Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• compositions containing a soluble form of HLA-G in the treatment of pathologies of the blood and of the circulatory system (anemias and ischemias).
• MHC antigens are divided into several classes, class I antigens (HLA-A, HLA-B and HLA-C) which have 3 globular domains ( ⁇ l, ⁇ 2 and ⁇ 3), and whose ⁇ 3 domain is associated with ⁇ 2 microglobulin, class II antigens (HLA-DP, HLA-DQ and HLA-DR) and class III antigens (complement).
• class I antigens include, in addition to the abovementioned antigens, other antigens, called non-classical class I antigens, and in particular the HLA-E, HLA-F and HLA-G antigens.
• this gene comprises 8 exons, 7 introns and a 3 'untranslated end; the 8 exons correspond respectively to: exon 1: signal sequence, exon 2: extracellular domain ⁇ 1, exon 3: extracellular domain ⁇ 2, exon 4: extracellular domain ⁇ 3, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and 3 'untranslated region (GERAGHTY et al., supra; ELLIS et al., J. Im unol., 1990, 144, 731-735 ; KIRSZENBAUM M.
• exon 1 signal sequence
• exon 2 extracellular domain ⁇ 1
• exon 3 extracellular domain ⁇ 2
• exon 4 extracellular domain ⁇ 3
• exon 5 transmembrane region
• exon 6 cytoplasmic domain I
• exon 7 cytoplasmic domain II (un
• HLA-G gene differs from other class I genes in that the translation termination codon, in phase, is located at the second codon of exon 6; consequently, the cytoplasmic region of the protein encoded by this HLA-6.0 gene is shorter than that of the cytoplasmic regions of the HLA-A, -B and -C proteins.
• HLA-G antigens are mainly expressed by cytotrophoblastic cells of the placenta and are considered to play a role in the protection of the fetus (absence of rejection by the mother).
• the HLA-G antigen is monomorphic, it may also be involved in the growth or function of placental cells (KONATS et al., Science, 1990, 248, 220-223).
• Other research concerning this non-classical class I antigen has shown that the primary transcript of the HLA-G gene can be spliced in several ways and produces at least 3 distinct mature AR ⁇ m: the primary transcript of HLA-G provides a complete copy (Gl) of 1200 bp, a fragment of 900 bp (G2) and a fragment of 600 bp (G3).
• the Gl transcript does not include exon 7 and corresponds to the sequence described by ELLIS et al. (cited above), that is to say that it codes for a protein which comprises a signal sequence, three external domains, a transmembrane region and a cytoplasmic sequence.
• AR ⁇ m G2 does not include exon 3, that is to say that it codes for a protein in which the domains ⁇ l and ⁇ 3 are directly joined;
• AR ⁇ G3 contains neither exon 3 nor exon 4, that is to say that it codes for a protein in which the ⁇ 1 domain and the transmembrane sequence are directly joined.
• the splicing which prevails for obtaining the HLA-G2 antigen involves the joining of an adenine (A) (originating from the domain coding for ⁇ 1) with an AC sequence (originating from the domain coding for ⁇ 3), which leads to the creation of an AAC codon (asparagine) in place of the GAC codon (aspartic acid), encountered at the start of the sequence coding for the ⁇ 3 domain in HLA-G 1.
• AAC codon asparagine
• GAC codon aspartic acid
• HLA-G molecule plays a fundamental role in protecting the fetus from a maternal immune response (induction of immune tolerance). Some of the inventors have confirmed this role: the HLA-G molecules, expressed on the surface of the trophoblasts, effectively protect fetal cells from lysis by natural killer ( ⁇ K) cells. maternal (CAROSELLA ED et al., CR Acad. Sci., 318, 827-830; CAROSELLA ED et al; Immunol. Today, 1996, 407-409).
• HLA-G mRNA the HLA-G4 transcript, which does not include exon 4; the HLA-G5 transcript, which includes intron 4, between exons 4 and 5, thus causing a modification of the reading frame, during the translation of this transcript and in particular the appearance of a stop codon, after l amino acid 21 of intron 4; the HLA-G6 transcript, having intron 4, but having lost exon 3 (KIRSZENBAUM M. et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 4209-4213; European Application EP 0 677 582; KIRSZENBAUM M.
• HLA-G mRNAs which potentially encode 7 isoforms of HLA-G including 4 membranes (HLA-G1, G2, G3 and G4) and 3 soluble (HLA-G5, G6 and G7).
• HLA-G antigen is restricted to privileged immune sites and in particular to the feto-maternal interface.
• membrane-bound protein HLA-G1 is mainly expressed by extraviral cytotrophoblast cells in which it protects the fetus from immune cells of maternal origin.
• Both membrane-bound isoforms and soluble isoforms are immunotolerant, that is, they inhibit cytolysis mediated by NK cells and CTLs as well as the alloproliferative T response; in addition, they induce apoptosis in T cells and NK CD8 + cells.
• the HLA-G protein exercises its function locally, both when it is expressed on the surface of cells and when it is secreted (action to distance); it thus ensures the immunosurveillance of the organism (Teyssier Em. Et al., Nat. Immunol, 1995, 14, 262-270).
• these soluble isoforms of HLA-G are present in the erythroid cells of the placental vessels of the 1 st trimester of pregnancy as well as during the early vascularization of the embryo and throughout erythropoiesis.
• the inventors have shown that the soluble isoforms of HLA-G also exert non-immunological functions. It is therefore an object of the invention to provide new applications of the soluble isoforms of HLA-G, directly linked to their non-immunological functions, in particular in pathologies of the blood circulation such as anemia or 'ischemia.
• a subject of the present invention is therefore the use of a composition comprising at least one soluble HLA-G isoform and at least one pharmaceutically acceptable vehicle, for the preparation of a medicament intended for the treatment of pathologies of the blood circulation .
• pathologies of the blood circulation is meant both pathologies of the blood and pathologies of the circulatory system (means of circulation).
• said pathology is selected from the group consisting of anemias and ischemias.
• the inventors have shown the interest of soluble isoforms of HLA-G: 1. in the proliferation, differentiation and maturation of erythroblasts into reticulocytes.
• compositions are in one of the following forms: - liquid form, suitable for parenteral or oral administration, - solid form, suitable for parenteral administration after dissolution or suspension, or oral administration.
• excipients suitable for these two forms are preferably: water,
• the present invention also relates to a method for detecting and optionally sorting cells with a non-immunological function expressing a soluble isoform of HLA-G (cells of erythrocyte and endothelial lines) in a biological sample, which method is characterized in that that it comprises at least the following steps: (a) bringing the biological sample to be tested into contact with a panel of antibodies selected from the group consisting of antibodies directed against the following markers: soluble HLA isoform -G, CD71, CD34 and CD45, and (b) detecting and optionally sorting cells corresponding to different stages of differentiation of erythrocyte or endothelial lines, according to their expression profile for the markers defined in (a).
• the biological sample comprises: (a) bringing the biological sample to be tested into contact with a panel of antibodies selected from the group consisting of antibodies directed against the following markers: isoform soluble in HLA-G, CD71, CD34 and CD45, (a ') the selection of cells expressing the soluble isoform of HLA-G and (b) the detection of the cell type using the marker CD71.
• the biological sample is advantageously: a blood sample (detection of circulating erythroid cell cells) or a bone marrow sample.
• HSC hematopoietic stem cells
• P-Sp / AGM para-aortic splanchnopleura / aorta-gonad-mesonephros intra-embryonic region
• - Erythroid cells first hematopoietic cells to appear during intraembryonic life, which will give rise to mature red blood cells.
• yolk sac yolk sac
• islets of blood that appear in the yolk sac as groups of cells between 15 and 24 days from development of the embryo and which differentiate into endothelial cells from the external cells of the clusters and into hematopoietic cells from the internal cells.
• Hemangioblast common precursor of endothelial cells and hematopoietic cells present in the yolk sac.
• Primary hematopoietic organs embryonic liver, fetal bone marrow.
• the cells of said biological sample are permeabilized; this step allows the antibodies to effectively access the markers to be detected, which are secreted in the cytoplasm, before their passage into the general circulation (case of the soluble isoforms of HLA-G).
• the soluble isoform of HLA-G is expressed in all the cells of the erythroid line and in all the hematopoietic organs, from the embryo to the adult, whereas it is not found in mature erythrocytes.
• no expression of a soluble isoform of HLA-G is observed in the hematopoietic stem cells (HSC) CD34 + CD45 + ; this implies that the soluble isoform of HLA-G has no action on multipotent stem cells.
• HSC hematopoietic stem cells
• the soluble isoform of HLA-G is produced in vitro during the differentiation of the erythroid line, whatever the stage of maturation.
• the soluble HLA-G isoform is involved in the proliferation and / or maturation of the precursors of the erythroid line.
• the soluble HLA-G isoform is expressed in endothelial cells at an early stage in the embryo, whereas it is not found in the endothelial cells of mature vessels. In particular, it is detected in the budding vessels of the chorionic villi and of the juxta-allantoic part of the yolk sac.
• FIG. 1 illustrates the specificity of the antibody 5A6G7 vis-à-vis the soluble forms of HLA-G.
• Figure 1A illustrates the results obtained by Western blot. The specificity of the 5A6G7 antibody was tested on the M8-pcDNA, M8-HLA-Gl, M8-HLA-G5 and M8-HLA-G6 lines; in parallel, the 4H84 antibody (McMaster et al., J. immunol., 1998 and Paul et al., Hum.
• the 4H84 antibody reveals 3 bands corresponding to HLA-G 1, HLA-G5, and HLA-G6; the 5A6G7 antibody reveals 2 bands corresponding to HLA-G5 and HLA-G6.
• the M8-pcDNA line, not expressing HLA-G, is not labeled with either the 4H84 antibody or the 5A6G7 antibody.
• Figure 1B shows the results obtained by immunocytochemistry. The specificity of the 5A6G7 antibody was tested on the M8-pcDNA, M8-HLA-G1, and M8-HLA-G5 lines, in parallel with the 4H84 antibody and an isotypic control. Positive markings are characterized by a gray color.
• line 1 the line M8-pcDNA, not expressing HLA-G, is neither marked by the antibody 4H84, nor by the antibody. 5A6G7.
• lane 2 the line M8-HLA-G1 which does not have the epitope recognized by the antibody 5A6G7 is only marked by the antibody 4H84.
• lane 3 the M8-HLA-G5 line is labeled with the two antibodies 4H84 and 5A6G7. (magnification x40).
• Figure IC illustrates the results obtained by immunofluorescence analyzed by confocal microscopy.
• the specificity of the 5A6G7 antibody was tested on the lines M8-pcDNA, M8-HLA-G1, M8-HLA-G5, FO and FO-HLA-G6. As expected, the M8-HLA-G5 and FO-HLA-G6 cells are labeled with the antibody 5A6G7B12; whereas the M8-pcDNA, M8-HLA-G1 and FO cells which do not have the epitope recognized by the antibody 5A6G7 are not labeled.
• the 4H84 antibody is a reference antibody which specifically recognizes all of the HLA-G isoforms.
• soluble isoforms HLA-G5 and G6 in the erythroblasts of the trophoblasts of the first trimester of pregnancy
• a expression of the soluble isoforms by the cytotrophoblastic islets (cell island trophoblast or CIT) but not by the PVTs ( perivillous trophoblast), determined by immunohistochemical analysis using the antibody 5A6G7 on sections of trophoblasts embedded in paraffin
• b analysis of the expression of the following markers: HLA-G5 / 6, CD34, CD71 and CD45 on the developing vessels, present in the trophoblast of the first trimester by immunohistochemistry
• c immunohistochemical staining of a differentiated vessel from trophoblast sections of a 32-day embryo with antibodies directed against HLA-G5 / G6 (5A6G7), CD34 (which marks the endothelial cells (ED) of the vessels, CD71 which identifies erythroid cells (ER)
• HLA-G5 is detected in erythroid cells which come from the yolk sac and are present in the lumen of the sinusoids (capillaries of the embryo).
• the endothelial cells are CD45 +.
• Some cells associated with hepatocytes are CD34 + and CD45 + and can be considered as the first progenitor cells from the AGM.
• - Figure 4 illustrates the stage of the embryo, the fetus of children and adults analyzed by immunohistochemistry.
• EXAMPLE 1 Production of a Monoclonal Antibody Called 5A6G7, Directed Specifically against Soluble Isoforms of HLA-G 1.1 Obtaining Ascites The monoclonal antibody 5A6G7 is produced using conventional protocols from splenocytes of Balb / c immunized mice by a synthetic 21-mer peptide corresponding to the C-terminal part encoded by intron 4 of the soluble forms of HLA-G, the sequence of which is: SKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 1), coupled to the ovalbumin carrier protein (OVA).
• OVA ovalbumin carrier protein
• the 5A6G7 monoclonal antibody is purified from ascites using protein A-Sepharose affinity chromatography and can be used both for detection, titration and purification of soluble forms of HLA-G.
• HLA-G5 and HLA-G6 The criteria required to obtain an antibody with good specificity are as follows: - detection of proteins of 37 kDa HLA-G5 and 28 Da HLA-G6 but not of the others HLA-G isoforms and other HLA class I molecules present in protein lysates from M8 cells (cells from the M8 melanoma cell line) transfected respectively by the pcDNA vector alone, HLA-G 1 cDNAs , -G2, -G3, -G4, -G5, or -G6; M8-pcDNA, M8- HLA-G1, M8-HLA-G5 and M8-HLA-G6 are cells derived from the melanoma cell line M8, transfected with the empty vector, HLA-G1, HLA-G5, or HLA- G6, respectively.
• FO and FO-HLA-G6 are cells from the FO melanoma cell line, transfected by the empty vector or HLA-G6, respectively.
• This monoclonal antibody makes it possible to discriminate significantly between the soluble HLA-G proteins generated by unhooking the membrane HLA-G forms which do not contain the epitope encoded by intron 4 and the HLA-G5 / -G6 proteins produced from Alternative spliced mRNAs which contain intron 4.
• Example 2 Detection of soluble HLA-G in circulating cells of chorionic vessels originating from placenta at the first trimester stage
• the antibody 4H84 is a reference antibody which recognizes all the isoforms of HLA-G.
• Monoclonal antibodies directed against the CD71 transferrin receptor (Novocastra Laboratories, UK) were used to identify erythroid cells.
• the monoclonal antibodies directed against CD34 (Novocastra Laboratories) and CD45 (Dako, FR) make it possible to detect hematopoietic progenitor cells whereas only the monoclonal antibody directed against CD34 makes it possible to recognize endothelial progenitor cells. 2.L3.
• Erythroid cell cultures Cultures of erythroid cells (1.10 5 umbilical cord blood cells or bone marrow cells) are spread on a semi-solid medium containing methylcellulose, 10% MCL (lymphocytic conditioned medium) (Stem cell , Vancouver) and 2 U / ml EPO (Roche, FR). These cultures are incubated at 37 ° C in an atmosphere containing 5% CO 2 , for 14 days. 2-phase liquid culture is used and is defined as follows.
• the cells are isolated by centrifugation on a gradient of Ficoll 1077 (SIGMA), and cultured at a density of 10 6 cells / ml in minimal alpha essential medium ( ⁇ -MEM, Sigma) supplemented with 10% fetal calf serum (FCS, Sigma), 1 ⁇ g / ml of cyclosporin A (Novartis) and 10% of medium recovered from cultures of bladder carcinoma cell lines 5637 negative for HLA-G (ATCC n ° HTB-9). The cultures are incubated for 7 days at 37 ° C., under an atmosphere of 5% CO 2 , in air with 98% humidity.
• SIGMA Ficoll 1077
• phase II culture After this phase I culture, the non-adherent cells are recovered, washed, and seeded at a density of 0.4 ⁇ 10 6 cells / ml in ⁇ -MEM, 30% FCS, 1% bovine serum albumin, 10 "5 M of ⁇ -mercaptoethanol, 15 mM of L-glutamine, 10 " 6 M of dexamethasone, and 1 U / ml of recombinant erythropoietin (Epo, Roche) for 5-6 days (called phase II culture). 2.1.4.
• Immunohistochemical analysis Dewaxed tissue sections are subjected to high temperature epitope recovery treatment in 10 mM sodium citrate buffer (pH 6.0) using a commercial microwave device to optimize the immunoassay -reactivity. Tissue sections were permeabilized using PBS IX with 0.1% saponin and 10 mM Hepes buffer. Peroxidase activity endogenous is inhibited by treating the sections for 5 minutes at room temperature with 3% hydrogen peroxide in water. Non-specific fixation is avoided by adding 50 mM Tris, 3% BSA (Sigma), 40% human serum for 20 minutes before staining with the primary antibody for 30 minutes at room temperature.
• the expression of the HLA-G protein is then evaluated on a series of tissue sections or cells using the universal biotin detection system UltraTech HRP (Immunotech, Coulter, France), according to the manufacturer's instructions.
• the immunocytochemical analysis of the expression of HLA-G by the BFU-E cells is carried out on cytocentrifuged BFU-E cells, which were recovered from the cultures in semi-solid medium and washed in PBS, before the centrifugation. 2.2 Results As shown in FIG. 2a, the proteins HLA-G5 and HLA-G6 are localized in the cells of extravoid trophoblasts.
• these soluble HLA-G molecules were detected by staining using the 5A6G7 monoclonal antibodies either in cells in close contact with budding vessels associated with cytotrophoblasts, or in cells devoid of axes villous, or either in cells located in the lumen of mature vessels ( Figures 2b and 2c).
• This new cellular localization of HLA-G is confirmed by using another anti-HLA-G antibody, the reference monoclonal antibody 87G.
• the reference monoclonal antibody 87G In order to identify more precisely these cells expressing the protein
• HLA-G which morphologically resemble erythroblasts
• an immunohistochemical analysis was performed on a series of trophoblast sections included in paraffin from 32-day embryos by detection of the following markers: the transferrin receptor CD71: expressed from erythroids (BFU-E, burst-forming unit) to reticulocytes CD34: expressed by both endothelial and hematopoietic progenitor cells - CD45: expressed from pro hematopoietic progenitor cells to mature cells of lymphoid and myeloid lines the soluble proteins HLA-G5 and HLA-G6.
• BFU-E burst-forming unit
• CD34 expressed by both endothelial and hematopoietic progenitor cells
• CD45 expressed from pro hematopoietic progenitor cells to mature cells of lymphoid and myeloid lines the soluble proteins HLA-G5 and HLA-G6.
• EXAMPLE 3 Identification of the Soluble HLA-G5 Isoform in Fetal Erythroblasts
• the organ in which erythroblasts are the main constituents to know the liver which is the main producer of red cells in the fetus, was used.
• 3.1.1 Tissues and monoclonal antibodies Proteins are extracted from the liver obtained from 2 separate embryos of 9 and 12 weeks respectively. The tissues to which the analyzes relate are those described in Example 2. The monoclonal antibody 5A6G7 described in Example 1 is used for the immunohistochemical analysis.
• the proteins are then electro-transferred onto a nitrocellulose membrane (Hybond-C extra).
• the membrane is then treated against non-specific bonds with 5% of freeze-dried skimmed milk in PBS containing 0.2% of Tween 20 (PBST) overnight at 4 ° C. After washing in PBST, the membrane is incubated with anti-mouse antibodies conjugated to HRP for 30 minutes at room temperature and washed in PBST.
• the HLA-G proteins are detected by chemiluminescence (ECL Plus Kit) (Menier et al., Hum. Immunol, 2003, 64, 315-326).
• EXAMPLE 4 Distribution of the Soluble HLA-G5 Isoform to All the Embryonic and Fetal Hematopoietic Organs
• the distribution of the soluble HLA-G5 Isoform to all the embryonic and fetal hematopoietic organs was analyzed by performing immunohistochemical experiments on sections of embryos and fetal tissue embedded in paraffin, using monoclonal antibodies against HLA-G5, CD71, CD34 or CD45.
• Materials and Methods The tissues analyzed come from embryonic and fetal hematopoietic organs as described in Example 2.
• the monoclonal antibodies directed against HLA-G5 (5A6G7) and the markers CD71, CD34 and CD45 of Example 1 were used under the conditions specified in Example 2.
• HLA-G + cells also express the CD71 receptor and this co-localization exists throughout the duration of embryonic and fetal development: in the yolk sac, in the 16 day embryo, in all the hematopoietic organs of '' a 32-day embryo, in the liver of 12, 34 and 36 weeks, in the bone marrow of 12, 14 and 16 weeks), as well as in the bone marrow in adults. They are therefore indeed a marker for the erythroid line. These results therefore illustrate the role of the soluble isoform of HLA-G in erythropoiesis as a marker in the differentiation of the erythroid line.
• the detection antibody a biotinylated W6 / 32 monoclonal antibody (Leinco Technologies Inc., Ballwin) recognizing a monomorphic determinant of the heavy chains of HLA class I associated with microglobulin B2, previously diluted 1/250, was incubated for 1 hour at 37 ° C.
• HLA-G5 concentration of HLA-G5 is understood between 25.5 ng / ml and 44.3 ng / ml.
• An immunocytochemical analysis of the corresponding cells confirms that the two-phase liquid culture allows the differentiation of the erythroid cells which are positive for CD71 and co-express CD36 (thrombospondin receptor and marker for identifying the CFU-E and erythroblast stages of erythroid line) at 3-1 of the second culture phase. From these results, it can therefore be concluded that the HLA-G5 molecules detected by ELISA in culture supernatants were indeed produced by erythroid cells during their differentiation.
• Example 6 Adult bone marrow erythroblasts express HLA-G5
• HLA-G5 is detected in BFU cells -E by immunocytochemical analysis using several anti-HLA-G monoclonal antibodies such as 5A6G7, MEM-G / 9, and 4H84. In addition, during an immunochemical analysis on sections of bone included in paraffin, HLA-G5 is then localized in erythroblasts from adult bone marrow.
• EXAMPLE 7 Expression of the Soluble HLA-G Isoform by the Embryonic Endothelial Cells
• the soluble HLA-G isoform is also identified in the endothelial cells in the budding vessels in two places: in the mesenchymal heart of the chorionic villi and in the juxta-allantoid part of the yolk sac of the early embryo. In each case, the HLA-G isoform co-locates with the CD34 marker.
• the presence of the soluble isoform of HLA-G in the endothelial cells of the budding vessels of chorionic villi and of the juxta-allantoid part of the yolk sac shows its involvement in the angiogenic process.
• Example 8 Effect of HLA-G5 on Erythroid Differentiation
• the effect of HLA-G5 on erythroid differentiation was analyzed on an erythroleukemic line (K562, ATCC) expressing or not HLA-G5, namely: a K562 line. transfected with an HLA-G5 expression plasmid and secreting HLA-G5 (K562- ⁇ cDNA-HLA-G5) and, as a control, a K562 line transfected with the empty vector (K562-pcDNA).
• glycophorin A and CD36 differentiation markers which appear in the last stages of erythroid differentiation (erythroblast, reticulocyte, mature red blood cell) was analyzed by flow cytometry using commercial antibodies directed against these markers. The results are illustrated below:
• HLA-G5 is made up of a greater number of mature erythroid cells, which express in greater quantity markers of erythrocyte differentiation.
• the effect of HLA-G5 on erythroid differentiation is confirmed by co-culture experiments between the two lines, in order to verify that the HLA-G5 form secreted by K562-pcDNA-HLA-G5 induces the differentiation of the line. K562-pcDNA.

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