Stéphane Fouquet

Early T Cell Signalling Is Reversibly Altered in PD-1+ T Lymphocytes Infiltrating Human Tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Progressive Upregulation of PD-1 in Primary and Metastatic Melanomas Associated with Blunted TCR Signaling in Infiltrating T Lymphocytes

Programmed death-1 (PD-1) is involved in T-cell tolerance to self-antigens. For some cancers, it has been suggested that the expression of a ligand of PD-1, namely PD-L1, could contribute to tumor escape from immune destruction. Nevertheless, the relationship between PD-1 expression on tumor-infiltrating T lymphocytes (TILs), disease stage, and TIL responsiveness is still poorly documented. In this study, we show that freshly isolated CD4(+) and CD8(+) TILs express substantial levels of PD-1 in primary melanomas. The expression of PD-1 was further increased at later stages in distant cutaneous metastases, especially on CD8(+) TILs. The expression of PD-1 ligands was frequent only in metastases, on both tumor cells and tumor-derived myeloid cells. TILs isolated from these cutaneous tumors are poorly reactive ex vivo, with blunted calcium response and IFN-γ production after TCR stimulation. Surprisingly, in distinct parts of a primary melanoma, either invasive or regressing, we show that TILs similarly express PD-1 and remain dysfunctional. The expressions of PD-1 and PD-L1 in metastatic melanoma lesions could be considered as witnesses of an unsuccessful anti-tumoral immune response, but the direct involvement of PD-1 in the severity of the disease, and the importance of TILs in tumor regression, remain to be established.

The Cellular Prion Protein PrPc Is Involved in the Proliferation of Epithelial Cells and in the Distribution of Junction-Associated Proteins

Background The physiological function of the ubiquitous cellular prion protein, PrPc, is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrPc is targeted to cell–cell junctions of polarized epithelial cells, where it interacts with c-Src. Methodology/Findings We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrPc is differentially targeted either to the nucleus in dividing cells or to cell–cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrPc interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrPc, desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrPc is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrPc knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. Conclusions/Significance From these results, PrPc could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.

Taurine deficiency damages photoreceptors and retinal ganglion cells in vigabatrin-treated neonatal rats

The anti-epileptic drug vigabatrin induces an irreversible constriction of the visual field, but is still widely used to treat infantile spasms and some forms of epilepsy. We recently reported that vigabatrin-induced cone damage is due to a taurine deficiency. However, optic atrophy and thus retinal ganglion cell degeneration was also reported in children treated for infantile spasms. We here show in neonatal rats treated from postnatal days 4 to 29 that the vigabatrin treatment triggers not only cone photoreceptor damage, disorganisation of the photoreceptor layer and gliosis but also retinal ganglion cell loss. Furthermore, we demonstrate in these neonatal rats that taurine supplementation partially prevents these retinal lesions and in particular the retinal ganglion cell loss. These results provide the first evidence of retinal ganglion cell neuroprotection by taurine. They further confirm that taurine supplementation should be administered with the vigabatrin treatment for infantile spasms or epilepsy.

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone‐treated mice. The analysis of the optic nerve and anterior commissure of Sema6A‐deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild‐type oligodendrocytes. In myelinating co‐cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation.

Src family kinases involved in CXCL12-induced loss of acute morphine analgesia.

Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occurred in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu-(MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1μg) significantly attenuated the subcutaneous morphine (4mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5μg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10μg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12-induced loss of acute morphine analgesia is linked to Src family kinases activation.

Multicolor analysis of oligodendrocyte morphology, interactions, and development with Brainbow

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single‐out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre‐lox recombination, previously used for visualizing axons and neurons. We used tamoxifen‐inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single‐cell resolution. GLIA 2015;63:699–717

Mutations in GREB1L Cause Bilateral Kidney Agenesis in Humans and Mice

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l-/- embryos and a slight decrease in ureteric bud branching in Greb1l+/- embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8

Recently, we described a modified AAV2 vector—AAV2‐7m8—having a capsid‐displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high‐level gene delivery into deep layers of the retina when virus is delivered into the eyes vitreous. Here, we further characterize AAV2‐7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2‐7m8 and AAV9‐7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV. Biotechnol. Bioeng. 2016;113: 2712–2724.