δγ T Cells Kill Plasmodium falciparum in a Granzyme- and Granulysin-Dependent Mechanism during the Late Stage

This information is current as Maria Andrea Hernández-Castañeda, Katharina Happ, of September 28, 2021. Filippo Cattalani, Alexandra Wallimann, Marianne Blanchard, Isabelle Fellay, Brigitte Scolari, Nils Lannes, Smart Mbagwu, Benoît Fellay, Luis Filgueira, Pierre-Yves Mantel and Michael Walch

J Immunol published online 17 February 2020 Downloaded from http://www.jimmunol.org/content/early/2020/02/14/jimmun ol.1900725

Supplementary http://www.jimmunol.org/content/suppl/2020/02/16/jimmunol.190072 http://www.jimmunol.org/ Material 5.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 17, 2020, doi:10.4049/jimmunol.1900725 The Journal of Immunology

gd T Cells Kill Plasmodium falciparum in a Granzyme- and Granulysin-Dependent Mechanism during the Late Blood Stage

Maria Andrea Herna´ndez-Castan˜eda,* Katharina Happ,* Filippo Cattalani,* Alexandra Wallimann,* Marianne Blanchard,* Isabelle Fellay,* Brigitte Scolari,* Nils Lannes,* Smart Mbagwu,* Benoıˆt Fellay,† Luis Filgueira,* Pierre-Yves Mantel,* and Michael Walch*

Plasmodium spp., the causative agent of , have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control Downloaded from of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular subset of gd T cells contributes to protective during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that gd T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-g. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several http://www.jimmunol.org/ vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by gd T cells to kill late-stage blood-residing P. falciparum. The Journal of Immunology, 2020, 204: 000–000.

alaria remains one of the major health problems that release of merozoites from the liver into the blood stream, fol- challenge developing countries. More than 200 million lowed by a rapid invasion of uninfected RBCs. Merozoites dif- people are annually infected worldwide with Plasmodium ferentiate into a ring form that grows into a trophozoite. In the

M by guest on September 28, 2021 spp. parasites (1). In the human host, Plasmodium spp. have a subsequent schizont stage, the nucleus undergoes multiple divi- complex life cycle, including a liver and blood stage. However, it sions to give rise to several daughter merozoites. These repeated is accepted that clinical malaria is caused by the intraerythrocytic cycles of invasion, replication, and egress from RBCs lead to replication of the parasites. These replication cycles start with the exponential growth of the parasites in the blood, responsible for almost all the clinical symptoms of malaria and the associated morbidity and mortality. Therefore, to efficiently prevent malaria *Anatomy Unit, Department of Oncology, Microbiology and Immunology, Faculty of pathogenesis and progression toward severe disease, tight control Science and Medicine, University of Fribourg, 1700 Fribourg, Switzerland; and †Cantonal Hospital of Fribourg, 1752 Villars-sur-Glaˆne, Switzerland of parasitemia is essential (2). ORCIDs: 0000-0002-7151-3546 (M.A.H.-C.); 0000-0001-8392-0715 (F.C.); 0000- Protective immune responses to blood-stage malaria are highly 0001-6755-3679 (A.W.); 0000-0002-2926-6182 (N.L.); 0000-0003-4297- complex, requiring the interplay of innate and adaptive mecha- 6303 (S.M.); 0000-0001-5270-629X (B.F.); 0000-0001-9526-9309 (P.-Y.M.); 0000- nisms of humoral (3) and cellular immunity (4, 5). Abs inhibit 0001-7284-3291 (M.W.). parasite invasion at several levels, such as through phagocytosis Received for publication June 26, 2019. Accepted for publication January 15, 2020. and complement activation (6, 7). However, less is known about This work was supported by the Swiss National Science Foundation (Grant cytotoxic immune cell mechanisms during the blood stage. A 310030_169928 to M.W.), the Bangerter-Rhyner-Foundation (to M.W. and P.-Y.M.), and the Research Fund of the University of Fribourg (to M.W.). particular subset of T , bearing the gd TCR, has Address correspondence and reprint requests to Dr. Pierre-Yves Mantel or Prof. been demonstrated to be of importance in defending the host Michael Walch, Department of Oncology, Microbiology and Immunology, Faculty against a broad range of pathogens (8). In patients suffering from of Science and Medicine, University of Fribourg, PER03.I.15, Route Albert Gockel Plasmodium falciparum infection, gd T cells, particularly cells 1, 1700, Fribourg, Switzerland (P.-Y.M.) or Department of Oncology, Microbiology and Immunology, Faculty of Science and Medicine, University of Fribourg, bearing the Vg9Vd2 TCR (9), expand massively in the peripheral PER03.I.14, Route Albert Gockel 1, 1700, Fribourg, Switzerland (M.W.). E-mail blood (10, 11). Nevertheless, their inhibitory mechanisms remain addresses: [email protected] (P.-Y.M.) or [email protected] (M.W.) ill defined (12). The online version of this article contains supplemental material. Cytotoxic lymphocytes kill infected or malignantly transformed Abbreviations used in this article: BCECF-AM, 2,7-bis(2-carboxyethyl)-5(6)- cells by the release of their cytotoxic granule content. Target cell carboxyfluorescein acetoxymethyl ester; CMA, concanamycin A; DCI, 3,4- dichloroisocoumarin; GNLY, granulysin; Gzm, granzyme; GzmB, Gzm B; HCT, death is mediated by cytotoxic serine proteases, the granzymes hematocrit; hu, hemolytic unit; iRBC, infected RBC; MCM, malaria culture medium; (Gzms), that are delivered into the target cell by the pore-forming PFN, perforin. protein perforin (PFN) (13). Cytotoxic granules of some mammals This article is distributed under The American Association of Immunologists, Inc., contain another cytolytic protein, granulysin (GNLY), that pref- Reuse Terms and Conditions for Author Choice articles. erentially targets prokaryotic cholesterol-poor membranes, such as Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 of bacteria, fungi, and parasites (14, 15). In line with that, it has

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900725 2 gd T CELLS KILL PLASMODIUM FALCIPARUM been demonstrated that the antiplasmodial activity of gd T cells sporadically assessed for LPS (ToxinSensor Chromogenic LAL Endotoxin depended on GNLY (16, 17). Assay Kit; GenScript). For indicated experiments, after 7 d of activation, We have recently discovered that cytotoxic lymphocytes (by the effector cell population was enriched for gd T cells using a MACS kit (Positive MACS Selection; Miltenyi) following manufacturer’s recom- the concerted action of PFN, GNLY, and the Gzms) kill intra- mendations, routinely yielding a cell purity of .95%. To get rid of the cellular bacteria (18) and certain unicellular parasites, such as bound Abs after the MACS purification, the gd T cells were further ex- Trypanosoma cruzi (19). In this study, we followed up on this line panded with culture supernatant and IL-2 for another 6 d before their use of research and addressed the question of how gd T cells restrict in reinvasion assays. Cell purity after the further expansion phase was reassessed by flow cytometry (see Fig. 3D). the growth of blood-residing P. falciparum. gd phenotypization Materials and Methods For phenotyping, cells were stained with fluorescently conjugated anti- Parasites CD45 and anti-CD3 (for flow cytometry gating) as well as with anti- ab2TCR, gd2TCR, anti-CD56 Abs (all BD), and/or Vd2-TCR (Miltenyi) Cultures of the 3D7 strain P. falciparum were used in the experiments. on ice before analysis by flow cytometry (MACSQuant; Miltenyi). + Parasites were cultured in human A RBCs (obtained from healthy vol- Wherever available, appropriate control Abs were used to establish unteers) in malaria culture medium (MCM) composed of RPMI 1640 the staining protocols and test the specificity of the primary Abs. (25 mM HEPES, low bicarbonate, no glutamine; Sigma-Aldrich) supple- For cytometry assays, cell viability was assessed with Zombie Aqua mented with 1% heat-inactivated human serum, Albumax II (Life Tech- (BioLegend). For intracellular assessment, cells were pretreated nologies), gentamicin (Sigma-Aldrich), 20% glucose, and hypoxanthine, with the cell activation mixture (BioLegend) containing brefeldin (5 mg/ml), as previously described (20, 21). The parasites were maintained at 37˚C PMA (81 mM), and ionomycin (1.34 mM) for 4 h at 37˚C before staining in 5% CO2, 5% O2, and 90% N2. Hematocrit (HCT) was adjusted to 2%, for surface markers on ice. After fixation and permeabilization (Fixation/ Downloaded from except where specified otherwise. Permeabilization Kit; BD Biosciences), the cells were stained with an anti– IFN-g Ab (BioLegend) for assessment by flow cytometry, and cell viability Stage-specific parasite enrichments was assessed using Zombie Aqua (BioLegend). For imaging, the speci- An enrichment of ring stages was achieved as previously described (22). mens were counterstained with DAPI (1 mg/ml; Sigma-Aldrich) or with Briefly, a culture with high proportion of later-stage parasites and with Hoechst dye (1 mg/ml; Sigma-Aldrich), mounted in Vectashield (Vector parasitemia between 3 and 10% was centrifuged at 240 3 g for 10 min, Laboratories), and analyzed by confocal microscopy (SP5; Leica). The supernatant was removed, and pellet was resuspended in 20 vol of 0.5% IFN-g levels were assessed by ELISA (IFN-g dual kit; R&D Systems) gelatin in RPMI and incubated at 37˚C for 30–60 min. After the incuba- following manufacturer’s recommendations. The dilutions of the super- http://www.jimmunol.org/ tion, the supernatant was transferred to a fresh tube, centrifuged at 240 3 g natants before the ELISA, including the splitting dilutions during the ex- for 4 min, and supernatant was discarded. The pellet was washed twice, pansion and activation of the cells, were respected in the calculation and HCT was adjusted to 0.5% by adding appropriate volume of MCM and of IFN-g levels to reflect the total production of the cytokine during the activation. To confirm their cytolytic potential, the cells were stained incubated at 37˚C in 5% CO2 for 18–20 h. For experiments requiring late stages (trophozoites and schizonts), a with fluorescently conjugated anti-GzmB, GNLY, and PFN Abs (BD culture with high proportion of ring stage and with parasitemia .5% was Biosciences) using a kit for intracellular protein staining (Fixation/ centrifuged at 1800 rpm for 4 min, and supernatant was removed. Pellet Permeabilization Kit; BD Biosciences); counterstained with DAPI; moun- ted in Vectashield; and assessed by confocal microscopy. Additionally, their was treated with 5% D-sorbitol (Sigma-Aldrich) for 10 min at 37˚C. After the incubation, the culture was centrifuged at 1800 rpm for 4 min, su- cytotoxic potential was tested using K562 cells in 2,7-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Sigma-Aldrich) pernatant was discarded, pellet was washed twice, and HCT was adjusted by guest on September 28, 2021 to 0.5% by adding appropriate volume of complete medium and incubated release assays, as described (27, 28). As controls, killer cells were at 37˚C in 5% CO for 18–20 h. pretreated with 3,4-dichloroisocoumarin (DCI) (25 mM; Sigma- 2 Aldrich) or with concanamycin A (CMA, 100 nM; Sigma-Aldrich) Purification of cytotoxic effector molecules for 30 min. Native GNLY, Gzm B (GzmB), and PFN were purified from the human NK gd T cells—iRBC cocultures cell line YT-Indy as described previously (23–25). The hemolytic activity Effector cells activated with supernatant of iRBC were incubated with of PFN was determined using serial dilutions in assay buffer (10 mM ∼ HEPES, 0.15 M NaCl, and 0.1% BSA, pH 7.5) that were incubated with an P. falciparum late stage ( 10% parasitemia) for indicated times and at equal volume of 0.2% human RBCs in assay buffer containing 5 mM indicated E:T ratios in MCM. For indicated experiments, a blocking gd TCR Ab (2 mg/ml, clone 11F2; Miltenyi) was added to the effector cells CaCl2 at 37˚C for 20 min in round-bottom microtiter plates (Nunc, Rochester, NY). After the incubation, plates were centrifuged at 400 3 g before the coincubation with the iRBCs. for 3 min, and the supernatants were transferred to a second flat-bottom To visualize the conjugates, the killer cells–iRBCs mixture was washed plate. The hemoglobin released into the supernatant was detected with a twice with MCM before fixation with 1.5% paraformaldehyde in PBS. The microplate reader at a wavelength of 405 nm. One hemolytic unit (hu) conjugates were stained with a fluorescently conjugated anti-GzmB Ab was defined as the amount resulting in 50% lysis of 0.1% RBCs in a (BD Biosciences) using a kit for intracellular marker staining (BD Bio- volume of 0.2 ml. sciences), counterstained with Hoechst, mounted in Vectashield, and For some experiments, GzmB was fluorescently labeled using the Alexa assessed by confocal microscopy. Some conjugates were stained with a Fluor 488 Microscale Protein Labeling Kit (Thermo Fisher Scientific) fluorescently conjugated anti-gdTCR Ab (BD Biosciences) or anti-CD3 following manufacturer’s recommendations. Ab (Miltenyi). gd T cell expansion and activation Treatment of iRBCs with cytotoxic effector molecules P. falciparum gd T cells were generated from PBMC isolated from whole blood of –infected RBCs were washed with PBS and resuspended at healthy donors (blood bank, Bern, Switzerland) by Ficoll-Paque PLUS 1% HCT in hypotonic cell buffer (40 mM HEPES, pH 7.4, 50 mM NaCl, 4 mM CaCl , 1% BSA, and 5 mM glucose). iRBCs were treated for 30 min (GE Healthcare) density centrifugation. PBMCs were cultured in H10 2 with GzmB in combination with PFN or GNLY (prediluted in hypotonic (RPMI supplemented with 10% heat-inactivated human serum and 1% antibiotic/antimycotic solution [Thermo Fisher Scientific]) for 1 h at 37˚C. assay buffer: 40 mM HEPES, pH 7.4, 50 mM NaCl). After the incubation, the nonadherent suspension cells were collected and Mitochondrial membrane potential further cultured in H10 supplemented with a 1/50 dilution of infected RBC (iRBC) culture supernatants (mixed stages, 10% parasitemia) + 100 U/ml The mitochondrial membrane potential of P. falciparum–infected RBCs recombinant human IL-2 (Miltenyi) for 14 d (26). As controls for the was demonstrated by the staining of iRBCs with 2 mM JC-1 (Abcam) dye activation conditions, the supernatant of non-iRBCs (cultured using the in MCM. Staining was performed for 15 min at 37˚C followed by washes same protocol for iRBCs) + IL-2 and IL-2 alone were used. The cells were with PBS. Stained iRBCs were coincubated for 1 h with activated gd diluted and supplemented with fresh supernatant/IL-2 every 3–4 d. For T cells in MCM, then stained with Hoechst for assessment by live cell some experiments, the iRBC supernatant was size fractionated by passing confocal microscopy. through a spin filter column (Amicon Ultra, 10-kDa cutoff; Merck). In In contrast, iRBCs were treated in hypotonic cell buffer with cytotoxic addition, to exclude endotoxin contamination, iRBC supernatants were effector molecules (in assay buffer) for 30 min before being stained with The Journal of Immunology 3

2 mM JC-1 and 1 mg/ml Hoechst, washed with PBS + 5 mM glucose, and Results then assessed live by confocal microscopy or flow cytometry. gd T cells expand and develop cytolytic potential in response Reinvasion assays to P. falciparum–infected RBC supernatants The killer cells were prestained with 2 mg/ml BCECF-AM for 30 min at In humans in vivo (10, 29) and under in vitro (26) conditions using 37˚C, then washed three times with PBS and coincubated with iRBCs at PBMCs, P. falciparum infection increases the percentage and indicated E:T ratios for 2 h. The cocultures were performed in triplicates in absolute number of gd T cells. When we treated PBMCs with round-bottom 96-well plates with 50,000 killer cells in 100 ml MCM. As controls, the killer cells were additionally pretreated (during the 2,7- culture supernatant of P. falciparum–infected RBCs and IL-2, we Bis(2-carboxyethyl)-5(6)-carboxyfluorescein [BCECF-AM] staining) with found an expansion of gd T cells from ,2% to .50% after 14 d DCI (25 mM) or with CMA (100 nM). After the coincubation, the cells of coincubation, whereas ab T cells were less affected. Impor- were pelleted by centrifugation and diluted by adding 200 ml of non- tantly, to exclude donor-specific variations, all the experiments iRBCs at 1% HCT in complete medium. After 48 h, the cultures were in this study used PBMCs of at least four donors. In control counterstained with 5 mM DRAQ5 (Thermo Fisher Scientific), and para- sitemia was measured by flow cytometry. cultures, activated with the supernatant of non-iRBCs + IL-2 or P. falciparum–infected RBCs in cell buffer, treated with the cytotoxic with IL-2 alone, gd T cells were far less expanded (Fig. 1A, effector molecules in assay buffer, were pelleted by centrifugation and then 1B). CFSE staining for flow cytometry demonstrated that diluted by adding 200 ml of non-iRBCs at 1% HCT in complete medium. .90% of the gd T cells proliferated upon stimulation with After 48 h, the cultures were counterstained with 1 mM SYBR green (Thermo Fisher Scientific) and assessed by flow cytometry. Reinvasion was calcu- iRBC supernatant and IL-2 (Supplemental Fig. 1A). Consistent lated using the following formula: (parasitemiatreated/parasitemiacontrols)3100. with previous work (26), size fractionation of the iRBC su- ,

pernatant revealed a small molecule of 10 kDa responsible for Downloaded from GzmB uptake the activation of gd T cells (Supplemental Fig. 1B), most likely For the assessment of GzmB uptake, GzmB-AF488 was used. After the a soluble phosphoantigen. Confocal microscopy showed that incubation with effector molecules for 30 min, the cells were washed with the cells, activated with iRBC supernatant and IL-2, grow in PBS, fixed with 1.5% paraformaldehyde, and counterstained with Hoechst gd before assessing by confocal microscopy. big clusters, most of which highly expressed the TCR, and only a few displayed surface CD56 staining, normally in- Dextran uptake creased in IL-2–activated PBMC cultures (30) (Fig. 1C, 1D). http://www.jimmunol.org/ P. falciparum–infected RBCs were treated with low concentrations of PFN Strikingly, most of these clustered cells highly expressed the (0.25 hu) or GNLY (0.5 mM) for 5 min at room temperature in the presence major cytotoxic effector proteins, in particular GzmB (Fig. 1E) of dextran 4kD-AF488 (0.1 mM; Sigma-Aldrich) and counterstained with but also GNLY and PFN (Fig. 1F), and displayed Gzm- and Hoechst. Cells were directly assessed live by confocal microscopy. PFN-dependent cytolytic activity against the cell line K562 Measurement of ATP content (Fig.1G).Ahallmarkofgd T cells in human malaria is the early production of IFN-g (31). PBMCs, which were activated P. falciparum–infected RBCs treated with cytotoxic proteins were mixed 1:1 with the lysis/reaction buffer of the BacTiter-Glo Cell Viability Assay with iRBC supernatant and IL-2, produced significantly higher kit (Promega). The mixture was incubated at room temperature for 20 min amounts of IFN-g in the second week of activation as compared before the luminescence was measured by plate reader. with control PBMCs activated with noninfected supernatant or by guest on September 28, 2021 Transmission electron microscopy with IL-2 alone (Fig. 1H). In addition, intracellular cytokine staining in flow cytometry revealed the major source of IFN-g P. falciparum–infected RBCs treated with cytotoxic proteins were prefixed to be gd T cells upon activation with iRBC supernatant and IL-2 in 0.1 M cacodylate buffer (2.5% glutaraldehyde and 0.8% parafor- (Fig. 1I). maldehyde), postfixed with an aqueous solution (1% OsO4 and 1.5% K4Fe[CN]6),andembeddedintoepon.Ultrathinsections(50nm)were contrasted with lead citrate and uranyl acetate and analyzed with a CM gd T cells bind P. falciparum–infected RBCs to transfer GzmB 100 (Philips). To test the possible role of gd T cells as killer cells against P. Assessment of plasmodial substrate cleavage falciparum–infected RBCs, we incubated the activated effector population with P. falciparum–infected RBCs and stained for Escherichia coli Bl21 harboring GST-fusion protein in pGEX4Ti were induced for60minat37˚Cwith0.25mMisopropylb-D-1-thiogalactopyranoside T cell surface markers and GzmB. Abs against the pan-gd TCR (IPTG) in Luria-Bertani broth containing 100 mg/ml ampicillin and 2% and CD3 surface staining indicated that gd T cells formed con- glucose, washed, and resuspended in 20 mM NaCl, 10 mM Tris (pH 7.4) jugates with iRBCs (Fig. 2A). Early and late parasite stages were before adding Gzms and sublytic GNLY, as described (18). Reactions were found conjugated to these effector cells. Allele-specific staining stopped by boiling in SDS-PAGE loading buffer. Samples were analyzed by immunoblot using anti-GST mAb (BD Biosciences). demonstrated the presence of the Vd2 chain at the surface of the conjugated effector cells (Fig. 2B). Double staining of the con- Flow cytometry jugates with Abs against the d2 chain and intracellular GzmB Cells were analyzed using multicolor flow cytometer: MACSQuant indicated polarization of the cytolytic granules in the gVd2 T cells (Miltenyi) or Accuri C6 (BD Biosciences). Data were analyzed using toward the immunological synapse (Fig. 2C), typically observed FlowJo software (Ashland, OR) in immune conjugates between effector and target cells (32). Confocal laser scanning microscopy High-resolution confocal microscopy clearly revealed that within many of these conjugates GzmB was transferred into the bound Fluorescent-labeled specimens were examined using a confocal laser scanning microscope (SP5; Leica). Image acquisition was performed with iRBCs to be found in close proximity to parasite DNA (Fig. 2D). 403 and 633 objectives. Images were analyzed with Imaris 9.1 software To estimate the frequency of