in
γδ T cells expressing other T cell-receptor chains such as Vγ4Vδ5 or Vδ3VγI,
or in the αβ-T4A.5 T cell clone, suggesting specific Vγ9Vδ2 TCR involvement
in degranulation (Figure 4C). To confirm this, we performed CD107a assays in
the presence of an anti-Vδ2 blocking antibody31An anti-NKG2D blocking
antibody was used as a relevant isotype control,15 as NKG2D is another
activating receptor expressed by the S1 and JT cell lines (Figure S2). The antiVδ2 antibody, but not the anti-NKG2D, antibody inhibited the expression of
CD107a triggered by schizonts and merozoites in a dose-dependent manner in
both T cell lines (Figure 4D). Thus, both intraerythrocytic-stage (i.e.,
schizonts) and extracellular-stage parasites (i.e., merozoites) specifically
induced Vγ9Vδ2 degranulation in a TCR-dependent manner.
RBC-invasive merozoites are the targets of the Vγ9Vδ2 T cell antiparasitic activity
16
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Vγ9Vδ2 T cell-mediated parasite inhibition may reflect cytotoxicity targeting
either the developing intracellular trophozoites/schizonts or the invasive
extracellular merozoites. Examination of Giemsa-stained smears failed to
identify intracellular parasites with abnormal morphology or pyknotic forms,
and schizont maturation appeared un-delayed (data not shown) suggesting that
trophozoites/schizonts were insensitive to the Vγ9Vδ2 T cell anti-parasitic
activity. To exclude the possibility that schizonts could release impaired or less
invasive merozoites after contact with Vγ9Vδ2 T cells, we removed the cells
from the culture 6 hours after co-culture (just before schizont rupture), and
incubated the culture for an additional 24 hours (Figure 5Ai), which abrogated
the inhibitory activity of the S1 and JT cell lines completely (Figure 5Aii and
5Aiii). These results indicate that Vγ9Vδ2 T cells do not inhibit schizont
maturation or merozoite development, as merozoites were invasive after
egress; however, granulysin was exocytosed during the 6 hours of contact with
the maturing schizonts, as the intracellular amount of granulysin after 6 hours
of co-culture (T6hr) was lower than that before the incubation period (T0)
(Figure 5Aiii).
Together, these data suggest that Vγ9Vδ2 T cell anti-parasitic activity
targets merozoites. To demonstrate targeting of the merozoites, we performed a
merozoite invasion assay in the presence of the γδ T cell lines. Invasive
merozoites (prepared as described in the Materials and Methods and Figure S7)
were incubated with Vγ9Vδ2 T cell lines or with the αβ T cell clone T4A.5 for
28 hours, after which parasitemia was measured (Figure 5Bi). Both the S1 and
JT lines inhibited merozoite reinvasion, whereas the T4A.5 clone did not
(Figure 5Bi and 5Bii). In addition, short-term Vγ9Vδ2 T cells lines displayed
17
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an inhibition of merozoite invasion ranging from 30% to 60% (Table S1).
Taken
together,
these
data
show
that
the
intra-erythrocytic-stage
(trophozoite/schizont) parasites are insensitive to the Vγ9Vδ2 T cell
granulysin-mediated cytotoxic machinery and that the invasive extracellular
stage merozoites are the only targets vulnerable to Vγ9Vδ2 T cell anti-parasitic
activity.
High frequency of granulysin-producing Vδ2 T cells in malaria patients
To investigate Vγ9Vδ2 T cell granulysin-triggering during human infection,
we first assessed the presence of granulysin in circulating γδ T cells.
Intracytoplasmic granulysin was detected ex vivo in Vδ2 T cells from patients
with acute P.falciparum infection. The percentage of Vδ2 granulysin-positive
cells was much higher in infected patients than in control donors (median
48.9% versus 8.5%, p=.001), indicating that P.falciparum infection primed
Vγ9Vδ2 T cells to produce granulysin (Figure 6A). To confirm the primed
status of Vδ2 cells in vivo, we examined the expression of cell surface markers
specific for different effector/memory subpopulations.32The ratio of Vδ2 T
cells exhibiting an effector memory (i.e., TEM:CD27-CD45RA-) versus a
central memory (i.e., TCM:CD27+CD45RA-) phenotype was reversed in
infected patients compared to that of healthy controls (Figure 6Bi and 6Bii).
Furthermore, the total proportion of effector cells, including TEM:CD27CD45RA- and TEMRA:CD27-CD45RA+ cells, was higher in infected patients
compared to controls (65.7±14.3 vs. 26.5±10.9, p=0.001). Granulysin plasma
levels were higher in malaria patients (n=12) than in healthy controls (n=13)
(median 95.6 versus 25.3 ng/mL, p=.00001, Figure 6Ci) and correlated
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positively with the percentage of Vδ2 T cells (Spearman's rho=0.785, p=.036,
Figure 6Cii). This suggests that Vδ2 T cells are an important source of plasma
granulysin. For two patients (P1 and P2), we could measure the in vitro antiparasitic potential of the circulating Vδ2 cells. We performed a CD107a assay
with PBMCs from these patients and observed that a significant proportion of
Vδ2+ cells degranulated upon schizont stimulation (14.3% for P1 and 10.7%
for P2), whereas uiRBC induced only minimal expression of CD107a (Figure
6D). Of note, Vδ2-negative T cells did not express significant levels of
CD107a under the same conditions, highlighting the specific involvement of
Vδ2 T cells in parasite-triggered degranulation. Together, these data
demonstrate that Vδ2 T cell activation in vivo is associated with granulysin
expression, which enables the cells to contribute to the control of parasitemia
through a granulysin-dependent mechanism.
19
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Discussion
Human immune effectors mechanisms able to promptly and swiftly limit
parasite blood-stage multiplication remain largely unknown. Here, we confirm
and elucidate the anti-plasmodial activity of human Vγ9Vδ2 T cells, an innatelike lymphocyte subset. We show in vitro that i) activated γδ T cells contribute
to PBMCs parasite growth inhibition ii) γδ T cell lines inhibit parasite growth
iii) granulysin, but not perforin, is essential for this anti-parasitic activity iv)
both the intra-erythrocytic parasitic stages and the extracellular free merozoites
trigger TCR-specific Vγ9Vδ2 activation, degranulation and granulysin release
v) the vulnerable target of Vγ9Vδ2 T cells is the extracellular merozoite.
Finally we provide in vivo data showing that during a primary P.falciparum
infection, circulating Vγ9Vδ2 T cells express granulysin and degranulate when
they encounter P.falciparum parasites. Altogether, this work demonstrates that
the extracellular merozoite is an activator and a target of unconventional
cytotoxic T cells and provides strong indication for a direct contribution of
Vγ9Vδ2 T cells in the early control of parasite density.
In vitro inhibition of P.falciparum growth by Vγ9Vδ2 T cells required
degranulation and contact between γδ T cells and parasites, confirming
previous observations26. The short-term degranulation assay used here, allowed
us to show for the first time that intact live P.falciparum blood stages (either
intracellular (P.falciparum-infected RBC) or extracellular (merozoites) may
directly trigger γδ T cell activation/degranulation through a γδ TCR-dependent
mechanism without intervention from accessory cells. These two stages
contain DOXP phosphoantigen metabolites known to stimulate Vγ9Vδ2 T
cells,33 however, thus far, they have only been shown to stimulate γδ T cells as
20
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lysates/semi-purified extracts6,34,35 or in the presence of accessory cells.25,36
The data presented here provide a new model for the identification of
molecular mechanisms involved in the still enigmatic activation process of
Vγ9Vδ2 T cell by eukaryotic microorganisms.
Perforin and granulysin have been implicated in the anti-microbial
activity of γδ T cells. Granulysin is a positively charged cytolytic protein that
can be detected in T- and NK-cell granules in humans and has no equivalent in
mice.37 It displays anti-microbial activity against different microorganisms,
including bacteria, fungi, and parasites.28 Intracellular microorganisms killing
by granulysin has been shown to be dependent on perforin,29,30 which allows
granulysin to penetrate host membranes in a manner similar to granzymes,
leaving the cell intact.38 In contrast, perforin is not required to kill extracellular
bacteria,29 as granulysin is sufficient to cause lysis.39 The generation of
granulysin-silenced and perforin-deficient Vγ9Vδ2 T cell lines provided us
with powerful tools to investigate the relative contribution of these molecules
to the anti-parasitic activity. Granulysin-silencing reduced anti-plasmodial
activity dramatically, demonstrating that granulysin is required for the antiplasmodial activity of γδ T cells in vitro. The essential role of granulysin in this
process is further supported by previous reports that showed that antigranulysin antibodies abolished γδ T cell inhibitory activity.26 In contrast,
perforin seems to play a minor role in anti-parasitic activity, including against
the intracellular forms of P.falciparum. Indeed, although trophozoites and
schizonts specifically triggered degranulation and granulysin release, their
intracellular development was not impaired when co-cultured with Vγ9Vδ2 T
cells. As the erythrocytic membrane is resistant to granulysin,40 perforin does
21
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not appear sufficient to allow granulysin to pass this barrier and reach the
intravacuolar parasite. Remaining protected from cytotoxic molecules within
the erythrocyte for the duration of the intracellular development can be viewed
as a Plasmodium evasion strategy that has not been previously appreciated. In
contrast, the extracellular merozoite comes in direct contact with cytotoxic
molecules such as granulysin, which impairs its invasiveness for RBC and
thereby decreases parasitemia. How granulysin reduces P.falciparum
merozoite infectivity remains unclear. Granulysin induces osmotic lysis of
bacteria by altering membrane permeability.39 In eukaryotic tumor cells,
granulysin destabilizes membrane integrity through its positive charges,
causing a rapid increase in cytoplasmic calcium that leads to apoptosis.41 The
merozoite membrane differs both from the outer wall/membrane of bacteria
and from the classical eukaryotic membrane bilayer,42 and further investigation
is required to elucidate the molecular mechanisms by which granulysin
damages merozoites.
High plasma levels of granulysin and a high frequency of circulating
Vγ9Vδ2 T cells containing granulysin were detected in P.falciparum-infected
patients,
further
indicating
that
natural
infection
primes
Vγ9Vδ2 T cells for granulysin production. The positive correlation between
the plasmatic granulysin concentration and the frequency of Vγ9Vδ2 T cells,
suggests that this subset might be an important contributor to the circulating
granulysin in malaria patients. This conclusion is supported by the observation
that Vγ9Vδ2 T cells were the only T lymphocyte population triggered by
P.falciparum-infected RBC to degranulate. Further studies are required to
investigate whether granulysin expression correlates with in vivo control of
22
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parasite density and protection against severe malaria. As perforin has been
involved in experimental malaria pathology,43 it will be of interest to examine
whether perforin and granulysin expression are parallel or diverge depending
on the malaria clinical form, as has been shown in leprosy patients.44
An intriguing question raised by our data, is how and where do
activated effector Vγ9Vδ2 T cells target extracellular merozoites? Although
binding of γδ T cells with merozoites could be observed in vitro, it is not clear
whether this leads to a contact-oriented granule release. Even if this does not
occur, it is possible that γδ T cells degranulate towards schizonts, creating a
locally-enriched granulysin environment into which merozoites are released.
Although not required for anti-parasitic activity, perforin might favor the
egress of merozoites similarly to the recently described falciparum-perforinlike protein45 and thus promote a targeted action of granulysin on egressing
merozoites. Further studies are required to address this point.
A plausible in vivo scenario is that sequestration of trophozoites and
schizonts into micro-vessels creates a local inflammatory environment,
eliciting the recruitment of primed Vγ9Vδ2 T cells. This could constitute a
favorable niche for direct contact between infected RBC and γδ T cells, leading
to granulysin release and targeting of merozoites. These events may occur
along the micro-vessel endothelial cells of deep organs or in the red pulp of the
spleen, the site of P.falciparum-infected RBC retention and γδ T cell
accumulation during malaria infection.46,47Recent data from studies of patients
with Stevens-Johnson syndrome have highlighted massive concentrations of
granulysin in blisters (up to 10,000 ng/mL), suggesting that high granulysin
concentrations compatible with toxic activity may be achieved in confined
23
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surroundings.22 In addition to its direct cytotoxic role, granulysin may also
exacerbate local inflammation and indirectly promote parasite clearance
through its chemotactic activity for monocytes/macrophages and its proinflammatory properties.48
The present work identified the RBC-invasive merozoite as a novel
activator and target for unconventional cytotoxic T cells and adds Vγ9Vδ2 T
cell-mediated granulysin release to the list of effector mechanisms operating in
concert at early points in P.falciparum infection. In addition, our data extend to
malaria - a blood perpetuating infection -, the indications for γδ T cell-based
immunotherapy, which have yielded encouraging results in the treatment
hematological malignancies.49
24
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Acknowledgments
The authors kindly thank the cytometry core facility and the
vectorology platform (Bordeaux University), S. Netzer, M. Taillepierre, J.
Dallennes for technical assistance. The authors are grateful to G. Milon, L.
Couzi, and M. Mamami-Matsuda for valuable discussions. This work received
funding from the Centre National de la Recherche Scientifique (UMR CNRS
5164), the Ministère de la Défense (AGENPPAL), the Region Aquitaine
(PATHINF), the Fondation pour la Recherche Médicale (FRM), the European
Community's 6th Framework Program under grant agreement (LSHP-CT2004-N°503578- BioMalPar Network of Excellence) and 7th Framework
Program (FP7/2007-N°242095- EVIMALAR. G.C. is a fellow of the
MalParTraining FP6-Marie Curie Action (contract number MEST-CT-2005020492).
25
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Authorships contributions
G.C. performed experiments, analyzed data, and wrote the manuscript.
S.L., M.G. and I.M. performed experiments and/or analyzed data. F.H., G.S.B.
and V.P. provided vital reagents used in this work. J.D.M., J.F.M. and M.T.B.
reviewed the manuscript and provided critical discussion. O.P. reviewed the
manuscript, provided critical discussion, and secured funding. C.B. supervised
the entire project, designed research, wrote the manuscript, and secured
funding.
Conflict of interest disclosure
The authors declare no competing financial interest.
26
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Figure Legends
Figure 1. Anti-plasmodial activity of Vγ9Vδ2 T cells in PBMC and as
purified T cell lines .
(A) Undepleted PBMCs (total PBMCs) or Vδ2+ T cell-depleted PBMCs (Vδ2depleted PBMCs) from 8 different healthy donors were activated with BrHPP
(400 nM) or left untreated for 40 hours and then co-cultured in a standard
parasite inhibition assay at a 4/1 E/T ratio with a synchronized trophozoite
culture for 24 hours. At the end of this period, parasitemia in co-cultured
samples was compared to that in synchronized trophozoite cultures incubated
without PBMCs by hydroethidine staining. The percentage of Vδ2+ CD3+
cells among total PBMCs varied among the donors (from 0,5% to 1,9%). Data
represent the parasitemia (means of duplicates) of co-cultures with PBMCs of
each donor tested (n=8). * : p ≤ .05 by the Wilcoxon signed-ranked test. (B)
Short-term Vγ9Vδ2 T-cell lines (STL) were tested after a 24 hour priming with
no cytokine added, or with IL-2 (20 IU/mL) only or with IL-15 (50 ng/mL) and
co-cultured with the parasites at a 4/1 E/T ratio in a standard parasite inhibition
assay. Data represent the mean parasitemia in the various co-cultures
conditions (n=10 STL). ** p≤.01 for Wilcoxon signed-ranked test
comparisons. (C) The S1 and the JT long-term Vγ9Vδ2 T-cell lines were (i)
primed for 24 hours with IL-2 (20 IU/mL) and increasing doses of IL-15 and
co-cultured with the parasites at a 4/1 E/T ratio or (ii) primed for 24 hours with
IL-2 (20 IU/mL) with or without added IL-15 (50 ng/mL) and co-cultured with
the parasites at increasing E/T ratios as indicated. Graphs represent the mean of
parasitemia ± SD of four independent experiments (n=4). * : p ≤ .05 and ** : p
≤ .01 by the Mann-Whitney rank sum test. ns: no significant.
Figure 2. Perforin is dispensable for Vγ9Vδ2 anti-parasitic activity.
A Vγ9Vδ2 T-cell line was generated from a perforin-deficient familial
hemophagocytic lymphohistiocytosis (FLH) patient, and its anti-parasitic
activity assessed in a standard parasite inhibition assay. (A) Flow cytometry
histograms showing perforin and granulysin expression in Vγ9Vδ2 T-cell lines
generated from 2 different donors (S1 and JT) and from an FLH patient
(PFNdef). Mean fluorescence intensity (MFI) for perforin and granulysin
intracellular staining is indicated in the upper right corner of each panel. Dotted
lines represent isotype control antibody. (B) Comparison of the anti-parasitic
activity of the PFNdef , S1 and JT cell lines. The αβ T4A.5 T-cell clone was
32
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used as a negative control. Anti-parasitic activity (%) was calculated as follows
(100 - [(average % parasitemia of duplicate with cells in the presence of IL2+IL-15/average % parasitemia of duplicate with cells in the presence of IL-2)
x100]. Data represent the mean ± SD of four independent experiments (n=4). *
: p ≤ .05 or ns : not significant by the Mann-Whitney rank sum test comparing
the PFNdef cell line with the S1, JT or T4A.5 T-cell lines.
Figure 3. Granulysin is essential for Vγ9Vδ2 anti-parasitic activity.
The S1 T cell line was infected with the lentiviral vector containing the
granulysin-specific shRNA sequences GNLYsh1, GNLYsh2, or a nonsilencing shRNA sequence, and their anti-parasitic activity has been assessed
in a standard parasite inhibition assay. (A) RT-PCR assessment of granulysin
mRNA levels in the non-infected S1 cell line (Ctrl) or S1 cell line infected with
non-silencing (NS), GNLYsh1 (Gnly1) or GNLYsh2 (Gnly2) shRNA
contructs. β2-microglobulin mRNA levels were measured as a control. (B)
Granulysin and perforin protein levels were detected in S1 (Ctrl), NS, Gnly1
and Gnly2 cell lines by Western Blotting. Actin expression was measured as a
loading control. (C) Intracellular granulysin expression was measured using
flow cytometry in the NS, Gnly1 and Gnly2 cell lines. The percentage of
granulysin-positive cells and the mean fluorescence intensity (MFI) of
granulysin intracellular staining are indicated in the upper right corner of each
panel. Dotted lines represent isotype control antibody. (D) Anti-parasitic
activity of the NS, Gnly1 and Gnly2 cell lines. Anti-parasitic activity (%) was
calculated as follows (100 - [(average % parasitemia of duplicate with cells in
the presence of IL-2+IL-15/average % parasitemia of duplicate with cells in the
presence of IL-2) x 100]. Data represent the mean ± SD of two independent
experiments (n=2). * : p ≤ .05 by the Mann-Whitney rank sum test comparing
NS with Gnly1 and Gnly2 T-cell lines.
33
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Figure 4. Vγ9Vδ2 T cells degranulate in the presence of trophozoites and
merozoites in a TCR-dependent mechanism
The S1 and JT Vγ9Vδ2 T-cell lines were incubated for 6 hours with uninfected
red blood cells (uiRBC), purified schizonts (Schiz) or merozoites (Mero) and
CD107a surface expression was detected as described in Materiels and
Methods. (A) Representative flow cytometry dot-plot showing the gating
strategy for Vδ2+ T cells and for detection of CD107a surface expression on
gated cells after incubation with uiRBCs or schizonts. (B) Percentage of
CD107a+ cells from S1 and JT cell lines incubated in complete medium (CM)
or with uiRBC, purified schizonts or merozoites at decreasing E/T ratios (1/10,
1/5 and 1/2.5). Data represent mean ± SD of four independent experiments
(n=4) and were obtained by collecting of 10,000 Vδ2+ events. * : p ≤ .05 or ns :
not significant by the Mann–Whitney rank sum test comparing cells incubated
with uiRBC, schizonts or merozoites to cells incubated with complete medium.
(C) Percentage of CD107a+ cells among S1 and JT cells, or among control Tcells (a Vγ4Vδ5 T-cell line, a Vδ3VγI T-cell and an αβ T4A.5 T-cell clone)
incubated with uiRBCs, purified schizonts or merozoites at 1/10 E/T ratio. The
figure is representative of three independent experiments (n=3). (D) Percentage
of CD107a+ cells among S1 (upper panel) and JT cells (lower panel) preincubated for 1 hr with 1, 0.5, 0.1 and 0.01 μg/mL of anti-Vδ2 (IgG1, clone
immu389) or anti-NKG2D (IgG1, clone 149810) blocking antibodies and then
incubated with uiRBCs, purified schizonts or merozoites at 1/10 E/T ratio.
Data represent mean ± SD of four independent experiments (n=4) and were
obtained by collecting 10,000 Vδ2+ events. * : p ≤ .05 or ns : not significant by
the Mann-Whitney rank sum test comparing cells incubated with different
concentration of anti-Vδ2 or anti-NKG2D blocking antibodies to untreated
cells upon schizont or merozoite stimulation.
Figure 5. Merozoites, not trophozoites, are the target of Vγ9Vδ2 antiparasitic activity.
(A) Primed S1 or JT Vγ9Vδ2 T cells were co-cultured with purified
trophozoites (E/T ratio 4/1) for either 24 hours until merozoite re-invasion (no
removal of γδ) or were removed by magnetic depletion after 6 hours of coculture (just before schizont rupture) (removal of γδ T6h). Mock-treated
parasites cultured without cells mock were used as a control. After cell
removal, the parasitemia was similar under the various conditions. (i)
Schematic representation of the experimental design. (ii) Anti-parasitic activity
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was calculated as follows (100 - [(average % parasitemia of duplicate with
cells in the presence of IL-2+IL-15/average % parasitemia of duplicate with
cells in the presence of IL-2)x100]. Data represents the mean ± SD of the antiparasitic activity observed in three independent experiments performed in
duplicate. * : p ≤ 0.05 for Mann-Whitney rank sum test comparing the
conditions “removal of γδ” and “no removal of γδ” for each T cell line. (iii)
Histograms represent the mean of parasitemia ± SD observed in one
representative experiment performed in duplicate with the S1 and the JT cell
lines. No cells: parasites cultured in the absence of cells. * : p ≤ .05 by the
Mann-Whitney rank sum test comparing the parasitaemia of parasites cultured
in the presence of T cells (S1 or JT) to the parasitaemia in absence of T cells
(no cells), for each condition, (i.e“removal of γδ” or “no removal of γδ”); ns:
not significant. Flow cytometry histograms show granulysin expression in the
S1 and JT cell lines before (T0) and after 6 hours (T6hr) of co-culture with
trophozoite-stage parasites. Dotted lines represent isotype control antibody. (B)
Purified merozoites were incubated with fresh uiRBCs alone (no cells) or in
the presence of S1 or JT T-cells or the αβ T4A.5 T-cell clone and the
parasitemia was assessed after 28 hours. (i) Schematic representation of the
experimental design (ii) Inhibition of merozoite reinvasion (%) was calculated
as follows: 100 – [(average % parasitemia with cells/average % parasitemia
without cells)x100]. Data represent the mean ± SD of the inhibition of
merozoite invasion observed in 3 independent experiments performed in
duplicate (n=3). * : p ≤ .05 by the Mann-Whitney rank sum test comparing S1
or JT to T4A.5 (iii) Parasitemia from one representative experiment is shown
(mean ± SD). * : p ≤ .05 by the Mann-Whitney rank sum test comparing the
parasitaemia in the presence of T cells (S1 or JT or T4A.5) to the parasitaemia
in absence of T cells (no cells); ns: not significant.
Figure 6. Granulysin and Vδ2+ T-cells phenotype in primary infected
patients.
PBMCs from P.falciparum primary-infected patients and healthy donors were
stained ex-vivo for intracellular granulysin and effector/memory surface
markers (A)(i) Representative flow cytometry dot-plot outlining the gating
strategy for Vδ2+ T-cells (CD3+Vδ2+ lymphocytes). Flow cytometry
histograms showing granulysin expression in Vδ2+ gated T cells (black line)
compared to isotype control antibody (dotted line) in a representative patient
and healthy donor (control). The percentage of positive cells for granulysin
35
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intracellular staining is indicated in the upper right corner of each panel. (ii)
Percentages of CD3+Vδ2+ cells positive for granulysin in malaria patients and
healthy donors (controls). The box represents the 75th and 25th percentiles,
and the bar represents the median. p values were determined using the Mann–
Whitney rank sum test to compare patients (n=7) with controls (n=10). Data
were obtained by collecting 300,000 total events. (B) (i) Representative
example of flow cytometry data showing the gating strategy for Vδ2+ T-cells
(CD3+Vδ2+ lymphocytes). The expression of the CD27 and CD45RA cell
surface markers in the gated Vδ2+ T cells define distinct effector/memory
subpopulations (TNAIVE, TCM, TEM and TEMRA). (ii) The data represent the mean
± SD of the percentage of Vδ2+ T cells of each effector/memory subset from
patients or controls. * : p ≤ .05 by the Mann–Whitney rank sum test comparing
patients (n=6) to controls (n=10). ns: not significant. Data were obtained by
collecting 300,000 total events. (C) (i) Plasma granulysin levels in patients and
controls were detected by ELISA. The box represents the 75th and 25th
percentiles and the bar represents the median. The p value was determined
using the Mann–Whitney rank sum test to compare patients (n=12) with
controls (n=13). (ii) Log-transformed levels of plasma granulysin plotted
against the percentage of CD3+ T cells expressing Vδ2 in patients. Statistical
analysis was performed using the Spearman's rank correlation test, and the rho
and the p value are indicated (n=7). (D) Fresh PBMCs from two patients (P1
and P2) were co-cultured with purified trophozoites (iRBCs) or uninfected red
blood cells (or uiRBCs) at a 1/5 E/T ratio. CD107a surface expression detected
after 6 hours of co-culture was measured. The percentage of CD3+Vδ2+ T cells
or CD3+Vδ2- T cells expressing CD107a is shown. Data were obtained by
collecting 300,000 total events.
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Prepublished online November 1, 2011;
doi:10.1182/blood-2011-08-376111
Control of Plasmodium falciparum erythrocytic cycle: gamma-delta T cells
target the red blood cell-invasive merozoites
Giulia Costa, Séverine Loizon, Marianne Guenot, Iulia Mocan, Franck Halary, Geneviève de Saint-Basile,
Vincent Pitard, Julie Déchanet-Merville, Jean-François Moreau, Marita Troye-Blomberg, Odile
Mercereau-Puijalon and Charlotte Behr
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Copyright 2011 by The American Society of Hematology; all rights reserved.