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International Immunology Advance Access originally published online on January 5, 2007
International Immunology 2007 19(2):193-201; doi:10.1093/intimm/dxl136
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

TLR9 ligand enhances proliferation of rat CD4+ T cell and modulates suppressive activity mediated by CD4+ CD25+ T cell

Elise Chiffoleau, Jean-Marie Heslan, Michele Heslan, Cedric Louvet, Thomas Condamine and Maria-Cristina Cuturi

Institut National de la Santé et de la Recherche Médicale Unité 643 and Institut de Transplantation Et de Recherche en Transplantation, CHU Hotel Dieu, 30 Boulevard Jean Monnet, 44093 Nantes Cedex 01, France

Correspondence to: E. Chiffoleau; E-mail: elise.chiffoleau{at}univ-nantes.fr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLRs) play a crucial role in the initiation of innate responses following microbial infection and also in adaptive immune responses by orchestrating the activation of different cell populations. TLRs are expressed at high levels in antigen-presenting cells and recent studies have demonstrated the expression and biological role of TLRs in mouse and human CD4+ T cells. In this study, we analyzed TLR mRNA expression in rat CD4+ T cells using stringent quantitative reverse transcription–PCR conditions enabling a direct comparison of the levels of each TLR. We show that TLR3, 5, 6 and 9 mRNAs are the highest TLRs expressed in rat CD4+ T cells and that TLR5 mRNA is highly expressed in regulatory CD4+ CD25+ T cells. In addition, we show that the TLR9 ligand (TLR9L), CpG oligodeoxynucleotide, synergizes with anti-CD3 to induce proliferation of both CD4+ CD25– and regulatory CD4+ CD25+ T cells and that TLR9L partially abrogates the suppressive activity mediated by regulatory CD4+ CD25+ T cells. This loss of suppression is in part due to the direct effect of TLR9L on effector T cells that are rendered more resistant to the regulation exerted by regulatory T cells. To our knowledge, this is the first study describing the expression of TLR mRNA in rat CD4+ T cells and the capacity of TLR9L to directly regulate rat T cell responses. Thus, TLR9L may rapidly increase the host's adaptive immunity by expanding effector cells and also by attenuating the suppressive activity mediated by regulatory T cells.

Keywords: co-stimulation, regulation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLRs) recognize a set of conserved molecular structures, the so-called pathogen-associated molecular patterns (PAMPs), that allow them to sense and initiate innate and adaptive immune responses. TLRs expressed by vertebrates are type 1 transmembrane proteins bearing an intracellular Toll/IL1R homology domain. Although the exact gene numbers may differ between species, it is likely that most mammalian species have 10–15 TLRs that differ in ligand specificities and expression patterns (1). TLRs are key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses. In addition, TLRs are also dedicated to the control of adaptive immunity by orchestrating the responses of different cell populations. Upon binding of their cognate ligands, TLRs recruit adaptor molecules to their intracellular signaling domain, leading to the activation of several kinases, nuclear factor-kB, and direct regulation of immune-responsive genes (2). TLR expression has been mostly described in antigen-presenting cells (APCs), neutrophils and epithelial cells. Nevertheless, several reports have demonstrated the expression and a biological role for TLRs in mouse and human CD4+ T cells (3–9). However, discrepancies exist in the literature concerning TLR expression in CD4+ T cells and the role of their respective ligands. Several studies have demonstrated that TLR ligands increase the proliferation of activated CD4+ T cells (6–8). A role of TLR ligands in the expansion and suppressive properties of regulatory T cells has also been reported (3, 7–9).

In this study, we show that rat CD4+ T cells express at the highest level TLR3, 5, 6 and 9 mRNAs and a preferential expression of TLR5 mRNA in regulatory CD4+ CD25+ T cells. The same pattern of mRNA expression was observed in two different T cell clones. Moreover, we showed that TLR9 ligand (TLR9L) synergizes with anti-CD3 to induce proliferation of both CD25– and CD25+ T cells, resulting in partially abrogation of the suppressive activity mediated by regulatory CD4+ CD25+ T cells. We show here that, in keeping with what has been observed in mice and humans, TLR ligands have the capacity to directly regulate rat CD4+ T cell responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
Lewis (LEW) rats (6–8 weeks old) were obtained from the Centre d'Elevage Janvier (Le Genest Saint-Isles, France). The Study was approved by our Institutional Review Board.

Antibodies
A number of hybridomas were obtained from the European Collection of Cell Culture (Salisbury, UK) and mAbs were purified from supernatants and labeled with FITC or PE (Bioatlantic, Nantes, France) or to AlexaFluor-647 or -488 (from Invitrogen, Molecular Probes, Cergy Pontoise, France): R7-3–FITC (anti-T), PE-cyanin 7 (Cy7)-conjugated Ox35 (anti-CD4), Ox8–PE (anti-CD8), allophycocyanin-Alexa-647-conjugated Ox39 (anti-CD25) and R7-3, Alexa-488-conjugated Ox22 (anti-CD45RC). Purified anti-rat CD3 was purchased from BD PharMingen (San Diego, CA, USA) and allophycocyanin-conjugated anti-mouse/rat FoxP3 from Clinisciences (Montrouge, France). Anti-CD28 antibodies were provided by J. Bluestone (San Francisco, CA, USA).

TLR ligands
The phosphodiester oligodeoxynucleotides (ODNs) containing the CpG motifs CpG-ODN2006 (5'-TCGTCGTTTTGTCCGTTTTGTCGTT-3') and CpG-ODN1688 (5'-TCCATGACGTTCCTGATGCT-3') and the non-CpG-ODN2006 (5'-TGCTGCTTTTGTTGCTTTTGTGCTT-3') were synthesized by Sigma-Genosys (Saint Quentin Fallavier, France) and used at concentrations from 1 to 0.06 µM. Heat-killed Listeria monocytogenes (HKLM) (108 particles ml–1), peptidoglycan (PGN) (10 mg ml–1), LPS (0.5 mg ml–1) and flagellin (100 ng ml–1) were purchased from Invivogen (Toulouse, France).

Cell purification and stimulation
CD4+ CD45RC low, CD4+ CD45RC high, CD4+ CD25– and CD4+ CD25+ T cells were purified from splenocytes and lymph node cells by positive selection using a FACSAria flow cytometer (BD Biosciences, Mountain View, CA, USA). Splenocytes were stained with R7-3–allophycocyanin, Ox35–Cy7 and Ox22–Alexa-488 for CD4+ CD45RC low and CD4+ CD45RC high T cell purification and by R7-3–FITC, Ox8–PE, Ox35–Cy7 and Ox39–allophycocyanin–Alexa-647 for CD4+ CD25– and CD4+ CD25+ T cell purification. Purity was >99%. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA) as previously described (10).

Stimulation assays.
CD4+ CD25– or CD4+ CD25+ T cells from naive rats (5 x 104 cells per well) were pre-incubated or not with CpG-ODN (16 h), extensively washed and/or directly stimulated in 96-well flat-bottom plates (NUNCTM, Merck, Eurolab, France) coated with anti-CD3 (0.1–1 mg ml–1) (2 h at 37°C) together with soluble anti-CD28 (0.1–1 mg ml–1) and with different TLR ligands at different doses. Cells were cultured in a final volume of 200 µl of RPMI 1640 (GIBCO) supplemented with 2 mM L-glutamine, 5 x 10–5 M2-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, 100 U ml–1 penicillin, 0.1 mg ml–1 streptomycin and 10% heat-inactivated FCS (GIBCO). The cultures were incubated at 37°C in 5% CO2 for 72 or 96 h and analyzed for CFSE and for intracellular Foxp3 expression (fixation/permeabilization) by FACSCalibur (BD Biosciences) and for IFN{gamma}, IL2 and IL10 production in supernatants by ELISA according to the manufacturer's instructions (BD PharMingen).

Quantitative reverse transcription–PCR
Total RNA from CD4+ CD45RC low, CD4+ CD45RC high, CD4+ CD25– and CD4+ CD25+ T cells and from the T cell clones A2b and Z1a was prepared by TRIzol® extraction (Invitrogen, Cergy Pontoise, France). Genomic DNA was removed by DNase treatment (Roche, Indianapolis, IN, USA) and mRNA was reverse transcribed into first-strand cDNA using polydT oligonucleotide and Moloney murine leukemia virus reverse transcriptase (Invitrogen).

For standard construction of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and TLR genes, the target sequence was amplified by PCR from a spleen cDNA library, and then electrophoresed and purified by phenol–chloroform extraction and ethanol precipitation. Subsequent dilutions of this standard DNA were performed to obtain 107, 106, 105, 104, 103 and 102 copies per well.

Real-time quantitative/PCR was carried out according to the TaqMan® procedure including, in addition to both primers, a gene-specific fluorescent probe of 20- to 30-mers. Only when the probe binds to the target gene as a double-strand DNA can the 5' exonuclease activity of the Taq polymerase release the fluorescence of the 5'carboxyfluorescein-labeled nucleotide. Such a three ‘primer party’ PCR ensures a very high specificity toward the target gene.

All TLR and HPRT primers and probes used were designed and synthesized by Applied Biosystems (Table 1) whose software selects for the best pair of primers and probes according to many criteria of sequence specificity, Tm, amplicon size and cross-hybridization (dimer formation). In addition, exon–intron boundaries were defined in order to have the probe spanning over one or more large introns so as to avoid amplification of the genomic DNA along with the RNA-derived gene product. All amplicon sizes ranged from 61 to 90 bp in our study. All PCR efficiencies were >95% as checked by the slope of the Ct values of 10-fold serial dilutions of positive samples and the log(1/dilution) (slopes ranged from –3.32 to –3.43, corresponding to 100 to 96% efficiency, respectively).


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  		Table 1. Sequences of primers and probes

 
Prior to any target gene measurement, all samples were tested for HPRT gene expression over three 5-fold dilutions so as to detect the presence of any PCR inhibitors. None of the samples included for study had detectable inhibitors.

Due to the small sizes of the amplicons (<100 bp), their exact size could not be determined using a standard 1.5% agarose gel. Dissociation curves of the amplicons obtained by PCR in the presence of SyberGreen were therefore used as markers of specificity of the amplified target gene.

Statistical analysis
Statistical evaluation was performed using the Student's t-test for unpaired data and results were considered significant for P-values <0.05. Data were expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Differential expression of mRNA encoding TLRs in naive, memory, CD25– and regulatory CD25+ CD4+ T cells
We evaluated mRNA encoding TLRs in rat naive CD45RC high, memory CD45RC low, CD25– and regulatory CD25+ CD4+ T cells by quantitative reverse transcription (RT)–PCR since no anti-TLR antibodies are available for the rat. These cells had been sorted by FACS (purity >99%). A high level of purity was necessary to exclude the presence of contaminating APCs that express TLR at high levels and which could therefore bias the results. We used specific and stringent primers and a specific probe for each TLR, ensuring high specificity of amplification as described in Methods. Moreover, we used standards allowing for a direct comparison of TLR expression. We also assessed the expression of mRNA encoding TLRs in total spleen that contains numerous APCs. We observed very low levels of TLR1, 2, 4, 7, 8 and 10 mRNAs in rat CD4+ T cells whereas the expression of these TLRs, with the exception of TLR10, was high in the spleen (Fig. 1A). We observed moderate levels of TLR3, 6 and 9 mRNA in naive CD45RC high, in memory CD45RC low or in the CD25– and regulatory CD4+ CD25+ T cell sub-populations. Interestingly, we observed specific expression of TLR4 mRNA in naive CD45RC high CD4+ T cells and a high and specific expression of TLR5 mRNA in the regulatory CD25+ sub-population. Expression of TLR5 mRNA was even higher in regulatory CD4+ CD25+ T cells than in the spleen.


Figure 1
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  		Fig. 1. TLR mRNA expression in rat CD4+ T cells. (A) Naive CD45RC high, memory CD45RC low, CD25– and regulatory CD25+ CD4+ T cells were FACS sorted (purity >99%) and analyzed for TLR mRNA expression by quantitative RT–PCR as described in Methods. A pair of stringent primers and a specific probe was used for each TLR, thus enabling high specificity toward the target gene. We compared mRNA expression with that measured in total spleen. The use of a standard meant that we could directly compare the mRNA level of each TLR. Data are expressed as mRNA levels relative to HPRT and are representative of three independent experiments. (B) A2b and Z1a rat CD4+ T cell clones were analyzed for TLR mRNA expression by quantitative RT–PCR as described in Methods. Data are expressed as mRNA levels relative to HPRT and are representative of three independent experiments.

 
These results demonstrate that naive, memory or regulatory CD25+ CD4+ T cells express moderate levels of mRNA encoding TLR3, 6 and 9 and that regulatory CD4+ CD25+ T cells express high levels of TLR5 mRNA. The same results were obtained in two different rat strains, LEW and Brown Norway (BN) (data not shown).

Analysis of TLR expression by CD4+ T cells requires the exclusion of contaminating cells expressing high levels of TLRs, such as APCs. The purity of the cell populations studied herein was >99%, as determined by FACS (data not shown). To confirm that the TLR mRNA expressions assessed in purified T cells were not due to contaminating APCs (albeit at <1%), we evaluated TLR mRNA expression in two different CD4+ T cell clones. A2b T cells are derived from an arthritogenic T cell line obtained from an arthritic LEW rat specific for the epitope of heat-shock protein 60 (11). Z1a cells are derived from a LEW rat-derived T cell line specific for the MBP (12). We found these two CD4+ T cell clones to express mRNA encoding TLR3, 5, 6 and 9 (Fig. 1B). This mRNA expression matched that measured in fresh conventional CD4+ T cells, thus confirming the preferential expression of TLR3, 5, 6 and 9 in CD4+ T cells.

Analysis of the biological role of TLRs expressed by CD4+ T cells
Proliferation.
To test whether the repertoire of TLRs expressed by CD4+ T cells correlated with their responsiveness to specific ligands, we analyzed the effect of several TLR ligands on the proliferation induced by plate-bound anti-CD3 antibody ± soluble anti-CD28 antibody.

CD4+ CD25– or regulatory CD4+ CD25+ T cells were stained with CFSE and stimulated for 3 days with plate-bound anti-CD3 (1 µg ml–1) in the presence of flagellin, CpG-ODN2006, LPS, PGN or HKLM, the respective ligands of TLR5, 9, 4, 2/6 and 2. We used physiological doses of TLR ligands that are routinely used in studies and have been shown to efficiently stimulate APCs in rats (13). CFSE staining showed that 59% of CD4+ CD25– T cells had proliferated in the presence of plate-bound anti-CD3 after 3 days of culture and that the addition of flagellin (TLR5L), LPS (TLR4L), PGN (TLR2/6L) or HKLM (TLR2L) did not modify their proliferation (Fig. 2A). About the same percentage of cells had proliferated and had performed the same number of divisions (52% with flagellin, 51% with LPS, 63% with PGN and 59% with HKLM). These histograms are representative of three independent experiments performed in duplicates. In contrast, the addition of CpG-ODN2006 (TLR9L) increased the number of CD4+ CD25– T cells that had proliferated (89 versus 59% in the controls). The same results were observed for regulatory CD4+ CD25+ T cells (Fig. 2B). These cells were hypoproliferative at this dose of stimulation as few cells had proliferated (13%), in contrast to CD4+ CD25– T cells (59%). The presence of flagellin, LPS, PGN or HKLM did not modify their proliferation (8, 10, 11 and 8% of cells proliferated, respectively). However, addition of CpG-ODN2006 strongly increased the proliferation of regulatory CD4+ CD25+ T cells as ~64% of the cells had proliferated.


Figure 2
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  		Fig. 2. CpG-ODN-enhanced proliferation of anti-CD3-stimulated CD4+ CD25– and regulatory CD4+ CD25+ T cells: (A) CD4+ CD25–and (B) regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%), CFSE labeled and stimulated with plate-bound anti-CD3 (1 µg ml–1) with flagellin (TLR5L, 100 ng ml–1), CpG-ODN (TLR9L, 10 µM), LPS (TLR4L, 0.5 µg ml–1 ), PGN (TLR2/6L, 10 µg ml–1) or HKLM (TLR6L, 108 particles ml–1) for 3 days. CFSE staining of cells was then analyzed by FACS. Data are representative of three independent experiments performed in duplicates.

 
Cytokine secretion.
To assess whether TLR ligands modulate cytokine secretion, we analyzed IFN{gamma}, IL2 and IL10 secretion by ELISA in supernatants from unstimulated or anti-CD3/anti-CD28 (1 µg ml–1)-stimulated CD4+ CD25– and regulatory CD4+ CD25+ T cells incubated in the presence of TLR ligands. At this dose of stimulation, CD4+ CD25– and regulatory CD4+ CD25+ T cells proliferated to the same extent (data not shown) and cytokines were assessed by ELISA. We found that stimulated CD4+ CD25– T cells expressed specifically IL2 and higher levels of IFN{gamma} compared with stimulated regulatory CD4+ CD25+ T cells and that the presence of TLR ligands did not modify the secretion of these cytokines in either sub-population (Fig. 3). On the contrary, stimulated CD4+ CD25– T cells secreted less IL10 than stimulated regulatory CD4+ CD25+ T cells and the addition of TLR ligands did not modify IL10 production.


Figure 3
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  		Fig. 3. TLR ligands do not modify IFN{gamma}, IL2 and IL10 secretion by optimally activated CD4+ CD25–and regulatory CD4+ CD25+ T cells: CD4+ CD25–and regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%) and stimulated with plate-bound anti-CD3 (1 µg ml–1) and soluble anti-CD28 (1 µg ml–1) with flagellin (TLR5L, 100 ng ml–1), CpG-ODN (TLR9L, 10 µM), LPS (TLR4L, 0.5 µg ml–1), PGN (TLR2/6L, 10 µg ml–1) or HKLM (TLR6L, 108 particles ml–1) for 3 days. IFN{gamma}, IL2 and IL10 secretion were analyzed by ELISA. Data are expressed in picograms per milliliter as mean ± SD of duplicates and are representative of two independent experiments.

 
Synergistic effect of CpG-ODN with anti-CD3 and anti-CD28 stimulation.
To determine the potency of the synergistic effect of CpG-ODN2006 on proliferation, CD4+ CD25– and CD4+ CD25+ T cells were stimulated with different doses of plate-bound anti-CD3 and soluble anti-CD28. Whereas at very low doses of anti-CD3 (0.1 µg ml–1), CD4+ CD25– T cells did not proliferate (4%), the presence of CpG-ODN2006 strongly enhanced the proliferation (56% of the cells had proliferated) (Fig. 4A). These histograms are representative of three independent experiments performed in duplicates. We show that the effect of CpG-ODN2006 was dose dependent and that non-CpG-ODN2006 had no effect on the proliferation of CD4+ T cells (1 µM) (Table 2). The addition of a low dose of anti-CD28 (0.1 µg ml–1) increased the proliferation in the presence of CpG-ODN2006 to 73%, whereas cells without CpG-ODN2006 did not proliferate at all (6%) (Fig. 4A). Similarly, the addition of CpG-ODN2006 to a higher dose of anti-CD3 (1 µg ml–1) that induced the proliferation of 54% of the cells increased proliferation even further (81%) and the same effect was observed upon the addition of soluble anti-CD28 (99%).


Figure 4
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  		Fig. 4. CpG-ODN synergizes with anti-CD3 and anti-CD28 to enhance proliferation of CD4+ CD25–and regulatory CD4+ CD25+ T cells: (A) CD4+ CD25–and (B) regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%), CFSE labeled and stimulated with different doses of plate-bound anti-CD3 (0.1 or 1 µg ml–1) and different doses of soluble anti-CD28 (0.1 or 1 µg ml–1) in the presence of CpG-ODN (TLR9L, 10 µM) for 4 days. CFSE staining of cells was then analyzed by FACS. Data are representative of three independent experiments performed in duplicates.

 

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  		Table 2. Dose effect of CpG-ODN: CFSE-stained CD4+ CD25– or CD4+ CD25+ regulatory T cells were stimulated with pb{alpha}CD3 (1 µg ml) in the presence of non-CpG-ODN2006 (1 µM) or CpG-ODN2006 (from 1 to 0.06 µM)

 
Exactly the same results were obtained for regulatory CD4+ CD25+ T cells (Fig. 4B). Whereas the regulatory CD4+ CD25+ T cells did not proliferate when stimulated with low doses of anti-CD3 (0.1 µg ml–1) (1%), CpG-ODN2006 induced their proliferation (15%) and the presence of anti-CD28 enhanced this proliferation further (27%). The effect of CpG-ODN2006 on the proliferation of CD4+ CD25+ T cells was dose dependent and was not observed with control non-CpG-ODN2006 (Table 2). Therefore, our results demonstrate that CpG-ODN2006 synergizes with anti-CD3 and anti-CD28 to enhance the proliferation of both CD4+ CD25– and regulatory CD4+ CD25+ T cells.

Effect of CpG-ODN on the suppressive activity mediated by regulatory T cells.
In order to determine whether TLR ligands could modulate the suppressive capacity of regulatory T cells, we co-incubated CFSE-labeled CD4+ CD25– T cells with regulatory CD4+ CD25+ T cells (ratio 1:1) (Fig. 5B) or with CD4+ CD25– T cells (ratio 1:1) as a control (Fig. 5A). These histograms are representative of three independent experiments performed in duplicates. CD4+ CD25+ T cells regulated the proliferation of CD4+ CD25– T cells since only 11% of the cells proliferated compared with 53% of the controls. The presence of flagellin, LPS, PGN or HKLM did not abrogate the suppressive activity of regulatory CD4+ CD25+ T cells (data not shown). In contrast, in the presence of CpG-ODN2006, the suppression mediated by regulatory CD4+ CD25+ T cells on CD4+ CD25– T cells was abrogated, as 57% of the cells proliferated (versus 11% in the controls). The same results were obtained with another oligonucleotide, CpG-ODN1668 (Fig. 5A and B). Therefore, CpG-ODN could modulate the capacity of regulatory CD4+ CD25+ T cells to suppress proliferation of CD4+ CD25– T cells or alternatively may render CD4+ CD25– T cells resistant to the suppression mediated by regulatory CD4+ CD25+ T cells.


Figure 5
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  		Fig. 5. CpG-ODN inhibits the suppressive properties exerted by regulatory CD4+ CD25+ T cells: CD4+ CD25–and regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%). CFSE-labeled CD4+ CD25–T cells were stimulated with plate-bound anti-CD3 (1 µg ml–1) in the presence of (A) CD4+ CD25–T cells (ratio 1: 1) or (B) regulatory CD4+ CD25+ T cells (ratio 1: 1) and in the presence of CpG-ODN2006 or CpG-ODN1668 (TLR9L, 10 µM) for 3 days. CFSE staining of cells was then analyzed by FACS (gated only in CFSE-stained CD4+ CD25–T cells). Data are representative of three independent experiments performed in duplicates. (C) CFSE-labeled regulatory CD4+ CD25+ T cells were stimulated with plate-bound anti-CD3 (1 µg ml–1) in the presence of CpG-ODN2006 (TLR9L, 10 µM) for 3 days and then stained for intracellular FoxP3 protein as described in Methods. CFSE and FoxP3 staining of cells were analyzed by FACS. Data are representative of three independent experiments performed in duplicates.

 
To test these hypotheses, we assessed FoxP3 protein expression in regulatory CD4+ CD25+ T cells stimulated in the presence of CpG-ODN2006. We previously demonstrated that FoxP3 is specifically expressed by regulatory CD4+ CD25+ T cells in rat (14). We observed that the addition of CpG-ODN2006 increases the proliferation of both FoxP3+ and FoxP3– CD4+ CD25+ T cells as, respectively, 21 and 23% of the cells proliferated versus 7 and 9% without CpG-ODN2006 (Fig. 5C) (these dot plots are representative of three independent experiments performed in duplicates). These data demonstrate that CpG-ODN2006 has a direct effect on the proliferation of FoxP3+ regulatory T cells and does not seem to modulate the expression of FoxP3.

Moreover, we pre-incubated CD4+ CD25– or regulatory CD4+ CD25+ T cells with CpG-ODN2006 for 16 h, washed them extensively and used them for stimulation and for the suppression assay. Interestingly, we observed that pre-incubation with CpG-ODN2006 still increases the proliferation of stimulated CD4+ CD25– T cells as 30% of the cells proliferated versus 14% in control (Fig. 6A) (these histograms are representative of three independent experiments performed in duplicates). However, pre-incubation of regulatory CD4+ CD25+ T cells with CpG-ODN2006 is not sufficient to increase their proliferation (4% of the cells proliferated versus 4% in control) (Fig. 4B). This contrasts with the increased proliferation observed when CpG-ODN2006 is present during the stimulation (Fig. 2B and 4B) and suggests that CD4+ CD25– T cells are more sensitive to CpG-ODN than regulatory CD4+ CD25+ T cells. Moreover, the pre-incubation of regulatory CD4+ CD25+ T cells with CpG-ODN2006 does not modulate their suppressive capacities as these cells are still able to inhibit the proliferation of effector CD4+ CD25– T cells. Indeed, 29% of effector cells proliferated in the presence of regulatory CD4+ CD25+ T cells pre-incubated with CpG-ODN2006 versus 22% in the presence of regulatory CD4+ CD25+ T cells not pre-incubated with CpG-ODN2006 (Fig. 6C). However, we observed that pre-incubation of effector CD4+ CD25– T cells with CpG-ODN2006 renders these cells resistant to the suppression mediated by regulatory CD4+ CD25+ T cells as 51% of the cells proliferated versus 22% when effector cells were not pre-incubated with CpG-ODN2006 (Fig. 6C).


Figure 6
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  		Fig. 6. CD4+ CD25–T cells pre-incubated with CpG-ODN2006 are rendered resistant to the suppression to regulatory CD4+ CD25+ T cells: (A) CD4+ CD25– and (B) regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%), CFSE labeled, incubated 16 h with CpG-ODN2006 (TLR9L, 10 µM), extensively washed and then stimulated with plate-bound anti-CD3 (1 µg ml–1) for 3 days. CFSE staining of cells was then analyzed by FACS. Data are representative of three independent experiments performed in duplicates. (C) CD4+ CD25–and regulatory CD4+ CD25+ T cells were FACS sorted (purity >99%). CFSE-labeled CD4+ CD25 –T cells pre-incubated or not with CpG-ODN2006 (TLR9L, 10 µM) for 16 h were stimulated with plate-bound anti-CD3 (1 µg ml–1) in the presence of CD4+ CD25–T cells (ratio 1: 1) or regulatory CD4+ CD25+ T cells (ratio 1: 1) also pre-incubated or not with CpG-ODN2006 (TLR9L, 10 µM). CFSE staining of cells was then analyzed by FACS after 3 days (gated only in CFSE-stained CD4+ CD25–T cells). Data are representative of three independent experiments performed in duplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we demonstrated that rat CD4+ T cells express TLR mRNA and that CpG-ODN enhanced their proliferation independently of APCs. To our knowledge, this is the first report describing mRNA expression of TLR in rat CD4+ T cells associated with a biological effect. We show that the TLR mRNAs expressed at the highest level in rat CD4+ T cells are TLR3, 5, 6 and 9. TLR3, 6 and 9 mRNAs are expressed at the same level in naive, memory or regulatory CD25+ CD4+ T cells; however, TLR5 mRNA is specifically expressed and at high level in regulatory CD4+ CD25+ T cells. We also observed a low but specific expression of TLR4 mRNA in naive CD4+ T cells.

Several studies have demonstrated the expression of TLR mRNA in mouse or human CD4+ T cells but with differing results. Indeed, Caron et al. (6) demonstrated that human CD4+ T cells express TLR1, 2, 3, 4, 5, 7 and 9 but not TLR6, 8 and 10 whereas Moseman et al. (15) described an absence of TLR9 mRNA expression in CD4+ T cells. Crellin et al. (7) demonstrated that both human CD4+ CD25– and CD4+ CD25+ regulatory T cells express TLR5 whereas another study described TLR8 as being the only receptor consistently expressed by human CD4+ CD25+ regulatory T cells (5). Discrepancies also exist in the results from studies in mice. Caramalho et al. (3) showed the expression of TLR1, 2, 6 and 7 in both the CD4+ CD25– and the CD25+ T cell populations of C57BL/6 mice, a stronger expression of TLR4 and 5 in CD4+ CD25+ regulatory T cells and no TLR3 and 9 mRNA expression in either population. Gelman et al. (4) showed in BALB/c mice that activated CD4+ T cells express TLR3, 5 and 9 but not TLR2 and 4. These discrepancies could be due to the technique used and its sensitivity and/or to the purity of the T cells used. We designed specific primers and a probe for each TLR in order to have stringent conditions and to prevent non-specific amplification. Moreover, we used quantitative RT–PCR which is very sensitive and thus able to detect few copies of a target gene. The use of a standard for each TLR enabled us to directly compare the levels of TLR mRNA. The same pattern of TLR mRNA expression was observed in two different rat CD4+ T cell clones, suggesting that the expression assessed in FACS-sorted CD4+ T cells was not due to contaminating APCs.

Another reason for the discrepancy in the results published concerning mice could be due to the strain used. Indeed, it has previously been shown that TLR expression in dendritic cells differs between BALB/c and C57BL/6 mice (16). We observed the same pattern of TLR mRNA expression in CD4+ T cells of two different rat strains: LEW and BN (data not shown). In humans, the problem could be confounded by different type of memory cells that may express different TLRs (6). The generation of antibodies for all these TLRs in different species will help to clarify the issue of TLR expression in different cell subtypes.

We additionally analyzed whether the repertoire of TLR expressed by CD4+ T cells correlated with their responsiveness to specific ligands. We found that TLR9L CpG-ODN potentiated stimulation of both CD4+ CD25– and regulatory CD4+ CD25+ T cells at levels similar to those achieved by co-stimulation with CD28. Moreover, CpG-ODN abrogated the suppressive activity mediated by regulatory CD4+ CD25+ T cells. Despite specific mRNA expression of TLR5 on regulatory CD4+ CD25+ T cells, we saw no effect of its specific ligand, flagellin, on the proliferation, cytokine production or suppressive activity in the conditions tested. In our hands, CpG-ODN had no T cell stimulatory activity when used alone but synergized with TCR-dependent stimuli. This shows that APCs are more sensitive to TLR ligand-induced activation than T cells which need TCR stimulation. Several studies have demonstrated a synergistic effect of TLR ligands on CD4+ T cell stimulation and CpG-ODN has been shown to directly promote activated CD4+ T cell survival in mice (4). Caron et al. (6) demonstrated that flagellin (TLR5L) and R-848 (TLR7/8) synergized with sub-optimal concentrations of anti-CD3 to up-regulate proliferation of human CD4+ T cells. Human CD4+ CD25+ regulatory T cells have been shown to express TLR5, and its respective ligand, flagellin, has been shown to enhance their suppressive activity (7). Another study showed that exposure of CD4+ CD25+ regulatory T cells to LPS enhances survival and proliferation and increases their suppressor efficiency (3). Moreover, Peng et al. (5) showed that CpG-ODN can directly mediate reversal effect on human regulatory T cell function. We showed that CpG-ODN abrogated the suppressive activity mediated by regulatory CD4+ CD25+ T cells. This loss in suppression was not due to a preferential expansion of FoxP3– non-regulatory T cells in the CD4+ CD25+ sub-population or to a loss in FoxP3 expression but rather to the resistance of effector cells to the suppression mediated by regulatory CD4+ CD25+ T cells. We did not observe an enhanced IL2 secretion by CD4+ CD25– T cells in response to CpG-ODN that could have facilitated their escape from the regulation exerted by regulatory CD4+ CD25+ T cell. Nevertheless, we cannot rule out the possibility that this loss of suppression observed in the presence of CpG-ODN during the suppression assay was also in part due to the increased proliferation of regulatory CD4+ CD25+ T cells. Indeed, two recent studies in mice demonstrated that the TLR2L, Pam3Cys-SK4, enhanced proliferation of both CD25– and regulatory CD25+ CD4+ T cells, resulting in a transient loss of suppressive activity (8, 9). This temporal loss of suppression was described to be due to effector cells rendered resistant to the suppression to regulatory T cells by increasing IL2 secretion (8) and also to the loss of suppression exert by regulatory T cells in expansion (9).

Collectively, these results together with our own demonstrate that PAMP signals contribute to the activation and expansion of regulatory CD4+ CD25+ T cells and thereby help to fine-tune the suppressive activity of regulatory T cells and, consequently, the magnitude of immune responses. Therefore, following infection, regulatory T cells may proliferate concomitant to effector cells. This mechanism would efficiently stimulate effector T cells recruited at the site of micro-organism entry. During this expansion, regulatory T cells will also proliferate under the influence of TLR ligands and the suppressive properties will be attenuated to allow for efficient eradication by effector cells. Regulatory T cells will subsequently fully recuperate their suppressive properties and thereby regulate the strength of the immune response and allow for an efficient memory T cell response (17, 18).


   Abbreviations
 
APC, antigen-presenting cell
BN, Brown Norway
CFSE, carboxyfluorescein diacetate succinimidyl ester
Cy7, cyanin 7
HKLM, heat-killed Listeria monocytogenes
HPRT, hypoxanthine-guanine phosphoribosyltransferase
LEW, Lewis
ODN, oligodeoxynucleotides
PAMP, pathogen-associated molecular pattern
PGN, peptidoglycan
RT, reverse transcription
TLR, Toll-like receptor
TLR9L, TLR9 ligand

   Notes
 
Transmitting editor: P. Ohashi

Received 4 July 2006, accepted 27 November 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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