Research Article

Professional Antigen-Presentation Function by Human γδ T Cells

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Science  08 Jul 2005:
Vol. 309, Issue 5732, pp. 264-268
DOI: 10.1126/science.1110267


Human γδ T cells are considered to play a vital role in protective immunity through cytokine secretion and cytotoxic activity. We report that cells expressing the Vγ2Vδ2+–T cell receptor (Vδ2+ T cells) also display principal characteristics of professional antigen-presenting cells such as dendritic cells. Thus, when activated, these cells efficiently processed and displayed antigens and provided co-stimulatory signals sufficient for strong induction of naïve αβ T cell proliferation and differentiation. We suggest that, upon microbial activation, Vδ2+ T cells participate in the induction of adaptive immune responses and that these cells may be a useful tool in vaccine development and immunotherapy.

γδ T cells represent a distinct subset of T cells characterized by T cell receptors (TCRs) that possess unique structural and antigen-binding characteristics (14). In normal human peripheral blood, these T cells make up 2 to 10% of the total T cell pool, with the majority (>80%) expressing the Vγ2Vδ2+ TCR (hereafter referred to as Vδ2+ T cells) (5, 6). Vδ2+ T cells specifically recognize small nonpeptide antigens, derived mostly from microbes or necrotic host cells (5, 6). In clear contrast to recognition of antigen by αβ T cells, Vδ2+ T cell–specific antigens do not need to be processed by professional antigen-presenting cells (APCs) such as dendritic cells (DCs) and do not require binding and presentation by classical major histocompatibility complex (MHC) molecules (5, 6). Vδ2+ T cells are also unique to higher primates, and their absence in other mammals suggests that they may have evolved to provide protection against distinct, species-specific microbes (3, 4, 7). Vδ2+ T cells display a range of innate effector functions including the rapid secretion of chemokines and cytokines and target cell lysis, as well as contribution to adaptive immunity, for example through B cell help, DC maturation, and provision of memory γδ T cell function (811).

Activated Vd2+T cells display phenotypic characteristics of professional APCs. We recently showed that TCR-triggering in human Vδ2+ T cells leads to rapid and transient expression of the lymph node (LN)–homing receptor CCR7 (8, 12). Furthermore, Vδ2+ T cells have been shown to be present in LNs draining mucosal tissues, suggesting that they might directly interact with adaptive immune cells. We hypothesized that γδ T cells might play a role in processing and presenting antigens. To explore whether γδ T cells display any feature of professional APCs, we examined tonsillar γδ T cells. We observed signs of preactivation (cell surface CD69 expression) on these cells, which is in line with LN-homing properties present in in vitro stimulated rather than in resting γδ T cells (8, 12). Moreover, the antigen-presenting MHC-II molecule (human leukocyte antigen D locus-related protein) HLA-DR was also substantially up-regulated on these cells, along with a wide array of T cell co-stimulation and adhesion molecules (Fig. 1A and table S1). To investigate how these antigen-presenting features might be regulated, we stimulated freshly isolated resting blood Vδ2+ T cells with the prototype Vγ2Vδ2-TCR ligand, isopentenyl pyrophosphate (IPP), to selectively expand Vδ2+ T cells (5, 6, 13). As with fresh tonsillar γδ T cells IPP-stimulated but not resting, Vδ2+ T cells expressed a repertoire of antigen-presentation and co-stimulation molecules that was almost indistinguishable from lipopolysaccharide (LPS)-matured, monocyte-derived DCs (Fig. 1B and table S2). Other natural Vδ2+ T cell stimuli also strongly induced similar APC features, for example, Escherichia coli KM20 lysate and 4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), which is produced during microbial isoprenoid synthesis (5, 6) (Fig. 1C). Surface, versus intracellular, staining revealed de novo production of MHC-II during Vδ2+ T cells activation and contrasted with the cell surface relocation of preformed intracellular MHC-II in maturating DCs (Fig. 1E) (14, 15). Most of the activation-induced surface molecules were maintained at high concentrations during the proliferation of Vδ2+ T cells (fig. S1). In contrast, peripheral blood αβ T cells displayed a relatively modest and clearly delayed expression of MHC-II and co-stimulation molecules during stimulation (Fig. 1, B and D). Abundant adhesion molecules on IPP-stimulated Vδ2+ T cells may explain their clustering observed with naive αβ T cells, resembling early steps in T cell activation by DCs (fig. S2) (16, 17).

Fig. 1.

Stimulated Vδ2+ T cells express APC molecules. (A) Tonsillar γδ T cells are activated and express antigen-presentation, co-stimulation, and adhesion molecules. γδ T cells in fresh cellular extracts from tonsils were analyzed for expression of cell surface markers. Histograms show results for indicated markers on HLA-DR/γδ-TCR double-positive cells (gate in contour blot); bold and faint lines (in line graphs) represent stainings with specific and isotype control antibodies, respectively. The data are representative of three independent experiments. (B) Cell surface markers were determined on freshly isolated γδ T cells (γδ T), 18-hour IPP-stimulated Vδ2+ T cells, monocyte-derived LPS-matured DCs (DC), 18-hour superantigen-stimulated αβ T cells (αβ T), and freshly isolated monocytes (M) (representative for six experiments). Numbers refer to the time of in vitro culture: 0, freshly isolated; 18, 18 hours. (C) Induction of antigen-presentation features upon treatment of Vδ2+ T cells for 36 hours with E. coli KM20 lysate at 1:1000 dilution (KM20) with 0.5 μM HMBPP and with 50 μM IPP, in comparison with 48-hour LPS-matured monocyte-derived DCs [DC(LPS)]. Mean fluorescence intensities (MFI) ± SD; n = 2. (D) Moderate and delayed expression of APC molecules on superantigen-stimulated αβ T cells. Cell surface HLA-DR was examined in superantigen-stimulated αβ T cells at 18 hours, 3 days, or 7 days of culture. HLA-DR-positive αβ T cells were further analyzed for coexpression of APC markers as indicated. Vβ2+ or CD4+ αβ T cells were stimulated by LPS-DCs presenting TSST-1 or a superantigen cocktail [TSST-1, staphylococcal enterotoxin serotypes A (SEA) and B (SEB)] each at 100 ng/ml. Data combine results obtained with three donors. 18-hour IPP-Vδ2+ T cells from the same donors were included as controls. (E) De novo synthesis or potential intracellular stores of preformed HLA-DR were evaluated by staining intact and permeabilized Vδ2+ T cells, either freshly isolated [γδ T (0)] or 18h IPP-stimulated [γδ T (18)]. As controls, immature [DC(medium)] and 44-hour LPS-maturated DCs [DC(LPS)] were used. Expression was analyzed by flow cytometry.

To illustrate cellular distribution of MHC II and morphologic changes, we performed confocal and digital interference contrast (DIC) image analyses with resting and IPP-stimulated Vδ2+ T cells (Fig. 2A). Resting Vδ2+ T cells express high amounts of cell surface γδ-TCRs but lack cell surface and intracellular HLA-DR, in agreement with flow cytometry analysis (Fig. 1E). The late endosomal-lysosomal marker Lamp-1/CD107a is found in intracellular compartments. Cytospin sections document the de novo expression of HLA-DR during short-term Vδ2+ T cell stimulation. The cultures were heterogeneous, i.e., contained partially and fully activated Vδ2+ T cells, as evidenced by intermediate or low concentrations of γδ-TCR and inverse concentrations of HLA-DR. LAMP-1 appears to cluster intracellularly in activated γδ-TCRlowHLA-DRhigh Vδ2+ Tcells. Control staining with immature and mature DC revealed the expected stainings, including cell surface relocation of preformed HLA-DR and Lamp-1 clustering during DC maturation (10, 15). Of note, activated Vδ2+ T cells carry numerous dendrite-like cell protrusions (Fig. 2A) and feature an “amoeboid”-like cell morphology (Fig. 2B).

Fig. 2.

HLA-DR expression and morphologic changes during Vδ2+ T cells activation. (A) Marker expression and distribution analysis by confocal microscopy in cytospin preparations containing freshly isolated (resting) and 18-hour IPP-stimulated (IPP) Vδ2+ T cells. As control for HLA-DR and Lamp-1 but not γδ-TCR expression, immature (iDC) and mature (mDC) DCs were used. Scale bars correspond to 10 μm; single and double stars denote a Vδ2+ T cell and an unrelated mononuclear cell; white arrows point at filamentous cell protrusions. (B) Phase contrast images of cultured Vδ2+ T cells after 48 hours of IPP stimulation. Black arrows point at amoeboid cell extensions.

Activated Vd2+T cells induce proliferation and differentiation in naïve CD4+ ab T cells. The APC-like phenotype suggested that activated but not resting Vδ2+ T cells perform APC functions. This was tested by measuring the ability of IPP-stimulated Vδ2+ T cells to induce primary CD4+ αβ T cell responses to MHC alloantigens in mixed leukocyte reactions (MLRs) (13). Proliferation was readily observed in response to distinct alloantigens present on heterologous Vδ2+ T cells (Fig. 3A), and was similar to that obtained with the use of LPS-matured DCs, at APC-responder ratios of up to 1:100. In contrast, very limited responses were obtained with the use of superantigen-stimulated αβ T cells as APCs (Fig. 3A).

Fig. 3.

Vδ2+ T cells induce primary αβ T cell responses. (A) In MLR, IPP-stimulated Vδ2+ T cells (circles), LPS-maturated DCs (squares), or superantigen-stimulated αβ T cells (triangles) were mixed with heterologous, naïve CD4+ αβ T cells at indicated APC dilutions. T cell proliferation was determined after 6 days of culture. (B) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled naïve, CD4+ αβ T cell proliferation in response to autologous IPP-stimulated Vδ2+ T cells, LPS-matured DCs, anti-CD3/CD28 stimulated CD4+ αβ T cells, and freshly isolated monocytes (M) after loading with 10 ng/ml TSST-1. APC:responder cell ratio was 1:5, and proliferated cells were determined after 4 days of culture. (C) Vδ2+ T cells (circles), DCs (squares), αβ T cells (triangles), and monocytes (diamonds) were loaded with increasing concentrations of TSST-1, and proliferation of Vβ2+-αβ T cells was determined as described above. (D) Cell lines generated in response to TSST-1–presenting Vδ2+ T cells (top) or TSST-1–presenting DC (bottom), prepared with 10 ng/ml TSST-1, were examined for production of intracellular IL-4 and IFN-γ. Numbers (%) in quadrants refer to cytokine-producing cells; numbers above dot blots indicate the APC:responder cell ratio (n = 3).

The relative contribution of co-stimulation to the efficiency of antigen presentation function of Vδ2+ T cells was next tested. To measure this, we used the superantigen, toxic shock syndrome toxin 1 (TSST-1). Because TSST-1 binds directly to MHC-II molecules on the surface of APCs, it does not require uptake and processing as required for conventional protein antigens. Furthermore, TSST-1 is selective for αβ-TCRs containing the Vβ2 chain, which make up 4 to 10% of peripheral blood CD3+ T cells, allowing responder cells to be readily detected (18). IPP-stimulated Vδ2+ T cells pulsed with 10 ng/ml TSST-1 induced a strong expansion of naïve autologous Vβ2+ αβ T cells, corresponding well with the response obtained with the use of TSST-1–loaded DCs (Fig. 3B) (19). In contrast, activated αβ T cells and freshly isolated monocytes did not induce substantial proliferation. TSST-1 titration experiments revealed the high potency of TSST-1–presenting Vδ2+ T cells, exceeding by 100-fold the responses obtained with αβ T cells or monocytes (Fig. 3C). At high TSST-1 loading concentrations, Vδ2+ T cells were consistently more effective than DCs in induction of primary T cell proliferation, whereas the reverse was observed at lower TSST-1 concentrations (Fig. 3C).

During primary immune responses, naïve CD4+ αβ T cells differentiate into subsets of polarized effector T helper cells: Th1, Th2, or Th0 with the capacity to produce type 1 [interferon γ (IFN-γ)], type 2 [interleukin 4 (IL-4)], or both cytokines, respectively (20, 21). We thus examined whether any differentiation in CD4+ T cells occurred after proliferation in response to TSST-1–presenting Vδ2+ T cells. At an initial APC:responder cell ratio of 1:5, the majority of responder cells displayed a polarized response, with an equal representation of all three T helper subsets (Th1, Th2, and Th0) (Fig. 3D). At higher Vδ2+ T cell concentration, the polarization response shifted to Th1, similar to the results obtained with TSST-1–loaded DCs at an APC:responder cell ratio of 1:5. Also, the percentage of Th1 cells increased (at constant APC:responder cell ratio) with increasing TSST-1 loading concentration (fig. S3A). TSST-1–presenting Vδ2+ T cells had no obvious inhibitory effects, that is, did not induce abortive proliferation or secretion of anti-inflammatory cytokines in αβ T cells (Fig. 3D and fig. S3B) (2226).

Activated Vd2+T cells process and present soluble protein antigen. To test the ability of Vδ2+ T cells to take up, process, and present soluble antigens to αβ T cells (14, 23, 25, 26), we used two different types of protein antigens: the single-chain protein tetanus toxoid (TT) and the highly complex protein mixture Mycobacterium tuberculosis–purified protein derivative (PPD). Proliferation of autologous, TT- or PPD-specific CD4+ αβ T cell lines, which respond to less stringent activation regimens (27), was readily detected (fig. S4). Processing of these two model antigens was further examined by using freshly isolated blood CD4+ αβ T cells as responder cells together with PPD-presenting and TT-presenting Vδ2+ T cells, respectively, leading to strong proliferation in both cases (Fig. 4A). Chloroquine, an inhibitor of endosomal and lysosomal acidification required for efficient MHC class II antigen presentation, prevented responder cell proliferation, demonstrating that intracellular processing of PPD and TT is essential for triggering antigen-specific responses. These responses also could be blocked completely with a neutralizing antibody to HLA-DR, emphasizing the importance of MHC class II in antigen presentation (Fig. 4A). DCs stand out from all other APCs by their unparalleled efficiency in antigen uptake, processing, and presentation (15, 28). Competence of activated Vδ2+ T cells in these processes was examined by correlating proliferation responses in freshly isolated peripheral blood CD4+ αβ T cells to various concentrations of PPD previously added during the generation of APCs (Fig. 4B). In these experiments using PPD between 0.01 and 10 μg/ml, Vδ2+ T cells were at least as efficient as DCs. Vδ2+ T cell–induced αβ T cell responses depended on physical contact as evidenced by lack of proliferation in the absence of direct contact with TT- or TSST-1–presenting Vδ2+ T cells, excluding soluble mediators as main contributors to these responses (Fig. 4C).

Fig. 4.

Antigen processing and presentation by Vδ2+ T cells. (A) Proliferation of fresh peripheral blood CD4+ αβ T cells in response to PPD-presenting (PPD) or TT-presenting (TT) Vδ2+ T cells. As control, Vδ2+ T cells without antigen or antigen-presenting DCs and αβ T cells were used, and proliferation responses were determined at day 6 (PPD) or 14 (TT) of culture. Chloroquine treatment of Vδ2+ T cells included treatment before and during antigen addition and treatment after antigen processing for 2 hours (post-chloroquine) followed by washing. HLA-DR or control antibodies were added to TT- or PPD-presenting Vδ2+ T cells before mixing with responder cells (n = 2). (B) Efficiency of APC function in Vδ2+ T cells evaluated by concentration-dependent proliferation of bulk primary CD4+ αβ T cells. Responses to PPD-presenting Vδ2+ T cells (circles) were compared with those obtained by using antigen-presenting DC (squares) or αβ T cells (triangles) after 6 days (n = 2). (C) Proliferation of αβ T cells is dependent on contact with TT- or TSST-1–presenting Vδ2+ T cells. In a two-chamber system, the upper chambers contained either CFSE-labeled TT-specific (TT) cultured CD4+ αβ T or naive CD4+ αβ T (TSST-1) responder cells alone, and the lower chambers contained these responder cells together with TT- or TSST-1–presenting Vδ2+ T cells at an APC:responder cell ratio of 1:5. Proliferation (reduction in CFSE fluorescence) was determined after 5 days of culture; undivided responder cells are indicated by bars.

Activated Vd2+ T cells induced naive CD8+ ab T cell proliferation and differentiation into cytotoxic T lymphocytes (CTLs). To determine whether Vδ2+ T cells can also act as APCs in the induction of primary CD8+ T cell responses, we set up naïve CD8+ αβ T cells in proliferation assays with heterologous (MHC-mismatched), IPP-stimulated Vδ2+ T cells, mature DCs, or superantigen-stimulated αβ T cells as APCs. Vδ2+ T cells equaled or exceeded DCs in their capability to induce CD8+ T cell proliferation, whereas αβ T cells proved to be greatly inferior APCs (Fig. 5A). CD8+ effector cells (CTLs) taken from day 14 MLR cultures displayed alloreactive cytotoxic activity (Fig. 5B) (13). CTLs generated with the use of Vδ2+ APCs were indistinguishable from those generated with the use of DCs with respect to perforin expression, IFN-γ production, specific cytotoxicity, and migration reprogramming, evidenced by loss of the LN receptor CCR7 in the majority of CTLs (Fig. 5B and fig. S5).

Fig. 5.

Vδ2+ T cells induce primary CD8+ T cell responses. (A) MLR with naïve, CD8+ αβ T cells and heterologous IPP-stimulated Vδ2+ T cells (circles), LPS-matured DCs (squares), or superantigen-stimulated αβ T cells (triangles) at decreasing APC:responder cell ratios (n = 6). (B) CD8+ T cells generated in response to heterologous Vδ2+ T cells (top) or DCs (bottom) at 14 days MLR culture were examined for cytolytic activity with the use of a mixture of heterologous (true targets) and autologous (negative control) CD4+ T cells that were labeled with high and low dose of CFSE, respectively. Loss in CFSEhigh events (arrows) corresponds to percent (%) target cell killing (n = 3).

Conclusions. Our findings lead us to propose an additional and unexpected role for Vδ2+ T cells in the initiation of adaptive immune processes. Human blood Vδ2+ T cells rapidly and substantially expand in response to microbial infections, and in vivo this would most likely occur via the recognition of common nonpeptide antigens (37). Such microbial metabolites are a frequent target for responder γδ T cells (1, 2, 7) and induce highly efficient responses that may be due to superior signaling by γδ-TCR as compared with αβ-TCR (29, 30). Rapid and local γδ T cell responses result in innate effector functions (cytokine and chemokine secretion and target cell lysis) (35, 7) as well as induction of the LN-homing receptor CCR7 (8, 12). Our in vitro findings predict that contact of Vδ2+ T cells with microbes could also result in induction of a professional APC function that would serve to initiate strong adaptive responses by CD4+ and CD8+ αβ T cells. We suggest a scenario in which TCR-activated Vδ2+ T cells take up and process microbial antigens and, after their relocation to draining LNs, induce antimicrobial responses in αβ T cells. Central to this model is the observation that Vδ2+ T cells rapidly but transiently up-regulate CCR7 upon γδ-TCR–triggering (8, 12), resembling induction of LN homing properties in DCs when exposed to inflammatory stimuli (3133). We show here that αβ T cell responses generated in vitro by activated Vδ2+ T cells match those obtained with mature DCs, at least in vitro. Of note, APC functions in Vδ2+ T cells are expected to be restricted to infectious (inflammatory) settings, because resting Vδ2+ T cells (equivalent to those not responding to infection in vivo) completely lack these features. This absence of APC function in resting Vδ2+ T cells also indicates that these cells are unlikely to substitute for DCs in their other role of controlling of self-tolerance and immune regulation (14, 2326). Consistent with this, activated Vδ2+ T cells also appear to lack anti-inflammatory effects on αβ T cells under the conditions used in our experiments, suggesting a specific role for these novel APCs in the initiation of pro-inflammatory immune responses. Early but transient CCR7 expression further predicts that the APC functions in activated Vδ2+ T cells would be most effective in the early stage of antimicrobial immune processes. The physiological importance of the findings reported here now need to be explored and expanded within in vivo settings of human antimicrobial immunity. Considering the ease of Vδ2+ T cell isolation and ex vivo manipulation as compared with that for DCs, it is possible that Vδ2+ T cells may be useful targets for manipulation in vaccine research and immunotherapy (24, 25, 3436).

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Tables S1 and S2

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