The interactions between TLRs and their ligands have profound immune modulation properties. Attention has focused mostly on the impact of TLR ligands on peripheral innate and adaptive immunity during viral infections, whereas little impact of TLR activation has been shown on thymic development. Here we show that treatment of murine fetal thymic organ cultures (FTOCs) with TLR3 or TLR7 ligands induced rapid expression of IFN-α and -β mRNA, hallmarks of acute and chronic viral infections. This resulted in an early developmental blockade, increased frequencies of apoptotic cells, and decreased proliferation of thymocytes, which led to an immediate decrease in cellularity. FTOCs infected with vesicular stomatitis virus, known to act through TLR7, were similarly affected. Down-regulation of IL-7R α-chain expression, together with an increased expression of suppressor of cytokine signaling-1 and a concomitant decreased expression of the transcriptional regulator growth factor independence 1 were observed in TLR ligands or IFN-treated FTOCs. This indicates a role for these pathways in the observed changes in thymocyte development. Taken together, our data demonstrate that TLR activation and ensuing type I IFN production exert a deleterious effect on T cell development. Because TLR ligands are widely used as vaccine adjuvants, their immunomodulatory actions mediated mainly by IFN-α suggested by our results should be taken in consideration.

The TLRs are major components of the innate immune system in that they are key sensors of pathogens. In mice, 12 different TLRs have been identified, including TLR3, TLR7, and TLR9, that recognize viral and/or bacterial nucleic acids. TLR3 recognizes dsRNA and signals downstream through Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β, to induce the activation of IFN-regulatory factor (IRF)4 3 and transcription and activation of IRF7. In contrast, TLR7 and TLR9 recognize ssRNA or guanosine analogs and CpG oligodeoxynucleotides (ODN), respectively; they signal through MyD88, leading to a phosphorylation cascade and the activation of IRF7 (1) which promotes an amplification of IFNs synthesis. Hence, two different pathways lead to the production of type I IFNs, encompassing mostly IFN-α and IFN-β (2) upon engagement by TLR ligands. Even though most cell types are capable of producing type I IFNs upon microbial, viral, or TLR ligand challenge, plasmacytoid dendritic cells, crucial players in the early innate response to viral infections, are the most potent producers of type I IFNs (3, 4, 5, 6).

All type I IFNs share the same surface receptor (IFN-α/βR) which is coupled to a uniform signal transduction cascade (7) involving JAK1, tyrosine kinase 2, STAT1, and STAT2, as well as IRFs (1, 8). Very few differential responses to IFN-α subtypes have been observed (2, 9, 10), and as a whole IFN-α subtypes have qualitatively similar biological activities. They are endowed with antiviral activity and induce apoptosis of infected cells (11, 12). Virus-induced IFN-α/β are also recognized for their antiproliferative and immunomodulatory activities (6, 13). Indeed, type I IFNs are able to induce proteins such as the protein kinase RNA-activated, which is involved in controlling cell proliferation (14, 15), or the p200 family of proteins, important regulators of cell growth, immunomodulation, and host resistance to viral infections (16). The immunomodulatory functions of type I IFNs indicate that these cytokines constitute important links between innate and adaptive immune responses. Indeed, type I IFNs induce expression of IL-15, a key regulator of memory CD8 and CD4 T cell development (17, 18). Viral infection induced expansion of Ag-specific CD8+ T cells is also dependent on the activity of type I IFNs (19). Moreover, these cytokines induce NK cell responses and dendritic cell maturation (20).

High levels of type I IFNs are rapidly produced upon viral infection (T. Démoulins, A. Abdallah, N. Keltaf, C. Gerarduzzi, D. Gauchat, S. Gratton, and R. P. Sékaly, submitted for publication) in the thymus, the primary source of naive T cells. This implies that T cell precursors can be exposed to high levels of systemic and thymic-derived IFN-α/β produced during the early phase of viral infections (21). The mechanisms leading to the up-regulation of type I IFNs in the thymus are not well characterized, although the role of TLR ligands cannot be discounted. Moreover, the few outcomes of type I IFNs described thus far on thymocytes are very different from the properties ascribed to the impact of type I IFN on mature peripheral T lymphocytes (3, 6, 8, 11, 12, 13). IFN-α has been demonstrated to exert deleterious effects on thymic T cell development through the increased expression of the cyclin-dependent kinase inhibitor p27Kip1, inasmuch as down-regulation of the latter is a necessary step for normal thymic T cell development and proliferation (22, 23). A strong decrease in thymic cellularity, mostly characterized by a severe drop in the number of CD4+CD8+ double-positive (DP) cells, was evidenced in newborn mice treated with an active human IFN-α2/α1 hybrid molecule leading to decreased resistance to viral infections (24). Moreover, a recent study has also revealed the detrimental effect of type I IFNs on T cell development from thymic progenitors. This effect was associated with an inhibition of the IL-7R signal transduction pathway (25). Because type I IFNs appear to affect the generation of an efficient naive T cell pool (25, 26),5 we studied the impact of IFNs induced by TLR triggering on thymocyte development independently of peripheral T cells. To this end, TLR ligands and type I IFNs were exogenously added to fetal thymic organ cultures (FTOC) and to the OP9-δ like (DL)-1 system (27), and their impact on T cell differentiation was evaluated.

C57BL/6 mice were purchased from Charles River Laboratories, 129Sv/Ev from Taconic Laboratories, and IFN-α/βR−/− from B&K Universal Limited Laboratory (28). Growth factor independence (Gfi)1+/GFP knock-in mice were provided by Dr. Tarik Möröy (29). They were housed and bred in our animal facilities. All animal studies were done in accordance with approved institutional animal care committee protocols.

Individual IFN-α subtypes cloned in pcDNA3 (Invitrogen) were obtained from T. Michiels (2) and transfected into COS cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The rIFN concentrations were determined using an ELISA kit (PBL Biomedical Laboratories) with a sensitivity of 5 pg/ml.

Day 15.5 fetal thymic lobes were isolated from wild-type (WT) C57BL/6, WT 129Sv/Ev, or IFN-α/βR−/− mice. Five to eight lobes were transferred to a 0.8-μm pore size Nucleopore polycarbonate membrane (Whatman) floating on IMDM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine (Invitrogen), 10% v/v FBS (Sigma-Aldrich), and 50 μM 2-ME (Sigma-Aldrich) in a 12-well plate (Falcon). The plates were incubated at 37°C, 7.5% CO2 in air. Treatments were applied to FTOCs after 3 days of culture, for 3, 6, or 9 h for RNA preparation or for 48 h for flow cytometry. Subsequently, fetal thymic lobes were washed twice in IMDM and treated for 5 min at 37°C with 2.4 mg/ml collagenase D (Roche) and 1 mg/ml DNase (Sigma-Aldrich). Enzymatic reaction was stopped by the addition of FBS, and thymic lobes were mechanically disrupted.

In each experiment, five to eight fetal thymic lobes were treated. In the experiment reported in Fig. 1 A, FTOCs were treated with either 30 or 100 μg/ml polyinosinic-polycytidylic acid (poly(I:C); Invivogen) or 250 or 500 μM loxoribine (Invivogen); in other figures, concentrations that induced IFN-α/β production but did not result in excess mortality were chosen; i.e., 100 μg/ml for poly(I:C) and 250 μM for loxoribine. We used 1000 U/ml of rIFN-α or IFN-αA (Biosource). The neutralizing anti-IFN-α and -β Abs (PBL Biomedical Laboratories) were used at 1000 U/ml. LPS-free PBS, 100 μl (Sigma-Aldrich), or empty pcDNA3 vector was added to FTOCs as a general or specific control, for rIFN treatment, respectively. Vesicular stomatitis virus (VSV) AV1 was added at 5 × 107 U/ml. Gfi1+/GFP mice were injected i.p. with 200 μg of poly(I:C) (Invivogen) or PBS daily for 3 days. Animals were euthanized 3 h after the last injection.

FIGURE 1.

Increased type I IFN and IRF mRNA expression upon TLR triggering. A, Relative levels of IFN-α (top) and -β (bottom) mRNA expression obtained by qRT-PCR after treatment for 6 h with PBS or TLR ligands (poly(I:C), 4.2- to 80-fold increase for IFN-α and 34- to 70-fold increase for IFN-β; and loxoribine, 3- to 3.1-fold increase for IFN-α and 2.4- to 3.9-fold increase for IFN-β. B, Relative levels of IRF1, IRF3, IRF7, and IRF9 mRNA expression obtained by qRT-PCR after 6 or 9 h of treatment with PBS, poly(I:C), loxoribine, or IFN-αA in WT and IFN-α/βR−/− mice. Results were normalized to an endogenous control gene (β-actin for IFN mRNA expression and GAPDH for IRF mRNA expression) and compared with PBS control (fold increase). Each measure was performed on a pool of 5–11 thymic lobes. Results are the mean of triplicates and are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 1.

Increased type I IFN and IRF mRNA expression upon TLR triggering. A, Relative levels of IFN-α (top) and -β (bottom) mRNA expression obtained by qRT-PCR after treatment for 6 h with PBS or TLR ligands (poly(I:C), 4.2- to 80-fold increase for IFN-α and 34- to 70-fold increase for IFN-β; and loxoribine, 3- to 3.1-fold increase for IFN-α and 2.4- to 3.9-fold increase for IFN-β. B, Relative levels of IRF1, IRF3, IRF7, and IRF9 mRNA expression obtained by qRT-PCR after 6 or 9 h of treatment with PBS, poly(I:C), loxoribine, or IFN-αA in WT and IFN-α/βR−/− mice. Results were normalized to an endogenous control gene (β-actin for IFN mRNA expression and GAPDH for IRF mRNA expression) and compared with PBS control (fold increase). Each measure was performed on a pool of 5–11 thymic lobes. Results are the mean of triplicates and are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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Single-cell suspensions obtained from a pool of five to eight fetal thymic lobes were stained for 30 min on ice with saturating concentrations of the following Abs: anti-CD3-Alexa 700, anti-CD4-PE-Cy7, anti-CD8- allophycocyanin-Cy7, anti-CD25-allophycocyanin, anti-CD44-PE-Cy5, anti-CD45-PE-Cy5, anti-H2Db MHC class I-PE, anti-I-Ad MHC class II-FITC, anti-c-Kit-PE (BD Biosciences); and anti-CD127-PE (eBioscience). Seven-color analyses were performed on an LSRII flow cytometer (BD Biosciences), and at least 300,000 live events were collected. The results were analyzed using DIVA software (BD Biosciences). To quantify apoptotic cells, after surface staining, cells were resuspended in annexin V binding buffer (30) containing annexin V-FITC just before FACS analysis. Thymic epithelial cells were identified as high forward and side scatter, CD45, class II+ cells as previously described (31).

To measure cell proliferation, 10 μg/ml BrdU (BD Biosciences) were added to FTOCs 18 h before harvest. After surface staining, cells were fixed with Cytofix buffer (BD Biosciences) containing 0.01% Tween 20 (Sigma-Aldrich), incubated for 1 h with a DNase solution (500 Kunitz U/ml DNase I in 4.2 mM MgCl2, 0.15 M NaCl, pH 5), and stained for 30 min on ice using the FITC-conjugated BrdU staining kit (BD Biosciences).

RNAs were obtained from FTOCs using an RNAquous kit (Ambion). For IFN-α and -β mRNA expression analysis, first-strand cDNA was synthesized using 1–2 μg of total RNA, 1.5 μg of random nucleotide hexamers (Invitrogen), 1 μg of oligo(dT)12–18 (Invitrogen), and 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) at 37°C for 50 min. cDNA was used as template in the quantitative SYBR Green PCR mix according to the manufacturer’s standard protocol (Roche). The primer sequences were: IFN-α (forward), 5′-GCTAGGCHYTRTGCTTTCCT-3′, IFN-α (reverse), 5′-CACAGRGGCTGTGTTTCTTC-3′; and IFN-β (forward) 5′-TTCAAGTGGAGAGCAGTTGAG-3′, IFN-β (reverse) 5′-CATCAACTATAAGCAGCTCCA-3′. Quantitative PCR results are expressed relative to the expression of the β-actin gene with the following primers: forward, 5′-CGTACCACAGGCATTGTGA-3′; reverse, 5′-CTCGTTGCCAATAGTGATGA-3′. All other mRNAs were quantified as described (32) at genomics platform at the Institute for Research in Immunology and Cancer.

Western blotting was performed as described (33). A polyclonal rabbit anti- suppressors of cytokine signaling (SOCS)-1; 1/250; Zymed) or a monoclonal mouse anti-β-actin Ab (1/2500; clone AC-15; Sigma-Aldrich) were used.

T cell development on OP9 stromal cell lines was performed as previously described (34). Briefly, E14-E15 fetal liver cells obtained from time pregnant C57BL/6 mice were depleted of mature cells using anti-CD24 mAb (J11d.2) plus complement. These cells were cultured for 12–14 days in the presence of Flt-3 ligand and IL-7 at 5 ng/ml as previously described (34). At day 4 or 12 of coculture, 1000 U/ml of rIFN-α1 were added, and the effect on T cell differentiation was observed according to the generation of double-negative (DN) and DP subsets and overall T cellularity. Flow cytometry was performed on developing T cells as previously described (34).

Statistical analyses were performed using Excel software (Microsoft). Data are expressed as means ± SD. Statistical significance of differences was determined by the paired two-tailed Student t test: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Given that most viruses trigger one or several TLRs (35, 36) and that TLR ligands are used as vaccine adjuvants, we investigated the impact of TLR triggering on the production of type I IFNs in developing thymocytes. A synthetic analog of dsRNA, poly(I:C), a guanosine analog derivatized at position N-7 and C-8, i.e., loxoribine or CpG ODN were added to E15.5 FTOCs. These three molecules, respectively, bind and trigger TLR3, TLR7, and TLR9 (37, 38, 39) and are all potent inducers of type I IFNs (35, 37, 40, 41, 42). TLR ligands were added to FTOCs after 3 days of culture, when the CD4/CD8 profile was comparable with that of a neonatal thymus with DP cells representing ∼80% of thymocyte population. Two different concentrations were used for each TLR ligand: 30 and 100 μg/ml for poly(I:C), and 250 and 500 μM for loxoribine.

Using quantitative real-time RT-PCR and pan IFN-α and IFN-β primers, a dose-dependent up-regulation of IFN-α and IFN-β mRNA expression was observed after addition of TLR ligands to FTOCs. Poly(I:C), or loxoribine up-regulated the expression of type I IFNs upon treatment of FTOCs when compared with PBS controls (Fig. 1 A). This up-regulation was observed as early as 3 h after TLR ligation (data not shown) and continued to increase at 6 h.

Poly(I:C) reproducibly induced higher levels of type I IFN mRNA expression than loxoribine.

IRF transcription factors play a central role (43) in regulating induction levels of type I IFN expression. Levels of IRF1, IRF7, and to a lesser extent IRF9 were significantly increased upon interaction of the different ligands with their cognate receptors. IRF1 and IRF7 expression levels were up-regulated by at least 3-fold, whereas IRF9 expression was increased by 2-fold when FTOCs were treated with 100 μg/ml poly(I:C), 250 μM loxoribine, or 1000 U/ml IFN-αA for 6 or 9 h (Fig. 1,B). In contrast, up-regulation of IRF mRNA expression was not observed in IFN-α/βR−/− mice. There were no change in IRF3 expression levels upon TLR ligation because IRF3 is not transcriptionally regulated; its activation is rather triggered through phosphorylation by TNFR-associated factor family member-associated NF-κB activator-binding kinase, dimerization, and nuclear translocation upon viral infection or TLR activation (44, 45, 46). IFN-αA-induced-IRF mRNA overexpression was decreased before that induced by TLR ligands. The results illustrated in Fig. 1 B show that IFN-α up-regulation of IRF1 and IRF7 peaked at 6 h and was down-regulated at 9 h. In contrast, TLR-induced IRF7 was still expressed at high levels at 9 h. IFN-αA acts directly on IRF7, whereas TLR ligands need to induce a first wave of IFN production involving mostly IRF3 and IFN-β as well as IFN-α4 production. These secreted IFNs subsequently feed back through their cognate receptor in an autocrine and paracrine fashion, inducing the later waves of IFN production triggered upon IRF1 or IRF7/9 transcription (1, 46). Taken together, these results confirm at the transcriptional level that TLR activation and IFN-α induce IRF-mediated IFN production in FTOCs.

As a positive control for IFN treatment, we analyzed the expression of IL-15 and MHC class I given that these molecules are downstream targets of IFN (17, 47). We observed a large increase in IL-15 expression after TLR3 or TLR7 activation. Indeed, IL-15 was significantly up-regulated 3- to 7-fold following these treatments (Fig. 2,A). A significant increase in the MHC class I (H2Db) mean fluorescence intensity (MFI) after TLR3 or TLR7 activation or IFN-αA treatment was also observed. Indeed, MHC class I MFI was up-regulated at least 2-fold after these treatments (Fig. 2,B). This increased expression of MHC class I MFI was also evidenced in thymic epithelial cells identified as CD45MHC class II+ cells (Ref. 31 and Fig. 2,C). These increased expressions of IL-15 and MHC class I up-regulation were not observed when IFN-α/βR−/− FTOCs were placed in the presence of the same TLR ligands (Fig. 2). Altogether, these results clearly confirm that TLR ligands induce type I IFNs and their downstream signal transduction targets in the developing thymocytes.

FIGURE 2.

Treatment of FTOCs with poly(I:C), loxoribine, or IFN-αA leads to the increase of IL-15 and MHC class I expression. A, Relative levels of IL-15 mRNA expression obtained by qRT-PCR after treatment for 6 h with PBS or TLR ligands (poly(I:C), 3.2-fold increase in WT mice, no increase in IFN-α/βR−/− mice; and loxoribine, 7.4-fold increase in WT mice, 2.1-fold increase in IFN-α/βR−/− mice). B and C, Expression of MHC class I (MFI) assessed by flow cytometry analysis in all FTOC cells (poly(I:C), 1.8-fold increase; loxoribine, 2.5-fold increase; and IFN-αA, 2.1-fold increase) and in thymic epithelial cells (TECs; CD45MHC class II+) (poly(I:C), 1.7-fold increase; loxoribine, 2-fold increase; and IFN-αA, 1.6-fold increase), respectively. For IL-15, results were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 5 to 11 thymic lobes. Results are the mean of triplicates and are representative of two independent experiments. For class I, each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. Flow cytometric results are expressed as mean ± SDs. Number of experiments: WT (n = 7); IFN-α/βR−/− (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Treatment of FTOCs with poly(I:C), loxoribine, or IFN-αA leads to the increase of IL-15 and MHC class I expression. A, Relative levels of IL-15 mRNA expression obtained by qRT-PCR after treatment for 6 h with PBS or TLR ligands (poly(I:C), 3.2-fold increase in WT mice, no increase in IFN-α/βR−/− mice; and loxoribine, 7.4-fold increase in WT mice, 2.1-fold increase in IFN-α/βR−/− mice). B and C, Expression of MHC class I (MFI) assessed by flow cytometry analysis in all FTOC cells (poly(I:C), 1.8-fold increase; loxoribine, 2.5-fold increase; and IFN-αA, 2.1-fold increase) and in thymic epithelial cells (TECs; CD45MHC class II+) (poly(I:C), 1.7-fold increase; loxoribine, 2-fold increase; and IFN-αA, 1.6-fold increase), respectively. For IL-15, results were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 5 to 11 thymic lobes. Results are the mean of triplicates and are representative of two independent experiments. For class I, each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. Flow cytometric results are expressed as mean ± SDs. Number of experiments: WT (n = 7); IFN-α/βR−/− (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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Having confirmed the induction of type I IFN mRNAs expression after TLR ligation in FTOCs, we performed experiments to assess their impact on thymocyte development. Thymic cellularity was analyzed 48 h after addition of 100 μg/ml poly(I:C) or 250 μM loxoribine to E15.5 fetal lobes. We observed a significant reduction in cell numbers per lobe after poly(I:C) or loxoribine treatment. Indeed, treatment with poly(I:C) led to a 1.7-fold reduction in cellularity while a >3-fold decrease was observed upon FTOC treatment with loxoribine. This loss of cellularity was also noted when commercially available IFN (i.e., IFN-αA) was added to FTOCs (1.8-fold decrease). When we treated FTOCs from IFN-α/βR−/− mice with the same TLR ligands, we did not observe a comparable drop in cell numbers (Fig. 3,A). IFN-α/βR−/− mice being on an A129 background, controls were performed on WT A129 mice; results are similar to those obtained with C57BL/6 mice and show that treatment with poly(I:C) and loxoribine also led to a decrease in cell numbers (Fig. 3 B). Taken together, these results indicate that the decrease in FTOC cell numbers is a direct consequence of type I IFN production induced by TLR triggering.

FIGURE 3.

Addition of TLR ligands or IFN-αA to FTOCs leads to decrease cell number per thymic lobe and to a partial blockade of thymocyte at the early developmental stages. A, Relative cell numbers per thymic lobes after TLR ligand treatment in WT mice (poly(I:C), 1.7-fold decrease; loxoribine, 3-fold decrease; and IFN-αA, 1.8-fold decrease) and IFN-α/βR−/− mice. B, Relative cell numbers per thymic lobes after TLR ligand treatment in WT C57BL/6 and WT 129 Sv/Ev mice. C, Normalized frequency of thymocytes at the DN3 stage (poly(I:C), 3-fold decrease; loxoribine, 7-fold decrease; and IFN-αA, 2.5-fold decrease). D, Representative contour plots of DN subsets upon TLR ligand treatment in WT and IFN-α/βR−/− mice. DN thymocytes (CD4CD8) were analyzed from DN1 to DN4 stages (DN1, CD44+CD25; DN2, CD44+CD25+; DN3, CD44CD25+; DN4, CD44CD25) by flow cytometry. Each measure was performed on a pool of 5–6 thymic lobes, and at least 300,000 events were recorded for each analysis in C and D. Results in A–C are expressed as mean ± SDs. Number of experiments: cellularity, WT C57BL/6 (n = 4), IFN-α/βR−/−, and WT 129Sv/Ev (n = 3); DN3%, WT (n = 5), IFN-α/βR−/− (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

Addition of TLR ligands or IFN-αA to FTOCs leads to decrease cell number per thymic lobe and to a partial blockade of thymocyte at the early developmental stages. A, Relative cell numbers per thymic lobes after TLR ligand treatment in WT mice (poly(I:C), 1.7-fold decrease; loxoribine, 3-fold decrease; and IFN-αA, 1.8-fold decrease) and IFN-α/βR−/− mice. B, Relative cell numbers per thymic lobes after TLR ligand treatment in WT C57BL/6 and WT 129 Sv/Ev mice. C, Normalized frequency of thymocytes at the DN3 stage (poly(I:C), 3-fold decrease; loxoribine, 7-fold decrease; and IFN-αA, 2.5-fold decrease). D, Representative contour plots of DN subsets upon TLR ligand treatment in WT and IFN-α/βR−/− mice. DN thymocytes (CD4CD8) were analyzed from DN1 to DN4 stages (DN1, CD44+CD25; DN2, CD44+CD25+; DN3, CD44CD25+; DN4, CD44CD25) by flow cytometry. Each measure was performed on a pool of 5–6 thymic lobes, and at least 300,000 events were recorded for each analysis in C and D. Results in A–C are expressed as mean ± SDs. Number of experiments: cellularity, WT C57BL/6 (n = 4), IFN-α/βR−/−, and WT 129Sv/Ev (n = 3); DN3%, WT (n = 5), IFN-α/βR−/− (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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To characterize the thymic developmental stage targeted by IFN, thymocytes were stained with CD4 and CD8 as well as CD25-, CD44-, and c-Kit-specific Abs to define the distribution of the different thymic subsets. Analysis of CD4CD8 DN subsets showed that poly(I:C) and loxoribine led to a substantial reduction of the DN3 population (CD25+CD44) compared with PBS-treated FTOCs. In these experiments, the proportion of DN3 cells within the total DN population was significantly decreased upon treatment with poly(I:C), a 3-fold decrease, and loxoribine, a 7-fold decrease (Fig. 3, C and D). The decrease in the frequencies and absolute numbers of DN3 cells is most probably the consequence of the blockade of thymocyte development before the DN3 stage. TLR7 ligand reproducibly led to a greater drop in DN3 frequencies (twice fewer). This decrease in frequencies of the DN3 subset within the DN population upon addition of TLR ligands mimicked that obtained upon addition of IFN-αA to FTOC (2.5-fold decrease). Finally, the normalized frequency of cells at the DN3 stage in TLR ligand triggered IFN-α/βR−/− FTOCs was comparable with the frequency observed in PBS-treated IFN-α/βR−/− FTOCs (Fig. 3, C and D), confirming that this developmental thymic blockade is mostly a consequence of the high levels of type I IFNs.

The blockade observed on the development of thymocytes in FTOCs was incomplete in that some of the progenitor T cells had already differentiated beyond the DN2 stage. To circumvent this caveat, we analyzed the impact of distinct individual type I IFNs (IFN-α1 and -α4) in the OP9-DL1 coculture system (34), which supports the differentiation of T cells from hemopoietic progenitors. In this experimental model, administration of rIFN-α1 or -α4 on day 4 of culture, at a concentration of 1000 U/ml, induced a complete blockade of T cell development as shown from the aberrant distribution of the different subsets of thymocytes monitored by CD4/CD8 and CD25/CD44 staining (Fig. 4,A). At day 8 of culture, results from CD25/CD44 staining showed that the proportion of DN1 cells (CD25CD44+) within the total DN cells represented 96.6 or 95.7% when cells were treated with rIFN-α1 or -α4, respectively, whereas the proportion of this subset represented only 69.8% of the DN subset upon treatment with supernatant of cells transfected with empty pcDNA3 used as control. At day 12 of culture, subset distribution of total thymocytes confirmed the developmental blockade at the DN stage. Indeed, CD4/CD8 staining showed that the proportion of DP subset within the total thymocyte population represented <2% in rIFN-α1- or -α4-treated cells, whereas DP proportion represented 11.4% in cultures incubated with control supernatants. To further confirm the subset specificity of this blockade, rIFN-α was added after 12 days of culture on developing T cells. Two days later, cells accumulated at the DN1 stage did not pursue their differentiation (17.2% for α1 and 18.1% for α4 vs 7.5% in the control at day 14), whereas the ones already at a further developmental stage continued to progress and differentiated into DP and then into CD8+ single-positive (SP) cells (Fig. 4,D). Moreover, as shown in the lowest panel of Fig. 4,D, the percentage of CD8+ SP cells is even higher in IFN-treated cultures (7.2 and 7.3% for α1 and α4, respectively) as compared with control cultures (4.4%), most probably due to the fact that the induction of proliferation of CD8+ SP cells previously reported for peripheral T cells (19) already occurs at this stage. These results concur with our observations using BrdU incorporation in FTOCs (see next section). Total cellularity was also assessed and results showed a strong reduction of cell numbers at days 12 and 14 when rIFN-α1 or rIFN-α4 were applied to precursors developing on OP9-DL1 cells (up to 7.7-fold decrease in comparison with control cultures; Fig. 4, B and C). Altogether, these results indicate that this decreased cellularity could result from the accumulation of cells at the DN1 and DN2 stages of T cell differentiation and their lack of further progression to more differentiated thymic subsets.

FIGURE 4.

Addition of IFN-α1 to fetal liver-derived hemopoietic stem cells cocultured with OP9-DL1 cells leads to a complete blockade of T cell development at the DN1 stage, to reduced cellularity over time and to an increased apoptosis. A and D, Flow cytometric analysis of T cells developing on OP9-DL1 cells. Shown are developing T cell analyses from DN1 to DN4 stages and from DN to SP populations at days 8 and 12 (A) or at days 12 and 14 (D) of coculture with or without the addition of IFN-α1 or -α4. In A, IFN-α were added at day 4 and in D, they were added at day 12 (shown as control). B and C, Relative cellularity at days 8, 12, and 14 of coculture (IFN-α1, 3-fold decrease; IFN-α4, 7.7-fold decrease at day 12; IFN-α1, 2.5-fold decrease; IFN-α4, 3.4-fold decrease at day 14). E, Normalized frequency of apoptotic cells assessed by annexin V labeling of OP9-DL1-derived T cells 44 h after the addition of IFN-α1 or IFN-α4 at day 16 of coculture (IFN-α1, 2.3-fold increase; IFN-α4, 1.9-fold increase). A representative example of two independent experiments is shown.

FIGURE 4.

Addition of IFN-α1 to fetal liver-derived hemopoietic stem cells cocultured with OP9-DL1 cells leads to a complete blockade of T cell development at the DN1 stage, to reduced cellularity over time and to an increased apoptosis. A and D, Flow cytometric analysis of T cells developing on OP9-DL1 cells. Shown are developing T cell analyses from DN1 to DN4 stages and from DN to SP populations at days 8 and 12 (A) or at days 12 and 14 (D) of coculture with or without the addition of IFN-α1 or -α4. In A, IFN-α were added at day 4 and in D, they were added at day 12 (shown as control). B and C, Relative cellularity at days 8, 12, and 14 of coculture (IFN-α1, 3-fold decrease; IFN-α4, 7.7-fold decrease at day 12; IFN-α1, 2.5-fold decrease; IFN-α4, 3.4-fold decrease at day 14). E, Normalized frequency of apoptotic cells assessed by annexin V labeling of OP9-DL1-derived T cells 44 h after the addition of IFN-α1 or IFN-α4 at day 16 of coculture (IFN-α1, 2.3-fold increase; IFN-α4, 1.9-fold increase). A representative example of two independent experiments is shown.

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Type I IFNs are known for their antiproliferative activities (6, 13), which could also explain the reduced cell numbers at the DN3 stage. Thymocyte proliferation was therefore assessed by measuring the incorporation of BrdU in FTOCs in the presence of poly(I:C), loxoribine, or IFN-αA. A 2-fold decrease in BrdU-labeled cells in the DN compartment was noted 48 h after TLR3 or TLR7 engagement or IFN-αA addition (Fig. 5, A and B), i.e., during the earliest stage of thymocyte development. Proliferation was also strongly reduced at the DP (2- to 6-fold decrease in BrdU+ cells) and to a lesser extent at the CD4+ SP stage (up to 4-fold decrease in BrdU+ cells), indicating that type I IFNs also affect later stages of thymic differentiation, and in particular those stages at which thymic selection occurs (48, 49). The proliferation of CD8+ SP cells seemed to be less sensitive to TLR triggering or IFN-αA addition because little or no reduction in cell proliferation was observed. This result could be explained by the previously reported impact of type I IFNs on CD8+ T cells (19).

FIGURE 5.

Addition of poly(I:C) or loxoribine to FTOCs leads to a decrease in proliferation and IL-7Rα-chain (CD127) expression. Flow cytometry histograms of BrdU-labeled DN cells (A) and percentage of BrdU+ cells (B) in each thymocyte subset assessed by anti-BrdU labeling 18 h after BrdU addition. C, Histogram of CD127-labeled DN cells. D, Percentage of the CD127+ cells in the DN, DN1, and DN2 subsets. E, MFI of CD127-labeled cells among thymic populations. Each measure was performed on a pool of 5–6 thymic lobes, and at least 300,000 events were recorded for each analysis. Results are expressed as mean ± SDs (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. n/d, Not determined because measure could not be done due to severe drop in cell numbers.

FIGURE 5.

Addition of poly(I:C) or loxoribine to FTOCs leads to a decrease in proliferation and IL-7Rα-chain (CD127) expression. Flow cytometry histograms of BrdU-labeled DN cells (A) and percentage of BrdU+ cells (B) in each thymocyte subset assessed by anti-BrdU labeling 18 h after BrdU addition. C, Histogram of CD127-labeled DN cells. D, Percentage of the CD127+ cells in the DN, DN1, and DN2 subsets. E, MFI of CD127-labeled cells among thymic populations. Each measure was performed on a pool of 5–6 thymic lobes, and at least 300,000 events were recorded for each analysis. Results are expressed as mean ± SDs (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. n/d, Not determined because measure could not be done due to severe drop in cell numbers.

Close modal

Because IL-7 is critical for DN cell proliferation (50, 51), we assessed the expression of the IL-7R α-chain (CD127) to explain the above-described loss of the DN3 subset and the proliferative defect. CD127 MFI and the frequency of CD127+ cells were significantly reduced in most thymic subsets in TLR ligands and IFN-αA-treated FTOCs when compared with PBS-treated FTOCs (Fig. 5, C–E). The impact of type I IFNs on CD127 expression was mostly evident within the DN1 and DN2 subsets. Indeed, a 2-fold decrease was observed in the MFI of CD127 and in the percentage of CD127+ cells in the latter two populations upon poly(I:C) or loxoribine treatment (Fig. 5,D, right). These subsets are those in which CD127 expression plays the most important role in regulating survival and proliferation (52). Interestingly, IL-7 up-regulation was not observed using quantitative PCR (data not shown), suggesting that the observed reduced number of cells expressing CD127 was not due to the internalization of CD127 resulting from excess production of IL-7. Rather, it most likely arises from a TLR-induced loss of cells expressing CD127 (Fig. 5, C and D). IL-7R α-chain levels and proliferating cell frequencies as measured by BrdU incorporation could not be obtained in DN3 cells (Fig. 5, B and E, right) due to the severe drop in cell numbers within this subset as a consequence of TLR activation (see Fig. 3, C and D).

Because type I IFNs are also known to be potent inducers of apoptosis (53), experiments were conducted on FTOCs to determine whether apoptosis also contributed to the loss of cellularity after addition of poly(I:C), loxoribine, or IFN-αΑ. The results illustrated in Fig. 6 show a significant increase of the percentage of annexin V+ cells in the DN (Fig. 6,B), in which the blockade was observed, and DP subsets (Fig. 6, A and C), which constitute the vast majority of thymocytes (80%), after treatment of FTOCs with either poly(I:C) or loxoribine. Indeed, we observed a 1.4-fold increase of annexin V+ cells following treatment of FTOCs with poly(I:C) and a 1.3-fold increase with loxoribine in DN cells as well as a 1.2-fold increase with poly(I:C) and a 1.9-fold increase with loxoribine in DP cells. These increased frequencies of apoptotic cells were also observed upon IFN-αA treatment of FTOCs (1.4-fold increase in DN cells and 1.6-fold increase in DP cells). Finally, increased frequencies of apoptotic cells were not observed when IFN-α/βR−/− FTOCs were treated (Fig. 6, B and C), thereby confirming the direct involvement of type I IFNs in this process. Interestingly, the induction of apoptosis was also observed in the OP9-DL1 system. An increase in annexin V+ DN cells (2.3-fold) was measured 44 h after the addition of IFNs on T cells developing in the OP9-DL1 system at day 16 and was not observed when T cell precursors were isolated from IFN-α/βR−/− mice (Fig. 4 E).

FIGURE 6.

Treatment of FTOCs with poly(I:C), loxoribine, or IFN-αA leads to the increase of apoptotic cells. A, Histogram of annexin V-labeled cells. B and C, percentage of annexin V+ DN and DP thymocytes, respectively. D, Relative mRNA expression of TRAIL obtained by qRT-PCR 6 h after treatment of WT (poly(I:C), 7.5-fold increase; loxoribine, 3.4-fold increase; and IFN-αA, 2.7-fold increase) and IFN-α/βR−/− FTOCs with TLR ligands or IFN-αA. For apoptosis, each measure was performed on a pool of 5 to 6 lobes, and at least 300,000 events were recorded for each analysis. Results in B and C are expressed as mean ± SDs. Number of experiments: WT (n = 5 for DN cells and n = 6 for DP cells), IFN-α/βR−/− (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Results in D were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 11 thymic lobes. Results in B and C are the mean of two independent experiments; results in D are the mean of triplicates and are representative of two independent experiments.

FIGURE 6.

Treatment of FTOCs with poly(I:C), loxoribine, or IFN-αA leads to the increase of apoptotic cells. A, Histogram of annexin V-labeled cells. B and C, percentage of annexin V+ DN and DP thymocytes, respectively. D, Relative mRNA expression of TRAIL obtained by qRT-PCR 6 h after treatment of WT (poly(I:C), 7.5-fold increase; loxoribine, 3.4-fold increase; and IFN-αA, 2.7-fold increase) and IFN-α/βR−/− FTOCs with TLR ligands or IFN-αA. For apoptosis, each measure was performed on a pool of 5 to 6 lobes, and at least 300,000 events were recorded for each analysis. Results in B and C are expressed as mean ± SDs. Number of experiments: WT (n = 5 for DN cells and n = 6 for DP cells), IFN-α/βR−/− (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Results in D were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 11 thymic lobes. Results in B and C are the mean of two independent experiments; results in D are the mean of triplicates and are representative of two independent experiments.

Close modal

Given that apoptosis was increased and because TRAIL, a member of TNF receptor family, is a key player in the apoptosis pathway and an IRF3 and IRF7 transcriptional target (54), we quantified TRAIL mRNA by qRT-PCR and observed a marked up-regulation of its transcription (poly(I:C), 6.1; loxoribine, 6; IFN-αA, 7.5; 6 h after treatment). This increased expression of TRAIL was not evidenced in IFN-α/βR−/− FTOCs (Fig. 6 D) subjected to the same inducers. Our results clearly suggest that TRAIL is involved in and partially responsible for the observed increase in cell death.

To further confirm the implication of type I IFNs in the above observed defects, we have produced different individual rIFN-α subtypes and used them to treat FTOCs. We observed that rIFN-α1, rIFN-α4 or rIFN-α11 induce individually similar deleterious effects when compared with the commercially available IFN-αA and TLR ligands in the fetal thymic population (Fig. 7). Indeed, loss of the DN3 subset is observed when we treated FTOCs with rIFN-α1, rIFN-α4, or rIFN-α11 (Fig. 7,A). MFI of CD127 in DN1 and DN2 subsets was also reduced as well with these three different rIFN subtypes (Fig. 7,B). Finally, rIFN subtypes induced an increase in the frequency of thymic cells (including DP cells) undergoing apoptosis (Fig. 7 C). We have also observed that IFN subtypes act in a dose-dependent manner for all the phenotypes presented (data not shown). (We have used the concentration showing effects comparable with those induced by poly(I:C) and loxoribine, i.e., 10,000 U/ml for IFN-αA and 1,000 U/ml for rIFN-α4 as well as for rIFN-α1 and rIFN-α11). Finally, individual rIFN subtypes seemed to have a lesser impact than TLR ligands suggesting that poly(I:C) and loxoribine induce different IFN subsets at the same time, accruing the effects of each individual subset, or that TLR ligands have some targets other than type I IFNs that amplify the observed phenotypes.

FIGURE 7.

Addition of different IFN-α subtypes to FTOCs leads to similar effects than treatment with either poly(I:C) or loxoribine, and these effects are partly reverted by the addition of anti-type I IFN Abs. A, Normalized frequency of thymocytes at the DN3 (CD44 CD25+) stage; B, relative expression of CD127 in DN1 and DN2 cells; C, normalized frequency of apoptotic DP cells as measured by annexin V staining. The fact that the inhibition induced by the anti-IFN-neutralizing Abs was only partial could be due to the large and constant IFN-α production which could not be totally counteracted by these Abs. Each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. Results are representative of three independent flow cytometric analyses.

FIGURE 7.

Addition of different IFN-α subtypes to FTOCs leads to similar effects than treatment with either poly(I:C) or loxoribine, and these effects are partly reverted by the addition of anti-type I IFN Abs. A, Normalized frequency of thymocytes at the DN3 (CD44 CD25+) stage; B, relative expression of CD127 in DN1 and DN2 cells; C, normalized frequency of apoptotic DP cells as measured by annexin V staining. The fact that the inhibition induced by the anti-IFN-neutralizing Abs was only partial could be due to the large and constant IFN-α production which could not be totally counteracted by these Abs. Each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. Results are representative of three independent flow cytometric analyses.

Close modal

Levels of SOCS-1 expression were investigated in FTOCs, given that it has previously been shown that SOCS-1 overexpression prevents the progression of thymic progenitors beyond the earliest stages of T cell development (55). Interestingly, the observed early developmental blockade (Fig. 3) was accompanied by an increased expression of SOCS-1 mRNA (poly(I:C), 3.3; loxoribine, 3.7; and IFN-αA, 5.3-fold increase) 6 h after the addition of the different TLR ligands. This up-regulation was not observed in IFN-α/βR−/− mice (Fig. 8,A). The up-regulation of SOCS-1 expression was confirmed at the protein level (Fig. 8,C, top) in that a 2-fold increase was reproducibly observed by densitometry analysis of Western blots (Fig. 8 C, bottom).

FIGURE 8.

Addition of TLR ligands or IFN-αA leads to the up-regulation of SOCS-1 expression and to the down-regulation of Gfi1 expression. A and B, Relative mRNA expression of SOCS-1 (poly(I:C), 3.3-fold increase; loxoribine, 3.7-fold increase; and IFN-αA, 5.3-fold increase) and Gfi1 (poly(I:C), 1.5-fold decrease; loxoribine, 2-fold decrease; and IFN-αA, 1.4-fold decrease) obtained by qRT-PCR 6 h after addition of poly(I:C), loxoribine, or IFN-αA in WT or IFN-α/βR−/− FTOCs, respectively. C, SOCS-1 and β-actin Western blot scan are shown as well as the fold increase of SOCS-1 protein levels (band quantification) 6 h after treatments. D, Histograms of GFP expression in an individual mice (MFI are shown); E, MFI of GFP expressed in thymocytes of Gfi1+/GFP mice injected for 3 days with poly(I:C) or PBS (DN3 and DN4: GFP MFI in PBS, 304 ± 7.5 and 253 ± 12.5, vs GFP MFI in poly(I:C), 210 ± 24 and 160 ± 23.6, respectively; and CD3low DP subset, GFP MFI in PBS, 148 ± 8 vs GFP MFI in poly(I:C), 109 ± 4). Results in A and B were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 11 thymic lobes. Results are the mean of duplicates for IFN-α/βR−/− and more than triplicates for WT and are representative of two independent experiments. Western blot results in C are representative of two independent experiments. E, Results are expressed as mean of five to six mice ± SDs and are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

Addition of TLR ligands or IFN-αA leads to the up-regulation of SOCS-1 expression and to the down-regulation of Gfi1 expression. A and B, Relative mRNA expression of SOCS-1 (poly(I:C), 3.3-fold increase; loxoribine, 3.7-fold increase; and IFN-αA, 5.3-fold increase) and Gfi1 (poly(I:C), 1.5-fold decrease; loxoribine, 2-fold decrease; and IFN-αA, 1.4-fold decrease) obtained by qRT-PCR 6 h after addition of poly(I:C), loxoribine, or IFN-αA in WT or IFN-α/βR−/− FTOCs, respectively. C, SOCS-1 and β-actin Western blot scan are shown as well as the fold increase of SOCS-1 protein levels (band quantification) 6 h after treatments. D, Histograms of GFP expression in an individual mice (MFI are shown); E, MFI of GFP expressed in thymocytes of Gfi1+/GFP mice injected for 3 days with poly(I:C) or PBS (DN3 and DN4: GFP MFI in PBS, 304 ± 7.5 and 253 ± 12.5, vs GFP MFI in poly(I:C), 210 ± 24 and 160 ± 23.6, respectively; and CD3low DP subset, GFP MFI in PBS, 148 ± 8 vs GFP MFI in poly(I:C), 109 ± 4). Results in A and B were normalized to an endogenous control gene (GAPDH) and compared with PBS control (fold increase). Each measure was performed on a pool of 11 thymic lobes. Results are the mean of duplicates for IFN-α/βR−/− and more than triplicates for WT and are representative of two independent experiments. Western blot results in C are representative of two independent experiments. E, Results are expressed as mean of five to six mice ± SDs and are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Close modal

Moreover, 6 h after TLR activation, we also observed a decreased expression of Gfi1 mRNA (poly(I:C), 1.5; loxoribine, 2; and IFN-αA, 1.4-fold decrease; Fig. 8,B), a SOCS-1 transcriptional inhibitor (56, 57). To further investigate the expression of Gfi1 after treatment with TLR ligands, we performed in vivo experiments using heterozygote Gfi:GFP knock-in mice (Gfi+/GFP; Ref. 29). In these mice, the coding region of a GFP reporter gene was inserted into the Gfi1 locus precisely at the translation initiation codon of Gfi1, placing the GFP directly under the transcriptional control of the upstream regulatory sequence of the Gfi1 gene. This allowed the temporal assessment of Gfi1 expression in T cell subsets by following GFP expression during T cell development using polychromatic flow cytometry. After three daily IP injections of poly(I:C) in Gfi+/GFP mice, we observed a significant decrease of Gfi1 as measured by GFP expression in all thymic cellular subsets except in CD3intermediate DPs (Fig. 8, D and E). Gfi1 down-regulation in poly(I:C)-treated Gfi+/GFP mice was very significant in the DN subset, and mostly in DN3 and DN4 cells (GFP MFI in PBS, 304 ± 7.5; 253 ± 12.5 vs GFP MFI in poly(I:C), 210 ± 24 and 160 ± 23.6, respectively) and the CD3low DP subset (GFP MFI in PBS: 148 ± 8 vs GFP MFI in poly(I:C): 109 ± 4). Importantly, these stages of thymic differentiation represent those where thymocytes proliferate after β-selection and when the down-regulation of SOCS-1 is therefore required (58). Moreover, treatment of these mice with poly(I:C) is associated with the same effects as those observed with poly(I:C)-treated FTOCs: loss of thymic cellularity as well as DN3 subset population was observed (data not shown). The down-regulation of Gfi1 expression most likely leads to the increased expression of SOCS-1 which has as consequence to stop T cell proliferation and T cell differentiation.

Experiments were performed on FTOCs to compare the impact of viral infection with those of loxoribine on thymic cellularity and thymocyte subset distribution. VSV was used in these experiments because it is recognized by TLR7 (59). The impact of VSV exposure on T cell development was comparable with that induced by type I IFNs (Fig. 9); although levels of IFN-α produced upon VSV infection were higher than those produced upon TLR ligation, they were even detectable at the protein level by ELISA (150 ng/ml compared with 40 ng/ml in the control). Hence, the loss of the DN3 subset previously observed with loxoribine was present as well (2-fold decrease; Fig. 9,A), and the down-regulation of CD127 expression as monitored by MFI was also reproduced in DN1 and DN2 thymic subsets (>1.5-fold decrease; Fig. 9,C) as well as in other subsets (data not shown). In addition, our results showed that thymocytes from FTOCs infected with VSV were characterized by an increase in apoptosis at all developmental stages, the DP stage in particular (a >2-fold increase; Fig. 9,B). These defects are similar to those reported above with TLR7 triggering by loxoribine (Fig. 3, C and D; Fig. 5, C and D; and Fig. 6 C). Taken together, these results show that loxoribine exerts qualitative and quantitative impacts on thymic development similar to those reported with viruses known to trigger TLR7.

FIGURE 9.

VSV infection inhibits T cell development similarly to TLR7 engagement by loxoribine. A, Normalized frequency of thymocytes at the DN3 stage. B, Normalized frequency of DP apoptotic cells as measured by annexin V staining. C, CD127 MFI in DN1 and DN2 cells assessed by flow cytometry. Each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. A representative example of two independent flow cytometric analyses is shown.

FIGURE 9.

VSV infection inhibits T cell development similarly to TLR7 engagement by loxoribine. A, Normalized frequency of thymocytes at the DN3 stage. B, Normalized frequency of DP apoptotic cells as measured by annexin V staining. C, CD127 MFI in DN1 and DN2 cells assessed by flow cytometry. Each measure was performed on a pool of 5–6 lobes, and at least 300,000 events were recorded for each analysis. A representative example of two independent flow cytometric analyses is shown.

Close modal

Herein, we show that type I IFNs produced upon the activation of TLRs in FTOCs exert deleterious effects on T cell development. Indeed, these TLR ligands induce the loss of the DN3 subset, and this was associated with a decrease of thymocyte proliferation, as well as an increase in apoptosis in all T cell subsets. These results were reproduced upon the direct exposure of developing thymocytes to IFN-αA and other rIFN-αs such as rIFN-α1, rIFN-α4, and rIFN-α11. We have not been able to evidence any differential impact of the IFN subtypes tested on T cell development. All tested subtypes led to most of the described deleterious effects in a dose-dependent manner (Fig. 7). These effects on T cell development were not observed when TLRs were activated in cells from IFN-α/βR−/− FTOCs (Figs. 1–3, 6, and 8) and were partially abrogated by the addition of anti-IFN-α and anti-IFN-β neutralizing Abs (Figs. 7 and 8,B). Moreover, IL-15 mRNA and class I expression increase (Fig. 2 A) were observed and confirmed IFN implication in our system because these molecules have been shown to be induced by type I IFN (13, 60). Altogether, these data confirm that the impairment of T cell development that we observed in FTOCs can be directly imparted to the induction of IFN production upon TLR triggering or addition of type I IFNs.

Most experiments were performed by incubating FTOCs with molecules that stimulate TLR3, TLR7, or TLR9. The results presented here focused on TLR3 (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β pathway) and TLR7 (MyD88 pathway) triggering as the very closely related TLR7, TLR8, and TLR9 form a functional subgroup within the TLR family (38). Moreover, we observed similar results when we treated FTOCs with either loxoribine or CpG ODN (data not shown). Quantitative differences between TLR3 and TLR7 were observed with regard to their capacity to induce IFN production and their deleterious impact on thymic differentiation. Poly(I:C) induced higher levels of IFN production, whereas loxoribine had a more drastic impact on thymocyte development, suggesting that loxoribine might trigger mechanisms other than type I IFNs which could be responsible for this increased cytotoxicity.

Moreover it is important that the cytoplasmic helicases retinoic acid-inducible protein-I and melanoma differentiation-associated gene 5 pathways participate to the IFN production in response to poly(I:C) (61), in contrast to loxoribine. Despite the higher levels of type I IFN produced upon TLR3 triggering, which could be due to the implication of retinoic acid-inducible protein-I and melanoma differentiation-associated gene 5, the impact on thymocyte differentiation was less drastic. Given that type I IFNs are produced in waves, we cannot exclude differential effects arising from synergies attributed to different IFN subtype combinations produced by different TLR triggering. It is difficult to rule out that thymocytes, which express much lower levels of TLRs than plasmacytoid dendritic cells (data not shown), could be also involved in type I IFN production upon TLR ligation (62, 63, 64).

Several steps of T cell development are affected by TLR activation and IFN production. The early T cell development blockade was observed in two experimental systems that support T cell development and differentiation, the FTOCs and the OP9-DL1 system. A complete blockade at the DN1 stage was observed in the OP9-DL1 system when fetal liver progenitor cells were allowed to differentiate for 4 days before the addition of IFN. In FTOCs, this developmental blockade resulted in a loss of the DN3 subsets as well as a decreased level of proliferation. These deficiencies in early T cell development are consistent with the down-regulation of the expression of the α-chain of the IL-7R, known to play an essential role in protecting thymocytes from apoptosis and in inducing thymocyte proliferation upon IL-7 stimulation (52). Hence, a decreased number of cells expressing the IL-7Rα-chain could lead to the observed decreased proliferation (Fig. 5, A and B) and the increased apoptosis reported in the DN population after TLR-induced IFN-α production (Fig. 6 B). Similarly, a partial deficiency of IL-7Rα has been shown to be sufficient in abrogating T cell development in humans (65). Hence, down-regulation of IL-7R expression could lead to the developmental blockade that we have reported and which occurs before the DN3 stage in the FTOC system.

In addition, we also showed that Gfi1 is down-regulated when SOCS-1 is up-regulated after TLR triggering or IFN-α treatment. These results support the fact that transcription of SOCS-1 is repressed by Gfi1-b, a paralog of Gfi1 (56, 57). Gfi1 and 1-b act equivalently on hemopoiesis (66). Furthermore, lack of Gfi1 is known to affect development of early CD4CD8cKit+ T cell progenitors (67), thereby confirming our results (Fig. 3). Moreover, it has already been shown that SOCS-1, which is normally expressed throughout thymocyte development, needs to be transiently suppressed, to allow DN3 expansion in response to signals from the pre-TCR, and that SOCS-1 overexpression in fetal liver-derived hemopoietic progenitors prevented their progression beyond the earliest stages of T cell development (55), an observation similar to what we report herein (Figs. 3, C and D, and 4, A and D). Because Gfi1 is down-regulated at all stages of T cell development including DN cells (Fig. 8 E), it will augment SOCS-1 expression leading to an inhibition of proliferation. Importantly, SOCS-1 actively suppresses IL-7R signal transduction in DP thymocytes that have not received TCR-mediated positive selection signals (58). Hence, SOCS-1 up-regulation could explain both the early developmental blockade and the effects observed on the later stages of thymopoiesis.

Type I IFNs activate the JAK-STAT pathway that up-regulates the expression of genes related to antiviral functions, thereby inducing a positive loop through enhanced expression of TLRs and IRFs. In contrast, type I IFNs also up-regulate SOCS-1 (Fig. 7), which blocks signaling through the JAK-STAT pathway and IRF expression (68, 69). SOCS-1 could, for instance, block the Stat-5 signaling downstream of IL-7R. As such, up-regulation of SOCS-1 associated with the down-regulation of IL-7R could exert a negative impact on IL-7 prosurvival signals such as Bcl-2 and Bcl-xL (70, 71). Our results suggest that the induced overexpression of SOCS-1, most likely as a result of Gfi1 down-regulation, could be responsible for the thymic defects we observed in FTOCs following TLR triggering and IFN production. Altogether, these observations could be linked with the role of the cdk inhibitor p27Kip1 in thymopoiesis. Indeed, like SOCS-1, this factor must be down-regulated to ensure T cell development and proliferation (72, 73).

As previously indicated, increased apoptosis was observed not only in the DN population, but also in later T cell developmental stages. This general increase of apoptosis as observed by annexin V staining (Fig. 6, A–C) clearly explains the loss in cellularity in developing FTOCs. Thus, type I IFNs exert a profound impact on thymocyte development in that they could affect specifically T cell development before β selection as well as later at the DP and SP stages. The up-regulation of TRAIL (Fig. 6 D), an IRF3 transcriptional target (74), suggests that TRAIL plays a role in the increased apoptosis that we observed after IFN production. This is consistent with the described induction of TRAIL following viral infections and exposure to type I IFN (53, 75, 76, 77).

Type I IFNs have been shown to exert a negative impact on the resistance to infectious agents as they increase susceptibility to bacterial infections (78, 79). Moreover, viral infection-induced type I IFNs affect the recruitment of naive T cells needed to generate a broad antiviral CD8 T cell response during persistent infection (80). Also, type I IFNs lead to the depletion of recirculating thymocytes, most likely by inhibiting lymphocyte responses to type 1 sphingosine 1-phosphate G protein-coupled receptor, including recent thymic emigrants and therefore promoting decreased thymic output5 and lymphocyte retention in lymphoid organs (26, 81, 82). Because IFN-α is widely used for treatment of viral infections (hepatitis C) and tumors such as melanoma (83, 84), it is important to consider that the therapeutic use of exogenous IFN-α may show deleterious effects such as lymphopenia. For instance, it has been reported that IFN-α therapy induces lymphopenia in hepatitis C virus-infected or hepatitis C virus and HIV-coinfected patients (85, 86). The impact of exogenous IFNs on T cell development that we describe here portrays the side effects of IFN-α treatment. Therefore, these side effects must be considered when applying IFN-α therapy to patients, particularly when they are immunocompromised as is the case for HIV-infected or cancer patients. TLR ligands are also increasingly considered for their use in adjuvantation of vaccines. Our results clearly indicate that the impact of TLR ligands on the generation of naive T cells must be assessed when developing these new strategies.

We thank Caroline Riel for the animal care and technical help, Raphaëlle Lambert and Pierre Chagnon from the genomics platform at the Institute for Research in Immunology and Cancer for the qR-PCR, and Sylvain Gimmig for help with cytometry. We also thank Dr. Claude Perreault, Dr. Daniel Lamarre, and Dr. Karine Maisnier-Patin for critically reading the manuscript and Dr. Thomas Démoulins for helpful discussion. The Gfi+/GFP mice were a gift from Dr. Tarik Möröy, and the VSV AV1 was from Dr. Ehssan Sharif-Askari.

The authors have no financial conflict of interest.

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 grants from the Canadian Institutes of Health Research (to R.-P.S. and J.-C.Z.-P.), from the Canadian Network for Vaccine and Immunotherapeutics (to R.-P.S.), and from the Ontario HIV Treatment Network (to J.-C.Z.-P.). R.-P.S. is the Canada Research Chair in Human Immunology and J.-C.Z.-P. is the Canada Research Chair in Developmental Immunology. R.L.M.-M. is supported by a Postdoctoral Fellowship from the Ontario HIV Treatment Network.

4

Abbreviations used in this paper: IRF, IFN-regulatory factor; ODN, oligodeoxynucleotide; DL, delta like; DN, double negative; DP, double positive; FTOC, fetal thymic organ culture; Gfi, growth factor independence; MFI, mean fluorescence intensity; poly(I:C), polyinosine-polycytidylic acid; SOCS, suppressors of cytokine signaling; SP, single positive; VSV, vesicular stomatitis virus; WT, wild type.

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