Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets

BACKGROUND: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. METHODS: In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. RESULTS: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. CONCLUSIONS: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer

Allogeneic hematopoietic stem cell transplantation for malignant disease: how to prevent graft-versus-host disease without jeopardizing the graft-versus-tumor effect ?

The graft-versus leukemia (GVL) effect, closely related to allogeneic stem cell tranplantation, plays a crucial role in curing the disease but is often associated with a deleterious reaction, called graft-versus-host disease (GVHD). So far, most strategies aiming at reducing GVHD also diminish the GVL effect. We try here to give an overview of strategies that could reduce GVHD without affecting GVL, or augment GVL without increasing GVHD or achieve both at the same time. © 2006 Elsevier Ltd. All rights reserved.SCOPUS: re.jinfo:eu-repo/semantics/publishe

The regulatory mechanisms of oncomiRs in cancer

Cancer development is a complex process that primarily results from the combination of genetic alterations and the dysregulation of major signalling pathways due to interference with the epigenetic machinery. As major epigenetic regulators, miRNAs are central players in the control of many key tumour development factors. These miRNAs have been classified as oncogenic miRNAs (oncomiRs) when they target tumour suppressor genes and tumour suppressor miRNAs (TS miRNAs) when they inhibit oncogene protein expression. Most of the mechanisms that modulate oncomiR expression are linked to transcriptional or posttranscriptional regulation. However, non-transcriptional processes, such as gene amplification, have been described as alternative processes that are responsible for increasing oncomiR expression. The current review summarises the different mechanisms controlling the upregulation of oncomiR expression in cancer cells and the tumour microenvironment (TME). Detailed knowledge of the mechanism underlying the regulation of oncomiR expression in cancer may pave the way for understanding the critical role of oncomiRs in cancer development and progression.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Freezing of dendritic cells, generated from cryopreserved leukaphereses, does not influence their ability to induce antigen-specific immune responses or functionally react to maturation stimuli.

It is of practical clinical importance to be able to reinfuse into patients dendritic cells which have been previously frozen in aliquots. However, there are few studies comparing the function of fresh and frozen dendritic cells. We therefore decided to perform a systematic immunophenotypic and functional comparison of fresh and frozen dendritic cells. We chose to assess functional properties using proliferation tests and evaluating the preservation of specific antigens presentation in the context of MHC class II. Dendritic cells, generated from leukaphereses of normal volunteers, were loaded with proteins by a 2-h incubation at a protein concentration of 50 microg/ml, and were thereafter used fresh or after freeze-thawing in an IFNgamma Elispot assay. The IFNgamma release from antigen specific T cells was not affected by liquid nitrogen storage of pulsed immature dendritic cells. In the same way, the storage did not alter their stimulatory properties for antigen specific autologous T cells or for allogeneic CD8+ T lymphocytes in a proliferation assay. We also showed that freezing non-pulsed immature dendritic cells did not alter their capacity to capture, process and generate antigen-specific reactions once thawed, nor did it impair their capacity to acquire fully mature characteristics using CD40L and IFNgamma, with respect to immunophenotype and bioactive IL-12 secretion.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mixed autologous leukocytes reactions performed using matured CML dendritic cells and CDS or CD4 T-cells results in colony inhibition only with CD4 cultured with IL-12 throughout and is not leukemia specific

CD34+ cells were isolated from the initial aphérèses from 3 CML patients before any treatment. All these patient had the b3a2 junction and MHC complexes for which CTL generation had been described using peptides (A2, DR201 - A2, DR1101 -All, DR2). DCs were generated from cytaphereses from the same patients, harvested when they had normalized their counts to have a sufficient proportion of monocytes. DCs were generated by classical adherence enrichment followed by a 6 days culture in RPMI, autologous serum 2%, GM-CSF and IL-4. At day 6. CD40L was added to mature the DCs, which were harvested for the MLRs after 48 hrs. We first assess the functional capacity of CML DCs using immunophenotyping, allogeneic and autologous proliferative tests and specificic antigen (Influenza) presentation in short MLR followed by ELISPOT assays. We could conclude that these DCs were functionally normal. We then performed autologous MLRs: mature DCs and responder cells (ratiol/10) were cultured the first week in RPMI, autologous serum 10%, IL-12 and 1L-6. Three types of responders were used (CDS, CDS + CD4 and CD4). The mixture CD8/CD4 was used to test for a potential help] effect. In one half of the conditions, IL-12 was added throughout the 6 weeks culture which led to six different culture conditions. IL-12 was added to reverse potential tolerance. The cells were restimulated each week by the same DCs and cultured with IL2 and IL-7, +/- IL 12. At the end, T-cells from the mixed CD8/CD4 cultures were separated into CD4 and CDS cells, to assess the potential of CD8+ cells cultured in the presence of CD4+ cells. T-cells were then co-cultured with CD34-positive cells for 24 hrs with IL2, SCF, IL3 and GCS-F. They were then seeded in a soft agar CFU-GM inhibition assay. After 2 weeks, colonies were counted and individual colonies tested for BCR-ABL expression using RT-PCR. Results: a significant (50%) inhibition of the number of colonies was observed only when CD4+ cells, from the MLRs with IL-12 added throughout, were co-cultured with CD34+ cells. However, the proportion of Ph+/Ph- colonies was unchanged in comparison with the control. We conclude that in our experimental conditions, inhibition of colonies occurs only when CD4+ cells, cultured with IL-12 throughout the whole MLR, are used. This inhibition is not leukemic specific and seems to be directed against autologous hematological determinants. None of our patients being HLA A3 or B8 positive, the absence of CDS response should be interpreted with caution. However, our observations should lead to careful reflection when designing immunotherapy protocols in CML patients.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.

Dendritic cells (DC) are professional antigen-presenting cells that play a central role in the control of immunity. Mature DC are characterized by high expression levels of MHC and co-stimulatory molecules, and by the secretion of IL-12, a key cytokine for the priming of cytotoxic T lymphocytes. Here, we have compared different maturation stimuli to reproducibly generate stable mature DC secreting high amounts of bioactive IL-12p70. We have compared soluble human trimeric CD40 ligand (sCD40L) combined with IFN-gamma, poly(I:C), a cocktail of cytokines (IL-1beta, IL-6 and tumor necrosis factor-alpha) with prostaglandin E(2) and lipopolysaccharide. A major concern, however, is whether DC, that have already produced high amounts of IL-12p70 during the maturation step, are still capable of secreting IL-12p70 after in vivo administration at the time of interaction with the targeted T cells. To mimic that situation, mature DC generated by those methods were compared for their ability to secrete IL-12p70 in the absence of IFN-gamma, using sCD40L. We observed a second consistent secretion of bioactive IL-12p70 upon subsequent sCD40L stimulation only when poly(I:C) was used as the maturating agent. Our data suggest that, for clinical use, poly(I:C) may be one of the most appropriate agents to generate stable mature DC. These mature DC might generate in vivo effective immune responses after injection, because they retain the ability to secrete bioactive IL-12 after CD40 ligation.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Acute myeloid leukemia-derived exosomes deliver miR-24-3p to hinder the T-cell immune response through DENN/MADD targeting in the NF-κB signaling pathways

Abstract Background microRNAs (miRNAs) are known as potent gene expression regulators, and several studies have revealed the prognostic value of miRNAs in acute myeloid leukemia (AML) patient survival. Recently, strong evidence has indicated that miRNAs can be transported by exosomes (EXOs) from cancer cells to recipient immune microenvironment (IME) cells. Results We found that AML blast-released EXOs enhance CD3 T-cell apoptosis in both CD4 and CD8 T cells. We hypothesized that miRNAs present in EXOs are key players in mediating the changes observed in AML T-cell survival. We found that miR-24-3p, a commonly overexpressed miRNA in AML, was present in released EXOs, suggesting that EXO-miR-24-3p was linked to the increased miR-24-3p levels detected in isolated AML T cells. These results were corroborated by ex vivo-generated miR-24-3p-enriched EXOs, which showed that miR-24-3p-EXOs increased apoptosis and miR-24-3p levels in T cells. We also demonstrated that overexpression of miR-24-3p increased T-cell apoptosis and affected T-cell proliferation by directly targeting DENN/MADD expression and indirectly altering the NF-κB, p-JAK/STAT, and p-ERK signaling pathways but promoting regulatory T-cell (Treg) development. Conclusions These results highlight a mechanism through which AML blasts indirectly impede T-cell function via transferred exosomal miR-24-3p. In conclusion, by characterizing the signaling network regulated by individual miRNAs in the leukemic IME, we aimed to discover new nonleukemic immune targets to rescue the potent antitumor function of T cells against AML blasts. Video Abstrac

Humanized Mice as a Valuable Pre-Clinical Model for Cancer Immunotherapy Research.

Immunotherapy with checkpoint inhibitors opened new horizons in cancer treatment. Clinical trials for novel immunotherapies or unexplored combination regimens either need years of development or are simply impossible to perform like is the case in cancer patients with limited life expectancy. Thus, the need for preclinical models that rapidly and safely allow for a better understanding of underlying mechanisms, drug kinetics and toxicity leading to the selection of the best regimen to be translated into the clinic, is of high importance. Humanized mice that can bear both human immune system and human tumors, are increasingly used in recent preclinical immunotherapy studies and represent a remarkably unprecedented tool in this field. In this review, we describe, summarize, and discuss the recent advances of humanized mouse models used for cancer immunotherapy research and the challenges faced during their establishment. We also highlight the lack of preclinical studies using this model for radiotherapy-based research and argue that it can be a great asset to understand and answer many open questions around radiation therapy such as its presumed associated "abscopal effect".info:eu-repo/semantics/publishe

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe