Effect of methotrexate and anti-TNF on Epstein-Barr virus T-cell response and viral load in patients with rheumatoid arthritis or spondylarthropathies

INTRODUCTION: There is a suspicion of increased risk of Epstein-Barr virus (EBV)-associated lymphoproliferations in patients with inflammatory arthritides receiving immunosuppressive drugs. We investigated the EBV load and EBV-specific T-cell response in patients treated with methotrexate (MTX) or anti-TNF therapy. METHODS: Data for patients with rheumatoid arthritis (RA) (n = 58) or spondylarthropathy (SpA) (n = 28) were analyzed at baseline in comparison with controls (n = 22) and after 3 months of MTX or anti-TNF therapy for EBV load and EBV-specific IFNγ-producing T cells in response to EBV latent-cycle and lytic-cycle peptides. RESULTS: The EBV load and the number of IFNγ-producing T-cells after peptide stimulation were not significantly different between groups at baseline (P = 0.61 and P = 0.89, respectively). The EBV load was not significantly modified by treatment, for RA with MTX (P = 0.74) or anti-TNF therapy (P = 0.94) or for SpA with anti-TNF therapy (P = 1.00). The number of EBV-specific T cells was not significantly modified by treatment, for RA with MTX (P = 0.58) or anti-TNF drugs (P = 0.19) or for SpA with anti-TNF therapy (P = 0.39). For all patients, the EBV load and EBV-specific T cells were significantly correlated (P = 0.017; R = 0.21). For most patients, short-term exposure (3 months) to MTX or anti-TNF did not alter the EBV load or EBV-specific T-cell response but two patients had discordant evolution. CONCLUSIONS: These data are reassuring and suggest there is no short-term defect in EBV-immune surveillance in patients receiving MTX or anti-TNF drugs. However, in these patients, long term follow-up of EBV-specific T-cell response is necessary and the role of non-EBV-related mechanisms of lymphomagenesis is not excluded

Interleukin-7 Influences FOXP3+CD4+ Regulatory T Cells Peripheral Homeostasis

Mechanisms governing peripheral CD4+ FOXP3+ regulatory T cells (Treg) survival and homeostasis are multiple suggesting tight and complex regulation of regulatory T cells homeostasis. Some specific factors, such as TGF-β, interleukin-2 (IL-2) and B7 costimulatory molecules have been identified as essentials for maintenance of the peripheral Treg compartment. Conversely, Treg dependency upon classical T cell homeostatic factors such as IL-7 is still unclear. In this work, we formally investigated the role of IL-7 in Treg homeostasis in vivo in murine models. We demonstrated that IL-7 availability regulated the size of peripheral Treg cell pool and thus paralleled the impact of IL-7 on conventional T cell pool. Moreover, we showed that IL-7 administration increased Treg cell numbers by inducing thymic-independent Treg peripheral expansion. Importantly the impact of IL-7 on Treg expansion was detected whether conventional T cells were present or absent as IL-7 directly participates to the peripheral expansion of Treg after adoptive transfer into lymphopenic hosts. Our results definitively identify IL-7 as a central factor contributing to Treg peripheral homeostasis, thus reassembling Treg to other T cell subsets in respect of their need for IL-7 for their peripheral maintenance

Causes and consequences of type I IFN activation in autoimmune diseases. Study in the Sjögren's syndrome model.

Le syndrome de Sjögren primitif (SSp) est une maladie auto-immune (MAI) systémique ayant des caractéristiques communes avec le lupus érythémateux. Ces caractéristiques incluent des mécanismes physiopathologiques et des facteurs de predispositions génétiques. Notre équipe et d’autres groupes ont pu mettre en evidence une signature interféron (IFN) dans les glandes salivaires et les PBMCs de patients ayant un SSp. Cette découverte a permis de mettre en évidence de nouvelles voies à explorer dans la pathogénie du SLE et SSp en permettant la focalisation des recherches sur le rôle de l’immunité innée et de la voie IFN.Nous avons confirmé le rôle de 2 gènes importants dans le SSp, impliqués dans les voies des IFN. Le premier est IRF5 sur la voie IFN de type I et STAT4 sur la voie IFN de type II. Nous avons pu mettre en évidence une fonctionnalité de l’allèle à risque d’IRF5 (Polymorphisme Indel situé dans le promoteur). Concernant STAT4, son expression n’était pas altérée par le SNP associé à la maladie. Toutefois, l’ARNm de STAT4 était corrélé à l’expression des gènes IFN de type I. Les dérégulations épigénétique pourraient jouer un rôle important dans la pathogénie de nombreuses MAI, en particulier la méthylation de l’ADN qui est hautement liée à l’extinction de l’expression des gènes. Nous avons étudié la méthylation du promoteur d’IRF5 et nous n’avons pas trouvé de régulation de ce promoteur par le méthylation. Une analyse de la méthylation avec une approche globale du méthylome est en cours dans notre équipe et permettra d’identifier de gènes cibles d’une dérégulation épigénétique pouvant être impliqués dans les MAI.Nous avons essayé de comprendre la relation entre STAT4 et gènes IFN de type I. Ainsi, nous rapportons que l’IL-12 induit spécifiquement l’IFN de type I par intéraction entre deux partenaires cellulaires, les lymphocytes T CD4+ et les cellules dendritiques plasmacytoïdes. Ces résultats pourraient expliquer l’implication des polymorphismes de STAT4 dans les MAI dépendantes de l’IFN de type I. Ces résultats suggèrent également que les MAI dépendantes des IFN de type I et II ne s’opposent pas. Elles seraient seulement le Yin et le Yang d’un facteur d’activation commun, STAT4, capable d’induire les IFNs de type I et II.Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease (AID) that presents similar characteristics to systemic lupus erythematosus. These characteristics include pathophysiology and genetic factors. Our team and other groups have highlighted an interferon (IFN) signature in salivary glands and PBMCs from patients with Sjögren syndrome. This signature demonstrates new pathways in pSS and lupus, focusing research on innate immunity and in the IFN pathway.We have confirmed the implication of 2 genes in the pSS, and these genes are involved in the IFN pathway. The first gene is IRF5 which is in the type I IFN pathway and the second is STAT4 which is in the type II IFN pathway. We have shown a functional consequence of IRF5 at-risk allele. Regarding STAT4, the associated SNP did not altered STAT4 mRNA expression but was highly correlated with type I IFN genes expression.The epigenetic deregulation could play a triggering role in autoimmune diseases, particularly through DNA methylation which is highly implicated in the suppression of gene expression. We studied the methylation of IRF5 promoter and found no methylation. Our team is currently undertaking a global approach with methylome analysis. This methylome study will assess specific gene methylation patterns and will allow a better understanding of the role of these genes in autoimmune diseases.We further demonstrated that IL-12 specifically induces a type I IFN signature through a CD4+ T cells and pDCs crosstalk. These results could explain the implication of STAT4 polymorphism not only in type II IFN-dependent AIDs but also in type I IFN-dependent AIDs. Our data confirm that type I IFN- and type II IFN-mediated AIDs do not have to be opposed. They are only the yin and the yang of a common STAT4 activation which may induce secretion of both cytokines

Causes et conséquences de l’activation de l’interféron de type I dans les maladies auto-immunes. Étude dans le modèle du syndrome de Sjögren

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease (AID) that presents similar characteristics to systemic lupus erythematosus. These characteristics include pathophysiology and genetic factors. Our team and other groups have highlighted an interferon (IFN) signature in salivary glands and PBMCs from patients with Sjögren syndrome. This signature demonstrates new pathways in pSS and lupus, focusing research on innate immunity and in the IFN pathway.We have confirmed the implication of 2 genes in the pSS, and these genes are involved in the IFN pathway. The first gene is IRF5 which is in the type I IFN pathway and the second is STAT4 which is in the type II IFN pathway. We have shown a functional consequence of IRF5 at-risk allele. Regarding STAT4, the associated SNP did not altered STAT4 mRNA expression but was highly correlated with type I IFN genes expression.The epigenetic deregulation could play a triggering role in autoimmune diseases, particularly through DNA methylation which is highly implicated in the suppression of gene expression. We studied the methylation of IRF5 promoter and found no methylation. Our team is currently undertaking a global approach with methylome analysis. This methylome study will assess specific gene methylation patterns and will allow a better understanding of the role of these genes in autoimmune diseases.We further demonstrated that IL-12 specifically induces a type I IFN signature through a CD4+ T cells and pDCs crosstalk. These results could explain the implication of STAT4 polymorphism not only in type II IFN-dependent AIDs but also in type I IFN-dependent AIDs. Our data confirm that type I IFN- and type II IFN-mediated AIDs do not have to be opposed. They are only the yin and the yang of a common STAT4 activation which may induce secretion of both cytokines.Le syndrome de Sjögren primitif (SSp) est une maladie auto-immune (MAI) systémique ayant des caractéristiques communes avec le lupus érythémateux. Ces caractéristiques incluent des mécanismes physiopathologiques et des facteurs de predispositions génétiques. Notre équipe et d’autres groupes ont pu mettre en evidence une signature interféron (IFN) dans les glandes salivaires et les PBMCs de patients ayant un SSp. Cette découverte a permis de mettre en évidence de nouvelles voies à explorer dans la pathogénie du SLE et SSp en permettant la focalisation des recherches sur le rôle de l’immunité innée et de la voie IFN.Nous avons confirmé le rôle de 2 gènes importants dans le SSp, impliqués dans les voies des IFN. Le premier est IRF5 sur la voie IFN de type I et STAT4 sur la voie IFN de type II. Nous avons pu mettre en évidence une fonctionnalité de l’allèle à risque d’IRF5 (Polymorphisme Indel situé dans le promoteur). Concernant STAT4, son expression n’était pas altérée par le SNP associé à la maladie. Toutefois, l’ARNm de STAT4 était corrélé à l’expression des gènes IFN de type I. Les dérégulations épigénétique pourraient jouer un rôle important dans la pathogénie de nombreuses MAI, en particulier la méthylation de l’ADN qui est hautement liée à l’extinction de l’expression des gènes. Nous avons étudié la méthylation du promoteur d’IRF5 et nous n’avons pas trouvé de régulation de ce promoteur par le méthylation. Une analyse de la méthylation avec une approche globale du méthylome est en cours dans notre équipe et permettra d’identifier de gènes cibles d’une dérégulation épigénétique pouvant être impliqués dans les MAI.Nous avons essayé de comprendre la relation entre STAT4 et gènes IFN de type I. Ainsi, nous rapportons que l’IL-12 induit spécifiquement l’IFN de type I par intéraction entre deux partenaires cellulaires, les lymphocytes T CD4+ et les cellules dendritiques plasmacytoïdes. Ces résultats pourraient expliquer l’implication des polymorphismes de STAT4 dans les MAI dépendantes de l’IFN de type I. Ces résultats suggèrent également que les MAI dépendantes des IFN de type I et II ne s’opposent pas. Elles seraient seulement le Yin et le Yang d’un facteur d’activation commun, STAT4, capable d’induire les IFNs de type I et II

Interleukin-7 Optimizes FOXP3+CD4+ Regulatory T Cells Reactivity to Interleukin-2 by Modulating CD25 Expression

<div><p>The vast majority of Foxp3 regulatory T cells (Treg) exhibits constitutive expression of CD25 (IL-2Rα), which allows the constitution of the high affinity IL-2Rαβγ receptor, ensuring efficient IL-2 binding by Treg. Maintenance of CD25 expression at Treg surface depends on both cell intrinsic factors and environmental stimuli such as IL-2 itself. Whether other factors can participate to maintenance of CD25 expression <i>in vivo</i> is at present unknown. In the present work we demonstrated that IL-7, a gamma-chain cytokine exerting a crucial role in T cell development and homeostasis, is able and necessary to sustain the expression of high levels of CD25 at Treg surface. We demonstrated that, during <i>in vitro</i> cultures performed in the absence of IL-2, IL-7 is able to sustain CD25 expression at Treg surface through a transcriptional mechanism. By studying mice in which IL-7 signaling is either genetically impaired or increased and by employing adoptive transfer murine models, we demonstrated that IL-7 is necessary for sustained expression of CD25 at Treg surface <i>in vivo</i>. To ascertain the biological impact of IL-7 mediated modulation of CD25 expression, we demonstrated that IL-7 modulation of CD25 expression at Treg surface affected their ability to efficiently bind IL-2 and transduce IL-2 signaling. Finally, we demonstrated that IL-7 dependent modulation of CD25 associated with potentiated IL-2 induced expansion of Treg <i>in vivo</i>. Collectively, our results identify IL-7 as a necessary factor contributing to sustained CD25 expression at Treg surface <i>in vivo</i> thereby affecting their ability to efficiently react to IL-2.</p></div

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IL-7 availability affected IL-2 binding capacity by Treg.

<p>(A) Splenocytes from FOXP3/GFP mice were incubated for 30 min with biotinylated IL-2. Bound biotinylated IL-2 was revealed with fluorochrome-conjugated streptavidine. Shown are representative profiles of IL-2 fluorescence on CD4+GFP- cells (open thick line) or CD4+GFP+ cells (gray shaded profiles). Cells not exposed to biotinylated IL-2 and directly stained with fluorochrome-conjugated streptavidine are shown as negative controls (open thin lines) (B) Total CD4+ cells isolated from FOXP3/GFP mice were cultured in the absence (NT) or in the presence of IL-7 at 10 ng/ml (IL-7). After 24 hours cells were harvested and stained with biotinylated IL-2 as described above. Shown are representative profiles of IL-2 fluorescence on CD4+GFP+ cells cultured with (gray shaded profiles) or without (open thick line) IL-7. Cells not exposed to biotinylated IL-2 and directly stained with fluorochrome-conjugated streptavidine are shown as negative controls (open thin lines). MFI of triplicates ± SD are shown. Data are representative of three independent experiments performed in triplicate. (C) Splenocytes from WT, IL-7Rα-/-, IL-7-/- and IL-7Tg mice were analyzed directly <i>ex vivo</i> for IL-2 binding capacity as in (A). Shown are IL-2 MFI on CD4+TCRβ+FOXP3+ cells. Results are representative of two independent experiments with four to six mice per group.</p