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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 18 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. REFERENCES 1. Cohen S, Mc GI, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961;192:733-737. 2. Sabchareon A, Burnouf T, Ouattara D, et al. Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am J Trop Med Hyg. 1991;45(3):297-308. 3. Stevenson MM, Riley EM. Innate immunity to malaria. Nat Rev Immunol. 2004;4(3):169-180. 4. Moser B, Eberl M. gammadelta T cells: novel initiators of adaptive immunity. Immunol Rev. 2007;215:89-102. 5. Morita CT, Jin C, Sarikonda G, Wang H. Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens. Immunol Rev. 2007;215:59-76. 6. Behr C, Poupot R, Peyrat MA, et al. Plasmodium falciparum stimuli for human gammadelta T cells are related to phosphorylated antigens of mycobacteria. Infect Immun. 1996;64(8):2892-2896. 7. Eberl M, Hintz M, Reichenberg A, Kollas AK, Wiesner J, Jomaa H. Microbial isoprenoid biosynthesis and human gammadelta T cell activation. FEBS Lett. 2003;544(1-3):4-10. 8. Roussilhon C, Agrapart M, Ballet JJ, Bensussan A. T lymphocytes bearing the gamma delta T cell receptor in patients with acute Plasmodium falciparum malaria. J Infect Dis. 1990;162(1):283-285. 9. Ho M, Tongtawe P, Kriangkum J, et al. Polyclonal expansion of peripheral gamma delta T cells in human Plasmodium falciparum malaria. Infect Immun. 1994;62(3):855-862. 10. Elloso MM, van der Heyde HC, vande Waa JA, Manning DD, Weidanz WP. Inhibition of Plasmodium falciparum in vitro by human gamma delta T cells. J Immunol. 1994;153(3):1187-1194. 11. Troye-Blomberg M, Worku S, Tangteerawatana P, et al. Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium 27 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. falciparum parasite express cytolytic and proinflammatory molecules. Scand J Immunol. 1999;50(6):642-650. 12. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193(4254):673-675. 13. Pasvol G, Wilson RJ, Smalley ME, Brown J. Separation of viable schizont- infected red cells of Plasmodium falciparum from human blood. Ann Trop Med Parasitol. 1978;72(1):87-88. 14. Salmon BL, Oksman A, Goldberg DE. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl Acad Sci U S A. 2001;98(1):271-276. 15. Halary F, Pitard V, Dlubek D, et al. Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells. J Exp Med. 2005;201(10):1567-1578. 16. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, et al. Perforin gene defects in familial hemophagocytic lymphohistiocytosis. Science. 1999;286(5446):1957-1959. 17. Bonneville M, Moreau JF, Blokland E, et al. T lymphocyte cloning from rejected human kidney allograft. Recognition repertoire of alloreactive T cell clones. J Immunol. 1988;141(12):4187-4195. 18. Zheng CF, Ma LL, Jones GJ, et al. Cytotoxic CD4+ T cells use granulysin to kill Cryptococcus neoformans, and activation of this pathway is defective in HIV patients. Blood. 2007;109(5):2049-2057. 19. Sena-Esteves M, Tebbets JC, Steffens S, Crombleholme T, Flake AW. Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods. 2004;122(2):131-139. 20. Betts MR, Brenchley JM, Price DA, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003;281(1-2):65-78. 21. van der Heyde HC, Elloso MM, vande Waa J, Schell K, Weidanz WP. Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum. Clin Diagn Lab Immunol. 1995;2(4):417-425. 22. Chung WH, Hung SI, Yang JY, et al. Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med. 2008;14(12):1343-1350. 28 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 23. Caccamo N, Meraviglia S, Ferlazzo V, et al. Differential requirements for antigen or homeostatic cytokines for proliferation and differentiation of human Vgamma9Vdelta2 naive, memory and effector T cell subsets. Eur J Immunol. 2005;35(6):1764-1772. 24. Elloso MM, Wallace M, Manning DD, Weidanz WP. The effects of interleukin-15 on human gammadelta T cell responses to Plasmodium falciparum in vitro. Immunol Lett. 1998;64(2-3):125-132. 25. D'Ombrain MC, Hansen DS, Simpson KM, Schofield L. gammadelta-T cells expressing NK receptors predominate over NK cells and conventional T cells in the innate IFN-gamma response to Plasmodium falciparum malaria. Eur J Immunol. 2007;37(7):1864-1873. 26. Farouk SE, Mincheva-Nilsson L, Krensky AM, Dieli F, Troye-Blomberg M. Gamma delta T cells inhibit in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that requires granulysin. Eur J Immunol. 2004;34(8):2248-2256. 27. Barry M, Bleackley RC. Cytotoxic T lymphocytes: all roads lead to death. Nat Rev Immunol. 2002;2(6):401-409. 28. Stenger S, Hanson DA, Teitelbaum R, et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science. 1998;282(5386):121-125. 29. Dieli F, Troye-Blomberg M, Ivanyi J, et al. Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes. J Infect Dis. 2001;184(8):1082-1085. 30. Walch M, Latinovic-Golic S, Velic A, et al. Perforin enhances the granulysin- induced lysis of Listeria innocua in human dendritic cells. BMC Immunol. 2007;8:14. 31. Lang F, Peyrat MA, Constant P, et al. Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands. J Immunol. 1995;154(11):5986-5994. 32. Dieli F, Poccia F, Lipp M, et al. Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites. J Exp Med. 2003;198(3):391-397. 33. Ralph SA, van Dooren GG, Waller RF, et al. Tropical infectious diseases: metabolic maps and functions of the Plasmodium falciparum apicoplast. Nat Rev Microbiol. 2004;2(3):203-216. 29 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 34. Behr C, Dubois P. Preferential expansion of V gamma 9 V delta 2 T cells following stimulation of peripheral blood lymphocytes with extracts of Plasmodium falciparum. Int Immunol. 1992;4(3):361-366. 35. Goerlich R, Hacker G, Pfeffer K, Heeg K, Wagner H. Plasmodium falciparum merozoites primarily stimulate the V gamma 9 subset of human gamma/delta T cells. Eur J Immunol. 1991;21(10):2613-2616. 36. Waterfall M, Black A, Riley E. Gammadelta+ T cells preferentially respond to live rather than killed malaria parasites. Infect Immun. 1998;66(5):2393-2398. 37. Pena SV, Hanson DA, Carr BA, Goralski TJ, Krensky AM. Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins. J Immunol. 1997;158(6):2680-2688. 38. Pipkin ME, Lieberman J. Delivering the kiss of death: progress on understanding how perforin works. Curr Opin Immunol. 2007;19(3):301-308. 39. Ernst WA, Thoma-Uszynski S, Teitelbaum R, et al. Granulysin, a T cell product, kills bacteria by altering membrane permeability. J Immunol. 2000;165(12):7102-7108. 40. Li Q, Dong C, Deng A, et al. Hemolysis of erythrocytes by granulysin-derived peptides but not by granulysin. Antimicrob Agents Chemother. 2005;49(1):388-397. 41. Krensky AM, Clayberger C. Granulysin: a novel host defense molecule. Am J Transplant. 2005;5(8):1789-1792. 42. Garcia CR, de Azevedo MF, Wunderlich G, Budu A, Young JA, Bannister L. Plasmodium in the postgenomic era: new insights into the molecular cell biology of malaria parasites. Int Rev Cell Mol Biol. 2008;266:85-156. 43. Nitcheu J, Bonduelle O, Combadiere C, et al. Perforin-dependent brain- infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis. J Immunol. 2003;170(4):2221-2228. 44. Ochoa MT, Stenger S, Sieling PA, et al. T-cell release of granulysin contributes to host defense in leprosy. Nat Med. 2001;7(2):174-179. 45. Roiko MS, Carruthers VB. New roles for perforins and proteases in apicomplexan egress. Cell Microbiol. 2009;11(10):1444-1452. 30 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 46. Bordessoule D, Gaulard P, Mason DY. Preferential localisation of human lymphocytes bearing gamma delta T cell receptors to the red pulp of the spleen. J Clin Pathol. 1990;43(6):461-464. 47. Safeukui I, Correas JM, Brousse V, et al. Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen. Blood. 2008;112(6):2520-2528. 48. Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM. Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol. 2005;174(9):5243-5248. 49. Wihelm M, Kunzmann V, Eckstein S, et al. γδ T cells for immune therapy of patients with lymphoid malignancies. Blood. 2003;102:200-206. 31 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 34 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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. 36 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 37 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 38 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 39 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 40 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 41 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 42 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 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 Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include digital object identifier (DOIs) and date of initial publication. Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.