Transcripts of the MHC class I HLA-G gene and their applications

Abstract

Transcripts of the major histocompatibility complex (MHC) Class I HLA-G gene which are present in foetal trophoblasts and/or in adult circulating mono-nuclear cells, as well as their applications. Either the said transcripts comprise, in succession in the 5′ to 3′ direction: a fragment encoding the signal peptide (exon 1), a fragment encoding the α1 domain (exon 2), a fragment encoding the α2 domain (exon 3), a fragment encoding the trans-membrane TM domain (exon 5), a fragment encoding the cytoplasmic domain (exon 6) and the 3′ untranslated fragment (exon 8), which sequence is designated HLA-G4, or the said transcripts comprise intron 4.

1 .cDNA sequence derived from an mRNA of the human MHC HLA-G gene, characterized in that it comprises, in succession on the 5′ to 3′ direction: 
 a fragment encoding the signal peptide (exon 1),  
 a fragment encoding the α1 domain (exon 2),  
 a fragment encoding the α2 domain (exon 3),  
 a fragment encoding the transmembrane TM domain (exon 5),  
 a fragment encoding the cytoplasmic domain (exon 6) and  
 the 3′ untranslated fragment (exon 8), which sequence is designated HLA-G4 and is present in trophoblasts of the first trimester of gestation as well as in adult circulating monouclear cells.  
 
     
     
         2 . Sequence according to claim  1 , characterized in that it comprises, in succession in the 5′ to 3′ direction: 
 the fragment encoding the α2 domain (exon 3),  
 the fragment encoding the transmembrane TM domain (exon 5),  
 the fragment encoding the cytoplasmic domain (exon 6), and  
 the 3′ untranslated fragment (exon 8).  
 
     
     
         3 . Sequence according to  claim 2 , characterized in that it encodes a protein in which the α2 domain and the transmembrane sequence of HLA-G are directly linked, it presents the SEQ ID NO:1 and it comprises 0.43 kb.  
     
     
         4 . Transcription product of the human MHC HLA-G gene, characterized in that it includes, proceeding from the 5′ end: 
 a fragment encoding the signal peptide (exon  
 a fragment encoding the α1 domain (exon 2),  
 a fragment encoding the α2 domain (exon 3),  
 a fragment encoding the transmembrane TM domain (exon 5), and  
 a fragment encoding the cytoplasmic domain (exon 6) of HLA-G, and such that it comprises 0.43 kb.  
 
     
     
         5 . Oligonucleotide, characterized in that it consists of a fragment of the sequence according to any one of claims  1  to  4 ,and in that it exhibits the SEQ ID NO:2.  
     
     
         6 . Oligonucleotide, characterized in that it consists of a fragment of the sequence according to any one of claims  1  to  4 , and in that it exhibits the SEQ ID NO:3.  
     
     
         7 . Oligonucleotide, characterized in that it consists of a fragment of the sequence according to any one of claims  1  to  4 , and in that it exhibits the SEQ ID NO:4.  
     
     
         8 . Oligonucleotide, characterized in that it consists of a fragment of the sequence according to any one of claims  1  to  4 , and in that it exhibits the SEQ ID NO:5.  
     
     
         9 . Oligonucleotide, characterized in that it consists of a fragment of the sequence according to any one of claims  1  to  4 , and in that it exhibits the SEQ ID NO:6.  
     
     
         10 . Nucleotide probes, characterized in that they consist of a nucleotide sequence according to any one of claims  1  to  9 , or a fragment thereof, which is labelled with a label such as a radioactive isotope, an appropriate enzyme or a fluorochrome.  
     
     
         11 . Probe according to  claim 10 , characterized in that it exhibits the SEQ ID NO:6 according to  claim 9 .  
     
     
         12 . Probe according to  claim 10 , characterized in that it exhibits the SEQ ID NO:4 according to  claim 7 .  
     
     
         13 . Pairs of primers for synthesizing a sequence according to any one of claims  1  to  4 , characterized in that each primer comprises a sequence or a sequence fragment according to any one of  claims 5  to  9 .  
     
     
         14 . Pair of primers according to  claim 13 , characterized in that it consists of an oligonucleotide of the SEQ ID NO:2 according to  claim 5  which is paired with an oligonucleotide of the SEQ ID NO:5 according to  claim 8 .  
     
     
         15 . Pair of primers according to  claim 13 , characterized in that it consists of an oligonucleotide of the SEQ ID NO:3 according to  claim 6  which is paired with an oligonucleotide of the SEQ ID NO:5 according to  claim 8 .  
     
     
         16 . Use of a cDNA sequence, designated HLA-G5 and derived from an mRNA of the human MHC HLA-G gene of adult circulating mononuclear cells, comprising, in succession in the 5′ to 3′ direction: 
 a fragment encoding the signal peptide (exon  
 a fragment encoding the α1 domain (exon 2),  
 a fragment encoding the α2 domain (exon 3),  
 a fragment encoding the α3 domain (exon 4), intron 4,  
 a fragment encoding the transmembrane TM domain (exon 5),  
 a fragment encoding the cytoplasmic domain (exon 6) and  
 the untranslated 3′ fragment (exon 8), for the preparation of a drug for its use as immunomodulating agent.  
 
     
     
         17 . Use of a cDNA sequence, designated HLA-G6 and derived from an mRNA of the human MHC HLA-G gene of adult circulating mononuclear cells, comprising, in succession in the 5′ to 3′ direction: 
 a fragment encoding the signal peptide (exon 1),  
 a fragment encoding the α1 domain (exon 2),  
 a fragment encoding the α3 domain (exon 4), intron 4,  
 a fragment encoding the transmembrane TM domain (exon 5),  
 a fragment encoding the cytoplasmic domain (exon 6), and  
 the 3′ untranslated fragment (exon 8), for the preparation of a drug for its use as immunomodulating agent.  
 
     
     
         18 . Use of a sequence according to  claim 16  or  17 , without exon 6 and/or without exon 8.  
     
     
         19 . Method for obtaining immunomodulating soluble proteins, characterized it comprises a expression vector construction in which a sequence with intron 4 is inserted, derived from adult circulating mononuclear cells, as defined according to any one of  claims 16  to  18  and expression of said soluble proteins.  
     
     
         20 . Peptides or peptide fragments, characterized in that they are encoded by at least one fragment according to any one of claims  1  to  4  or  16  to  18 .  
     
     
         21 . Peptide according to  claim 20 , characterized in that it corresponds to the SEQ ID NO:7.  
     
     
         22 . Peptide according to  claim 20 , characterized in that it is encoded by a fragment of HLA-G5 transcript and it corresponds to the SEQ ID NO:8.  
     
     
         23 . Peptide according to  claim 20 , characterized in that it is encoded by a fragment of HLA-G6 transcript and it corresponds to the SEQ ID NO:9.  
     
     
         24 . Procedure for selecting and enriching with undifferentiated haematopoietic cells (immature blood cells or stem cells), characterized in that it comprises: 
 (a) withdrawing a sample which is selected, as the case may be, from peripheral blood, umbilical cord blood or bone marrow,    (b) bringing the said sample into contact with anti-CD34 antibodies,    (c) separating the cell complexes formed which contain a CD34 antigen-anti-CD34 antibody,    (d) carrying out an in-situ RT PCR on the cells obtained in step (c) in the presence of primers which are labelled with a fluorochrome according to any one of  claims 13  to  15 ,    (e) separating the fluorescent cells which are obtained, and    (f) selecting the pluripotent, immature non-fluorescent cells.    
     
     
         25 . Procedure for detecting cells which express the transcript according to any one of claims  1  to  4 , characterized in that it comprises the in-situ implementation of an RT PCR, by: 
 (a) bringing blood cells into contact with a pair of primers which are labelled in accordance with any one of  claims 13  to  15 , and  
 (b) separating the labelled cells by any appropriate means.  
 
     
     
         26 . Procedure for detecting blood cells and more particularly adult circulating mononuclear cells, expressing a transcript with intron 4, characterized in that it comprises the in-situ implementation of an RT PCR, by: 
 (a) bringing blood cells into contact with a pair of labelled primers according to the invention, selected from among the pairs (1)-(2) and (3)-(4) according to any one of  claims 13  to  15 ,    (b) selecting transcripts comprising intron 4, by hybridization with a probe, eventually labelled, selected among the SEQ ID NO:10 probe and the SEQ ID NO:11 probe and    (b) separating the labelled cells by any appropriate means, in particular by cytofluorometry.    
     
     
         27 . Antibodies, characterized in that they are directed against the HLA-G proteins according to any one of  claims 20  to  23 .  
     
     
         28 . Procedure for separating nucleated foetal cells, starting from a sample of maternal blood, characterized in that it comprises: 
 (1) bringing the sample of maternal blood into contact with antibodies directed against the HLA-G proteins according to  claim 27 , and    (2) separating the foetal cell-antibody complexes which are obtained.    
     
     
         29 . Procedure for specifically separating circulating mononuclear cells, characterized in that it comprises the in-situ implementation of an RT PCR, by: 
 (a) bringing blood cells into contact with a pair of primers which are labelled according to any one of  claims 13  to  15 , and    (b) separating the labelled cells in a suitable manner.    
     
     
         30 . Medicament, characterized in that it comprises a peptide according to  claim 20  or  claim 21 .  
     
     
         31 . Immunomodulating compositions, characterized in that they comprise a peptide according to  claim 20  or  claim 21  which is combined with an excipient or a support which is appropriate from the pharmaceutical point of view.  
     
     
         32 . Use of peptides according to  claim 22  or  claim 23 , for preparation of a drug having a immunomodulating activity.  
     
     
         33 . Method of detection of CD8 receptors, which procedure is characterized in that it comprises bringing mononuclear cells into contact with a peptide according to any one of  claims 20  to  23  and detecting CD8-peptide complexes by any appropriate means.