Human Effector CD8+ T Lymphocytes Express TLR3 as a Functional Coreceptor

Human Effector CD8+ T Lymphocytes Express TLR3 as a Functional Coreceptor This information is current as of December 20, 2015.

Julie Tabiasco, Estelle Devêvre, Nathalie Rufer, Bruno Salaun, Jean-Charles Cerottini, Daniel Speiser and Pedro Romero J Immunol 2006; 177:8708-8713; ; doi: 10.4049/jimmunol.177.12.8708 http://www.jimmunol.org/content/177/12/8708

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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References

The Journal of Immunology

Human Effector CD8ⴙ T Lymphocytes Express TLR3 as a Functional Coreceptor1 Julie Tabiasco,* Estelle Deveˆvre,* Nathalie Rufer,†‡ Bruno Salaun,* Jean-Charles Cerottini,* Daniel Speiser,*‡ and Pedro Romero2*‡

T

he receptor Toll was initially identified in Drosophila, where it plays an essential role in antifungal responses (1). Since then, at least 10 mammalian homologues, called TLR, have been cloned, and these receptors have rapidly emerged as crucial players in the detection of invading pathogens and in the mounting of antimicrobial immune responses (reviewed in Refs. 2 and 3). TLR all bear a Toll/IL-1R motif on their intracellular tail, which is essential for downstream signaling, and many leucin-rich repeats in their extracellular part, which are most probably involved in ligand binding. TLR recognize so-called pathogen-associated molecular patterns, including, among others, LPS (ligand for TLR4), DNA containing unmethylated CpG motifs (ligand for TLR9), viral dsRNA (ligand for TLR3), or ssRNA (ligand for TLR7 and 8) (4 –7). TLR-induced signaling spreads to several adaptors and downstream effectors, and usually culminates in activation of NF-␬B, MAPKs, and IFN-responsive factors (IRF),3 as well as in cytokine production (reviewed in Refs. 8 and 9). Of note, TLR3 is the only TLR identified to date that does not use the adaptor MyD88 to transmit signals, but rather relies on Toll/IL-1R domain-containing adaptor-inducing IFN-␤ to initiate the intracellular signaling cascade triggered by specific ligand binding (10, 11).

*Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland; †Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; and ‡National Center for Competence in Research, Molecular Oncology, Lausanne, Switzerland Received for publication March 6, 2006. Accepted for publication September 28, 2006. 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 J.T. was supported in part by a grant from the Fondation pour la Recherche Me´dicale (Paris, France; code FRM SPE 20021213061). B.S. was supported in part by Oncosuisse Grant OCS-01596-08-2004. 2 Address correspondence and reprint requests to Dr. Pedro Romero, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (Centre Hospitalier Universitaire Vaudois), Avenue PierreDecker, 4, 1005 Lausanne, Switzerland. E-mail address: [email protected] 3

Abbreviations used in this paper: IRF, IFN-responsive factor; DC, dendritic cell.

Copyright © 2006 by The American Association of Immunologists, Inc.

Due to the expression pattern and the ligand specificity of these receptors that enable pathogen recognition, many of the studies to elucidate TLR biology have primarily focused on cells involved in the innate immune response, such as dendritic cells (DC), macrophages, or monocytes (12, 13). However, by modulating the maturation of DCs (14), TLR agonists also indirectly shape adaptive immunity, as evidenced, for example, by the ability of the TLR9 ligand CpG to skew the T cell response toward a Th1 type of response (15, 16). Interestingly, recent reports have highlighted an unsuspected direct role of TLR in T lymphocyte biology. Several TLR have indeed been found to be expressed and to function in both conventional and regulatory CD4⫹ T lymphocytes. TLR2 has been described as a costimulatory receptor able to increase proliferation and IFN-␥ secretion induced by TCR stimulation in conventional CD4⫹ T cells (17), whereas its engagement on regulatory T cells induces proliferation and transient loss of suppressive capacity (18). Similarly, the TLR5 agonist flagellin has been shown to synergize with TCR stimulation in conventional memory CD4⫹ T cells (19), and to increase proliferation and suppressive capacity of their regulatory counterparts (20). TLR8 has been shown to directly modulate T regulatory cell functions (21), whereas TLR3 agonists have been suggested to prolong CD4⫹ T cell survival (22). However, besides mRNA expression-based studies (23, 24), only scarce functional data are available regarding TLR function in CD8⫹ T cells (25). Although these cells are considered as the main actors of antitumoral responses, they are often anergized in the tumor environment (26, 27). As the optimization of their activation is therefore one of the main goals of cancer immunotherapies, TLR agonists have been used as adjuvants in conjunction with Ag to increase CTL responses, e.g., in antimelanoma vaccines (28). Several murine models have indeed shown that TLR agonists can considerably enhance vaccine-induced, Ag-specific immune responses. In this respect, TLR3 and TLR9 agonists are the most potent adjuvants (29). Notably, TLR3 stimulation in vivo promotes Ag cross-presentation by DC (30) and dramatically increases Ag-specific effector and memory CD8⫹ T cell responses (31, 32). However, it is not known whether this effect is direct or mediated by APCs such as DC. 0022-1767/06/$02.00

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TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8ⴙ T cell subsets. We report that a subpopulation of CD8ⴙ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8ⴙ T cells increased IFN-␥ secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-␥ production by Ag-specific CD8ⴙ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy. The Journal of Immunology, 2006, 177: 8708 – 8713.

The Journal of Immunology TLR3 mRNA has been found to be expressed in human CD8⫹ T cells (23, 24). In the absence of functional data, we sought to investigate this issue in more detail. We therefore analyzed TLR3 expression in different human CD8⫹ T cell subsets, and could show that TLR3 is present in effector memory and effector cells, but not in naive or central memory cells. Moreover, functional assays clearly demonstrated that the engagement of TLR3 by specific agonists increased the production of IFN-␥ induced by TCRdependent and -independent stimulation, but did not affect cytolytic activity. Taken together, these results indicate that functional TLR with costimulatory activity can be expressed not only by CD4⫹, but also by CD8⫹ T cells. These data therefore suggest that CD8⫹ T cells could become a new direct target of TLR-based adjuvants in T cell vaccination strategies, to maximize the activation signals provided by DC activated through their own TLR.

Materials and Methods Cells

mAbs and reagents mAbs were from BD Biosciences, except anti-CD45-ECD (Beckman Coulter) and goat anti-rat IgG allophycocyanin (Caltag Laboratories). AntiCCR7 rat IgG mAb 3D12 was provided by M. Lipp (Max Delbru¨ck Institute, Berlin, Germany). Anti-CD3 Ab used in redirected lysis assay was LAU T3 Ab. Poly(I:C) was purchased from Amersham Biosciences and InvivoGen; poly(dI:dC) was from Roche Diagnostics.

Cell separation Peripheral blood samples were collected from healthy donors. PBMCs were obtained by density centrifugation using Ficoll-Hypaque (Pharmacia). Our experimental procedures involve two steps designed to exclude contamination with NK cells. First, CD8⫹ T cells were positively enriched from cryopreserved or fresh PBMCs using anti-CD8⫺-coated magnetic microbeads (Miltenyi Biotec), a procedure that eliminates most NK cells because they are not efficiently retained by the magnet. Immediate reanalysis of the isolated populations revealed ⬎93% purity (CD3⫹CD8⫹). Second, flow cytometry-based analysis and sorting were performed on gated CD3⫹CD8⫹ T cells, allowing the exclusion of any residual contaminating NK cells in the sorted populations (typically ⬍0.3%) on a FACSVantage SE using CellQuest software (BD Biosciences).

Flow cytometry Cells were stained with appropriate mAbs in PBS, 0.2% BSA, and 50 ␮M EDTA for 20 min at 4°C and either directly analyzed by flow cytometry with a FACScan or sorted with a FACSVantage SE into defined populations. Both are equipped with CellQuest software (BD Biosciences). For intracellular staining, cells were first labeled with mAbs to cell surface markers, fixed and permeabilized in PBS/1% formaldehyde/2% glucose/5 mM Na-azide for 20 min at room temperature, and incubated with mAbs to intracellular proteins in PBS/0.1% saponin for 20 min at 4°C.

Cytolytic activity Sorted CD8⫹ T cell subsets were kept overnight in complete medium and used in mAb-redirected 51Cr release assays at a defined lymphocyte to target cell ratio (10:1). FcR␥-bearing P815 target cells were labeled with Na51CrO4 and used in the presence or the absence of anti-CD3 mAb (LAU T3, various concentrations). Cytolytic activity of Ag-specific CD8⫹ T cell clones was assessed using target cells (T2) labeled with 51Cr for 1 h at 37°C and washed twice. Labeled target cells (1000 cells in 50 ␮l) were incubated in presence of various concentrations of cognate peptide (50 ␮l) for 10 min at room temperature before the addition of effector cells. Chromium release

was measured after incubation for 4 h at 37°C. The percentage of specific lysis was calculated as: 100 ⫻ ((experimental ⫺ spontaneous release)/ (total ⫺ spontaneous release)). Radioactivity was measured with TopCount (PerkinElmer Life and Analytical Sciences).

Cytokine analysis Cell-free supernatants were analyzed for IFN-␥ content using an ELISA kit (BioSource International), according to the manufacturer’s instructions.

RNA isolation and RT-PCR Total RNA was isolated from sorted CD8⫹ T cells or Ag-specific CTL clones using an RNA Microprep Kit (Stratagene). The DNase treatment step (20 min at 37°C) was performed before column elution. Reverse transcription was performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). Amplifications were performed with platinum TaqDNA polymerase (Invitrogen Life Technologies) for 38 cycles: 30 s at 94°C; 45 s at 58°C; 60 s at 72°C. The amplification products were resolved in a 2% agarose gel containing ethidium bromide (Sigma-Aldrich). The primer pairs used for PCR analysis were as follows: TLR3 forward, 5⬘-TCGAGAGTGCCGTCTATTTGC-3⬘ and reverse, 5⬘-ATGCACACAGCATCCCAAAG-3⬘.

Results

TLR3 mRNA expression by activated CD8⫹ T cells To investigate TLR expression by defined human T lymphocyte subsets, we sorted CD4⫹ and CD8⫹ T cells and evaluated TLR expression patterns by RT-PCR. CD8⫹ T cells, but not CD4⫹ T cells, expressed TLR3, but not TLR2, TLR4, or TLR9 (data not shown). To determine whether TLR3 expression was linked to CD8⫹ T cell differentiation, we next isolated by magnetic and flow cytometry-based cell sorting highly purified CD8⫹ T cell subsets based on their cell surface expression of CD45RA and CCR7 (Fig. 1A). As previously reported, simultaneous staining for CD45RA and CCR7 defines four functionally distinct subsets of CD8⫹ T cells, which are designated as naive (C45RA⫹CCR7⫹), effector (CD45RA⫹CCR7⫺), effector memory (CD45RA⫺CCR7⫺), and central memory (CD45RA⫺CCR7⫹), respectively (33). RT-PCR analysis of total RNA isolated from the four different CD8 subsets clearly revealed expression of TLR3 mRNA in effector and effector memory cells, but not in naive or central memory cells (Fig. 1B). Both the inclusion of cDNA dilutions and the use of CD8 and GAPDH primers allowed to exclude artifacts due to a higher mRNA content in the effector and effector memory subsets. Purified CD8⫹ T cell subsets from six healthy donors showed the same expression pattern (data not shown). Thus, TLR3 expression in human CD8⫹ T cells appears to depend on their differentiation status. Poly(I:C) costimulates IFN-␥ secretion by activated Ag-experienced CD8⫹ T cells To explore the functional role, if any, of TLR3 in effector CD8⫹ T cells, we used the synthetic copolymer polyinosinic-polycytidylic acid, poly(I:C), which mimics dsRNA, a well-defined TLR3 agonist. Highly purified CD8⫹ T cells (⬎99% CD8⫹/CD3⫹) were sorted into naive, effector, and effector memory subsets (with a purity ⱖ95% upon reanalysis of sorted cells by flow cytometry). After overnight rest in culture medium without cytokines, individual T cell subsets were stimulated with various doses of PHA, a mitogen that binds to a number of glycoproteins expressed on the plasma membrane and can activate strong T cell responses. The IFN-␥ content in cell-free supernatants harvested 48 h after PHA stimulation was measured by ELISA. Correlating with TLR3 expression, only effector and effector memory CD8⫹ T cells secreted IFN-␥ in response to short-term PHA stimulation (Fig. 2). Addition of the TLR3 agonist poly(I:C) significantly increased the quantity of IFN-␥ released by effector and/or effector memory CD8⫹ T cells in response to PHA, in a dose-dependent manner

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Sorted CD8⫹ T cells were kept in RPMI 1640 containing 1% nonessential amino acids, 1% sodium pyruvate, 1% L-glutamine, 0.1% 2-ME, and 8% human serum (complete medium). Viral or tumor Ag-specific CD8⫹ T cell clones were isolated and propagated in in vitro culture, as described elsewhere (26), and grown in complete medium plus 150 U/ml human rIL-2 (Proleukin; Chiron). The cells used in the assays described in this study were harvested at least 8 days after the last in vitro stimulation. Cell lines used as targets in cytotoxicity or APCs in cytokine production assays were: P815 (murine Fc␥R⫹ mastocytoma) and the HLA-A2⫹ human mutant cell line CEM ⫻ 721.T2 (T2) cultured, respectively, in DMEM medium and RPMI 1640 medium containing 10% FCS.

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FIGURE 1. TLR3 gene expressed by activated human CD8⫹ T cells. A, Magnetic bead-sorted CD8⫹ T cells from a healthy donor were labeled with anti-CD45RA and anti-CCR7 fluorescent conjugates and sorted on a flow cytometer into naive (N), central memory (CM), effector memory (EM), and effector (E) cell subsets. B, Sorted CD8⫹ T cell subsets were lysed, and total RNA was purified and then reverse transcribed. The indicated cDNA 10-fold serial dilutions were amplified under nonsaturating conditions with specific primers corresponding to the indicated genes. The starting undiluted material (lane 0) contained the cDNA from 10,000 sorted cells. PCR products were then resolved by 2% agarose gel electrophoresis and stained with ethidium bromide.

Poly(I:C) has no detectable effect on CTL activity Effector CD8⫹ T cells exert cytolytic activity and kill target cells after productive TCR ligation. For freshly isolated bulk CD8⫹ T cells, triggering of lytic function can be achieved by cross-linking with anti-CD3 Abs in a redirected lysis assay. As expected, antiCD3 Ab triggered readily detectable lytic activity among CD8⫹ T cells (Fig. 3). Addition of poly(I:C), at concentrations that co-

⫹

FIGURE 2. Poly(I:C) stimulation increases CD8 T cell IFN-␥ responses in a dose-dependent manner. Sorted naive (A), effector memory (B), effector (C), and pooled effector memory plus effector (D) CD8⫹ T cells from a healthy donor were cultured at 2.5 ⫻ 105 cells/ml for 48 h in medium supplemented with the indicated concentrations of the mitogen PHA in the absence or presence of various concentrations of poly(I:C) (closed symbol) or of poly(dI:dC) (open symbol). All samples were assayed in triplicates. IFN-␥ concentration in cell culture supernatants was measured by ELISA.

stimulate IFN-␥ secretion, had no effect on CTL activity (Fig. 3A). Moreover, no effect was observed when a pool of sorted effector and effector memory CD8⫹ T cells was used as a CTL source (Fig. 3B). Likewise, addition of the control poly(dI:dC) did not induce any changes in the levels of CTL activity (Fig. 3). Exposure to poly(I:C) increases IFN-␥ secretion, but does not affect the lytic activity of Ag-specific cloned CD8⫹ T cells To test the function of TLR3 in well-defined Ag-specific T cells, we used HLA-A2-restricted CTL clones isolated from healthy donors or melanoma patients. TLR3 transcripts could be amplified in all six Ag-specific CD8 T cell clones tested. These included clones specific for immunodominant epitopes from influenza, EBV, and CMV (data not shown). Addition of poly(I:C) during Ag triggered specific lysis assay (Fig. 4A) or during redirected lysis assay

FIGURE 3. Poly(I:C) stimulation does not increase effector CD8⫹ T cell cytolytic activity. Redirected killing by effector cells was investigated using anti-CD3 (LAU T3 Ab, at different concentrations) and 51Cr-labeled P815 target cells at a 10:1 E:T cell ratio, for 4 h in the absence or presence of various concentrations of poly(I:C) (closed symbol) or poly(dI:dC) (open symbol). A, Total sorted CD8⫹ T cells were treated with graded amounts of poly(I:C) or poly(dI:dC) during redirected lysis assay. B, Pooled effector and effector memory CD8⫹ T cells, obtained by flow cytometry-based cell sorting, were treated with graded amounts of poly(I:C) or poly(dI:dC) during redirected lysis assay.

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(Fig. 2, B–D). However, poly(I:C) by itself did not detectably induce IFN-␥ release by any of the purified CD8⫹ T cell subsets even at the highest concentration tested (Fig. 2, B–D). Moreover, addition of poly(I:C) during stimulation of naive CD8⫹ T cells with PHA did not induce any detectable IFN-␥ secretion (Fig. 2A). The costimulatory effect of poly(I:C) was specific, as indicated by the lack of activity, even at high concentrations, of the control poly(dI:dC), which does not engage TLR3 (Fig. 2, B–D).

The Journal of Immunology

(Fig. 4B) did not detectably modulate target cell lysis. Titration curves for both antigenic peptide and cross-linking anti-CD3 Ab were identical in the presence or absence of graded amounts of poly(I:C). In contrast, IFN-␥ concentrations in 48-h coculture supernatants were significantly enhanced (data not shown). To better quantitate this effect, CD8⫹ T cells from the same clone were stimulated with specific peptide-pulsed APCs at a 1:1 ratio during 48 h. As expected, cloned CD8⫹ T cells secreted IFN-␥ in a peptide concentration-dependent manner. In contrast to lytic activity, addition of poly(I:C) during the stimulation period increased the quantity of IFN-␥ released in a dose-dependent fashion (Fig. 4C). It is noteworthy that T2 cells used as APCs in this experiment expressed TLR3 transcripts, as assessed by RT-PCR. However, they did not produce detectable IFN-␥ when incubated in the same assay conditions, except for the inclusion of CD8 T cells (data not shown).

FIGURE 5. Pretreatment with poly(I:C) does not change CD8⫹ T cell IFN-␥ secretion or cytolytic capacity. A, Purified CD8⫹ T cells were preincubated in IL-2-containing culture medium in absence (䡺) or in presence of 100 ␮g/ml poly(I:C) (f) for 48 h. After treatment, cells were stimulated during 48 h with graded amounts of PHA. IFN-␥ concentration in cell-free supernatant was measured by ELISA. B, Purified CD8⫹ T cells were cultured during 48 h in IL-2-containing culture medium in absence (䡺) or in presence of 100 ␮g/ml poly(I:C) (f). After treatment, cells were stimulated during 48 h with 10 ␮g/ml PHA and graded amount of poly(I:C). IFN-␥ concentration in cell-free supernatant was measured by ELISA. C, Cloned CD8⫹ T cells were preincubated in IL-2-containing culture medium in absence or in presence of 100 ␮g/ml poly(I:C) for 20 h. After treatment, specific killing of 51Cr-labeled T2 target cells by a CD8⫹ T cell clone was assessed using graded amounts of specific peptides at a 10:1 E:T cell ratio.

Pretreatment of CD8⫹ T cells with poly(I:C) does not affect IFN-␥ secretion or cytolytic activity Purified CD8⫹ T cells were preincubated with 100 ␮g/ml poly(I:C) or with medium during 48 h before stimulation with PHA. Poly(I:C)- or medium-preincubated CD8⫹ T cells secreted comparable amounts of IFN-␥ in response to PHA at any concentration (Fig. 5A). However, further addition of graded amounts of poly(I:C) during the assay did increase IFN-␥ secretion by both populations (Fig. 5B). Moreover, preincubation of cloned CD8⫹ T cells with 100 ␮g/ml poly(I:C) during 20 h had no effect on their lytic activity (Fig. 5C). Altogether, these results indicate that poly(I:C) exposure does not sensitize CD8⫹ T cells to subsequent stimulation. Instead, poly(I:C) has to be present at time of stimulation to exert its effect on IFN-␥ secretion. Additional experiments showed that neither Ag- nor mitogen-driven CD8⫹ T cell proliferation was modified by inclusion of poly(I:C) in the culture medium even at concentrations as high as 100 ␮g/ml (data not shown). This lack of effect

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FIGURE 4. Poly(I:C) stimulation increases IFN-␥ secretion, without modulation of cytolytic activity, by Ag-specific CD8⫹ T cell clones. A, Specific killing of 51Cr-labeled T2 target cells by a CD8⫹ T cell clone was determined using graded amounts of specific peptides, at 10:1 E:T cell ratio for 4 h in the absence or presence of poly(I:C) (closed symbol) or poly(dI:dC) (open symbol). B, Redirected killing by a CD8⫹ T cell clone was investigated using anti-CD3 (LAU T3 Ab, at different concentrations) and 51Cr-labeled P815 target cells at a 10:1 E:T cell ratio for 4 h in the absence or presence of poly(I:C) (closed symbol) or poly(dI:dC) (open symbol). C, A CD8⫹ T cell clone was cocultured with specific peptidepulsed APCs for 48 h at a 1:1 CD8⫹ T cell to APC ratio. IFN-␥ concentration in cell-free supernatant was measured by ELISA. All samples were assayed in duplicates.

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was documented using purified CD8⫹ T cell subsets freshly isolated from PBLs as well as Ag-specific CD8⫹ CTL clones propagated by serial restimulation in vitro.

gand. As in other reports, we used the same range of poly(I:C) concentrations, and transfection was not used. Thus, it is conceivable that effective concentration of ligand at the endosomal compartment is likely to be much lower and closer to physiologically relevant levels. Interestingly, TLR2 has been shown to relocalize to the cell surface of CD4⫹ T cells upon stimulation with anti-CD3 Ab and IFN-␣ (17). Similarly, TLR3 was detected at the cell surface of ␥␦ and ␣␤ T lymphocytes after activation (34). It remains to be determined whether such a relocalization of TLR3 occurs upon CD8⫹ T cell activation. However, because the binding of dsRNA to TLR3 was shown to require an acidic microenvironment (48), it is more likely to occur in TLR3-containing intracellular vesicles. The elucidation of the mechanisms leading to the delivery of dsRNA to these structures awaits further investigations. Wesch et al. (34) recently suggested that the moderate increase of cytokine production observed after stimulation of ␣␤ T cells with poly(I:C) might be explained by the responsiveness of only a subset of this population. We indeed provide strong evidence in the present work that among CD8⫹ T cells, only the effectors, but not their naive or central memory counterparts, express TLR3 and are functionally responsive to stimulation with poly(I:C). TLR ligands raise a great deal of interest in the field of cancer immunotherapy, because their immunostimulatory properties could be exploited to increase the efficacy of cancer vaccines (49 – 51). For instance, recently, TLR9 ligands have been successfully used in conjunction with peptide-based vaccines to induce strong Ag-specific CTL responses in cancer patients (28). Although not yet tested in humans, TLR3 ligands are also promising in this respect, as they have been shown to promote Ag cross-presentation, to favor Th1 type of responses, and to activate NK cells (29, 30, 40). Murine tumor models using immunization with poly(I:C) as adjuvant have demonstrated the potency of this compound to enhance both primary and memory CD8⫹ T cell responses (31, 32). Our results suggest that, in addition to their well-characterized effects on DCs, TLR3 agonists might also directly influence some CD8⫹ T cell effector functions. The increased IFN-␥ production provided by TLR3 signaling in CD8⫹ effector T cells could be beneficial in therapeutic vaccines, and may lead to better responses against tumors or chronic viral infections.

Discussion

The present results demonstrate that human effector CD8⫹ T cells express functional TLR3, which can enhance TCR-induced IFN-␥ secretion upon engagement with specific agonists. The study of TLR expression and functions has initially mainly focused on innate immune cells, as these cells are the first line of defense against pathogen infection. However, it is becoming increasingly clear that TLR can also be expressed by adaptive immune cells such as T lymphocytes, and that they can modulate the functions of these cells (17–20, 34). The physiological relevance of these observations is not clear yet, but expression of functional TLR3 in CD8⫹ T cells is reminiscent of that of other germlineencoded activatory receptors such as NKG2D, which is involved in NK-mediated antiviral innate immunity (35) and which can also be expressed by cells of the adaptive immune system such as CD8⫹ T cells (36, 37). However, in contrast with NKG2D, TLR3 directly recognizes viral components, whereas NK-activating receptor ligands are expressed by target cells upon viral infection, cellular stress, or transformation (35, 38). Interestingly, because functional TLR3 is also present in NK cells (39, 40), both NKG2D and TLR3 may represent complementary arms of the antiviral arsenal developed by the innate immune system. Our results suggest that this might also be true for adaptive immunity, if TLR3 and NKG2D can be simultaneously engaged in CD8⫹ T cells. This issue remains to be addressed. One of the main consequences of TLR3-induced signaling in cells of the innate immune system is the secretion of massive quantities of type I IFNs (10, 11), which are antiviral cytokines. Because effector CD8⫹ T cells are involved in the clearance of viral infections, it is of interest that they express receptors endowed with the capacity to detect viral components. It is indeed conceivable that upon CTL-induced lysis, virally infected cells release viral genetic material such as dsRNA in the extracellular space. As suggested by our results, detection of dsRNA by TLR3 expressed in effector CD8⫹ T cells would then enhance their IFN-␥ production, which can in turn induce a strong antiviral state (41, 42) propagating to the cells in the vicinity of the CTL:infected target cell conjugates. This scenario would constitute an efficient amplification system of the overall antiviral effect that can be mediated by Ag-specific CD8⫹ T cell responses. The understanding of the precise involvement of TLR3 expressed by CD8⫹ T cells in viral infections will obviously need further investigations. Viral infections require a Th1 type of response to be cleared. Most TLR ligands have the capacity to promote cell-mediated immunity through their action on DC (14, 16). Interestingly, several IRF, which are downstream targets of TLR signaling (8), have been reported recently to influence the differentiation of Th cells through their expression in both APCs and T lymphocytes themselves (43, 44). As shown in this study, TLR3 expression in CD8⫹ T cells is restricted to Ag-experienced cells, and thus seems to be associated with a particular state of differentiation. IRF3, which is strongly activated by TLR3 signaling, is also involved in the regulation of TLR3 expression (45, 46). It could therefore be interesting to investigate the role of IRF3 in CD8⫹ T cell differentiation, and to correlate its expression with that of TLR3. The subcellular localization of TLR3 has not been investigated in this study, but it is generally believed to be mainly intracellular in immune cells, including resting human T lymphocytes (34, 47). The costimulatory effect of poly(I:C) observed in our study and in others (34) was induced by exogenous addition of the TLR3 li-

Acknowledgments We acknowledge N. Montandon, P. Corthe´sy, S. Reynard, V. Rubio-Godoy, and C. Baroffio for excellent technical help, and Drs. D. Rimoldi, Y. Mahnke, and G. Bioley for insightful discussions and reagents. We also thank Martine Van Overloop for excellent secretarial assistance.

Disclosures The authors have no financial conflict of interest.

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