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The Journal of Immunology, 2006, 176: 1348-1354.
Copyright © 2006 by The American Association of Immunologists

Direct Costimulatory Effect of TLR3 Ligand Poly(I:C) on Human {gamma}{delta} T Lymphocytes1

Daniela Wesch2, Susann Beetz, Hans-Heinrich Oberg, Matthias Marget, Kirsten Krengel and Dieter Kabelitz

Institute of Immunology, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLR3 recognizes viral dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). TLR3 expression is commonly considered to be restricted to dendritic cells, NK cells, and fibroblasts. In this study we report that human {gamma}{delta} and {alpha}beta T lymphocytes also express TLR3, as shown by quantitative real-time PCR, flow cytometry, and confocal microscopy. Although T cells did not respond directly to poly(I:C), we observed a dramatic increase in IFN-{gamma} secretion and an up-regulation of CD69 when freshly isolated {gamma}{delta} T cells were stimulated via TCR in the presence of poly(I:C) without APC. IFN-{gamma} secretion was partially inhibited by anti-TLR3 Abs. In contrast, poly(I:C) did not costimulate IFN-{gamma} secretion by {alpha}beta T cells. These results indicate that TLR3 signaling is differentially regulated in TCR-stimulated {gamma}{delta} and {alpha}beta T cells, suggesting an early activation of {gamma}{delta} T cells in antiviral immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A small fraction of peripheral blood CD3+ T cells expresses the TCR{gamma}{delta} instead of the conventional TCR{alpha}beta. {gamma}{delta} T cells recognize Ags without requirement for Ag processing and independently of classical MHC molecules (1, 2). In the blood of adult humans, a majority (50–95%) of {gamma}{delta} T cells expresses V{gamma}9V{delta}2 TCR. These cells primarily recognize phosphorylated intermediates of the nonmevalonate pathway of the bacterial isoprenoid biosynthesis pathway (phosphoantigens) (3, 4) and thus sense microbial infection at a very early stage. After ligand recognition, {gamma}{delta} T cells rapidly release cytokines such as IFN-{gamma} and TNF-{alpha}, thereby activating innate immune cells and facilitating adaptive immune responses by {alpha}beta T cells (1, 5). Therefore, it is assumed that {gamma}{delta} T cells may serve as protection against infections as a first line of defense before Ag-specific {alpha}beta T cells expand. Moreover, {gamma}{delta} T cells play a not precisely defined role during antiviral immunity. Increased numbers of {gamma}{delta} T cells are present in the peripheral blood of EBV-infected (V{delta}2) or HIV-infected (V{delta}1) individuals and CMV-infected patients (V{delta}1) after kidney transplantation (6, 7, 8).

TLRs are pattern recognition receptors involved in the innate immune response to infection, which also contribute to the regulation of adaptive immune responses. To date, TLR have been considered to be expressed mainly in APC, including monocytes, macrophages, and dendritic cells (DCs).3 In APC, TLR ligands induce the up-regulation of costimulatory molecules, including CD80, CD86, and CD40 and the production of proinflammatory cytokines, thereby indirectly costimulating T cell activation (9, 10, 11, 12, 13). Recent studies suggested that {gamma}{delta} T lymphocytes are also stimulated indirectly via TLR-mediated activation of immature myeloid DC (via TLR3) or plasmacytoid DC (via TLR9) (14, 15).

TLR3 recognizes viral dsRNA and a synthetic analog, polyinosinic-polycytidylic acid (poly(I:C)), which both lead to the production of type I and type II IFN via NF-{kappa}B activation and IFN regulatory factor-3 (16, 17, 18, 19, 20). In addition to TLR3, the RNA helicase retinoic acid-inducible gene I (RIG-I) has recently been shown to interact with cytoplasmic dsRNA, leading to the activation of NF-{kappa}B and IFN regulatory factor-3 (21, 22). Furthermore, cellular mRNA functions as a host-derived TLR3 ligand (23). TLR3 was shown to be expressed in myeloid BDCA-1+ (CD1c+)/CD11c+ DC, NK cells, fibroblasts, and intestinal epithelial cells and more recently in human T lymphocytes using quantitative PCR (24, 25, 26, 27). Although TLR3 function and signal transduction have been well studied in nonlymphoid cells, the functional relevance of TLR3 in T cells is enigmatic.

In the present study we focused on the expression of TLR3 in {alpha}beta and {gamma}{delta} T cells and their functional responsiveness after stimulation with poly(I:C). Our results show that TCR-activated {gamma}{delta} T cells, but not {alpha}beta T cells, produce large amounts of IFN-{gamma} when costimulated with poly(I:C).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and activation of {gamma}{delta} T cell clones and lines

{gamma}{delta} T cell clones and lines were established and cultured with occasional restimulation, as previously described (28). V{delta}1 {gamma}{delta} T cell clones and lines were cultured in 96-well microculture plates coated with 0.5 µg/ml anti-CD3 mAb OKT3, and V{delta}2 {gamma}{delta} T cell clones and lines were stimulated with optimal concentrations of bromohydrin pyrophosphate (BrHPP; 200 nM; provided by Innate Pharma), 0.5 µg/ml anti-CD3 mAb, or anti-V{gamma}9 mAb 7A5 (29). Both {gamma}{delta} T cell subsets were stimulated in the absence or the presence of different concentrations of poly(I:C) (Calbiochem/Merck or Amersham Biosciences) 10–12 days after restimulation in serum-free X-VIVO 15 medium (Cambrex BioScience). Proliferation was measured by uptake of [3H]TdR during the last 8 h of a 3-day culture period in the presence of 50 U/ml rIL-2 (Chiron). IFN-{gamma} secretion was determined in the absence of IL-2 after 12 h using a commercially available ELISA kit (Quantikine; R&D Systems) according to the manufacturer’s instructions.

Isolation of leukocyte subpopulations

{gamma}{delta} T cells, {alpha}beta T cells, and CD14+ monocytes/macrophages were positively separated from freshly isolated PBMC using the MACS system (Miltenyi Biotec). To avoid examination of in vivo (pre)activated {gamma}{delta} T cells, care was taken to select blood donors in whom {gamma}{delta} T cells accounted for <6%. Briefly, PBMC were pretreated with Fc-blocking reagents (Miltenyi Biotec) to avoid unspecific binding of Abs to FcR-bearing cells. To isolate {gamma}{delta} T cells, PBMC were stained with haptenated anti-TCR{gamma}{delta} mAb, followed by anti-hapten microbeads-FITC; for {alpha}beta T cells, PBMC were incubated with anti-TCR{alpha}beta mAb BMA031, followed by goat anti-mouse IgG microbeads, and for isolation of CD14+ monocytes/macrophages, PBMC were incubated with CD14 microbeads. The purity of the positively selected cells was ≥98%. CD56+ NK cells were purified by negative selection procedures using NK Cell Isolation Kit II (Miltenyi Biotec). Immature DCs were generated from the adherent cell fraction of PBMC. Adherent cells were cultured in serum-free X-VIVO 15 medium (Cambrex BioScience) supplemented with IL-4 (1000 U/ml; R&D Systems) and GM-CSF (1000 U/ml; R&D Systems) for 4–5 days.

Flow cytometry and confocal laser scanning microscopy

The following mAb were used to analyze the purity of the freshly isolated lymphocytes and monocytes/macrophages: anti-CD3, pan anti-TCR{gamma}{delta}, pan anti-TCR{alpha}beta, anti-CD56, anti-CD14, anti-CD11c, and anti-CD1c (all from BD Biosciences). To determine the CD69 expression on {gamma}{delta} T cells within PBMC, PE-labeled CD69 mAb and FITC-labeled pan anti-TCR{gamma}{delta} mAb (BD Biosciences) were used. The positively separated {gamma}{delta} T cells, which were labeled with haptenated anti-TCR{gamma}{delta} mAb, followed by FITC-conjugated anti-hapten microbeads during separation, were stained only with PE-labeled CD69 mAb. PE-conjugated anti-TLR3 mAb TLR3.7 (Biosciences) was used for surface and intracellular staining. For intracellular detection, cells were washed, fixed, and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen) following the manufacturer’s instructions. Thereafter, cells were stained with mAb TLR3.7 or isotype control, and measured on a FACScan or FACSCalibur flow cytometer (BD Biosciences) using CellQuest software. For laser scanning microscopy, established {gamma}{delta} T cell clones were allowed to adhere to poly-L-lysine-coated glass slides. The cells were fixed and permeabilized with 100% methanol (Merck), then washed extensively three times with PBS. After blocking with 0.5% BSA, cells were stained with unconjugated anti-TLR3 mAb TLR3.7 or isotype control for 1 h, followed by the second step Ab Alexa 546-conjugated goat anti-mouse (Invitrogen Life Technologies, Inc.) for 1 h. The stained cells were visualized at x630 magnification with a confocal laser scanning microscope (Zeiss).

Determination of cytokine production

Positively isolated, highly purified {gamma}{delta} or {alpha}beta T cells (4 x 106/well) were cultured in wells coated with rabbit anti-mouse Ig (to cross-link the TCR) in the presence or in the absence of 50 µg/ml poly(I:C) without IL-2 in serum-free X-VIVO 15 medium. Isolated T cells were also stimulated with poly(I:C) alone or via TCR cross-linking in the presence of 1 µg/ml soluble anti-CD28 mAb (BD Pharmingen). Supernatants were collected after 24 h and were stored at –20°C until use. Supernatants were screened for cytokines and chemokines using the RayBio Human Cytokine Ab Array VI and 6.1 Map, and VII and 7.1 Map (Hoelzel Diagnostic), which allow simultaneous detection of 2 x 60 cytokines and chemokines. Signals were detected by chemiluminescence, followed by semiquantitative analysis with AIDA software (Ray-Test). To determine the intensity, local background was subtracted from each value and normalized against the positive controls of each membrane. Normalization was performed by Excel calculation. To confirm and quantify the results of the cytokine array, IFN-{gamma} was also measured by intracellular flow cytometry and ELISA (Quantikine; R&D Systems). For analysis of intracellular IFN-{gamma}, 1 x 106/well positively isolated {gamma}{delta} or {alpha}beta T cells were stimulated for 24 h with immobilized rabbit anti-mouse Ig with or without poly(I:C). During the last 4 h of stimulation, 3 µM monensin was added to the cultures. The cells were harvested, washed, fixed, and permeabilized using the Cytofix/Cytoperm kit according to the manufacturer’s instructions. Subsequently, the cells were washed and stained with PE-labeled mouse anti-human IFN-{gamma} or isotype-matched Ig control (BD Pharmingen) for 25 min. After washing steps, cells were analyzed on a FACSCalibur flow cytometer.

Quantitative RT-PCR

RNA was isolated using the NucleoSpin RNA II kit (Macherey & Nagel). cDNA was synthesized from 1 µg of total RNA in a 20-µl reaction volume using random hexamers as primer (first-strand cDNA synthesis kit; Amersham Biosciences). PCR was performed with Platinum SYBR Green quantitative PCR SuperMix UDG (Invitrogen Life Technologies) on an iCycler (Bio-Rad) with the following cycling conditions: 2 min at 50°C; 2 min at 95°C; and 45 cycles of 15 s at 95°C, 15 s at 62°C, and 30 s at 72°C. Two microliters of cDNA was used in each amplification reaction. Primers were designed using GeneFisher software for TLR3 (forward, 5'-ACAGCCAGCTGTCCACCA-3'; reverse, 5'-TCCATGTTAAGGTGCTCCAA-3') and for RNA polymerase II as the housekeeping gene for normalization (forward, 5'-GCACCACGTCCAATGACAT-3'; reverse, 5'-GTGCGGCTGCTTCCATAA-3'). Primer specificity was confirmed by melting curve analysis. No unspecific products were observed. Serial dilutions of plasmids containing the cloned PCR products were used to obtain a standard curve.

Statistical analysis

Student’s t test (paired data) was used to analyze the statistical significance of differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR3 ligand poly(I:C) enhances {gamma}{delta} T cell proliferation

In our first set of experiments we investigated whether the TLR3 ligand poly(I:C) has a direct effect on {gamma}{delta} T cells. We observed a reproducible and significant increase in proliferation (as measured by [3H]TdR uptake) in the absence of APC when established V{delta}2 or V{delta}1 {gamma}{delta} T cell clones and lines were cultured in the presence of poly(I:C) alone or in combination with a TCR stimulus such as anti-V{gamma}9 mAb for V{delta}2 T cells and anti-CD3 for V{delta}1 T cells (Fig. 1a). Comparable results were obtained with BrHPP or anti-CD3 mAb for V{delta}2 T cell clones and lines instead of anti-V{gamma}9 mAb (data not shown). The proliferation in response to TCR stimulus alone was not significantly increased in the majority of {gamma}{delta} T cell clones and lines due to the partially overlapping effect of activation-induced cell death after TCR triggering (29). Furthermore, IFN-{gamma} production by these {gamma}{delta} T cell clones and lines stimulated with BrHPP or anti-V{gamma}9 mAb for V{delta}2 T cell, or anti-CD3 mAb for V{delta}1 T cells was enhanced by poly(I:C) (Fig. 1b and data not shown). These results suggested that {gamma}{delta} T cells, like NK cells (18, 30), might indeed directly respond to poly(I:C) in addition to the previously described indirect effects mediated via myeloid DC. Therefore, we investigated in detail the expression of TLR3 in freshly isolated resting and short-term activated (12–24 h) {gamma}{delta} T cells and activated {gamma}{delta} T cell clones and lines.


Figure 1
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  		FIGURE 1. Effects of poly(I:C) on the proliferation and IFN-{gamma} production of {gamma}{delta} T cell clones and lines. a, V{delta}1 or V{delta}2 {gamma}{delta} T cell clones or lines (5 x 104 cells) were cultured in medium alone or stimulated with poly(I:C) or via TCR with 0.5 µg/ml immobilized anti-V{gamma}9 mAb (V{delta}2 T cells; {diamondsuit}, {blacktriangleup}, and {blacksquare}) or 0.5 µg/ml immobilized anti-CD3 mAb (V{delta}1 T cells; {diamond}, {triangleup}, and {square}), indicated as TCR, in the absence or the presence of 10 µg/ml poly(I:C) and exogenous IL-2. Each symbol represents the data from one donor, and each point presents the mean value of triplicate determinations. Proliferation was measured by [3H]TdR incorporation after 3 days. The increase in proliferation was significant (**, p < 0.01). b, A V{delta}2 {gamma}{delta} T cell line of one representative donor was cultured in medium or with different concentrations of poly(I:C), as indicated, in the absence ({square}) or the presence ({blacksquare}) of anti-V{gamma}9 mAb without IL-2. IFN-{gamma} secretion was determined by ELISA after 24 h. The mean ± SD of triplicate determinations are shown.

 
{gamma}{delta} and {alpha}beta T cells express TLR3

TLR3 expression was previously found in total T lymphocytes by Northern blot analysis and RT-PCR (24, 25, 26, 27), but no data are available for purified human {gamma}{delta} T cells. We observed the expression of TLR3 by RT-PCR in four established {gamma}{delta} T cell clones and in six newly generated {gamma}{delta} T cell lines expressing various V{gamma}- and V{delta}-chains (data not shown). Next, we investigated TLR3 mRNA by quantitative RT-PCR in freshly isolated, highly purified (>98%) {gamma}{delta} T cells in comparison with other subsets of human PBMC (Fig. 2a). No other potentially TLR3-expressing cells (neither CD56+ NK cells nor CD11c+ CD1c+ myeloid DCs) were detected in the purified {gamma}{delta} or {alpha}beta T cells. In line with published data, TLR3 expression was nearly absent in freshly isolated monocytes/macrophages and was prominent in NK cells. Surprisingly, highly purified {gamma}{delta} and {alpha}beta T lymphocytes gave strong TLR3 signals. In all four tested blood samples, however, TLR3 mRNA expression was 1.5–4 times higher in {gamma}{delta} T cells compared with {alpha}beta T cells (Fig. 2). TLR3 expression was confirmed in cell sorter-purified (99.9% purity) {gamma}{delta} T cells and {alpha}beta T cells (Fig. 2b, {triangleup}). Next, we analyzed TLR3 expression at the protein level. We detected TLR3 protein in {gamma}{delta} T cells by flow cytometry and confocal laser scanning microscopy. In line with the results of Matsumoto et al. (17), we found that TLR3 is expressed intracellularly, but not on the cell surface, in monocyte-derived immature DC (Fig. 3a). We observed the same pattern in freshly isolated (resting) {gamma}{delta} and {alpha}beta T lymphocytes, where TLR3 was predominantly localized intracellularly and not on the cell surface (Fig. 3a). Peripheral blood {gamma}{delta} T cells preferentially express V{gamma}9 V{delta}2, whereas V{delta}1-expressing cells are under-represented. However, all {gamma}{delta} T cells expressed TLR3 independently of the V{gamma}/V{delta}-chain in the six tested donors (data not shown). Additionally, we investigated TLR3 expression in several V{delta}1 and V{delta}2 {gamma}{delta} and {alpha}beta T cell clones and T cell lines by confocal laser scanning microscopy and flow cytometry, again with positive results. Intracellular TLR3 expression of one representative V{delta}1 {gamma}{delta} T cell clone is shown in Fig. 3a (upper right, laser scanning microscopy). Additionally, we observed modest TLR3 expression on the surface of all tested {gamma}{delta} T cell clones and lines (Fig. 3b). These results unambiguously demonstrate that resting T lymphocytes and T cell clones and lines, including {gamma}{delta} T cells, express TLR3 intracellularly, whereas only {gamma}{delta} T cell clones and lines display weak cell surface expression. For this reason, we decided to examine the potential up-regulation of cell surface TLR3 on resting {gamma}{delta} T cells after stimulation. We observed an up-regulation of TLR3 on the surface of positively isolated {gamma}{delta} T cells after TCR cross-linking, which was increased even more 24 h after TCR cross-linking in the presence of poly(I:C) (Fig. 3c). We obtained comparable results when {gamma}{delta} T cells were analyzed within PBMC stimulated with BrHPP in the presence or the absence of poly(I:C) (Fig. 3c). This prompted us to investigate possible direct effects of TLR3 ligation on freshly isolated, highly purified {gamma}{delta} T cells.


Figure 2
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  		FIGURE 2. Quantitative analysis of TLR3 mRNA expression in subsets of human PBMC. The expression of TLR3 mRNA in highly purified monocytes/macrophages, NK cells, and {gamma}{delta} and {alpha}beta T cells was analyzed by quantitative real-time PCR. The results are depicted as the number of transcripts per 1000 copies of the housekeeping gene RNA polymerase II, indicated as the expression rate. a, Comparison of {alpha}beta T cells, {gamma}{delta} T cells, monocytes/macrophages, and NK cells from one representative donor. b, TLR3 mRNA expression in {alpha}beta and {gamma}{delta} T cells from three additional donors. {triangleup}, Data from sorted cells (99.9% purity).

 

Figure 3
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  		FIGURE 3. Intracellular and surface expressions of TLR3 in {gamma}{delta} and abeta T cells. a, The purity of MACS-isolated {alpha}beta and {gamma}{delta} T cells is shown in the upper row. The expression of TLR3 in these cells was determined on the cell surface (middle row) and intracellularly (lower row) by PE-conjugated anti-TLR3 mAb TLR3.7. Open histograms indicate isotype controls. Immature DC served as a positive control for intracellular TLR3 expression (lower row, right). Data from one representative donor (of six) are shown. TLR3 expression in one representative V{delta}1 T cell clone was analyzed by confocal laser scanning microscopy with the unconjugated anti-TLR3 mAb TLR3.7, followed by Alexa 546-conjugated goat anti-mouse Ab (middle row, right),with the appropriate isotype control (upper row, right). b, TLR3 surface expression is shown on one representative V{delta}1 and one V{delta}2 T cell clone (bold line) in comparison with the isotype control (dotted line). c, Up-regulation of cell surface TLR3 24 h after stimulation was analyzed in positively isolated {gamma}{delta} T cells and {gamma}{delta} T cells within PBMC. The dotted lines indicate the isotype control, and the gray histograms depict the medium control, whereas the thin lines indicate TLR3 up-regulation after TCR stimulation, and the bold lines show TLR3 up-regulation after TCR stimulation in the presence of poly(I:C).

 
Differential responses of {gamma}{delta} and {alpha}beta T cells to poly(I:C)

Highly purified, positively selected {gamma}{delta} and {alpha}beta T cells from three healthy donors were stimulated by TCR cross-linking (via rabbit anti-mouse Ig) in the presence or the absence of 50 µg/ml poly(I:C) or with poly(I:C) alone. CD69 expression and secretion of cytokines and chemokines in the supernatants were analyzed after 24 h (Figs. 4 and 5). In contrast to NK cells and myeloid immature DCs, freshly isolated {gamma}{delta} T lymphocytes did not respond to poly(I:C) alone in the absence of APC. As shown in Fig. 4, we compared CD69 expression on highly purified {gamma}{delta} T cells and {gamma}{delta} T cells within PBMC. Similar to the findings reported by Kunzmann et al. (14), we observed CD69 up-regulation on {gamma}{delta} T cells by poly(I:C) within PBMC stimulated, or not, by BrHPP (Fig. 4a). Additionally, CD69 expression on highly purified {gamma}{delta} T cells was increased when cells were cultured for 24 h in medium alone (possibly due to the effects of positive magnetic separation) and was further up-regulated after TCR cross-linking or TCR cross-linking in the presence of poly(I:C)), whereas poly(I:C) alone did not enhance CD69 on purified {gamma}{delta} T cells (Fig. 4b). In these experiments, cytokine and chemokine productions were measured in parallel after 24 h by a human cytokine Ab array. The results showed that poly(I:C) significantly increased the TCR-mediated IFN-{gamma} production of positively selected, highly purified {gamma}{delta} T lymphocytes, whereas poly(I:C) alone did not induce IFN-{gamma} production (Fig. 5a). The costimulatory effect of poly(I:C) on IFN-{gamma} production was confirmed by intracellular flow cytometry (Fig. 5b) and ELISA (Fig. 6a). Additionally, several other cytokines (TNF-{alpha}, IL-1beta, IL-6, and GM-CSF) and chemokines (RANTES and MCP-1) were moderately modulated by poly(I:C) (Fig. 5a). To selectively activate V{gamma}9V{delta}2 T cells, highly purified {gamma}{delta} T cells were stimulated with BrHPP. Again, poly(I:C) drastically enhanced BrHPP-stimulated IFN-{gamma} production (Fig. 6a). Furthermore, the addition of anti-TLR3 Ab to these cultures partially inhibited IFN-{gamma} production from 20–30% in three donors (Fig. 6b). In striking contrast to {gamma}{delta} T cells, poly(I:C) did not stimulate an IFN-{gamma} response in {alpha}beta T cells, neither alone nor in combination with a TCR stimulus. As expected, positively selected and TCR cross-linked {alpha}beta T cells produced a variety of cytokines and chemokines when costimulated with soluble anti-CD28 mAb (Fig. 5a). The productions of cytokines and chemokines, such as IL-2, IFN-{gamma}, TNF-beta, IL-13, and RANTES, were not modified when poly(I:C) was additionally added, whereas moderate effects were noted for IL-10, TNF-{alpha}, and IL-6 after TCR cross-linking with poly(I:C) in the absence as well as the presence of soluble anti-CD28 mAb (Fig. 5b and data not shown).


Figure 4
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  		FIGURE 4. Poly(I:C) enhances CD69 expression on {gamma}{delta} T cells. CD69 expression was compared on {gamma}{delta} T cells within PBMC (a) and on highly purified {gamma}{delta} T cells (b) from three donors before culture (0 h) and 24 h after culture in medium. PBMC were stimulated with BrHPP and highly purified positively selected {gamma}{delta} T cells via rabbit anti-mouse Ig (to cross-link the TCR) with or without poly(I:C) or with poly(I:C) alone as indicated. PBMC were stained with FITC-labeled pan-TCR {gamma}{delta}-specific mAb plus PE-labeled CD69 mAb. A gate was set on the total population of {gamma}{delta} T cells. Highly purified {gamma}{delta} T cells (with the FITC-labeled anti-TCR{gamma}{delta} mAb on the surface) were stained only with PE-conjugated CD69 mAb.

 

Figure 5
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  		FIGURE 5. Production of IFN-{gamma} in response to TCR cross-linking and poly(I:C). a, {gamma}{delta} and {alpha}beta T cells were purified by positive selection with MACS. The cells were cultured in the absence (medium) or the presence of rabbit anti-mouse Ig (TCR stimulation) and poly(I:C) or anti-CD28 mAb as indicated. Cytokines and chemokines were measured in the culture supernatants by RayBio cytokine Ab array. The numbers in brackets represent the normalized intensity. b, IFN-{gamma} was determined by intracellular flow cytometry in {gamma}{delta} T cells after TCR cross-linking (TCR stimulation) with or without poly(I:C) as indicated. The results of one representative of three experiments are shown.

 

Figure 6
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  		FIGURE 6. Poly(I:C) enhances IFN-{gamma} secretion in response to TCR cross-linking or BrHPP. Positively selected {gamma}{delta} T cells (2.5 x 105) were stimulated through the TCR via immobilized rabbit anti-mouse Ab (ram) or BrHPP in the presence or the absence of 10 µg/ml poly(I:C). IFN-{gamma} was determined in the supernatants by ELISA. a, The results of three experiments with different blood donors. b, Inhibition of IFN-{gamma} secretion in the presence of 25 µg/ml anti-TLR3 mAb. Anti-TLR4 mAb or control mouse IgG did not modulate IFN-{gamma} secretion.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we demonstrate that human {gamma}{delta} T cells express TLR3, the pattern recognition receptor for dsRNA. TLR3 is known to be expressed in immature myeloid DC, NK cells, fibroblasts, intestinal epithelial cells, and, to some extent in T cells (16, 17, 18, 24, 25, 26, 27). We observed that highly purified {gamma}{delta} T cells expressed similar levels of TLR3 mRNA as NK cells. In contrast to the results reported by Kunzmann et al. (14), we found that freshly isolated human {gamma}{delta} T cells as well as established {gamma}{delta} T cell lines and clones express TLR3 protein. The reason for this discrepancy is not clear, but might be related to the use of different Abs. We tested different commercially available anti-TLR3 mAbs. Only the anti-TLR3.7 mAb, which is suitable for cell surface and intracellular staining (31), yielded reliable results when directly fluorochrome-conjugated mAb were used. We did not detect TLR3 on the cell surface of resting T cells, but did find TLR3 intracellularly in {gamma}{delta} and also {alpha}beta T cells. After short-term stimulation, TLR3 was up-regulated on the surface of {gamma}{delta} T cells. Moderate expression of TLR3 was also detectable on the surface of {gamma}{delta} T cell clones and lines. This provides a basis for poly(I:C) ligand binding to TLR3 and subsequent signaling events. Moreover, we were able to partially inhibit the costimulatory effect of poly(I:C) by adding neutralizing anti-TLR3 mAb, which presumably antagonizes TLR3 on the cell surface. Furthermore, we have demonstrated a direct costimulatory effect of the TLR3 surrogate ligand poly(I:C) on the IFN-{gamma} production of freshly isolated {gamma}{delta}, but not {alpha}beta T cells. In the studies by Kunzmann et al. (14), poly(I:C) alone did not induce effects on purified {gamma}{delta} T cells and T cell clones, and the authors concluded that the costimulation was mediated by TLR3 expressing immature myeloid DC via the production of type I IFN. In agreement with these results, we observed that poly(I:C) alone did not induce any effect on freshly isolated {gamma}{delta} T cells, in line with the absence of TLR3 surface expression on unstimulated (resting) {gamma}{delta} T cells. Our data indicate that {gamma}{delta} T cells have to be activated via TCR to up-regulate TLR3. This fit well with the recent study by Brandes et al. (32), which demonstrated that resting isolated {gamma}{delta} T cells have to be activated to perform APC function, as shown by them, or to become directly sensitive to costimulatory effects, as shown by us. Furthermore, we observed that highly purified {gamma}{delta} T cells secrete several other cytokines and chemokines (e.g., TNF-{alpha}, GM-CSF, IL-1beta, IL-6, RANTES, and MCP-1) after TCR cross-linking, which seems to be modulated in the presence of poly(I:C). We noticed constitutive expression of RANTES, MCP-1, and IL-6 in {gamma}{delta} T cells cultured in medium alone as described previously for RANTES (33), whereas MCP-1 was not detected by Cipriani et al. (34) with ELISA using PBMC cultured in isopentenyl pyrophosphate plus IL-2. To confirm and detect marginal changes in expression levels, additional experiments with assays more sensitive than the human cytokine Ab array are required. In this study we have focused on IFN-{gamma} production, which is the most prominent cytokine produced after TCR cross-linking and poly(I:C) stimulation of freshly isolated {gamma}{delta} T cells. Poly(I:C) significantly increased TCR-stimulated IFN-{gamma} production by purified {gamma}{delta} T cells in the absence of other TLR3-expressing cells (no detectable CD1c+CD11c+ DC) and without added IL-2. IL-2 is dispensable when expression of cytokines and chemokines is analyzed in {gamma}{delta} T cells after short-term stimulation; however, IL-2 is essential for the proliferation of {gamma}{delta} T cells. Lamont et al. (35) reported that enhanced STAT4 DNA binding and increased IFN-{gamma} production are triggered by IL-2 in human V{gamma}9V{delta}2 T cells. Freshly isolated human peripheral blood {gamma}{delta} T cells are not able to produce IL-2 (36), which explains the low level of IFN-{gamma} after TCR stimulation in the absence of IL-2 (Fig. 5a, upper part). The addition of poly(I:C) might overcome the requirement for IL-2 in this regard. In contrast to resting {gamma}{delta} T cells, activated {gamma}{delta} T cell clones and lines are able to produce IL-2 and produce higher amounts of IFN-{gamma} upon TCR stimulation alone. In this study the addition of poly(I:C) caused only a moderate enhancement of IFN-{gamma} secretion due to the preactivated state of the {gamma}{delta} T cell clones (Fig. 1b). In contrast to freshly isolated {gamma}{delta} T cells, poly(I:C) did not costimulate IFN-{gamma} production in TLR3-expressing {alpha}beta T cells. As expected, {alpha}beta T cells required costimulatory signals, such as anti-CD28, to produce high amounts of cytokines and chemokines, which were not modified when poly(I:C) was also added (Fig. 5b and data not shown). Poly(I:C), instead of soluble anti-CD28, moderately enhanced some cytokines (e.g., TNF-{alpha}, IL-6, and IL-10), an observation that requires additional analysis and quantification by other methods.

Increasing evidence indicates that certain T cells can indeed express functional TLRs. TLR2, TLR5, and TLR7/8 ligands have been recently found to directly costimulate memory CD4+ T cells (37, 38), and TLR8 ligands have been shown to reverse suppression by regulatory T cells (39). These studies failed to detect an effect of poly(I:C) on CD4+ {alpha}beta T cells (37, 38). In accordance, we did not detect a costimulatory effect of poly(I:C) on IFN-{gamma}-producing {alpha}beta T cells. However, the previous studies did not examine whether CD4+ or perhaps CD8+ {alpha}beta T cells produced TNF-{alpha} or IL-6 after stimulation via TCR and poly(I:C). The moderately enhanced cytokine production of {alpha}beta T cells after TCR stimulation in the presence of poly(I:C) in our experiments might be due to the responsiveness of only a subset of {alpha}beta T cells, e.g., memory cells. In line with such an assumption, memory CD4+ T cells are known to be more sensitive to TLR-mediated activation than naive CD4+ T cells (38). Moreover, it might also be possible that TLR3 promotes {alpha}beta T cell survival, as described by Gellman et al. (40) for activated CD4+ {alpha}beta T cells in the mouse.

RIG-I was recently identified as another receptor for poly(I:C) and therefore for dsRNA (21, 22). RIG-I belongs to the antiviral host response mechanisms. It detects dsRNA, which accumulates in the cytoplasm in virus-infected cells. For our experiments, we cannot exclude that poly(I:C) stimulates an intracellular host response within {gamma}{delta} T cells. However, the incubation with poly(I:C) alone did not result in activation of {gamma}{delta} T cells detectable with the readout systems used in our study.

As we showed, {gamma}{delta} T cells express TLR3, which is an important determinant of cellular responses to external dsRNA, and therefore senses virus infection in other cells. Similar to human NK cells, {gamma}{delta} T cells respond to poly(I:C) with up-regulation of CD69 as well as increased IFN-{gamma} production (13, 18, 30); however, {gamma}{delta} T cells need a second stimulus provided via TCR to efficiently support antiviral immunity. As discussed by Caron et al. (38), non-T cells are more sensitive to pathogen-associated molecular pattern-mediated activation, whereas pathogen-associated molecular pattern activation of T cells would take place during massive entry of microorganisms. Our data clearly suggest that {gamma}{delta} T cells emerge as another cell population to guard and eliminate viral infections.

It has long been observed that {gamma}{delta} T cells are involved in antiviral defense reactions; however, their precise role is still elusive (1, 6, 7, 8). In CMV-infected patients after kidney transplantation, V{delta}2-negative {gamma}{delta} T cells show a long-lasting expansion and display a strong reactivity against CMV-infected cells (8, 41). This reactivity requires TCR engagement. Also, this subset of {gamma}{delta} T cells has been reported to recognize stress-induced MHC class I-related chains A and B on epithelial cells via their TCR (42). The additional signaling via TLR3 in the case of a virus infection might provide specific information for the cells to initiate the appropriate effector functions. In this context, it is very interesting that TLR9 and TLR3 are mainly involved in antiviral immunity against murine CMV infection. As demonstrated by Tabeta et al. (43), TLR3–/– and TLR9CpG1/CpG1 mice are susceptible to murine CMV infection, which resulted in impaired type-I IFN production. Their data suggest that both TLR pathways together are necessary for overall protection against murine CMV infection (43).

In summary, our results suggest that integrated signals from TLR3 and TCR together induce a strong antiviral effector function in {gamma}{delta} T cells and thus support a decisive role of {gamma}{delta} T cells in early defense against viral infection. Most likely, such a mechanism is not restricted to human {gamma}{delta} T cells, because bovine {gamma}{delta} T cells were also recently found to express TLR3 (44). Together, these results support the idea that {gamma}{delta} T cells form a link between the innate and adaptive immune systems (1, 45). Furthermore, our study supports the emerging concept that certain subsets of human T lymphocytes react directly to certain TLR ligands (37, 38, 39, 40).


   Acknowledgments
 
We gratefully acknowledge the technical assistance of Ina Martens, Hoa Ly, Katrin Köbsch, and Parvin Davarnia. We also thank Michaela Kaniess for sorting the {gamma}{delta} T cells, and Innate Pharma for the gift of BrHPP.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (Priority Program 1110 Innate Immunity). Back

2 Address correspondence and reprint requests to Dr. Daniela Wesch, Institute of Immunology, Universitätsklinikum Schleswig-Holstein Campus Kiel, Michaelisstrasse 5, D-24105 Kiel, Germany. E-mail address: wesch{at}immunologie.uni-kiel.de Back

3 Abbreviations used in this paper: DC, dendritic cell; BrHPP, bromohydrin pyrophosphate; poly(I:C), polyinosinic-polycytidylic acid; RIG-I, retinoic acid-inducible gene I. Back

Received for publication June 28, 2005. Accepted for publication November 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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