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

Julie C. Ribot, Ana deBarros, Liliana Mancio-Silva, Ana Pamplona and Bruno Silva-Santos
J Immunol August 1, 2012, 189 (3) 1202-1208; DOI: https://doi.org/10.4049/jimmunol.1200268
Julie C. Ribot
*Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal;
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Ana deBarros
*Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal;
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Liliana Mancio-Silva
*Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal;
†Unidade de Malária, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal; and
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Ana Pamplona
*Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal;
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Bruno Silva-Santos
*Unidade de Imunologia Molecular, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal;
‡Instituto Gulbenkian de Ciência, 2781-901 Oeiras, Portugal
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Abstract

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αβ counterparts, the costimulation requirements of γδ 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 γδ 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 γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ 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, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ–mediated and IL-17–mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ–producing γδ cells. Our data collectively show that CD28 signals are required for IL-2–mediated survival and proliferation of both CD27+ and CD27− γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.

Introduction

γδ T cells have been evolutionarily conserved as a lymphocyte lineage whose signature TCR (TCRγδ) does not obey the paradigm of MHC restriction (1). In fact, we know very little about Ag specificity and recognition via TCRγδ (2). Although this has not precluded advances in exploiting the functional properties of these lymphocytes, particularly in cancer immunotherapy (3), or in dissecting their pathological contribution to autoimmunity (4–6), it remains a priority to understand how γδ cells are activated in vivo. As part of our efforts to elucidate the contribution of costimulatory receptors to this process (7, 8), we addressed the role of the Ig superfamily protein CD28.

Much of what we know about T cell costimulation has come from studies on CD28 and its ligands, B7.1 (CD80) and B7.2 (CD86), which are typically found on professional APCs, such as dendritic cells (DCs) or B cells (9). B7–CD28 signaling in αβ T cells was shown to have both qualitative and quantitative effects that lower activation thresholds, promote cell proliferation, and enhance functional activity in vitro and in vivo (reviewed in Refs. 9–11).

In contrast to this well-established function in αβ T cells, the role of CD28 in γδ T cell activation has remained controversial because of discrepant results of previous studies (7). In particular, resting murine γδ splenocytes (12) and various intraepithelial lymphocytes subsets (13–15) were reported to be devoid of CD28 expression, which was observed, however, upon cellular activation (12). This pattern contrasted with that of human Vδ2+ PBLs, which significantly downregulated CD28 following activation (16). Moreover, human Vδ1+ cells, unlike their Vδ2+ counterparts, failed to express CD28 (17). Furthermore, although CD28 signals promoted the in vitro proliferation of mouse splenocytes (12, 15) and human γδ lymphocytes (18), the alloreactivity of murine Vγ2+ transgenic thymocytes was normal in the absence of CD28 signaling (19). Critically, none of these studies examined the role of CD28 costimulation during physiological γδ cell responses to infection.

In this study, we used the last generation of specific mAbs and genetically manipulated mice to unequivocally assess the impact of B7–CD28 signals in the context of γδ cell responses to Plasmodium parasites, the infectious agents that cause malaria. Infection by Plasmodium is known to cause striking expansions of γδ cells both in mice (20–22) and in humans (23–26). In fact, in patients infected with either Plasmodium falciparum (23, 24) or Plasmodium vivax (25), γδ cells frequently expand to 30–40% of all peripheral blood T cells (compared with 1–5% in healthy donors). Moreover, γδ cells were shown to be the major cellular source of the key proinflammatory cytokine IFN-γ in patients in endemic areas (27), and this correlated with reduced incidence of clinical episodes (28). Therefore, malaria constitutes one of the most relevant physiological contexts in which to investigate γδ cell activation. We performed a comprehensive series of gain-of-function and loss-of-function studies that demonstrates a critical role for B7–CD28 interactions in the activation and expansion of murine and human proinflammatory γδ T cell subsets in response to Plasmodium Ags.

Materials and Methods

Mice

All mice were adults 4–10 wk of age. C57BL/6 (B6), B6.TCRα-deficient (Tcra−/−), and B6.CD28-deficient (Cd28−/−) mice were described previously (29, 30) and were obtained from The Jackson Laboratory. B6.CD27-deficient (Cd27−/−) mice were a kind gift from Dr. Jannie Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands). Mice were bred and maintained in the specific pathogen-free animal facilities of Instituto de Medicina Molecular. When stated, mice were infected i.p. with 106 Plasmodium berghei ANKA (PbA)-infected erythrocytes and monitored as described (31). All experiments involving animals were performed in compliance with the relevant laws and institutional guidelines and were approved by the local ethics committees.

Preparation of supernatants from P. falciparum-infected erythrocyte cultures

P. falciparum 3D7 blood-stage parasites were cultured using modifications to the method described by Trager and Jensen (32). Parasites were grown in human erythrocytes (5% hematocrit, 5% parasitemia) in RPMI 1640 medium containing l-glutamine supplemented with 0.5% (w/v) AlbuMAX II, 25 mM HEPES, and 0.05 mg/ml gentamicin at 37°C in a 5% CO2 environment. Supernatant of synchronized cultures in mature late-stage schizonts (40–50 h after reinvasion) was obtained by centrifugation at 2000 rpm for 5 min. Synchronization of cultures consisted of treatment with 5% (w/v) d-sorbitol, one or two cycles before supernatant collection.

Culture of human PBLs

Human peripheral blood was collected from anonymous healthy volunteers in accordance with the guidelines of the Declaration of Helsinki. Total PBMCs were prepared and cultured as previously described (33, 34). For purification of γδ PBLs, cells were sorted on a MiniMACS separator using an anti-TCRγδ MicroBead Kit (Miltenyi Biotec). Specific Vγ9Vδ2 T cell activation was accomplished by continuous stimulation (up to 6 d) of total PBMCs or sorted γδ-PBLs with 10 nM 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP; Echelon Biosciences) and 25–100 U/ml IL-2 (Roche Applied Science). Alternatively, PBMCs or sorted γδ-PBLs were cultured in the presence of collected supernatant from P. falciparum cultures. When indicated, anti-human blocking mAbs (or isotype controls) were added at a concentration of 10 μg/ml.

Culture of murine lymphocytes

Cells from spleen, lymph nodes (LNs), or peritoneal cavity were prepared as described (21). Cells were cultured in RPMI 1640 medium with 10% FCS, 50 μM 2-ME, l-glutamine, nonessential amino acids, 10 mM HEPES, penicillin, and streptomycin in 96-well round-bottom plates. When indicated, 30–300 U/ml rIL-2 (Sigma) was added to the cultures. Cells were stimulated with 0.5 μg/ml soluble anti-CD3ε Ab, in the presence of 5 × 105 APCs (3000 rad-irradiated splenocytes)/5 × 104 responding cells. Alternatively, cells were cultured on plate-bound anti-CD3ε (0.1–10 μg/ml) and anti-CD28 (10 μg/ml) or soluble rCD70 (5 μg/ml; kindly provided by Dr. Jannie Borst). When indicated, anti-mouse agonist or blocking mAbs were added to the culture medium at a concentration of 1–5 μg/ml.

mAbs

The following anti-mouse mAbs were purchased from eBioscience (San Diego, CA) and used for flow cytometry: FITC-labeled anti-TCRδ (eBioGL3), anti-CD69 (H1.2F3), and anti–IL-17 (eBio17B7); PE-labeled anti-TCRδ (eBioGL3), anti-CD11c (N418), and anti-IFN-γ (XMG1.2); PerCP-Cy5.5–labeled anti-CD3ε (145.2C11); PE-Cy7–labeled anti-CD19 (eBio1D3); allophycocyanin-labeled anti-TCRδ (eBioGL3), anti-CD11b (M1/70), and anti–IL-17 (eBio17B7); eFluor 450-labeled anti-CD4 (RM4-5); biotin-conjugated anti-CD28 (37.51); and streptavidin-PE.

In murine cell cultures, anti-mouse mAbs (from eBioscience) specific for IL-2 (JES6-1A12), ICOS (7E.17G9), CD80 (16-10A1), and CD86 (GL1) were used as blocking reagents; anti-mouse mAbs for CD3ε (145.2C11) and CD28 (37.51) were used as agonists.

The following anti-human mAbs were used for flow cytometry: anti–CD28-FITC (CD28.2; eBioscience); anti–CD69-PE (FN50), anti–CD70-PE (ki-24), anti–CD80-PE (L307.4), and anti–CD86-allophycocyanin (2331) (all from BD Pharmingen, San Diego, CA); and anti–OX40 ligand (OX40L)-PE (11C3.1) and anti–ICOS ligand (ICOSL)-PE (2D3) (both from BioLegend, San Diego, CA).

The mAbs used as blocking reagents in human cell cultures were anti-CD70 (ki-24) and anti-CD86 (IT2.2) (both from BD Pharmingen); anti-ICOSL (9F.8A4; BioLegend); and anti-CD80 (37711) and anti-OX40L (159403) (both from R&D Systems). IgG1 (MOPC-21), IgG2 (MPC-11), and IgG3 (MG3-35) were used as isotype controls (all purchased from BioLegend).

Flow cytometry and cell sorting

Cells were sorted electronically using a FACSAria (BD Biosciences, San Jose, CA). For cell surface stainings, cells were incubated for 15 min on ice in 2.4G2 (anti-FcγR mAb) hybridoma supernatant and then incubated for 15 min with saturating concentrations of the indicated mAbs. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) (both from Sigma) for 4 h at 37°C; 10 μg/ml brefeldin A (Sigma) was added during the last 2 h. Cells were stained for the indicated cell surface markers, and intracellular staining was performed using fixation/permeabilization and permeabilization buffers (both from eBioscience), following the manufacturer’s instructions. Samples were analyzed using Fortessa or FACSCalibur (both from BD Biosciences).

In vitro functional assays

Lymphocyte activation and apoptosis were assessed between days 1 and 6 of culture by staining for CD69 and for annexin V (BD Pharmingen), respectively, according to the manufacturer’s instructions. IL-2 secretion was quantified using Cytometric Bead Array as described (33). For analysis of cell proliferation, cells were stained with 5 μM the cytoplasmic dye CFSE (Molecular Probes) for 5 min at 37°C, cultured for 3 d, and analyzed by flow cytometry for CFSE dilution. Alternatively, 1 μCi [3H]thymidine (Amersham) was added for the last 18 h, and [3H]thymidine incorporation was measured using the MicroBeta TriLux scintillation counter (PerkinElmer).

Quantitative real-time PCR

RNA was prepared and analyzed by quantitative real-time PCR, as previously described (21, 22). The primers used were: Il2-Fwd, 5′-GCTGTTGATGGACCTACAGGA-3′; Il2-Rev, 5′-TTCAATTCTGTGGCCTGCTT-3′; Efa1-Fwd, 5′-ACACGTAGATTCCGGCAAGT-3′; and Efa1-Rev, 5′-AGGAGCCCTTTCCCATCTC-3′. Transcripts were quantified by the standard curve method, normalized to Efa1, and expressed in arbitrary units.

Statistical analysis

Statistical significance of differences between populations was assessed using the Mann–Whitney test.

Results

B7–CD28 interactions promote the proliferation and survival of murine γδ T cells

This study began with the observation that lymphoid (spleen and LN) γδ cells, in contrast to their intraepithelial counterparts (13–15), constitutively express CD28 at similar levels to αβ cells (Fig. 1A). Moreover, in vitro activation with anti-CD3 (αCD3ε) mAb significantly upregulated CD28 levels on isolated (FACS-purified) γδ cells (Fig. 1B), whereas the addition of B7-expressing APCs provoked CD28 downregulation (Supplemental Fig. 1A). In contrast, the CD28-related Ig superfamily member, ICOS, was not expressed in either resting or activated γδ cells (Fig. 1B).

FIGURE 1.
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FIGURE 1.

CD28 signals promote murine γδ T cell activation and proliferation. (A) Cells pooled from spleens and LN of adult WT B6 mice were stained with mAbs specific for CD3ε, TCRγδ, and CD28. The graph shows the expression of CD28 on pregated CD3ε+ TCRγδ+ cells, CD3ε+ TCRγδ− cells, and CD3ε− cells. Peripheral lymphoid cells from Cd28−/− mice (shaded curve) are shown as a negative control. (B) γδ cells were sorted from pooled spleen and LNs of Tcra−/− 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) γδ cells were sorted by FACS from pooled spleen and LNs of Tcra−/− 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 (αB7), or agonist anti-CD28. (C) Representative forward scatter (FSC)/side scatter (SSC) plots. Numbers refer to percentages of cells within the indicated regions. (D) CD69 expression on γδ 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.

To functionally test the role of B7–CD28 interactions in γδ cell activation, we used CD28 agonists (anti-CD28 mAb) or antagonists (anti-B7.1/CD80 and anti-B7.2/CD86 mAbs) in cocultures of γδ cells (10%) and irradiated splenocytes (90%). After 3 d of activation (with αCD3ε mAb), the percentage of live γδ cells recovered from these cultures was markedly higher in the presence of CD28 agonists and lower in the presence of CD28 antagonists, whereas blocking ICOS had no effect (Fig. 1C). This correlated well with the activation phenotype of γδ cells, as evaluated by the expression of the activation marker CD69 (Fig. 1D).

To assess the precise role of CD28 signaling on γδ cell proliferation, we performed [3H]thymidine incorporation and CFSE-dilution assays. CD28 agonists enhanced γδ cell proliferation, whereas CD28 antagonists inhibited it completely (Fig. 1E, 1F). Consistent with this, CD28-deficient γδ cells showed impaired activation and proliferation in vitro (Supplemental Fig. 1B, 1C). Furthermore, CD28 signaling on γδ cells also affected their survival 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 γδ T cells.

CD28 costimulation is necessary for autocrine IL-2 production by γδ T cells

Because the previous (standard) assays used total irradiated splenocytes as “feeders,” which typically included ∼30% of αβ T cells that also express CD28, it was critical to assess whether B7–CD28 signals operated directly on γδ cells. For this, we established cultures of highly (>99%) purified γδ cells, activated by plastic-bound agonist Abs to TCR/CD3 and CD28. We observed that CD28 costimulation provided a marked proliferative advantage at low amounts of TCR/CD3 triggering (i.e., 0.1 μg/ml anti-CD3ε mAb) (Fig. 2A). This effect was lost at saturating (10-fold higher) concentrations of anti-CD3ε mAb (Fig. 2A).

FIGURE 2.
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FIGURE 2.

CD28 costimulation of γδ cells induces secretion of IL-2 required for proliferation. γδ cells were sorted using FACS from pooled spleen and LN of Tcra−/− mice and activated with the indicated amounts of plate-bound anti-CD3 mAb in the presence of 10 μg/ml plate-bound anti-CD28 mAb (αCD28; 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 μg/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 μg/ml anti-CD3. (C) Quantitative real-time PCR for Il2 expression in purified γδ cells cultured for 6 h with 0.1 μg/ml anti-CD3 mAb in media alone, media supplemented with soluble rCD70 (sCD70) or anti-CD28 mAb (αCD28), or isotype control (iso ctrl). Expression was normalized 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 μg/ml plate-bound anti-CD3 in the presence of 10 μg/ml isotype control (iso ctrl) (gray), 10 μg/ml plate-bound anti-CD28 (green), 5 μg/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.

To gain further mechanistic insight into the direct effects of CD28 on γδ cell activation, we considered that CD28 costimulation of αβ cells can enhance their production of IL-2 (35, 36), which is a key determinant of γδ cell expansion (33, 37). Therefore, we measured IL-2 protein levels in the supernatants of the γδ cell cultures. Strikingly, IL-2 was only detected when these were supplemented with CD28 agonists (Fig. 2B). The induction of IL-2 expression in CD28-costimulated γδ cells was also observed at the mRNA (transcriptional) level (Fig. 2C). Importantly, CD27 triggering using soluble recombinant ligand (7, 8) did not upregulate Il2 expression (Fig. 2C), suggesting that this mechanism is specifically downstream of CD28 signaling in γδ cells.

The interesting dynamics of IL-2 production by γδ cells and their proliferation following TCR/CD28 stimulation (Supplemental Fig. 1D) prompted us to test whether autocrine IL-2 production was essential for γδ cell proliferation. Upon addition of neutralizing Abs against murine IL-2, a strong reduction in γδ cell expansion was observed (Fig. 2D, 2E). Conversely, the addition of saturating doses of IL-2 bypassed the need for CD28 costimulation, both in isolated γδ cell cultures (Fig. 2D, 2E) and in cocultures with splenic APCs (Supplemental Fig. 1E). These results show that CD28 costimulation directly controls γδ cell expansion through the induction of IL-2 production.

CD28 signals are required for γδ cell responses to Plasmodium infection in vivo

To establish the importance of CD28 signaling on γδ cell activation in vivo, we resorted to a mouse model of severe malaria, induced by PbA infection of B6 mice (31). Importantly, we had previously documented very robust γδ cell responses in this animal model (8, 21, 22).

We followed the phenotype of γδ cells during the course of infection and detected a transient upregulation of CD28 at day 3, whereas ICOS was not induced on γδ cells (Fig. 3A). In PbA-infected mice, the modulation of CD28 expression on γδ cells was accompanied by the upregulation of its inducible ligand, CD86, on professional APCs, such as DCs (Fig. 3B). This led us to investigate the functional consequences of B7–CD28 costimulation in vivo. For this, we analyzed CD28-deficient mice and observed their failure to expand γδ cells on infection, in contrast to wild-type (WT) controls (Fig. 3C). This correlated with a comparatively small increase in the pool of CD69+ (activated) γδ cells in infected CD28-deficient mice (Fig. 3D). Importantly, the effects of CD28 deficiency on the numbers of total or activated γδ cells were not phenocopied in CD27-deficient animals (Fig. 3C, 3D), thus demonstrating that the two costimulatory pathways play distinct roles in γδ T cell expansion in vivo.

FIGURE 3.
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FIGURE 3.

CD28 signals are required for murine γδ cell responses to Plasmodium infection in vivo. B6 (WT), Cd28−/−, or Cd27−/− mice were infected with PbA, and their spleens were harvested and analyzed by flow cytometry at the indicated days postinfection. (A) Mean fluorescence intensities (MFI) for CD28 and ICOS expression in γδ T cells from WT mice. Dashed line represents isotype control staining. (B) MFI for CD86 expression in DCs from WT, Cd28−/−, or Cd27−/− mice at day 3 postinfection. Dashed line indicates reference MFI in naive WT animals. Absolute numbers of total γδ cells (C) and activated CD69+ γδ cells (D) at day 3 postinfection. (E and F) γδ cells were sorted using FACS and stained intracellularly for IFN-γ and IL-17. Absolute numbers of IFN-γ+ γδ cells (E) or IL-17+ γδ cells (F) are shown. Error bars represent SD. All data are from three independent experiments involving 6–10 mice. *p < 0.05, **p < 0.01, ***p < 0.001. ns, Not statistically significant (p ≥ 0.05).

In PbA-infected CD28-deficient animals, the reduced (compared with WT) γδ T cell compartment also contained smaller pools of both IFN-γ–producing and IL-17–producing subsets (Supplemental Fig. 2). In fact, the CD28 deficiency completely prevented the accumulation of IFN-γ+ (Fig. 3E) and IL-17+ (Fig. 3F) γδ cells upon malaria infection. We observed an additional difference in comparison with CD27-deficient animals: the expansion of IL-17+ γδ cells was selectively impaired in CD28−/− mice (Fig. 3F). In contrast, both mutant models exhibited a defect in the IFN-γ response (Fig. 3E). This led us to assess the combined effect of CD27/CD28 signals on IFN-γ production by γδ cells in vivo. For this, we decided to block B7–CD28 signaling in CD27−/− mice during the course of infection. The efficient mAb-mediated neutralization of CD80 and CD86 caused a significant decrease in the numbers of activated IFN-γ+ γδ cells in the CD27-deficient background (Supplemental Fig. 3). Thus, CD28 acts nonredundantly and synergistically with CD27 in the activation of IFN-γ+ γδ cells following malaria infection.

These data collectively demonstrate that CD28 signals are required for the expansion of murine proinflammatory γδ cell subsets in vivo.

Dynamic expression of CD28 and its B7 ligands on activated human γδ cells

We next investigated the role of CD28 costimulation on human γδ cells. Freshly isolated Vγ9Vδ2 PBLs were previously shown to express CD28 (18, 38). Moreover, Vγ9Vδ2 PBLs are known to be highly activated by soluble metabolites produced by apicomplexan protozoa like P. falciparum (39). To study the expression dynamics of CD28 and its B7 ligands, CD80 and CD86, on Vγ9Vδ2 PBLs exposed to microbial Ags, we established an experimental system in which either total PBMCs or purified γδ PBLs were incubated for 2–6 d with supernatants collected from P. falciparum-infected erythrocyte cultures. We observed a marked downregulation of CD28 after 2 d of culture with P. falciparum-derived supernatants (Fig. 4A), concomitantly with the induction of CD80 and CD86 expression on Vγ9Vδ2 PBLs (Fig. 4B). This induction was a direct effect of the Plasmodium Ags on Vγ9Vδ2 PBLs, because it was also observed on isolated Vγ9Vδ2 PBLs (Supplemental Fig. 4A).

FIGURE 4.
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FIGURE 4.

Dynamic expression of CD28, CD80, and CD86 in human Vγ9Vδ2 T cells. (A) MACS-sorted γδ PBLs of two healthy donors were analyzed by flow cytometry for CD28 expression (gated on Vγ9+ cells), either before (non-stim) or after 2 d of stimulation with supernatants of P. falciparum-infected erythrocyte cultures. The gates were set based on isotype control stainings. Numbers refer to percentages of cells within the indicated quadrants. Vγ9+ cells within total PBMC cultures were analyzed by flow cytometry for CD80 and CD86 expression before (ns) or after 2 d of stimulation with supernatants of P. falciparum (Pf)-infected erythrocyte cultures (B) or after the indicated days of stimulation with 10 nM HMB-PP (C). All data are representative of three independent experiments with similar results. ns, Nonstimulated.

A strong candidate P. falciparum Ag was HMB-PP, the most potent natural Vγ9Vδ2 TCR agonist known (33, 39), because it is the product of HMB-PP synthase (GcpE) present in all Plasmodium spp. and is predominantly expressed on mature blood-stage parasites (http://plasmodb.org; Gene ID: PF10_0221). Therefore, we established similar cultures of PBMCs or purified Vγ9Vδ2 PBLs on medium supplemented with HMB-PP. This resulted in the marked induction of both CD80 and CD86 on Vγ9Vδ2 PBLs from various healthy donors (Fig. 4C, Supplemental Fig. 4B). The upregulation of B7 ligands and downregulation of CD28 receptor on P. falciparum-activated or HMB-PP–activated Vγ9Vδ2 PBLs prompted us to test the functional consequences of CD28 engagement in human γδ cells.

CD28 costimulation controls survival and proliferation of activated human γδ cells

To directly assess the B7–CD28 costimulation requirements of HMB-PP–activated Vγ9Vδ2 PBLs, we used monoclonal blocking Abs specific to CD80 and CD86 in Vγ9Vδ2 PBL cultures. We observed a clear impairment in cell proliferation, as assessed by CFSE-dilution assays (Fig. 5A), compared with control cultures incubated with isotype Abs. Moreover, CD80/CD86 blockade also affected the survival of HMB-PP–activated Vγ9Vδ2 PBLs, as indicated by the accumulation of annexin V+ cells (Fig. 5B). This combined survival/proliferation effect of CD80/CD86 inhibition resulted in a highly significant reduction in thymidine incorporation in Vγ9Vδ2 PBL cultures (Fig. 5C). Moreover, and consistent with the murine data (Fig. 2D, 2E), the need for CD28 costimulation was bypassed by saturating amounts of exogenous IL-2 (Fig. 5C).

FIGURE 5.
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FIGURE 5.

B7–CD28 interactions are necessary for survival and proliferation of activated human Vγ9Vδ2 T cells. MACS-sorted γδ PBLs were stimulated for 4 d with 10 nM HMB-PP (in the presence of 25 U/ml recombinant human IL-2 [rhIL-2]). Blocking Abs specific for CD80 and CD86, ICOSL, OX40L, or CD70 (or isotype controls) were added at the beginning of the cultures. (A) Cells were labeled with CFSE before being cultured for 4 d and then analyzed by flow cytometry. Cells from isotype control cultures are shaded in gray. (B) Annexin V staining was performed on day 4 and analyzed by flow cytometry (gating on Vγ9+ cells). (C) [3H]thymidine was added during the last 18 h of culture, and its incorporation was measured in a scintillation counter (cpm). All data are representative of three independent experiments with similar results. Error bars represent SD. *p < 0.05, **p < 0.01. IL-2high, Cells cultured in the presence of 100 U/ml rhIL-2 (in addition to CD80/CD86 blockade).

Interestingly, the inhibition of ICOSL (CD275; B7-H2) or OX40L (CD252) had no impact on their proliferation (Fig. 5A, 5C). Conversely, and in agreement with our previous results (40), the inhibition of CD70–CD27 signaling prevented efficient Vγ9Vδ2 PBL expansion (Fig. 5A, 5C). Thus, CD27 and CD28 provide independent and nonredundant costimulatory signals that determine human Vγ9Vδ2 T cell survival and proliferation upon activation.

Discussion

A critical step toward the development of more efficient therapeutic strategies based on γδ T cells is improvement of our understanding of the molecular mechanisms that control their activation and expansion. In this study, we showed that γδ cell activation critically 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–CD28 interactions in γδ T cell activation. Importantly, the role of CD28 signaling was established through in vitro systems, as well as in vivo, in the context of the immune response to Plasmodium parasites.

Using a model of severe malaria induced by PbA infection of B6 mice (31), we previously showed robust γδ cell expansion and cytokine (IFN-γ and IL-17) production (8, 21, 22). In this study, we demonstrated that these in vivo γδ cell activities are strictly dependent on CD28 signaling, which promotes their survival and proliferation on activation. Furthermore, we showed that similar rules apply to human γδ cells incubated with soluble Ags derived from cultures of P. falciparum-infected erythrocytes. We believe that these results provide important novel knowledge for the therapeutic manipulation of γδ cells in the context of malaria.

In recent years, we have significantly improved our understanding on how γδ cells acquire and exert their cytokine-producing capacities (2, 41). In particular, we (21) and other investigators (42) demonstrated that murine γδ cells are programmed in the thymus to become either IFN-γ or IL-17 producers. Various molecular mechanisms, including TCR (42–44), CD27 (21), LTβR (45), and TGFβR (46) signaling, have been proposed to participate in the “developmental preprogramming” (2) of γδ cells. Once in the periphery, innate TLR-mediated signals are key for the expansion of IL-17+ γδ cells. Thus, TLR stimulation of myeloid cells led to the secretion of IL-1β and IL-23, which were the direct inducers of IL-17+ γδ cell proliferation (5, 8). By contrast, IFN-γ+ γδ cells were refractory to IL-1β/IL-23 stimulation but typically relied on TCR and CD27 signals for their expansion (8, 40, 47).

These data led us previously to propose a model in which IFN-γ+ γδ cells followed an “adaptive-like” mode of activation (requiring TCR plus costimulation), whereas IL-17+ γδ cells behaved as innate-like lymphocytes (8). Importantly, the data presented in this article call for a significant revision of this model. Thus, we provide clear evidence that IL-17+ γδ cells, like IFN-γ+ γδ cells, depend on CD28 costimulatory signals for their expansion in vivo. This costimulation requirement of IL-17+ γδ cells is also likely to reflect an underappreciated dependence on TCR-mediated activation (5, 48, 49). Thus, although hampered by the lack of known TCRγδ ligands, future research should readdress the role of TCR stimulation on the development and activation of IL-17+ γδ cells.

Of note, the effects of CD28 costimulation can be bypassed in vitro by saturating TCR (Fig. 2A) signals. Our finding that γδ cell expansion (on infection) in vivo is impaired in CD28-deficient mice importantly suggests that physiological TCRγδ triggering (by yet mostly unknown ligands) relies on intermediate avidities, at which the TCR acts synergistically with CD28.

Importantly, our detailed examination of CD28-deficient and CD27-deficient mice (Fig. 3) established that the two costimulatory pathways play independent (and nonredundant) roles in γδ cell activation. Mechanistically, we showed that the induction of IL-2 production is a major (Fig. 2B) and specific (Fig. 2C) function of CD28 (but not CD27) costimulation in γδ cells, which are known to strongly benefit from IL-2 signals for their expansion (33, 37). The fact that γδ cells themselves can produce high levels of IL-2 strictly upon CD28 costimulation defines important rules for their expansion in situ. This notwithstanding, B7–CD28 signals can additionally contribute to γδ cell expansion through costimulation of αβ cells, as we previously showed that coculture with IL-2–producing CD4+ CD25− cells enhanced γδ cell proliferation (22).

Finally, although this study focused on infection, γδ cells were also shown to be key players in autoimmunity, because their secretion of IL-17 promotes the development of experimental autoimmune encephalomyelitis (5, 6), collagen-induced arthritis (4), and psoriatic plaques (50) in animal disease models. Therefore, future research should examine the potential of manipulating pathogenic IL-17+ γδ cell responses through the inhibition of B7–CD28 costimulatory signals.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank M.M. Mota, T. Hänscheid, J.P. Simas, J. Decalf, A.E. Sousa, L. Graca, and J. Borst for reagents and suggestions and the staffs of the Flow Cytometry and Animal facilities of Instituto de Medicina Molecular for technical assistance.

Footnotes

  • This work was supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-MII/104158/2008) and the European Molecular Biology Organization (Young Investigator Programme). L.M.-S. is supported by a European Molecular Biology Organization fellowship (ALTF960-2009).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    B6
    C57BL/6
    DC
    dendritic cell
    HMB-PP
    4-hydroxy-3-methyl-but-2-enyl pyrophosphate
    ICOSL
    ICOS ligand
    LN
    lymph node
    OX40L
    OX40 ligand
    PbA
    Plasmodium berghei ANKA
    WT
    wild-type.

  • Received January 23, 2012.
  • Accepted May 24, 2012.
  • Copyright © 2012 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 189 (3)
The Journal of Immunology
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B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production
Julie C. Ribot, Ana deBarros, Liliana Mancio-Silva, Ana Pamplona, Bruno Silva-Santos
The Journal of Immunology August 1, 2012, 189 (3) 1202-1208; DOI: 10.4049/jimmunol.1200268

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B7–CD28 Costimulatory Signals Control the Survival and Proliferation of Murine and Human γδ T Cells via IL-2 Production
Julie C. Ribot, Ana deBarros, Liliana Mancio-Silva, Ana Pamplona, Bruno Silva-Santos
The Journal of Immunology August 1, 2012, 189 (3) 1202-1208; DOI: 10.4049/jimmunol.1200268
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Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

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Print ISSN 0022-1767        Online ISSN 1550-6606