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The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue‐specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA‐G molecule. Considering the immunomodulatory properties of Aire and HLA‐G, and considering that the role of the constitutive thymus expression of HLA‐G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA‐G expression in 4D6 thymic cells and in the HLA‐G‐positive JEG‐3 choriocarcinoma cells. Aire promoted the transactivation of HLA‐G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA‐G protein solely in 4D6 thymic cells. Luciferase‐based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5′‐untranslated regulatory region (5′‐URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire‐binding sites. These results indicate that the Aire‐induced upregulation of HLA‐G in thymic cells is likely to act through the interaction of Aire with specific HLA‐G 5′‐URR DNA‐binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G – cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

The HLA-G 5’URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5′URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5′URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5′URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5′URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-β and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5′URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5′URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

Background: Umbilical mesenchymal stem/stromal cells (MSCs), and especially those derived from Wharton’s jelly (WJ), are a promising engineering tool for tissue repair in an allogeneic context. This is due to their differentiation capacity and immunological properties, like their immunomodulatory potential and paracrine activity. Hence, these cells may be considered an Advanced Therapy Medicinal Product (ATMP). The purpose of this work was to differentiate MSCs from WJ (WJ-MSCs) into chondrocytes using a scaffold and to evaluate, in vitro, the immunomodulatory capacities of WJ-MSCs in an allogeneic and inflammatory context, mimicked by IFN-γ and TNF-α priming during the chondrogenic differentiation. Methods: Scaffolds were made from hydrogel composed by alginate enriched in hyaluronic acid (Alg/HA). Chondrogenic differentiation, immunological function, phenotype expression, but also secreted soluble factors were the different parameters followed during 28 days of culture. Results: During chondrocyte differentiation, even in an allogeneic context, WJ-MSCs remained unable to establish the immunological synapse or to induce T cell alloproliferation. Moreover, interestingly, paracrine activity and functional immunomodulation were maintained during cell differentiation. Conclusion: These results show that WJ-MSCs remained hypoimmunogenic and retained immunomodulatory properties even when they had undergone chondrocyte differentiation.

// Layale Yaghi 1, 2, 3 , Isabelle Poras 1, 2 , Renata T. Simoes 1, 2, 4 , Eduardo A. Donadi 5 , Jorg Tost 6, 7 , Antoine Daunay 6 , Bibiana Sgorla de Almeida 1, 2, 5 , Edgardo D. Carosella 1, 2 , Philippe Moreau 1, 2 1 Commissariat a l’Energie Atomique et aux Energies Alternatives, Institut des Maladies Emergentes et des Therapies Innovantes, Service de Recherches en Hemato-Immunologie, Hopital Saint-Louis, Paris, France 2 Universite Paris-Diderot, Sorbonne Paris-Cite, UMR E5, Institut Universitaire d’Hematologie, Hopital Saint-Louis, Paris, France 3 Lebanese University, School of Medicine, Hadath, Lebanon 4 Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, IEP/SCBH, Belo Horizonte, Minas Gerais, Brasil 5 Divisao de Imunologia Clinica, Departamento de Clinica Medica, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo, Brasil 6 Centre d’Etude du Polymorphisme Humain, Fondation Jean-Dausset, Laboratory for Functional Genomics, Paris, France 7 Commissariat a l’Energie Atomique et aux Energies Alternatives, Centre National de Genotypage, Laboratory for Epigenetics and Environment, Evry, France Correspondence to: Philippe Moreau, email: philippe.moreau@cea.fr Keywords: HLA-G, HIF-1, exon 2 HRE, glioma Received: July 02, 2016 Accepted: August 20, 2016 Published: August 26, 2016 ABSTRACT HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G . We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2’deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G . Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at –966 bp in the 5’UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

The HLA-G 5’URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5′URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5′URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5′URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5′URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-β and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5′URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5′URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

Made available in DSpace on 2018-12-11T17:23:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The immune checkpoint HLA-G prevents maternal rejection of the fetus and contributes in cancer invasion and acceptance of allografts. The 5′ and 3′ regulatory regions of the HLA-G gene are polymorphic and balancing selection probably maintains this variability. It is proposed that nucleotide variations may affect the level of HLA-G expression. To investigate this issue we aimed to analyze how haplotypes of the 3′ untranslated region (3′UTR) with highest worldwide frequencies, namely UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18 and UTR-7, impact the expression of a luciferase reporter gene in vitro. Experiments performed with the HLA-G positive cell lines JEG-3 (choricarcinoma) and FON (melanoma), and with the HLA-G negative cell lines M8 (melanoma) and U251MG (glioblastoma) showed that the HLA-G 3′UTR polymorphism influences the response to endogenous cellular factors and may vary according to the cell type. UTR-5 and UTR-7 impact the activity of luciferase the most whereas UTR-2, UTR-3, UTR-4, and UTR-18 have intermediate impact, and UTR-1 has the lowest impact. These results corroborate the previous associations between amounts of plasma sHLA-G levels and 3′UTR haplotypes in healthy individuals and reinforce that 3′UTR typing may be a predictor of the genetic predisposition of an individual to express different levels of HLA-G. Commissariat à l'Energie Atomique et aux Energies Alternatives Institut des Maladies Emergentes et des Thérapies Innovantes Service de Recherches en Hémato-Immunologie Hôpital Saint-Louis Université Paris-Diderot Sorbonne Paris-Cité UMR-E5 Institut Universitaire D'Hématologie Hôpital Saint-Louis Lebanese University School of Medicine Programa de Pósgraduação em Ciências da Saúde Centro de Ciências da Saúde Universidade Federal do Rio Grande do Norte Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto Universidade de São Paulo Departamento de Biologia Celular Embriologia e Genética Centro de Ciências Biológicas Universidade Federal de Santa Catarina Department of Genetics Ribeirão Preto Medical School University of São Paulo Divisão de Imunologia Clínica Departamento de Clínica Médica Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo Department of Pathology School of Medicine Universidade Estadual Paulista Unesp Department of Pathology School of Medicine Universidade Estadual Paulista Unesp CAPES: 014747/2013-8 FAPESP: 2014/18730-9 CNPq: 406594/2013-9

Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2′-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2′-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G–negative tumors.

Mouse/human somatic cell hybrids constitute a valuable resource for both genetic and physical mapping. In this report, we describe the production and characterization of a series of six monochromosomal hybrids generated by fusion of murine micro-cells with intact human recipient cells. The presence of each mouse chromosome was characterized by PCR analysis and the integrity of the mouse chromosome retained in the hybrids confirmed by fluorescence in situ hybridization (FISH) analysis.

Made available in DSpace on 2014-12-03T13:10:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-10-25Bitstream added on 2014-12-03T13:24:37Z : No. of bitstreams: 1 WOS000326155400002.pdf: 307856 bytes, checksum: 70a96b7f354c2328e9f6fc73981f14f0 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Commissariat a L'Energie Atomique (CEA), France Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ANR Agence Nationale de la Recherche Deutsche Krebshilfe HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5' and 3' untranslated (3' UTR) regions. At least three 3' UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3' UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3' UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P < 0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P

Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2′-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2′-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G–negative tumors.

To facilitate the identification of mouse metaphase chromosomes by fluorescence in situ hybridization (FISH), a complete collection of mouse chromosome-specific markers has been established. Yeast artificial chromosome libraries were screened by polymerase chain reaction using primers for known loci. DNAs from positive clones were then tested by FISH. One probe per chromosome was selected on the basis of high specificity (nonchimerism) and strong fluorescence.

Non-classical HLA-G and HLA-E molecules have well-recognized immunomodulatory effects on peripheral blood immune-competent cells; however, little is known regarding the effect of these molecules in central tolerance. Aire is responsible for promiscuous gene expression of tissue-related antigens by thymic cells during induction of central tolerance. We characterized the differential transcript profiles of murine MHC class Ib (Qa-1/HLA-E, Qa-2/HLA-G) and Aire genes during ontogeny of thymus, and identified that the transcriptional profile of the functional homolog of HLA-G, (H2-Q7/Qa2) was positively correlated with Aire during thymus fetal development, indicating that non-classical MHC class I genes could be under the transcriptional control of Aire. This study aimed to evaluate the transcriptional and post-transcriptional relation between Aire and HLA-G. Using previously validated Aire plasmid-based constructs, we transfected different cell-lines: HLA-G + Cells (JEG3), HLA-G- Cells (U251) and thymic cells (4D6 and LT-TEC2), and assessed HLA-G gene and protein expression. Using an in silico approach, we found 4 putative binding sites for Aire-complex at the 5’-promoter region of the HLA-G gene. We cloned, selected (enzyme digestion: EcoRI/HindIII) and validated (sequencing/alignment) the bacterial-vectors containing Aire. Initially, we observed that only JEG-3 cells were HLA-G + with transcript levels being 10.000-fold greater than the other cell lines and 50% of cell population presenting HLA-G in cell surface. After transient transfection, HLA-G transcript levels were increased only in 4D6 and LT-TEC2 cells (P = 0.03 and P = 0.007, respectively) compared to control (empty-vector). We did not observe any modification in the HLA-G cell surface expression in all cell lines. Concluding, Aire may play a role in the transcriptional regulation of HLA-G, which appears to be cell type-dependent, i. e., primarily inducted in thymic cell lines.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done1. Species for which radiation hybrid–based transcript maps have been established include human2, rat3,4,5, mouse6, dog7, cat8 and zebrafish9,10. We have established a comprehensive first-generation–placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Retinal neurons of virtually every type are coupled by gap-junctional channels whose pharmacological and gating properties have been studied extensively. We have begun to identify the molecular composition and functional properties of the connexins that form these 'electrical synapses,' and have cloned several that constitute a new subclass (gamma) of the connexin family expressed predominantly in retina and brain. In this paper, we present a series of experiments characterizing connexin36 (Cx36), a member of the gamma subclass that was cloned from a mouse retinal cDNA library. Cx36 has been localized to mouse chromosome 2, in a region syntenic to human chromosome 5, and immunocytochemistry showed strong labeling in the ganglion cell and inner nuclear layers of the mouse retina. Comparison of the developmental time course of Cx36 expression in mouse retina with the genesis of the various classes of retinal cells suggests that the expression of Cx36 occurs primarily after cellular differentiation is complete. Because photic stimulation can affect the gap-junctional coupling between retinal neurons, we determined whether lighting conditions might influence the steady state levels of Cx36 transcript in the mouse retina. Steady-state levels of Cx36 transcript were significantly higher in animals reared under typical cyclic-light conditions; exposure either to constant darkness or to continuous illumination reduced the steady-state level of mRNA approximately 40%. Injection of Cx36 cRNA into pairs of Xenopus oocytes induced intercellular conductances that were relatively insensitive to transjunctional voltage, a property shared with other members of the gamma subclass of connexins. Like skate Cx35, mouse Cx36 was unable to form heterotypic gap-junctional channels when paired with two other rodent connexins. In addition, mouse Cx36 failed to form voltage-activated hemichannels, whereas both skate and perch Cx35 displayed quinine-sensitive hemichannel activity. The conservation of intercellular channel gating contrasts with the failure of Cx36 to make hemichannels, suggesting that the voltage-gating mechanisms of hemichannels may be distinct from those of intact intercellular channels.

Background Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein. Results We have cloned the murine homologue of CTNS, Ctns, and the encoded amino acid sequence is 92.6% similar to cystinosin. We localised Ctns to mouse chromosome 11 in a region syntenic to human chromosome 17 containing CTNS. Ctns is widely expressed in all tissues tested with the exception of skeletal muscle, in contrast to CTNS. Conclusions We have isolated, characterised and localised Ctns, the murine homologue of CTNS underlying cystinosis. Furthermore, our work has brought to light the existence of a differential pattern of expression between the human and murine homologues, providing critical information for the generation of a mouse model for cystinosis.