Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d |
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Authors: | Yu Karl O A Im Jin S Illarionov Petr A Ndonye Rachel M Howell Amy R Besra Gurdyal S Porcelli Steven A |
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Affiliation: | Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. |
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Abstract: | The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells. |
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Keywords: | α-GalCer, α-galactosylceramide Ab, antibody AP, alkaline phosphatase β2m, β2-microglobulin DMSO, dimethylsulfoxide ELISA, enzyme-linked immunosorbent assay FACS, fluorescence-activated cell sorting FBS, fetal bovine serum hCD1d, human CD1d Ig, immunoglobulin iGb3, isoglobotrihexosylceramide iNKT cells, mouse Vα14 or human Vα24 invariant natural killer T cells mAb, monoclonal antibody mCD1d, mouse CD1d MW, molecular weight NGS, normal goat serum ON, overnight PBS, phosphate-buffered saline PE, phycoerythrin PPD, purified protein derivative of Mycobacterium tuberculosis strain H37Ra RT, room temperature TCR, T cell receptor TH1(2), T helper type 1 (or 2). |
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