Exploration of atypical Vκ2-Vκ rearrangements in ex vivo B cells subpopulations and in a in vitro B cell differentiation model

Au cours de la lymphopoïèse B, de nombreux mécanismes recombinatoires sont mis en place pour permettre la synthèse des immunoglobulines, qui sont composées de quatre chaînes protéiques : deux chaînes lourdes et deux chaînes légères identiques. Au sein du locus IGK des chaînes légères, des mécanismes entraînent l'association d'un fragment de gène de variabilité Vκ et de jonction Jκ : c’est la recombinaison V(D)J des gènes des immunoglobulines. Des travaux antérieurs du Pr Brigitte Gubler ont identifié des réarrangements atypiques associant deux gènes Vκ, appelés Vκ2-Vκ, dans les prélèvements de patients atteints de LAL-B pédiatrique. Durant ces travaux de thèse, nous avons poursuivi cette étude en nous concentrant sur l'apparition physiologique des réarrangements Vκ2-Vκ au sein de populations cellulaires triées de sang placentaire. Nous avons découvert que ces réarrangements atypiques apparaissent à une fréquence comprise entre 0,5% et 1%. Nos résultats montrent une utilisation préférentielle des gènes en fonction du stade de maturation. Dans un nouveau modèle de différenciation B in vitro développé à partir de cellules souches matures en coculture avec des cellules souches stromales issues de sang de cordon, nous avons constaté que ces réarrangements atypiques ont une fréquence inférieure (0,2%) mais qu'ils possèdent les mêmes caractéristiques d'utilisation préférentielle des gènes au cours de la différenciation B. Ces résultats démontrent l'existence de réarrangements atypiques Vκ2-Vκ physiologiques et démontrent de façon indirecte l'existence d'un enhancer fonctionnel responsable du contrôle des recombinaisons atypiques Vκ2-Vκ permettant la diversité et la révision du répertoire des chaînes légères κ des immunoglobulinesDuring B lymphopoiesis, many recombinant mechanisms are involved allowing the synthesis of immunoglobulins. The immunoglobulins are composed of two heavy and light identical chains. Within the IGK locus, mechanisms allow the association of a gene fragment of variability Vκ and junction Jκ. This mechanism is called V (D) J recombination of immunoglobulin genes. Previous work by Prof. Brigitte Gubler identified atypical rearrangements associating two Vκ genes, called Vκ2-Vκ in samples from patients with pediatric ALL-B. During this thesis work, we continued this study by focusing on the physiological appearance of Vκ2-Vκ rearrangements in sorted cell populations of cord blood. We have identified these atypical rearrangements at a frequency of between 0.5% and 1%. Our results show a preferential use of genes depending on the stage of maturation. In addition, in a new model of in vitro B-cell differentiation developed from mature stem cells in coculture with cord stromal stem cells, we found these rearrangements at a lower frequency, but having the same characteristics of preferential use of genes than previously demonstrated. These results demonstrate the existence of atypical physiological Vκ2-Vκ rearrangements and indirectly demonstrate the existence of a functional enhancer responsible for the control of atypical Vκ2-Vκ recombination, allowing diversity and revision of the repertoire IG kappa light chain

Exploration des réarrangements atypiques Vκ2-Vκ dans des sous-populations B ex vivo et dans un modèle de différenciation B in vitro

During B lymphopoiesis, many recombinant mechanisms are involved allowing the synthesis of immunoglobulins. The immunoglobulins are composed of two heavy and light identical chains. Within the IGK locus, mechanisms allow the association of a gene fragment of variability Vκ and junction Jκ. This mechanism is called V (D) J recombination of immunoglobulin genes. Previous work by Prof. Brigitte Gubler identified atypical rearrangements associating two Vκ genes, called Vκ2-Vκ in samples from patients with pediatric ALL-B. During this thesis work, we continued this study by focusing on the physiological appearance of Vκ2-Vκ rearrangements in sorted cell populations of cord blood. We have identified these atypical rearrangements at a frequency of between 0.5% and 1%. Our results show a preferential use of genes depending on the stage of maturation. In addition, in a new model of in vitro B-cell differentiation developed from mature stem cells in coculture with cord stromal stem cells, we found these rearrangements at a lower frequency, but having the same characteristics of preferential use of genes than previously demonstrated. These results demonstrate the existence of atypical physiological Vκ2-Vκ rearrangements and indirectly demonstrate the existence of a functional enhancer responsible for the control of atypical Vκ2-Vκ recombination, allowing diversity and revision of the repertoire IG kappa light chainsAu cours de la lymphopoïèse B, de nombreux mécanismes recombinatoires sont mis en place pour permettre la synthèse des immunoglobulines, qui sont composées de quatre chaînes protéiques : deux chaînes lourdes et deux chaînes légères identiques. Au sein du locus IGK des chaînes légères, des mécanismes entraînent l'association d'un fragment de gène de variabilité Vκ et de jonction Jκ : c’est la recombinaison V(D)J des gènes des immunoglobulines. Des travaux antérieurs du Pr Brigitte Gubler ont identifié des réarrangements atypiques associant deux gènes Vκ, appelés Vκ2-Vκ, dans les prélèvements de patients atteints de LAL-B pédiatrique. Durant ces travaux de thèse, nous avons poursuivi cette étude en nous concentrant sur l'apparition physiologique des réarrangements Vκ2-Vκ au sein de populations cellulaires triées de sang placentaire. Nous avons découvert que ces réarrangements atypiques apparaissent à une fréquence comprise entre 0,5% et 1%. Nos résultats montrent une utilisation préférentielle des gènes en fonction du stade de maturation. Dans un nouveau modèle de différenciation B in vitro développé à partir de cellules souches matures en coculture avec des cellules souches stromales issues de sang de cordon, nous avons constaté que ces réarrangements atypiques ont une fréquence inférieure (0,2%) mais qu'ils possèdent les mêmes caractéristiques d'utilisation préférentielle des gènes au cours de la différenciation B. Ces résultats démontrent l'existence de réarrangements atypiques Vκ2-Vκ physiologiques et démontrent de façon indirecte l'existence d'un enhancer fonctionnel responsable du contrôle des recombinaisons atypiques Vκ2-Vκ permettant la diversité et la révision du répertoire des chaînes légères κ des immunoglobuline

An Alginate-Based Hydrogel with a High Angiogenic Capacity and a High Osteogenic Potential

International audienceIn bone tissue engineering, autologous cells are combined with osteoconductive scaffolds and implanted into bone defects. The major challenge is the lack of post-implantation vascular growth into biomaterial. The objective of the present study was to develop a new alginate-based hydrogel that enhances the regeneration of bone defects after surgery. The viability of human bone marrow-derived mesenchymal stem cells (BM-MSCs) or human endothelial cells (ECs) cultured alone or together on the hydrogel was analyzed for 24 and 96 h. After seeding, the cells self-assembled and aggregated to form clusters. For functional validation, empty or cellularized hydrogel matrices were implanted ectopically at subcutaneous sites in nude mice. After 2 months, the matrices were explanted. Transplanted human cells were present, and we observed vessels expressing human von Willebrand factor (resulting from the incorporation of transplanted ECs into neovessels and/or the differentiation of BM-MSCs into ECs). The addition of BM-MSCs improved host vascularization and neovessel formation from human cells, relative to ECs alone. Although we did not observe bone formation, the transplanted BM-MSCs were able to differentiate into osteoblasts. This new biomaterial provided an appropriate three-dimensional environment for transplanted cells and has a high angiogenic capacity and an osteogenic potential

Rescuing SLAMF3 Expression Restores Sorafenib Response in Hepatocellular Carcinoma Cells through the Induction of Mesenchymal-to-Epithelial Transition

International audienceSimple Summary Sorafenib is a treatment for advanced HCC which demonstrated a poor objective response rate due to important induction of resistance. We demonstrated that induction of acquired-resistance to sorafenib in Huh-7 cell line leads to the loss of SLAMF3 expression, a tumor suppressor receptor in HCC. In these cells, the sorafenib-resistant phenotype is characterized by the increase of aggressiveness and induction of the epithelial-to-mesenchymal transition. Acquired-resistance to sorafenib induce a multipotent mesenchymal stem cells characteristic. Interestingly, SLAMF3 overexpression reversed the epithelial-to-mesenchymal transition and decreased metastatic potential in sorafenib-resistant cells through the control of ERK1/2 and mTOR signaling pathways. SLAMF3 seems to be a theranostics tools to the management of sorafenib treatment. Background: Acquired resistance to sorafenib in hepatocellular carcinoma (HCC) patients results in poor prognosis. Epithelial-to-mesenchymal transition (EMT) is the major mechanism implicated in the resistance to sorafenib. We have reported the tumor suppressor role of SLAMF3 (signaling lymphocytic activation molecules family 3) in HCC progression and highlighted its implication in controlling the MRP-1 transporter activity. These data suggest the implication of SLAMF3 in sorafenib resistance mechanisms. Methods: We evaluated the resistance to sorafenib in Huh-7 cells treated with progressive doses (Res cells). We investigated the link between acquired resistance to sorafenib and SLAMF3 expression by flow cytometry and Western blot methods. Furthermore, we analyzed the EMT and the stem cell potential of cells resistant to sorafenib. Results: Sorafenib resistance was confirmed in Res cells by analyzing the cell viability in the presence of sorafenib. The mesenchymal transition, in Res cells, was confirmed by high migratory index and the expression of EMT antigens. Interestingly, we found that loss of SLAMF3 expression corresponded to sorafenib-resistant phenotypes. The overexpression of SLAMF3 reversed EMT, decreased metastatic potential and inhibited mTOR/ERK1/2 in Res cells. Conclusions: We propose that rescuing SLAMF3 expression in resistant cells could represent a potential therapeutic strategy to enhance sorafenib efficacy in HCC patients

Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma

Abstract Background Epidermal growth factor receptor (EGFR) gene alterations and amplification are frequently reported in cases of glioblastoma (GBM). However, EGFR-activating mutations that confer proven sensitivity to tyrosine kinase inhibitors (TKIs) in lung cancer have not yet been reported in GBM. Case presentation Using next-generation sequencing, array comparative genomic hybridization and droplet digital PCR, we identified the p.L861Q EGFR mutation in a case of GBM for the first time. The mutation was associated with gene amplification. L861Q may be a clinically valuable mutation because it is known to sensitize non-small-cell lung cancers to treatment with the second-generation EGFR TKI afatinib in particular. Furthermore, we used slice culture of the patient’s GBM explant to evaluate the tumour’s sensitivity to various EGFR-targeting drugs. Our results suggested that the tumour was not intrinsically sensitive to these drugs. Conclusions Our results highlight (i) the value of comprehensive genomic analyses for identifying patient-specific, targetable alterations, and (ii) the need to combine genomic analyses with functional assays, such as tumour-derived slice cultures