Sophie Lefebvre

Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5, -G6, and -G7 transcripts in human transfected cells

The nonclassical HLA-G primary transcript is alternatively spliced to generate several mRNAs that have the capacity to encode four membrane bound isoforms, namely HLA-G1, -G2, -G3, and -G4 and two soluble isoforms HLA-G5 and -G6. We aimed at defining the capacity of full length and truncated soluble HLA-G transcripts to be translated in human cell lines. Our study of HLA-G alternative transcripts in various human tissues led us to identify a new splice variant of the HLA-G mRNA, named G7, in which open reading frame continues in intron 2. Due to the presence of a stop codon within intron 2, HLA-G7 transcripts retain the capacity to be translated as soluble truncated HLA-G proteins bearing the alpha1 domain linked to two specific aminoacids encoded by intron 2. Expression vectors containing cDNAs encoding HLA-G5, -G6, and -G7 isoforms were transfected into human cell lines. The presence of translated HLA-G5, -G6, and -G7 proteins was detected in protein extracts of transfected cells by Western blot and immunoprecipitation, but only the full length HLA-G5 soluble isoform could be clearly detected as a secreted protein in both transfected cells supernatants and body fluids.

Specific activation of the non-classical class I histocompatibility HLA-G antigen and expression of the ILT2 inhibitory receptor in human breast cancer

The HLA‐G molecule is a non‐classical HLA class I antigen selectively expressed by trophoblastic cells that invade the maternal decidua during human pregnancy. HLA‐G is believed to contribute to tolerance of the semi‐allogeneic fetus by inhibiting maternal immune responses. Similarly, HLA‐G expression in tumour cells may favour their escape from host immune surveillance. This study investigated HLA‐G expression in human mammary tumours. Immunohistochemical analysis of cryo‐preserved and paraffin‐embedded breast tissue biopsies, using two HLA‐G‐specific antibodies, revealed that unlike non‐cancerous breast tissue in the vicinity of the tumour, 14 out of 36 breast cancer lesions selectively expressed HLA‐G. HLA‐G expression was significantly more frequent in lesions that were highly infiltrated by host immune cells, thus correlating HLA‐G activation with inflammation. Further histological and double‐staining immunofluorescence analysis attributed HLA‐G expression mainly to tumour epithelial cells and to subsets of infiltrating CD68+ and CD8+ cells. RT‐PCR analysis suggested that HLA‐G was activated at the transcriptional level in breast tumours. The presence of ILT2 (Ig‐like transcript 2) killing inhibitory receptors known to interact with HLA‐G was also demonstrated in host immune cells that infiltrate breast cancer lesions. These results indicate that HLA‐G is up‐regulated at high frequencies in human breast cancer, where it may impair efficient anti‐tumour immunity. Copyright

THE HUMAN IMMUNOGLOBULIN HEAVY VARIABLE GENES

‘Human Immunoglobulin Heavy Variable Genes’, the fourth report of the ‘IMGT Locus on Focus’ section, comprises five tables entitled: (1) ‘Number of human germline IGHV genes at 14q32.33 and potential repertoire’; (2) ‘Human germline IGHV genes at 14q32.33’; (3) ‘Human IGHV orphons on chromosome 15 (15q11.2)’; (4) ‘Human IGHV orphons on chromosome 16 (16p11.2)’, and (5) ‘Human IGHV allele table’. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cnusc.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.

Modulation of HLA-G expression in human thymic and amniotic epithelial cells.

Expression of the nonclassical HLA class I antigen, HLA-G, is tightly regulated. HLA-G physiologic expression is mostly restricted to some placental and thymic cell types. Only few established cell lines express HLA-G in vitro. Cytokine-induced expression of HLA-G is hardly observed and also depends on the cell lineage. We assessed expression and cytokine regulation of HLA-G in primary cultures derived from human thymus and amnion epithelial cells, which also express HLA-G in vivo. We show that HLA-G cell surface expression is maintained, but decreases gradually, in primary cultures derived from human thymus and amnion epithelial cells. We also show that IFN-gamma re-induces HLA-G cell surface expression and upregulates classical class I gene expression in both primary cultures and in a thymus derived cell line. We further show that IFN-gamma also upregulates levels of HLA-G transcripts in TEC primary cultures. This study provides evidence that IFN-gamma induction of HLA-G expression occurs in the human amnion and the thymus, and is mediated at the transcriptional level in these tissues. These results also suggest a role for the microenvironment in regulating HLA-G in vivo gene expression in the thymus and amnion membrane.

Molecular mechanisms controlling constitutive and IFN-γ-inducible HLA-G expression in various cell types ☆

HLA-G molecule is thought to play a major role in down-regulating the maternal immune response by inhibiting NK and T cell cytolytic activities. We examined the molecular regulatory mechanisms that may control the restricted expression pattern of the HLA-G gene. We first analyzed protein interactions between nuclear extracts from the HLA-G-positive JEG-3 choriocarcinoma and the HLA-G-negative NK-like YT2C2 cell lines to a 244 bp regulatory element located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. This allowed characterization of cell-specific DNA-protein interactions that could account for differential cell-specific expression of the HLA-G gene. In particular two DNA-protein complexes were exclusively observed in YT2C2, suggesting that this HLA-G regulatory element is a target for putative cell-specific repressor factors. We further mapped nuclear factor binding sites to a 70 bp fragment in the upstream region of the regulatory element. We then investigated the effect of IFN-gamma on HLA-G gene expression. HLA-G cell surface expression was enhanced by IFN-gamma treatment in JEG-3 and U937 cell lines and peripheral blood monocytes while no effect was observed in tera-2 teratocarcinoma cell line. HLA-G transcriptional activity was increased only in JEG-3 and U937 cell lines. Activity of the 1.4-kb HLA-G promoter region was unchanged after IFN-gamma treatment in JEG-3 and Tera-2. These results suggest that both post-transcriptional and transcriptional mechanisms implicating IFN-responsive regulatory sequences outside the 1.4 kb-region are involved in IFN-gamma gene activation of the HLA-G gene.

Discovery of BAF312 (Siponimod), a Potent and Selective S1P Receptor Modulator

A novel series of alkoxyimino derivatives as S1P1 agonists were discovered through de novo design using FTY720 as the chemical starting point. Extensive structure-activity relationship studies led to the discovery of (E)-1-(4-(1-(((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (32, BAF312, Siponimod), which has recently completed phase 2 clinical trials in patients with relapsing-remitting multiple sclerosis.

SPECIFIC BINDING OF NUCLEAR FACTORS TO THE HLA-G GENE PROMOTER CORRELATES WITH A LACK OF HLA-G TRANSCRIPTS IN FIRST TRIMESTER HUMAN FETAL LIVER

The nonclassical MHC class I HLA-G antigen is expressed in cytotrophoblasts during pregnancy and may play a role in inhibiting lysis by maternal natural killer cells. HLA-G gene transcription was analyzed in human fetal liver of 6-8 wk of gestation, a development stage where classical HLA class I expression is very reduced. We demonstrated that HLA-G transcription is undetectable in these cells and we investigated the molecular mechanisms that control the lack of HLA-G gene transcription. We compared protein interactions of nuclear extracts from first trimester fetal livers, YT2C2-PR (HLA-G negative) and JEG-3 (HLA-G positive) cell lines to a 244-bp EcoR I/Hind III DNA region located 1.2 kb from the HLA-G gene, previously shown to direct HLA-G expression in transgenic mouse placenta. A strong specific C7-factor was specifically detected in first trimester fetal liver that could account for the inhibition of HLA-G transcription. Interaction of C7-factor and cell-specific factors previously detected in YT2C2 cell line (C5, C6) with two distinct regulatory regions identify this 244-bp EcoR I/Hind III fragment as a putative target for inhibition of HLA-G transcription.