B7−CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human δγ T Cells via IL-2 Production

This information is current as Julie C. Ribot, Ana deBarros, Liliana Mancio-Silva, Ana of September 24, 2021. Pamplona and Bruno Silva-Santos J Immunol 2012; 189:1202-1208; Prepublished online 25 June 2012; doi: 10.4049/jimmunol.1200268

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Supplementary http://www.jimmunol.org/content/suppl/2012/06/25/jimmunol.120026 Material 8.DC1 http://www.jimmunol.org/ References This article cites 49 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/189/3/1202.full#ref-list-1

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human gd T Cells via IL-2 Production

Julie C. Ribot,* Ana deBarros,* Liliana Mancio-Silva,*,† Ana Pamplona,* and Bruno Silva-Santos*,‡ gd T cells play key nonredundant roles in to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for gd activation and expansion in vivo. In striking contrast to their ab counterparts, the costimulation requirements of gd T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in gd T cell activation. We report in this article

that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid gd T cells and Downloaded from synergizes with the TCR to induce autocrine IL-2 production that promotes gd cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, gd cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, gd cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-g–mediated and IL-17–mediated gd cell responses, which contrasted with the selective effect of CD27 on IFN-g–producing gd cells. Our data http://www.jimmunol.org/ collectively show that CD28 signals are required for IL-2–mediated survival and proliferation of both CD27+ and CD272 gd T cell subsets, thus providing new mechanistic insight for their modulation in disease models. The Journal of Immunology, 2012, 189: 1202–1208.

d T cells have been evolutionarily conserved as a lym- dendritic cells (DCs) or B cells (9). B7–CD28 signaling in ab g phocyte lineage whose signature TCR (TCRgd) does not T cells was shown to have both qualitative and quantitative effects obey the paradigm of MHC restriction (1). In fact, we that lower activation thresholds, promote cell proliferation, and know very little about Ag specificity and recognition via TCRgd enhance functional activity in vitro and in vivo (reviewed in Refs. by guest on September 24, 2021 (2). Although this has not precluded advances in exploiting the 9–11). functional properties of these , particularly in In contrast to this well-established function in ab T cells, (3), or in dissecting their pathological contribu- theroleofCD28ingd T cell activation has remained contro- tion to (4–6), it remains a priority to understand versial because of discrepant results of previous studies (7). how gd cells are activated in vivo. As part of our efforts to elu- In particular, resting murine gd splenocytes (12) and various cidate the contribution of costimulatory receptors to this process intraepithelial lymphocytes subsets (13–15) were reported to be (7, 8), we addressed the role of the Ig superfamily protein CD28. devoid of CD28 expression, which was observed, however, upon Much of what we know about T cell costimulation has come cellular activation (12). This pattern contrasted with that of human from studies on CD28 and its ligands, B7.1 (CD80) and B7.2 Vd2+ PBLs, which significantly downregulated CD28 following (CD86), which are typically found on professional APCs, such as activation (16). Moreover, human Vd1+ cells, unlike their Vd2+ counterparts, failed to express CD28 (17). Furthermore, although *Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de CD28 signals promoted the in vitro proliferation of mouse Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal; †Unidade de Mala´ria, splenocytes (12, 15) and human gd lymphocytes (18), the allor- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, eactivity of murine Vg2+ transgenic was normal in the 1649-028 Lisboa, Portugal; and ‡Instituto Gulbenkian de Cieˆncia, 2781-901 Oeiras, Portugal absence of CD28 signaling (19). Critically, none of these studies Received for publication January 23, 2012. Accepted for publication May 24, 2012. examined the role of CD28 costimulation during physiological gd cell responses to infection. This work was supported by Fundac¸a˜o para a Cieˆncia e Tecnologia (PTDC/SAU-MII/ 104158/2008) and the European Molecular Biology Organization (Young Investiga- In this study, we used the last generation of specific mAbs and tor Programme). L.M.-S. is supported by a European Molecular Biology Organiza- genetically manipulated mice to unequivocally assess the impact of tion fellowship (ALTF960-2009). B7–CD28 signals in the context of gd cell responses to Plasmo- Address correspondence and reprint requests to Prof. Bruno Silva-Santos or Dr. Julie dium parasites, the infectious agents that cause . Infection Ribot, Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Avenida Prof. Egas Moniz, 1649-028 Lisboa, Portugal. E-mail addresses: [email protected] by Plasmodium is known to cause striking expansions of gd cells (B.S.-S.) and [email protected] (J.R.) both in mice (20–22) and in humans (23–26). In fact, in patients The online version of this article contains supplemental material. infected with either Plasmodium falciparum (23, 24) or Plasmo- Abbreviations used in this article: B6, C57BL/6; DC, ; HMB-PP, 4- dium vivax (25), gd cells frequently expand to 30–40% of all hydroxy-3-methyl-but-2-enyl pyrophosphate; ICOSL, ICOS ligand; LN, ; peripheral T cells (compared with 1–5% in healthy donors). OX40L, OX40 ligand; PbA, Plasmodium berghei ANKA; WT, wild-type. Moreover, gd cells were shown to be the major cellular source of Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the key proinflammatory IFN-g in patients in endemic www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200268 The Journal of Immunology 1203 areas (27), and this correlated with reduced incidence of clinical were used as blocking reagents; anti-mouse mAbs for CD3ε (145.2C11) and episodes (28). Therefore, malaria constitutes one of the most CD28 (37.51) were used as agonists. relevant physiological contexts in which to investigate gd cell The following anti-human mAbs were used for flow cytometry: anti– CD28-FITC (CD28.2; eBioscience); anti–CD69-PE (FN50), anti–CD70- activation. We performed a comprehensive series of gain-of- PE (ki-24), anti–CD80-PE (L307.4), and anti–CD86-allophycocyanin function and loss-of-function studies that demonstrates a critical (2331) (all from BD Pharmingen, San Diego, CA); and anti–OX40 ligand role for B7–CD28 interactions in the activation and expansion of (OX40L)-PE (11C3.1) and anti–ICOS ligand (ICOSL)-PE (2D3) (both murine and human proinflammatory gd T cell subsets in response from BioLegend, San Diego, CA). The mAbs used as blocking reagents in human cell cultures were anti- to Plasmodium Ags. CD70 (ki-24) and anti-CD86 (IT2.2) (both from BD Pharmingen); anti- ICOSL (9F.8A4; BioLegend); and anti-CD80 (37711) and anti-OX40L Materials and Methods (159403) (both from R&D Systems). IgG1 (MOPC-21), IgG2 (MPC-11), Mice and IgG3 (MG3-35) were used as controls (all purchased from BioLegend). All mice were adults 4–10 wk of age. C57BL/6 (B6), B6.TCRa-deficient (Tcra2/2), and B6.CD28-deficient (Cd282/2) mice were described previ- Flow cytometry and cell sorting ously (29, 30) and were obtained from The Jackson Laboratory. B6.CD27- 2/2 Cells were sorted electronically using a FACSAria (BD Biosciences, San deficient (Cd27 ) mice were a kind gift from Dr. Jannie Borst (Neth- Jose, CA). For cell surface stainings, cells were incubated for 15 min on ice erlands Cancer Institute, Amsterdam, The Netherlands). Mice were bred in 2.4G2 (anti-FcgR mAb) hybridoma supernatant and then incubated for and maintained in the specific pathogen-free animal facilities of Instituto 6 15 min with saturating concentrations of the indicated mAbs. For intra- de Medicina Molecular. When stated, mice were infected i.p. with 10 cellular cytokine staining, cells were stimulated with PMA (50 ng/ml) and Plasmodium berghei ANKA (PbA)-infected erythrocytes and monitored as ionomycin (1 mg/ml) (both from Sigma) for 4 h at 37˚C; 10 mg/ml bre- Downloaded from described (31). All experiments involving animals were performed in feldin A (Sigma) was added during the last 2 h. Cells were stained for the compliance with the relevant laws and institutional guidelines and were indicated cell surface markers, and intracellular staining was performed approved by the local ethics committees. using fixation/permeabilization and permeabilization buffers (both from Preparation of supernatants from P. falciparum-infected eBioscience), following the manufacturer’s instructions. Samples were analyzed using Fortessa or FACSCalibur (both from BD Biosciences). erythrocyte cultures P. falciparum 3D7 blood-stage parasites were cultured using modifications In vitro functional assays to the method described by Trager and Jensen (32). Parasites were grown activation and apoptosis were assessed between days 1 and 6 of http://www.jimmunol.org/ in human erythrocytes (5% hematocrit, 5% parasitemia) in RPMI 1640 culture by staining for CD69 and for annexin V (BD Pharmingen), re- medium containing L-glutamine supplemented with 0.5% (w/v) AlbuMAX spectively, according to the manufacturer’s instructions. IL-2 secretion was II, 25 mM HEPES, and 0.05 mg/ml gentamicin at 37˚C in a 5% CO2 quantified using Cytometric Bead Array as described (33). For analysis of environment. Supernatant of synchronized cultures in mature late-stage cell proliferation, cells were stained with 5 mM the cytoplasmic dye CFSE schizonts (40–50 h after reinvasion) was obtained by centrifugation at (Molecular Probes) for 5 min at 37˚C, cultured for 3 d, and analyzed by 2000 rpm for 5 min. Synchronization of cultures consisted of treatment flow cytometry for CFSE dilution. Alternatively, 1 mCi [3H]thymidine with 5% (w/v) D-sorbitol, one or two cycles before supernatant collection. (Amersham) was added for the last 18 h, and [3H]thymidine incorporation Culture of human PBLs was measured using the MicroBeta TriLux scintillation counter (Perki- nElmer).

Human peripheral blood was collected from anonymous healthy volunteers by guest on September 24, 2021 in accordance with the guidelines of the Declaration of Helsinki. Total Quantitative real-time PCR PBMCs were prepared and cultured as previously described (33, 34). For RNA was prepared and analyzed by quantitative real-time PCR, as previously purification of gd PBLs, cells were sorted on a MiniMACS separator using described (21, 22). The primers used were: Il2-Fwd, 59-GCTGTTGAT- an anti-TCRgd MicroBead Kit (Miltenyi Biotec). Specific Vg9Vd2 T cell GGACCTACAGGA-39; Il2-Rev, 59-TTCAATTCTGTGGCCTGCTT-39; activation was accomplished by continuous stimulation (up to 6 d) of total Efa1-Fwd, 59-ACACGTAGATTCCGGCAAGT-39; and Efa1-Rev, 59-AG- PBMCs or sorted gd-PBLs with 10 nM 4-hydroxy-3-methyl-but-2-enyl GAGCCCTTTCCCATCTC-39. Transcripts were quantified by the standard pyrophosphate (HMB-PP; Echelon Biosciences) and 25–100 U/ml IL-2 curve method, normalized to Efa1, and expressed in arbitrary units. (Roche Applied Science). Alternatively, PBMCs or sorted gd-PBLs were cultured in the presence of collected supernatant from P. falciparum cul- Statistical analysis tures. When indicated, anti-human blocking mAbs (or isotype controls) were added at a concentration of 10 mg/ml. Statistical significance of differences between populations was assessed using the Mann–Whitney test. Culture of murine lymphocytes Cells from spleen, lymph nodes (LNs), or peritoneal cavity were prepared as Results described (21). Cells were cultured in RPMI 1640 medium with 10% FCS, B7–CD28 interactions promote the proliferation and survival 50 mM 2-ME, L-glutamine, nonessential amino acids, 10 mM HEPES, of murine gd T cells penicillin, and streptomycin in 96-well round-bottom plates. When indi- cated, 30–300 U/ml rIL-2 (Sigma) was added to the cultures. Cells were This study began with the observation that lymphoid (spleen and stimulated with 0.5 mg/ml soluble anti-CD3ε Ab, in the presence of 5 3 LN) gd cells, in contrast to their intraepithelial counterparts (13– 105 APCs (3000 rad-irradiated splenocytes)/5 3 104 responding cells. Alternatively, cells were cultured on plate-bound anti-CD3ε (0.1–10 mg/ 15), constitutively express CD28 at similar levels to ab cells (Fig. ml) and anti-CD28 (10 mg/ml) or soluble rCD70 (5 mg/ml; kindly provided 1A). Moreover, in vitro activation with anti-CD3 (aCD3ε) mAb by Dr. Jannie Borst). When indicated, anti-mouse agonist or blocking significantly upregulated CD28 levels on isolated (FACS-purified) mAbs were added to the culture medium at a concentration of 1–5 mg/ml. gd cells (Fig. 1B), whereas the addition of B7-expressing APCs mAbs provoked CD28 downregulation (Supplemental Fig. 1A). In con- trast, the CD28-related Ig superfamily member, ICOS, was not The following anti-mouse mAbs were purchased from eBioscience (San expressed in either resting or activated gd cells (Fig. 1B). Diego, CA) and used for flow cytometry: FITC-labeled anti-TCRd (eBioGL3), anti-CD69 (H1.2F3), and anti–IL-17 (eBio17B7); PE-labeled To functionally test the role of B7–CD28 interactions in gd cell anti-TCRd (eBioGL3), anti-CD11c (N418), and anti-IFN-g (XMG1.2); activation, we used CD28 agonists (anti-CD28 mAb) or antago- PerCP-Cy5.5–labeled anti-CD3ε (145.2C11); PE-Cy7–labeled anti-CD19 nists (anti-B7.1/CD80 and anti-B7.2/CD86 mAbs) in cocultures of (eBio1D3); allophycocyanin-labeled anti-TCRd (eBioGL3), anti-CD11b gd cells (10%) and irradiated splenocytes (90%). After 3 d of (M1/70), and anti–IL-17 (eBio17B7); eFluor 450-labeled anti-CD4 ε (RM4-5); biotin-conjugated anti-CD28 (37.51); and streptavidin-PE. activation (with aCD3 mAb), the percentage of live gd cells In murine cell cultures, anti-mouse mAbs (from eBioscience) specific for recovered from these cultures was markedly higher in the presence IL-2 (JES6-1A12), ICOS (7E.17G9), CD80 (16-10A1), and CD86 (GL1) of CD28 agonists and lower in the presence of CD28 antagonists, 1204 CD28 COSTIMULATION OF gd T CELLS Downloaded from FIGURE 1. CD28 signals promote murine gd T cell activation and proliferation. (A) Cells pooled from spleens and LN of adult WT B6 mice were stained with mAbs specific for CD3ε, TCRgd, and CD28. The graph shows the expression of CD28 on pregated CD3ε+ TCRgd+ cells, CD3ε+ TCRgd2 cells, and CD3ε2 cells. Peripheral lymphoid cells from Cd282/2 mice (shaded curve) are shown as a negative control. (B) gd cells were sorted from pooled spleen and LNs of Tcra2/2 mice and cultured with plate-bound anti-CD3ε mAb. Mean fluorescence intensity (MFI) of CD28 or ICOS stainings at indicated time points are shown. Dashed line represents isotype control staining. (C–G) gd cells were sorted by FACS from pooled spleen and LNs of Tcra2/2 mice, labeled with CFSE, and cultured for 3 d in the presence of APCs and soluble anti-CD3 mAb, without mAbs (ctrl), or with the addition of the following

mAbs: blocking anti-ICOS, blocking anti-CD80 and anti-CD86 (aB7), or agonist anti-CD28. (C) Representative forward scatter (FSC)/side scatter (SSC) http://www.jimmunol.org/ plots. Numbers refer to percentages of cells within the indicated regions. (D) CD69 expression on gd cells at day 2 (upper panel) or at the indicated time points (lower panel). Color code is the same as used in (G). (E)[3H]thymidine incorporation for 18 h between days 2 and 3, measured in a scintillation counter (cpm). (F) CFSE dilution at day 3 (left panel) and extracted numbers of cells per division (right panel). (G) Percentage of annexin V+ cells at day 3. Error bars represent SD (n = 3). All data are representative of three to five independent experiments.*p , 0.05, **p , 0.01, ***p , 0.001. whereas blocking ICOS had no effect (Fig. 1C). This correlated dilution assays. CD28 agonists enhanced gd cell proliferation, well with the activation phenotype of gd cells, as evaluated by the whereas CD28 antagonists inhibited it completely (Fig. 1E, 1F). expression of the activation marker CD69 (Fig. 1D). Consistent with this, CD28-deficient gd cells showed impaired

To assess the precise role of CD28 signaling on gd cell pro- activation and proliferation in vitro (Supplemental Fig. 1B, 1C). by guest on September 24, 2021 liferation, we performed [3H]thymidine incorporation and CFSE- Furthermore, CD28 signaling on gd cells also affected their sur-

FIGURE 2. CD28 costimulation of gd cells induces secretion of IL-2 required for proliferation. gd cells were sorted using FACS from pooled spleen and LN of Tcra2/2 mice and activated with the indicated amounts of plate-bound anti-CD3 mAb in the presence of 10 mg/ml plate-bound anti-CD28 mAb (aCD28; in green) or isotype control (iso ctrl). (A) CFSE dilution profiles after 2 d under the indicated concentrations of anti-CD3 (left panel) and extracted numbers of cells/division at 0.1 mg/ml anti-CD3 (right panel). (B) IL-2 concentration was measured at the indicated time points in the supernatant of cultures stimulated with 0.1 mg/ml anti-CD3. (C) Quantitative real-time PCR for Il2 expression in purified gd cells cultured for 6 h with 0.1 mg/ml anti-CD3 mAb in media alone, media supplemented with soluble rCD70 (sCD70) or anti-CD28 mAb (aCD28), or isotype control (iso ctrl). Expression was nor- malized to the housekeeping gene Efa1 and expressed in arbitrary units (a.u.). CFSE dilution profiles (D) and extracted numbers of cells/division (E) after 3 d of stimulation with 0.1 mg/ml plate-bound anti-CD3 in the presence of 10 mg/ml isotype control (iso ctrl) (gray), 10 mg/ml plate-bound anti-CD28 (green), 5 mg/ml anti–IL-2 (white), or 300 U/ml rIL-2 (black). Error bars represent SD (n = 3). Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 1205 vival following activation, as indicated by annexin V staining of day-3 cultures (Fig. 1G). Collectively, these data demonstrate that B7–CD28 signals control the activation, survival, and proliferation of murine gd T cells. CD28 costimulation is necessary for autocrine IL-2 production by gd T cells Because the previous (standard) assays used total irradiated splenocytes as “feeders,” which typically included ∼30% of ab T cells that also express CD28, it was critical to assess whether B7–CD28 signals operated directly on gd cells. For this, we established cultures of highly (.99%) purified gd cells, activated by plastic-bound agonist Abs to TCR/CD3 and CD28. We ob- served that CD28 costimulation provided a marked proliferative advantage at low amounts of TCR/CD3 triggering (i.e., 0.1 mg/ml anti-CD3ε mAb) (Fig. 2A). This effect was lost at saturating (10- fold higher) concentrations of anti-CD3ε mAb (Fig. 2A). To gain further mechanistic insight into the direct effects of

CD28 on gd cell activation, we considered that CD28 costimu- Downloaded from lation of ab cells can enhance their production of IL-2 (35, 36), which is a key determinant of gd cell expansion (33, 37). Therefore, we measured IL-2 protein levels in the supernatants of the gd cell cultures. Strikingly, IL-2 was only detected when these were supplemented with CD28 agonists (Fig. 2B). The induction FIGURE 3. CD28 signals are required for murine gd cell responses to 2/2 2/2 of IL-2 expression in CD28-costimulated gd cells was also ob- Plasmodium infection in vivo. B6 (WT), Cd28 ,orCd27 mice were http://www.jimmunol.org/ served at the mRNA (transcriptional) level (Fig. 2C). Importantly, infected with PbA, and their spleens were harvested and analyzed by flow A CD27 triggering using soluble recombinant ligand (7, 8) did not cytometry at the indicated days postinfection. ( ) Mean fluorescence in- upregulate Il2 expression (Fig. 2C), suggesting that this mecha- tensities (MFI) for CD28 and ICOS expression in gd T cells from WT mice. Dashed line represents isotype control staining. (B) MFI for CD86 nism is specifically downstream of CD28 signaling in gd cells. 2 2 2 2 expression in DCs from WT, Cd28 / ,orCd27 / mice at day 3 post- The interesting dynamics of IL-2 production by gd cells and infection. Dashed line indicates reference MFI in naive WT animals. their proliferation following TCR/CD28 stimulation (Supple- Absolute numbers of total gd cells (C) and activated CD69+ gd cells (D)at mental Fig. 1D) prompted us to test whether autocrine IL-2 pro- day 3 postinfection. (E and F) gd cells were sorted using FACS and stained duction was essential for gd cell proliferation. Upon addition of intracellularly for IFN-g and IL-17. Absolute numbers of IFN-g+ gd cells neutralizing Abs against murine IL-2, a strong reduction in gd cell (E) or IL-17+ gd cells (F) are shown. Error bars represent SD. All data are by guest on September 24, 2021 expansion was observed (Fig. 2D, 2E). Conversely, the addition of from three independent experiments involving 6–10 mice. *p , 0.05, saturating doses of IL-2 bypassed the need for CD28 costimula- **p , 0.01, ***p , 0.001. ns, Not statistically significant (p $ 0.05). tion, both in isolated gd cell cultures (Fig. 2D, 2E) and in cocultures with splenic APCs (Supplemental Fig. 1E). These In PbA-infected CD28-deficient animals, the reduced (compared results show that CD28 costimulation directly controls gd cell with WT) gd T cell compartment also contained smaller pools of expansion through the induction of IL-2 production. both IFN-g–producing and IL-17–producing subsets (Supple- mental Fig. 2). In fact, the CD28 deficiency completely prevented CD28 signals are required for gd cell responses to the accumulation of IFN-g+ (Fig. 3E) and IL-17+ (Fig. 3F) gd Plasmodium infection in vivo cells upon malaria infection. We observed an additional difference To establish the importance of CD28 signaling on gd cell activation in comparison with CD27-deficient animals: the expansion of IL- in vivo, we resorted to a mouse model of severe malaria, induced 17+ gd cells was selectively impaired in CD282/2 mice (Fig. 3F). by PbA infection of B6 mice (31). Importantly, we had previously In contrast, both mutant models exhibited a defect in the IFN-g documented very robust gd cell responses in this animal model (8, response (Fig. 3E). This led us to assess the combined effect of 21, 22). CD27/CD28 signals on IFN-g production by gd cells in vivo. For We followed the phenotype of gd cells during the course of this, we decided to block B7–CD28 signaling in CD272/2 mice infection and detected a transient upregulation of CD28 at day 3, during the course of infection. The efficient mAb-mediated neu- whereas ICOS was not induced on gd cells (Fig. 3A). In PbA- tralization of CD80 and CD86 caused a significant decrease in the infected mice, the modulation of CD28 expression on gd cells was numbers of activated IFN-g+ gd cells in the CD27-deficient accompanied by the upregulation of its inducible ligand, CD86, on background (Supplemental Fig. 3). Thus, CD28 acts nonre- professional APCs, such as DCs (Fig. 3B). This led us to inves- dundantly and synergistically with CD27 in the activation of IFN- tigate the functional consequences of B7–CD28 costimulation g+ gd cells following malaria infection. in vivo. For this, we analyzed CD28-deficient mice and observed These data collectively demonstrate that CD28 signals are re- their failure to expand gd cells on infection, in contrast to wild- quired for the expansion of murine proinflammatory gd cell subsets type (WT) controls (Fig. 3C). This correlated with a compara- in vivo. tively small increase in the pool of CD69+ (activated) gd cells in infected CD28-deficient mice (Fig. 3D). Importantly, the effects of Dynamic expression of CD28 and its B7 ligands on activated CD28 deficiency on the numbers of total or activated gd cells were human gd cells not phenocopied in CD27-deficient animals (Fig. 3C, 3D), thus We next investigated the role of CD28 costimulation on human gd demonstrating that the two costimulatory pathways play distinct cells. Freshly isolated Vg9Vd2 PBLs were previously shown to roles in gd T cell expansion in vivo. express CD28 (18, 38). Moreover, Vg9Vd2 PBLs are known to be 1206 CD28 COSTIMULATION OF gd T CELLS highly activated by soluble metabolites produced by apicom- CD28 costimulation controls survival and proliferation of plexan protozoa like P. falciparum (39). To study the expression activated human gd cells dynamics of CD28 and its B7 ligands, CD80 and CD86, on To directly assess the B7–CD28 costimulation requirements of Vg9Vd2 PBLs exposed to microbial Ags, we established an ex- HMB-PP–activated Vg9Vd2 PBLs, we used monoclonal blocking perimental system in which either total PBMCs or purified gd Abs specific to CD80 and CD86 in Vg9Vd2 PBL cultures. We PBLs were incubated for 2–6 d with supernatants collected from observed a clear impairment in cell proliferation, as assessed by P. falciparum-infected erythrocyte cultures. We observed a CFSE-dilution assays (Fig. 5A), compared with control cultures marked downregulation of CD28 after 2 d of culture with incubated with isotype Abs. Moreover, CD80/CD86 blockade also P. falciparum-derived supernatants (Fig. 4A), concomitantly with affected the survival of HMB-PP–activated Vg9Vd2 PBLs, as the induction of CD80 and CD86 expression on Vg9Vd2 PBLs indicated by the accumulation of annexin V+ cells (Fig. 5B). This (Fig. 4B). This induction was a direct effect of the Plasmodium combined survival/proliferation effect of CD80/CD86 inhibition Ags on Vg9Vd2 PBLs, because it was also observed on isolated resulted in a highly significant reduction in thymidine incorpora- Vg9Vd2 PBLs (Supplemental Fig. 4A). tion in Vg9Vd2 PBL cultures (Fig. 5C). Moreover, and consistent A strong candidate P. falciparum Ag was HMB-PP, the most with the murine data (Fig. 2D, 2E), the need for CD28 costimu- potent natural Vg9Vd2 TCR agonist known (33, 39), because it is lation was bypassed by saturating amounts of exogenous IL-2 the product of HMB-PP synthase (GcpE) present in all Plasmo- (Fig. 5C). dium spp. and is predominantly expressed on mature blood-stage Interestingly, the inhibition of ICOSL (CD275; B7-H2) or parasites (http://plasmodb.org; Gene ID: PF10_0221). Therefore, OX40L (CD252) had no impact on their proliferation (Fig. 5A, we established similar cultures of PBMCs or purified Vg9Vd2 5C). Conversely, and in agreement with our previous results (40), Downloaded from PBLs on medium supplemented with HMB-PP. This resulted in the inhibition of CD70–CD27 signaling prevented efficient the marked induction of both CD80 and CD86 on Vg9Vd2 PBLs Vg9Vd2 PBL expansion (Fig. 5A, 5C). Thus, CD27 and CD28 from various healthy donors (Fig. 4C, Supplemental Fig. 4B). The provide independent and nonredundant costimulatory signals that upregulation of B7 ligands and downregulation of CD28 receptor determine human Vg9Vd2 T cell survival and proliferation upon on P. falciparum-activated or HMB-PP–activated Vg9Vd2 PBLs activation. prompted us to test the functional consequences of CD28 en- http://www.jimmunol.org/ gagement in human gd cells. Discussion A critical step toward the development of more efficient therapeutic strategies based on gd T cells is improvement of our understanding of the molecular mechanisms that control their activation and expansion. In this study, we showed that gd cell activation criti- cally requires CD28 costimulation in both mice and humans. The selective effect of CD28, but not of its related Ig superfamily

comember ICOS, emphasizes the biological relevance of B7– by guest on September 24, 2021 CD28 interactions in gd T cell activation. Importantly, the role of CD28 signaling was established through in vitro systems, as well

FIGURE 5. B7–CD28 interactions are necessary for survival and pro- liferation of activated human Vg9Vd2 T cells. MACS-sorted gd PBLs FIGURE 4. Dynamic expression of CD28, CD80, and CD86 in human were stimulated for 4 d with 10 nM HMB-PP (in the presence of 25 U/ml Vg9Vd2 T cells. (A) MACS-sorted gd PBLs of two healthy donors were recombinant human IL-2 [rhIL-2]). Blocking Abs specific for CD80 and analyzed by flow cytometry for CD28 expression (gated on Vg9+ cells), CD86, ICOSL, OX40L, or CD70 (or isotype controls) were added at the either before (non-stim) or after 2 d of stimulation with supernatants of P. beginning of the cultures. (A) Cells were labeled with CFSE before being falciparum-infected erythrocyte cultures. The gates were set based on cultured for 4 d and then analyzed by flow cytometry. Cells from isotype isotype control stainings. Numbers refer to percentages of cells within the control cultures are shaded in gray. (B) Annexin V staining was performed indicated quadrants. Vg9+ cells within total PBMC cultures were analyzed on day 4 and analyzed by flow cytometry (gating on Vg9+ cells). (C)[3H] by flow cytometry for CD80 and CD86 expression before (ns) or after 2 d thymidine was added during the last 18 h of culture, and its incorporation of stimulation with supernatants of P. falciparum (Pf)-infected erythrocyte was measured in a scintillation counter (cpm). All data are representative cultures (B) or after the indicated days of stimulation with 10 nM HMB-PP of three independent experiments with similar results. Error bars represent (C). All data are representative of three independent experiments with SD. *p , 0.05, **p , 0.01. IL-2high, Cells cultured in the presence of 100 similar results. ns, Nonstimulated. U/ml rhIL-2 (in addition to CD80/CD86 blockade). The Journal of Immunology 1207 as in vivo, in the context of the immune response to Plasmodium future research should examine the potential of manipulating parasites. pathogenic IL-17+ gd cell responses through the inhibition of B7– Using a model of severe malaria induced by PbA infection of B6 CD28 costimulatory signals. mice (31), we previously showed robust gd cell expansion and cytokine (IFN-g and IL-17) production (8, 21, 22). In this study, Acknowledgments we demonstrated that these in vivo gd cell activities are strictly We thank M.M. Mota, T. Ha¨nscheid, J.P. Simas, J. Decalf, A.E. Sousa, L. dependent on CD28 signaling, which promotes their survival and Graca, and J. Borst for reagents and suggestions and the staffs of the Flow proliferation on activation. Furthermore, we showed that similar Cytometry and Animal facilities of Instituto de Medicina Molecular for rules apply to human gd cells incubated with soluble Ags derived technical assistance. from cultures of P. falciparum-infected erythrocytes. We believe that these results provide important novel knowledge for the Disclosures therapeutic manipulation of gd cells in the context of malaria. The authors have no financial conflicts of interest. In recent years, we have significantly improved our under- standing on how gd cells acquire and exert their cytokine- producing capacities (2, 41). In particular, we (21) and other References investigators (42) demonstrated that murine gd cells are pro- 1. Hayday, A. C. 2000. [gamma][delta] cells: a right time and a right place for grammed in the to become either IFN-g or IL-17 pro- a conserved third way of protection. Annu. Rev. Immunol. 18: 975–1026. 2. Hayday, A. C. 2009. Gammadelta T cells and the lymphoid stress-surveillance ducers. 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