Approche différentielle de la prévention et du traitement de la maladie du greffon contre l’hôte expérimentale de type chronique sclérodermique : focus sur l’Imatinib et l’Azacytidine

Introduction : La maladie du greffon contre l’hôte (GVHD) demeure une des complications majeures des allogreffes de cellules souches hématopoïétiques. Parmi les formes de la maladie, on distingue notamment la forme aigue et la forme chronique. Au sein de la forme chronique, on distingue également la GVHD de type chronique sclérodermique caractérisée par une fibrose importante des tissus cibles. Les traitements actuels (corticothérapie standard en première ligne) demeurant insatisfaisants, plusieurs études tentent d’évaluer l’impact prophylactique/thérapeutique mais également les aspects immunomodulateurs de nouvelles molécules potentiellement intéressantes dans la gestion de la GVHD chronique sclérodermique. Parmi ces molécules, on retrouve notamment l’imatinib, un inhibiteur de tyrosines kinases capable d’inhiber les protéines c-Abl et le PDGF-récepteur intervenants dans la signalisation pro-fibrotique de la pathologie mais également l’azacytidine, un agent hypométhylant capable d’induire l’hypométhylation du gène FoxP3 permettant la conversion des lymphocytes T conventionnels vers un phénotype T régulateur essentiel dans le contrôle de la pathologie. Méthodes : Dans la présente thèse, nous avons utilisé un modèle murin de GVHD chronique sclérodermique bien connu. Des souris receveuses de souches Balb/cJ (H-2d) furent transplantées avec 10.106 de cellules de moelles et 70.106 splénocytes totaux issus de donneuses B10.D2 (H-2d) après irradiation corporelle totale (7 Gy). Après greffe, les receveuses furent traitées par imatinib (étude n°1) à raison de 150 mg/kg/jour entre les jours 10 et 52 post-greffe ou par azacytidine (Aza, étude n°2) à raison de 0.5 ou 2 mg/kg toutes les 48h entre les jours 10 et 30 post-greffe. Résultats : Au cours de la première étude, l’imatinib montra un effet limité avec une sévérité similaire entre les souris traitées ou non malgré une perte de poids plus faible chez les souris traitées au jour 52 (P=0,02). L’imatinib réduit la prolifération des cellules T totales (P=0.02), des cellules T CD8+ (P=0.01) et des cellules T régulatrices (Tregs) (P=0.02) dans la rate. De plus, les souris traitées à l’imatinib ont un niveau de phosphorylation du PDGF-R significativement plus bas que les souris contrôles au jour 29 post-greffe (P=0.008). Au cours de la seconde étude, les résultats montrent que l’administration d’Aza toutes les 48h du jour 10 au jour 30 post-greffe à une dose de 0,5 mg/kg ou 2 mg/kg atténue la GVHD chronique. De plus, les souris traitées à l’Aza ont une plus haute fréquence de Tregs dans le sang et le thymus au jour 35 post-greffe ainsi qu’une plus grande déméthylation de l’enhancer de FoxP3 et du promoteur de l’IL-2 dans les splénocytes au jour 52. De façon intéressante, les Tregs issus des souris traitées à l’Aza expriment également plus le marqueur d’activation CD103 au jour 52. Les souris traitées à l’Aza ont également un taux plus faible de lymphocytes T conventionnels CD4+ exprimant l’antigène Ki67 au jour 21 post-greffe démontrant l’effet anti-prolifératif de l’Aza sur les cellules T.Conclusions : L’imatinib a un impact limité sur la GVHD chronique sclérodermique murine malgré une inhibition significative du PDGF récepteur tandis que l’Aza prévient la GVHD dans le même modèle expérimental. Ces données pourraient servir de base pour une étude pilote sur l’administration d’Aza pour la prévention de la GVHD chronique chez les patients présentant un risque élevé de développer la maladie

Novel approaches for preventing acute graftversus- host disease after allogeneic hematopoietic stem cell transplantation

Introduction Allogeneic hematopoietic stem cell transplantation (alloHSCT) offers potential curative treatment for a wide range of malignant and nonmalignant hematological disorders. However, its success may be limited by post-transplant acute graft-versus-host disease (aGVHD), a systemic syndrome in which donor’s immune cells attack healthy tissues in the immunocompromised host. aGVHD is one of the main causes of morbidity and mortality after alloHSCT. Despite standard GVHD prophylaxis regimens, aGVHD still develops in approximately 40–60% of alloHSCT recipients. Areas covered In this review, after a brief summary of current knowledge on the pathogenesis of aGVHD, the authors review the current combination of a calcineurin inhibitor with an antimetabolite with or without added anti-thymocyte globulin (ATG) and emerging strategies for GVHD prevention. Expert opinion A new understanding of the involvement of cytokines, intracellular signaling pathways, epigenetics and immunoregulatory cells in GVHD pathogenesis will lead to new standards for aGVHD prophylaxis allowing better prevention of severe aGVHD without affecting graft-versus-tumor effects

L'azacytidine prévient la maladie du greffon contre l'hôte de type chronique sclérodermique expérimentale

Introduction: Graft-versus-host disease (GVHD) remains one major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Following unmanipulated peripheral-blood stem cell transplantation, 60% of the patients experience chronic GVHD while approximately 15% of them develop a sclerodermic form of chronic GVHD characterized by multiple organ fibrosis and loss of skin elasticity. Regulatory T cells (Tregs) play a pivotal role in the pathology of chronic GVHD by inhibiting alloreactive conventional T cells. Several studies have shown the hypomethylating agent Azacytidine (Aza) can demethylate the master transcription factor of Treg (Forkhead box protein 3 factor, FoxP3), thus promoting Treg differentiation of conventional T cells. This work investigates the impact of Aza in a classical murine model of sclerodermic chronic GVHD (B10.D2 BALB/cJ). Methods: Lethally irradiated BALB/cJ recipient mice were injected with 107 bone marrow cells + 7.107splenocytes from B10.D2 donor mice. Recipients were treated with subcutaneous injections of Aza at the dose of 0,5 or 2 mg/kg every two days from day 10 to 30 following transplantation. Mice GVHD was evaluated with five criteria (weight loss, activity, fibrosis, hair loss and mice posture; 0-1-2 points/criteria). Mice were sacrificed at a score of 8/10 (or > 20% weight loss). Results: Mice treated with Aza 0.5 mg/kg (n = 14) or 2 mg/kg (n = 17) had significant lower clinical scores compared to control ones (n = 15) after treatment. FACS analysis showed a higher proportion of Treg among CD4+ T cells in the blood of Aza 2 mg/kg mice than in control mice (P = 0.047), as well as a higher percentage of Tregs expressing the KI67 proliferative marker on the same day (P = 0.0005). Finally, analyses of the cellular blood components with Cell-dyn demonstrated that Aza 2 mg/kg treated mice were significantly lymphopenic as compared to control mice (P = 0.05). Conclusion : Aza prevented sclerodermic GVHD in this classical murine model of chronic GVHD.Impact of chronic graft-versus-host disease and immunosuppressive drugs on thymic immune reconstitution and Tregs cells after allo-hematopoietic stem cell transplantation (Allo-HSCT)

Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease

Background Previous studies have demonstrated that regulatory T cells (Tregs) play a protective role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Tregs constitutively express the gene of the transcription factor Foxp3 whose CNS2 region is heavily methylated in conventional CD4+ T cells (CD4+Tconvs) but demethylated in Tregs. Methods Here, we assessed the impact of azacytidine (AZA) on cGVHD in a well-established murine model of sclerodermic cGVHD (B10.D2 (H-2d) → BALB/cJ (H-2d)). Results The administration of AZA every 48 h from day +10 to day +30 at the dose of 0.5 mg/kg or 2 mg/kg mitigated chronic GVHD. Further, AZA-treated mice exhibited higher blood and thymic Treg frequencies on day +35, as well as higher demethylation levels of the Foxp3 enhancer and the IL-2 promoter in splenocytes at day +52. Interestingly, Tregs from AZA-treated mice expressed more frequently the activation marker CD103 on day +52. AZA-treated mice had also lower counts of CD4+Tconvs and CD8+ T cells from day +21 to day +35 after transplantation, as well as a lower proportion of CD4+Tconvs expressing the Ki67 antigen on day +21 demonstrating an anti-proliferating effect of the drug on T cells. Conclusions Our results indicate that AZA prevented sclerodermic cGVHD in a well-established murine model of cGVHD. These data might serve as the basis for a pilot study of AZA administration for cGVHD prevention in patients at high risk for cGVHD

L'imatinib améliore la survie de la maladie du greffon contre l'hôte de type chronique par inhibition du TGF-β et du PDGFR chez la souris

Introduction: Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Approximately 15% of the patients develop the sclerodermatous cGvHD (scl-cGvHD) form of the disease characterized by multiple organ fibrosis and loss of skin elasticity. A few studies have suggested potential benefits of imatinib, a tyrosine kinase inhibitor, as a treatment of fibrosis in scl-cGVHD due to its ability to inhibit simultaneously the PDGF receptor and TGF-β pathways (via ABL inhibition), which are both involved in fibrosis . Aim: This work investigates the impact of imatinib on fibrosis in the B10.D2 to BALB/cJ scl-cGvHD murine model. Lethally irradiated BALB/cJ recipient mice were injected with 107 bone marrow cells + 7.107 splenocytes from B10.D2 donor mice. Recipients were treated with imatinib 150 mg/kg/day (50 mg/kg in the morning followed by 100 mg/kg in the evening) by oral gavage or the same volume of sterile water. Mice health status was evaluated with a scoring system encompassing five criteria (weight loss, activity, fibrosis, hair loss and mice posture; 0-1-2 points/criteria). Mice were sacrificed at a score of 8/10 according to our local ethical committee. Results: Mice given daily 150 mg/kg imatinib had a better survival than control mice (42 versus 33 days, p = 0,0357). cGvhD scores were suggestively lower in imatinib-treated than in control mice (p ≤ 0,15). Further, histological analyses evidenced reduction in the levels of both PDGF receptor (p = 0,033) and c-Abl (p = 0,185) phosphorylation in imatinib as compared to control mice. Finally, no significant differences were observed in the number or frequency of lymphocyte subsets in the 2 groups of mice. Conclusion: Imatinib slightly decreased fibrosis and significantly improved survival in a severe scl-cGvHD murine model.Effets in vitro et in vivo de l'imatinib et du nilotinib, deux inhibiteurs de tyrosine kinase, dans la maladie du greffon contre l'hôte de type chronique sclérodermiqu