Mice reconstituted with a human immune system and bearing human tumors represent a promising model for developing novel cancer immunotherapies. Here, we used mass cytometry and multi-parametric flow cytometry to characterize human leukocytes infiltrating a human breast cancer tumor model in immunocompromised NOD.SCID.γc-null mice reconstituted with a human immune system and compared it to samples of breast cancer patients. We observed highly activated human CD4 and CD8 T cells in the tumor, as well as minor subsets of innate immune cells in both settings. We also report that ICOS CD4 regulatory T cells (Treg) were enriched in the tumor relative to the periphery in humanized mice and patients, providing a target to affect Treg and tumor growth. Indeed, administration of a neutralizing mAb to human ICOS reduced Treg proportions and numbers and improved CD4 + T cell proliferation in humanized mice. Moreover, a combination of the anti-ICOS mAb with cyclophosphamide reduced tumor growth, and that was associated with an improved CD8 to Treg ratio. Depletion of human CD8 T cells or of murine myeloid cells marginally affected the effect of the combination therapy. Altogether, our results indicate that a combination of anti-ICOS mAb and chemotherapy controls tumor growth in humanized mice, opening new perspectives for the treatment of breast cancer. One sentence summary: Targeting ICOS in combination with chemotherapy is a promising strategy to improve tumor immunity in humans.
MAIT cells are an evolutionarily conserved T cell subset recognizing ubiquitous microbial metabolites. Herein, we review recent literature showing that MAIT cells can be divided into type 1 and type 17 subsets, which acquire a tissue resident differentiation program in the thymus and localize in specific tissues. We also discuss the nature and in vivo availability of the different agonist and antagonist MAIT ligands with potential consequences for MAIT cell biology.
CD4 T follicular helper (Tfh) cells are essential for inducing efficient humoral responses. T helper polarization is classically orientated by dendritic cells (DCs), which are composed of several subpopulations with distinct functions. Whether human DC subsets display functional specialization for Tfh polarization remains unclear. Here we find that tonsil cDC2 and CD14 macrophages are the best inducers of Tfh polarization. This ability is intrinsic to the cDC2 lineage but tissue dependent for macrophages. We further show that human Tfh cells comprise two effector states producing either IL-21 or CXCL13. Distinct mechanisms drive the production of Tfh effector molecules, involving IL-12p70 for IL-21 and activin A and TGFβ for CXCL13. Finally, using imaging mass cytometry, we find that tonsil CD14 macrophages localize in situ in the B cell follicles, where they can interact with Tfh cells. Our results indicate that human lymphoid organ cDC2 and macrophages play complementary roles in the induction of Tfh responses.
The aim of this study was to show, for the first time, the effect of a hypercaloric diet on the mitochondrial reshuffle of hepatocytes during the progression from steatosis to steatohepatitis to cirrhosis in Psammomys obesus, a typical animal model of the metabolic syndrome. Metabolic and oxidative stresses were induced by feeding the animal through a standard laboratory diet (SD) for nine months. Metabolic parameters, liver malondialdehyde (MDA) and glutathione (GSH), were evaluated. The pathological evolution was examined by histopathology and immunohistochemistry, using CD3 and CD20 antibodies. The dynamics of the mitochondrial structure was followed by transmission electron microscopy. SD induced a steatosis in this animal that evolved under the effect of oxidative and metabolic stress by the appearance of adaptive inflammation and fibrosis leading the animal to the cirrhosis stage with serious hepatocyte damage by the triggering, at first the mitochondrial fusion-fission cycles, which attempted to maintain the mitochondria intact and functional, but the hepatocellular oxidative damage was increased inducing a vicious circle of mitochondrial alteration and dysfunction and their elimination by mitophagy. P. obesus is an excellent animal model of therapeutic research that targets mitochondrial dysfunction in the progression of steatosis.
Fluorescent polymersomes with both aggregation-induced emission (AIE) and CO2-responsive properties were developed from amphiphilic block copolymer PEG-b-P(DEAEMA-co-TPEMA) in which the hydrophobic block was a copolymer made of tetraphenylethene functionalized methacrylate (TPEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) with unspecified sequence arrangement. Four block copolymers with different DEAEMA/TPEMA and hydrophilic/hydrophobic ratios were synthesized and bright AIE polymersomes were prepared by nanoprecipitation in THF/water and dioxane/water systems. Polymersomes of PEG45-b-P(DEAEMA36-co-TPEMA6) were chosen to study the CO2-responsive property. Upon CO2 bubbling vesicles transformed to small spherical micelles, and upon Ar bubbling micelles returned to vesicles with the presence of a few intermediate morphologies. These polymersomes might have promising applications as sensors, nanoreactors or controlled release systems.
Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient’s tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM.
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.
In neuroblastoma (NB), genetic alterations in chromatin remodeling (CRGs) and epigenetic modifier genes (EMGs) have been described. We sought to determine their frequency and clinical impact. Whole exome (WES)/whole genome sequencing (WGS) data and targeted sequencing (TSCA®) of exonic regions of 33 CRGs/EMGs were analyzed in tumor samples from 283 NB patients, with constitutional material available for 55 patients. The frequency of CRG/EMG variations in NB cases was then compared to the Genome Aggregation Database (gnomAD). The sequencing revealed SNVs/small InDels or focal CNAs of CRGs/EMGs in 20% (56/283) of all cases, occurring at a somatic level in 4 (7.2%), at a germline level in 12 (22%) cases, whereas for the remaining cases, only tumor material could be analyzed. The most frequently altered genes were ATRX (5%), SMARCA4 (2.5%), MLL3 (2.5%) and ARID1B (2.5%). Double events (SNVs/small InDels/CNAs associated with LOH) were observed in SMARCA4 (n=3), ATRX (n=1) and PBRM1 (n=1). Among the 60 variations, 24 (8.4%) targeted domains of functional importance for chromatin remodeling or highly conserved domains but of unknown function. Variations in SMARCA4 and ATRX occurred more frequently in the NB as compared to the gnomAD control cohort (OR=4.49, 95%CI:1.63-9.97, P=0.038; OR 3.44, 95%CI:1.46-6.91, P=0.043, respectively). Cases with CRG/EMG variations showed a poorer overall survival compared to cases without variations. Genetic variations of CRGs/EMGs with likely functional impact were observed in 8.4% (24/283) of NB. Our case-control approach suggests a role of SMARCA4 as a player of NB oncogenesis. This article is protected by copyright. All rights reserved.
The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other’s TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.
G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.
Procedure for extra training
You are already autonomous on one or more microscopes and want to either have additional training or become autonomous on a new system. You must request a new training by following the step described in Access & Prices.
Training on an imaging system is done in four parts:
- Training which includes theoretical description of the system and the reminder of the basics of microscopy,
- A theoretical safety regulations training specific to the imaging centre, including if required laser safety and procedures for evacuations,
- A practical training including among others the start/stop procedure of the system, the acquisition of 2D images up to 4D if necessary, data backup, data transfer, procedures for cleaning, booking systems.
- A check of individual knowledge. The user is supervised during his first booking.
The duration of the training may vary according to the systems and knowledge of users. Training can be done individually or in small group of 3-4 people.
The booking system can only be done by users who have validated training. Each system requires proper training and authorization from a PICT team member. No exceptions will be accepted.
Once your training is confirmed, you can access the online booking system. To book a system, follow the procedure :
- Open a web browser (Firefox, Safari, etc.)
- go to the link: http://iris.science-it.ch/
- Click Sign-in
- Go to Scheduler and filter to find the resource you would like to book
- Select the timeslot you want to book.
Respect the rules of booking. Each system have their own booking restriction.
- Cancellation can be done anytime but Deletion are only allowed 24 hours in advance.
- Respect the material and other users !!
At the end of the session, the item must be returned in excellent condition:
- For microscopes: data should be transferred and removed, bench and microscope should be fully cleaned.
- For image processing computer: data should be transferred and removed, turn off the PC.
You are in charge of the system during all your session and you have to declare any problems that you could encounter. You can declare an incident online using OpenIRIS.
Feel free to give us any feedback concerning imaging systems.
Please report any incident by using the incident form in http://iris.science-it.ch/or if urgent matter contact the PICT staff in charge of the microscope. Thank you in advance for your participation in order to maintain the system in perfect working condition
The mission of the imaging team is to guide and assist users in acquiring and analyzing images. Thus, it’s not required to include any staff member of the imaging centre in the author list of the publication. However, when a staff member of the imaging centre has significantly contributed to a publication, it is logical and highly recommended to add this person in the list of authors.
Mention of the imaging centre in the publication
Users must indicate in the acknowledgment section that they have used the PICT imaging centre in any publication.
For example, “The authors wish to thank [xxx] from the PICT-IBiSA Imaging facility of Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR-10-INBS-04)”
List of publications
Users must submit, as soon as possible, the references of any publications that have used the equipment of the imaging centre. These informations highlight the use of the equipment of the imaging centre, the efficiency of investment and facilitate all requests for funding for the acquisition of new microscopes.
The Institut Curie IT provides storage space for images. Please contact the IT department for any request. Your data must be saved after each session with your care.
Attention, we are not responsible for any data stored on machines connected to the system. User data will be automatically deleted after a few days. This ensures that there is always storage space for subsequent experiments. In addition, computers of the imaging centre belong to the Institut Curie and are therefore subject to the use of the IT. Moreover, it is strictly forbidden to:
- Install or uninstall software
- To use computers for personal purpose
- Using computers to download music, video, etc …
Do assisted reproductive technologies alter DNA methylation and/or transcription of transposable elements and imprinted genes in cord blood and placenta?
The discovery of genomic imprinting by Davor Solter, Azim Surani and co-workers in the mid-1980s has provided a foundation for the study of epigenetic inheritance and the epigenetic control of gene activity and repression, especially during development. It also has shed light on a range of diseases, including both rare genetic disorders and common diseases. This article is being published to celebrate Solter and Surani receiving a 2018 Canada Gairdner International Award “for the discovery of mammalian genomic imprinting that causes parent-of-origin specific gene expression and its consequences for development and disease”.
During early mammalian development, the chromatin landscape undergoes profound transitions. The gene-involved in growth control-provides a valuable model to study this window: upon exit from naïve pluripotency and prior to tissue differentiation, it undergoes a switch from a distal to a proximal promoter usage, accompanied by a switch from polycomb to DNA methylation occupancy. Using a mouse embryonic stem cell (ESC) system to mimic this period, we show here that four enhancers contribute to the promoter switch, concomitantly with dynamic changes in chromatin architecture. In ESCs, the locus is partitioned to facilitate enhancer contacts with the distal promoter. Relieving the partition enhances proximal promoter activity, as observed during differentiation or with genetic mutants. Importantly, we show that 3D regulation occurs upstream of the polycomb and DNA methylation pathways. Our study reveals the importance of multi-layered regulatory frameworks to ensure proper spatio-temporal activation of developmentally important genes.
Do assisted reproductive technologies (ARTs) impact on the expression of transposable elements (TEs) in preimplantation embryos?