title: Multiplex bead-based immunoassay for the free soluble forms of the HLA-G receptors, ILT2 and ILT4.
abstract: Human leukocyte antigen (HLA)-G is an immune-inhibitory molecule that exerts its function via interaction with two main inhibitory receptors: ILT2 and ILT4. This interaction is considered to be an immune checkpoint. HLA-G can be found as a soluble molecule, but it is not known if its receptors can also be found as soluble molecules. In this work, we present a multiplex luminex-based assay to measure soluble ILT2 (sILT2) and soluble ILT4 (sILT4) molecules together. It is based on two antibody pairs, GHI/75 and HP-F1-PE for ILT2 and 27D6 and 42D1-PE for ILT4. The characterization of our method reveals that it specifically detects the free soluble forms of sILT2 and sILT4, and not those complexed to HLA Class I molecules such as their ligand of highest affinity HLA-G. A study on two small cohorts of cancer patients demonstrated that soluble ILT2 and ILT4 molecules were of low abundance in the plasma of healthy controls, but that elevated levels of plasmatic sILT2 were present in non-muscle-infiltrating bladder cancer patients. This demonstrated that the titration test is indeed working, and that soluble ILT2 molecules do exist in pathological contexts, which relevance may now be sought on larger cohorts and other pathologies.
authors.affiliation: Hemato-Immunology Research Department, CEA, Hospital Saint Louis, 1 Avenue Claude Vellefaux, Bat Lailler, 75475 Paris Cedex 10, France; University Paris Diderot, Sorbonne Paris Cité, UMR E_5 Institut Universitaire d'Hematologie, Saint-Louis Hospital, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
authors.forename: Ching-Lien
authors.initials: CL
authors.lastname: Wu
authors.affiliation: Department of Immunology and Microbiology, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.
authors.forename: Signe Goul
authors.initials: SG
authors.lastname: Svendsen
authors.affiliation: Hemato-Immunology Research Department, CEA, Hospital Saint Louis, 1 Avenue Claude Vellefaux, Bat Lailler, 75475 Paris Cedex 10, France; University Paris Diderot, Sorbonne Paris Cité, UMR E_5 Institut Universitaire d'Hematologie, Saint-Louis Hospital, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France; AP-HP, Saint-Louis Hospital, Department of Urology, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
authors.forename: Adrien
authors.initials: A
authors.lastname: Riviere
authors.affiliation: Hemato-Immunology Research Department, CEA, Hospital Saint Louis, 1 Avenue Claude Vellefaux, Bat Lailler, 75475 Paris Cedex 10, France; AP-HP, Saint-Louis Hospital, Department of Urology, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
authors.forename: François
authors.initials: F
authors.lastname: Desgrandchamps
authors.affiliation: Hemato-Immunology Research Department, CEA, Hospital Saint Louis, 1 Avenue Claude Vellefaux, Bat Lailler, 75475 Paris Cedex 10, France; University Paris Diderot, Sorbonne Paris Cité, UMR E_5 Institut Universitaire d'Hematologie, Saint-Louis Hospital, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.
authors.forename: Edgardo D
authors.initials: ED
authors.lastname: Carosella
authors.affiliation: Hemato-Immunology Research Department, CEA, Hospital Saint Louis, 1 Avenue Claude Vellefaux, Bat Lailler, 75475 Paris Cedex 10, France; University Paris Diderot, Sorbonne Paris Cité, UMR E_5 Institut Universitaire d'Hematologie, Saint-Louis Hospital, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Electronic address: joel.lemoult@cea.fr.
authors.forename: Joël
authors.initials: J
authors.lastname: LeMaoult
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D015331:Cohort Studies; D059951:HLA-G Antigens / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis*
chemical_list: D015703:Antigens, CD; D001798:Blood Proteins; D059951:HLA-G Antigens; C105299:LILRB1 protein, human; C491341:LILRB2 protein, human; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic
keywords: Human leukocyte antigen G (HLA-G); ILT2; ILT4; Immune check point; Immune regulation; Immunoassay