Principles and applications of CRISPR toolkit in virus manipulation, diagnosis, and virus-host interactions

Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions

The TNF/TNFR2 pathway as a new target for immune checkpoint therapy in allogeneic stem cell transplantation

Les lymphocytes T régulateurs (Treg) sont des cellules capables de moduler la réponse immunitaire allogénique dans les deux sens. En effet, il a d'abord été démontré dans des modèles expérimentaux de maladie du greffon contre l'hôte (GVHD) chez la souris que l’élimination des Treg pouvait accentuer la GVHD, alors qu’une thérapie cellulaire à base de Treg peut la contrôler. Ces modèles pré-cliniques ont conduit au développement d'essais cliniques de thérapie cellulaire avec des résultats très prometteurs dans l’allogreffe de cellules souches hématopoïétiques (alloHSCT). Cependant, les procédures d’élimination des Treg ainsi que leur production restent difficiles à développer en grade clinique limitant ainsi leur utilisation. Il est donc essentiel d'avoir une meilleure compréhension des facteurs impliqués in-vivo dans l’effet des Treg au cours de l’alloHSCT afin de développer des approches thérapeutiques alternatives.Plusieurs études ont montré qu'en l'absence de cellules T effectrices, l’effet suppresseur des Treg est altéré. Etant donné que le TNFαest une cytokine centrale abondamment produite par les cellules T au cours de la GVHD, nous avons testé si dans ce contexte les Treg étaient également dépendent d’une signalisation via le TNFα. Pour tester cette hypothèse, nous avons utilisé trois approches expérimentales différentes bloquant l’interaction TNF/TNFR2 dans notre modèle de GVHD expérimentale. Nous révélons pour la première fois une dépendance complète au TNFα des Treg en termes de capacités suppressives in-vivo. Nous montrons également que la seule inhibition de la production de TNFα par les cellules T du donneur est suffisante pour abolir complètement l'effet suppresseur des Treg. Nous pensons que nos résultats ouvrent la voie vers le développement d’une nouvelle approche thérapeutique ciblant les points de contrôle de la réponse immunitaires afin de moduler l’alloréactivité après alloHSCT. En effet, l'inhibition de la voie TNF/TNFR2 pourrait potentialiser l'effet GVL des lymphocytes T du donneur en bloquant l'effet des Treg. Inversement, l’alloréactivité pourrait être fortement réduite ce qui aboutirait au contrôle de la GVHD par l’utilisation d’une molécule agoniste du TNFR2 qui permettrait d’augmenter les Treg ex-vivo.Regulatory Tcells (Treg) are key target cells to modulate in both ways the allogeneic immune response. Indeed, it was initially demonstrated in experimental mouse models of graft-versus-host disease (GVHD) that Tregs depletion could intensify the disorder whereas cell therapy using Tregs allowed to efficiently prevent experimental aGVHD. These pre-clinical models led to the development of clinical trials of Treg-based cell therapy with already very promising results in allogeneic hematopoietic stem cell transplantation (alloHSCT). However, the procedures of ex-vivo Tregs depletion as well as Tregs production remain difficult to develop at clinical grade thus limiting their dissemination. It is therefore critical to have a better understanding of factors involved in-vivo in these Treg-dependent effects in alloHSCT to develop alternative therapeutic approaches.Previous studies showed that in the absence of effector Tcells, suppressive effect of Tregs was significantly altered. Since TNFα is a central cytokine abundantly produced by Tcells during aGVHD, we tested whether in the context of alloHSCT the Tregs effect also depends on TNFα signaling. To test this hypothesis, we used three different experimental approaches to prevent TNF/TNFR2 interaction in our model of experimental aGVHD. We revealed for the first time a complete TNFα dependency for Tregs in terms of suppressive capacity in-vivo. We also showed that the sole inhibition of TNFα production by donor Tcells was sufficient to completely abolish the Tregs suppressive effect. We believe that our results pave the way for a novel immune checkpoint therapy to modulate alloreactivity after alloHSCT. Indeed, TNFα inhibition could potentiate the GVL effect of donor lymphocyte by blocking Treg effect. On the other hand, the alloreactivity could be strongly reduced to control GVHD by using TNFR2 agonist molecules in order to expand Tregs ex-vivo

TNFR2 Is a Crucial Hub Controlling Mesenchymal Stem Cell Biological and Functional Properties

Mesenchymal stem cells (MSCs) have drawn lots of attention as gold standard stem cells in fundamental and clinical researches during the last 20 years. Due to their tissue and vascular repair capacities, MSCs have been used to treat a variety of degenerative disorders. Moreover, MSCs are able to modulate immune cells’ functions, particularly T cells while inducing regulatory T cells (iTregs). MSCs are very sensitive to inflammatory signals. Their biological functions could remarkably vary after exposure to different pro-inflammatory cytokines, notably TNFα. In this article, we have explored the importance of TNFR2 expression in a series of MSCs’ biological and functional properties. Thus, MSCs from wild-type (WT) and TNFR2 knockout (TNFR2 KO) mice were isolated and underwent several ex vivo experiments to investigate the biological significance of TNFR2 molecule in MSC main functions. Hampering in TNFR2 signaling resulted in reduced MSC colony-forming units and proliferation rate and diminished the expression of all MSC characteristic markers such as stem cell antigen-1 (Sca1), CD90, CD105, CD44, and CD73. TNFR2 KO-MSCs produced more pro-inflammatory cytokines like TNFα, IFNγ, and IL-6 and less anti-inflammatory mediators such as IL-10, TGFβ, and NO and induced Tregs with less suppressive effect. Furthermore, the TNFR2 blockade remarkably decreased MSC regenerative functions such as wound healing, complex tube formation, and endothelial pro-angiogenic support. Therefore, our results reveal the TNFα–TNFR2 axis as a crucial regulator of MSC immunological and regenerative functions

Current Preventions and Treatments of aGVHD: From Pharmacological Prophylaxis to Innovative Therapies

International audienceGraft versus host disease (GVHD) is one of the main causes of mortality and the reason for up to 50% of morbidity after hematopoietic stem cell transplantations (HSCT) which is the treatment of choice for many blood malignancies. Thanks to years of research and exploration, we have acquired a profound understanding of the pathophysiology and immunopathology of these disorders. This led to the proposition and development of many therapeutic approaches during the last decades, some of them with very promising results. In this review, we have focused on the recent GVHD treatments from classical chemical and pharmacological prophylaxis to more innovative treatments including gene therapy and cell therapy, most commonly based on the application of a variety of immunomodulatory cells. Furthermore, we have discussed the advantages and potentials of cell-free therapy as a newly emerging approach to treat GVHD. Among them, we have particularly focused on the implication of the TNFα-TNFR2 axis as a new immune checkpoint signaling pathway controlling different aspects of many immunoregulatory cells

Single-cell transcriptomic profiling and characterization of endothelial progenitor cells: new approach for finding novel markers

International audienceAbstract Background Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial and cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in silico approach for finding novel EPC markers. Methods We assembled five groups of chosen EPC-related genes/factors using PubMed literature and Gene Ontology databases. This shortened database of EPC factors was fed into publically published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs, and two adult endothelial cell types (ECs) from the skin and adipose tissue. Further, the database was used for functional enrichment on Mouse Phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors’ PBMCs (33 thousand single-cell transcriptomes) and analyzed the expression of these EPC factors. Results Transcriptome analyses showed that BMP2, 4, and ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 and VEGF-C were significantly higher in EPCs and HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs and skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 was significantly higher in EPCs and adipose ECs. Moreover, functional enrichment of EPC-related genes on Mouse Phenotype and STRING protein database has revealed significant relations between chosen EPC factors and endothelial and vascular functions, development, and morphogenesis, where ephrinB2, BMP2, and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single-cell RNA-sequencing analyses have revealed that among the EPC-regulated markers in transcriptome analyses, (i) ICAM1 and Endoglin were weekly expressed in the monocyte compartment of the peripheral blood; (ii) CD163 and CD36 were highly expressed in the CD14+ monocyte compartment whereas CSF1R was highly expressed in the CD16+ monocyte compartment, (iii) L-selectin and IL6R were globally expressed in the lymphoid/myeloid compartments, and (iv) interestingly, PLAUR/UPAR and NOTCH2 were highly expressed in both CD14+ and CD16+ monocytic compartments. Conclusions The current study has identified novel EPC markers that could be used for better characterization of EPC subpopulation in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications

Post-decellularization techniques ameliorate cartilage decellularization process for tissue engineering applications

International audienceDue to the current lack of innovative and effective therapeutic approaches, tissue engineering (TE) has attracted much attention during the last decades providing new hopes for the treatment of several degenerative disorders. Tissue engineering is a complex procedure, which includes processes of decellularization and recellularization of biological tissues or functionalization of artificial scaffolds by active cells. In this review, we have first discussed those conventional steps, which have led to great advancements during the last several years. Moreover, we have paid special attention to the new methods of post-decellularization that can significantly ameliorate the efficiency of decellularized cartilage extracellular matrix (ECM) for the treatment of osteoarthritis (OA). We propose a series of post-decellularization procedures to overcome the current shortcomings such as low mechanical strength and poor bioactivity to improve decellularized ECM scaffold towards much more efficient and higher integration

Weighted correlation network analysis revealed novel long non-coding RNAs for colorectal cancer

International audienceAbstract Colorectal cancer (CRC) is one of the most prevalent cancers worldwide, which after breast, lung and, prostate cancers, is the fourth prevalent cancer in the United States. Long non-coding RNAs (lncRNAs) have an essential role in the pathogenesis of CRC. Therefore, bioinformatics studies on lncRNAs and their target genes have potential importance as novel biomarkers. In the current study, publicly available microarray gene expression data of colorectal cancer (GSE106582) was analyzed with the Limma, Geoquery, Biobase package. Afterward, identified differentially expressed lncRNAs and their target genes were inserted into Weighted correlation network analysis (WGCNA) to obtain modules and hub genes. A total of nine differentially expressed lncRNAs (LINC01018, ITCH-IT, ITPK1-AS1, FOXP1-IT1, FAM238B, PAXIP1-AS1, ATP2B1-AS1, MIR29B2CHG, and SNHG32) were identified using microarray data analysis. The WGCNA has identified several hub genes for black (LMOD3, CDKN2AIPNL, EXO5, ZNF69, BMS1P5, METTL21A, IL17RD, MIGA1, CEP19, FKBP14), blue (CLCA1, GUCA2A, UGT2B17, DSC2, CA1, AQP8, ITLN1, BEST4, KLF4, IQCF6) and turquoise (PAFAH1B1, LMNB1, CACYBP, GLO1, PUM3, POC1A, ASF1B, SDCCAG3, ASNS, PDCD2L) modules. The findings of the current study will help to improve our understanding of CRC. Moreover, the hub genes that we have identified could be considered as possible prognostic/diagnostic biomarkers. This study led to the determination of nine lncRNAs with no previous association with CRC development

Differences and similarities between mesenchymal stem cell and endothelial progenitor cell immunoregulatory properties against T cells

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Human fetal liver MSCs are more effective than adult bone marrow MSCs for their immunosuppressive, immunomodulatory, and Foxp3+ T reg induction capacity

International audienceAbstract Background Mesenchymal stem cells (MSCs) exhibit active abilities to suppress or modulate deleterious immune responses by various molecular mechanisms. These cells are the subject of major translational efforts as cellular therapies for immune-related diseases and transplantations. Plenty of preclinical studies and clinical trials employing MSCs have shown promising safety and efficacy outcomes and also shed light on the modifications in the frequency and function of regulatory T cells (T regs). Nevertheless, the mechanisms underlying these observations are not well known. Direct cell contact, soluble factor production, and turning antigen-presenting cells into tolerogenic phenotypes, have been proposed to be among possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion and activity. We and others demonstrated that adult bone marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4 + helper and CD8 + cytotoxic T cells but also indirectly through the induction of T regs. In parallel, we demonstrated that fetal liver (FL)-MSCs demonstrates much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4 + and CD8 + T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs. Methods MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation, and their proliferation potential. Using different in vitro combinations, we performed co-cultures of FL- or BM-MSCs and murine CD3 + CD25 − T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs. Results We demonstrated that although both types of MSC display similar cell surface phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4 + and CD8 + murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4 + CD25 + Foxp3 + T regs compared to BM-MSCs. Conclusions These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction

TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

International audienceBackground: In addition to their multilineage potential, mesenchymal stem cells (MSCs) have a broad range of functions from tissue regeneration to immunomodulation. MSCs have the ability to modulate the immune response and change the progression of different inflammatory and autoimmune disorders. However, there are still many challenges to overcome before their widespread clinical administration including the mechanisms behind their immunoregulatory function. MSCs inhibit effector T cells and other immune cells, while inducing regulatory T cells (T regs), thus, reducing directly and indirectly the production of pro-inflammatory cytokines. TNF/TNFR signaling plays a dual role: while the interaction of TNFα with TNFR1 mediates pro-inflammatory effects and cell death, its interaction with TNFR2 mediates anti-inflammatory effects and cell survival. Many immunosuppressive cells like T regs, regulatory B cells (B regs), endothelial progenitor cells (EPCs), and myeloid-derived suppressor cells (MDSCs) express TNFR2, and this is directly related to their immunosuppression efficiency. In this article, we investigated the role of the TNFα/TNFR2 immune checkpoint signaling pathway in the immunomodulatory capacities of MSCs.Methods: Co-cultures of MSCs from wild-type (WT) and TNFR2 knocked-out (TNFR2 KO) mice with T cells (WT and TNFα KO) were performed under various experimental conditions.Results: We demonstrate that TNFR2 is a key regulatory molecule which is strongly involved in the immunomodulatory properties of MSCs. This includes their ability to suppress T cell proliferation, activation, and pro-inflammatory cytokine production, in addition to their capacity to induce active T regs.Conclusions: Our results reveal for the first time the importance of the TNFα/TNFR2 axis as an active immune checkpoint regulating MSC immunological functions