Are human V\u3b42pos T cells really resistant to aging and Human Cytomegalovirus infection?

In their recent paper, Weili Xu et al. [1] described the different behaviors of V\u3b41pos and V\u3b42pos T cell subsets in response to lifelong stress and claimed that V\u3b42pos T cells are not affected by aging and Human Cytomegalovirus (HCMV) infection. While we agree that these two \u3b3\u3b4 T cell subsets diverge both in phenotype/function and in tissue distribution, we are somewhat surprised that authors did not take into account the several previously published and contradictory experimental evidence in regards to senescence of V\u3b42pos T cells [2,3]. These latter studies reported that HCMV infection not only induces a clonal expansion of a distinct V\u3b39neg/V\u3b42pos T cell subset, but also determines a concomitant adaptive differentiation from CD27high na\uefve cells to CD27low/neg terminal-effectors. However, Weili Xu et al. argued that the expression and kinetics of both CD27 and CD45RA surface markers do not change and follow the homeostatic changes of V\u3b42pos T cells. This statement goes in the opposite direction to previously reported findings as the CD27/CD45RA phenotype has been shown to mark the maturation and differentiation (TNa\uefve, TCentral-Memory, Teffector-Memory and TEffectory-Memory RA) of V\u3b42pos T cells. Indeed, the different surface expression of both CD27 and CD45 parallel the progressive decrease of telomere length, the proliferative capacity as well as the different effector-functions and resistance to death of V\u3b42+ T cells in response to antigens and homeostatic cytokines [4,5]. Hence, we believe that these controversial issues require further discussion beyond the unilateral conclusion given by the study of Weili Xu et al

Chemotherapy accelerates immune-senescence and functional impairments of Vδ2pos T cells in elderly patients affected by liver metastatic colorectal cancer.

Human (gamma delta) γδ T cells are unconventional innate-like lymphocytes displaying a broad array of anti-tumor activities with promising perspectives in cancer immunotherapy. In this context, Vδ2pos T cells represent the preferential target of several immunotherapy protocols against solid tumors. However, the impact of both aging and chemotherapy (CHT) on Vδ2pos T cells is still unknown. The present study evaluates with multi-parametric flow cytometry the frequencies, terminal differentiation, senescence and effector-functions of peripheral blood and tumor infiltrating Vδ2pos T cells purified from liver metastases (CLM) of patients affected by colorectal cancer (CRC) compared to those of sex- and age-matched healthy donors. The peripheral blood of CLM patients underwent CHT is characterized by decreased amounts of Vδ2pos T cells showing a relative increase of terminally-differentiated CD27neg/CD45RApos (TEMRA) cells. The enrichment of this latter subset is associated with an increased expression of the senescent marker CD57. The acquisition of CD57 on TEMRA Vδ2pos T cells is also coupled with impairments in cytotoxicity and production of TNF-α and IFN-γ. These features resemble the acquisition of an immune-senescent profile by Vδ2pos T cells from CLM patients that received CHT, a phenomenon that is also associated with the loss of the co-stimulatory marker CD28 and with the induced expression of CD16. The group of CLM patients underwent CHT and older than 60 years old showed higher frequencies of CD57pos and TEMRA Vδ2pos T cells. Similar results were found for tumor infiltrating Vδ2pos T cell subset purified from CLM specimens of patients treated with CHT. The toxicity of CHT regimens also affects the homeostasis of Vδ2pos T cells by inducing higher frequencies of circulating CD57pos TEMRA subset in CLM underwent CHT and younger than 60 years old. Taken together, our data demonstrate that the enrichment of senescent Vδ2pos T cells in CLM patients is not only induced by patients' aging but also by the toxicity of CHT that further accelerates the accumulation of CD57pos TEMRA cells highly dysfunctional in their anti-tumor activities. These results are important to both predict the clinical outcome of CLM and to optimize those protocols of cell cancer immunotherapy employing unconventional Vδ2pos T cells

Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity \u2018\u2018sandwich\u2019\u2019 (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy application

Developing gene and cell therapies for rare diseases: An opportunity for synergy between academia and industry

For the last 20 years, academic research has been the major, and often only, driving force behind the spectacular development of gene transfer technology for the therapy of rare genetic diseases. Investors and industry became eventually interested in gene and cell therapy, due to the success of a series of pioneering clinical trials that proved efficacy and safety of last-generation technology, and to favorable orphan drug legislation in both Europe and the United States. Developing this forms of therapy is however complex and requires skills and knowledge not necessary available to the industry, which is better placed to develop processes and products and put them on the market. Cooperation between academia and industry is an opportunity to de-risk innovative approaches and ensure a faster and more economical development of therapies for diseases with high unmet medical needs and low-profit expectations

Gene Therapy For Inherited Blood Diseases, From Viral Vectors To Gene Editing

Twenty-five years ago, genetically modified bone marrow cells were administered for the first time to a child suffering from adenosine deaminase (ADA) deficiency, a rare disorder of the immune system. Since then, gene therapy has struggled to find its place in clinical medicine, amid a rollercoaster of successes and setbacks and hype and skepticism with little precedent in modern times. Recently, a series of authoritative clinical studies proved that transplantation of genetically modified hematopoietic stem cells can cure severe diseases like immunodeficiencies, hemoglobinopathies and metabolic diseases, contributing to transforming gene therapy into one of the hottest area of investment for the biotechnology and the pharma industry. The basic technology for the genetic modification of stem cells relies on retroviral vectors, and particularly on those derived from oncoretroviruses or lentiviruses, such as HIV-1. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. Addressing these issues brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. More recently, a new generation of technology emerged, aimed at correcting the genome rather than replacing defective gene function. This technology relies on designer nucleases capable of generating double- or single-stranded breaks in genomic DNA, which are then repaired either by error-prone non homologous end-joining or by the more precise homologous recombination. This allows generating knock-out mutations or repairing genes with remarkable precision and efficiency in many cell types. At Genethon, we are using lentiviral vector technology to correct Wiskott-Aldrich syndrome, X-linked SCID, chronic granulomatous disease and sickle-cell disease, while developing CRISPR/Cas9-based genome editing for a number of applications

Genome-wide definition of promoter and enhancer usage during neural induction of human embryonic stem cells

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate

TIMP3 interplays with apelin to regulate cardiovascular metabolism in hypercholesterolemic mice

Tissue inhibitor of metalloproteinase 3 (TIMP3) is an extracellular matrix (ECM) bound protein, which has been shown to be downregulated in human subjects and experimental models with cardiometabolic disorders, including type 2 diabetes mellitus, hypertension and atherosclerosis. The aim of this study was to investigate the effects of TIMP3 on cardiac energy homeostasis during increased metabolic stress conditions

Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of alpha v beta 3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with alpha v beta 3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR

Interactions between Retroviruses and the Host Cell Genome

Replication-defective retroviral vectors have been used for more than 25 years as a tool for efficient and stable insertion of therapeutic transgenes in human cells. Patients suffering from severe genetic diseases have been successfully treated by transplantation of autologous hematopoietic stem-progenitor cells (HSPCs) transduced with retroviral vectors, and the first of this class of therapies, Strimvelis, has recently received market authorization in Europe. Some clinical trials, however, resulted in severe adverse events caused by vector-induced proto-oncogene activation, which showed that retroviral vectors may retain a genotoxic potential associated to proviral integration in the human genome. The adverse events sparked a renewed interest in the biology of retroviruses, which led in a few years to a remarkable understanding of the molecular mechanisms underlying retroviral integration site selection within mammalian genomes. This review summarizes the current knowledge on retrovirus-host interactions at the genomic level, and the peculiar mechanisms by which different retroviruses, and their related gene transfer vectors, integrate in, and interact with, the human genome. This knowledge provides the basis for the development of safer and more efficacious retroviral vectors for human gene therapy