Abstract
Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E HC-affinity columns, suggesting that the HLA-I reactivity is due to antibodies formed against HLA-E. Hence, we examined whether anti-HLA-E antibodies can react to HLA-I alleles. Monoclonal IgG antibodies (mAbs) against HCs of two HLA-E alleles were generated in Balb/C mice. The antibodies were analyzed using multiplex bead assays on a Luminex platform for HLA-I reactivity. Beads coated with an array of HLA heterodimers admixed with HCs (LABScreen) were used to examine the binding of IgG to different HLA-Ia (31-HLA-A, 50-HLA-B, and 16-HLA-C) and Ib (2-HLA-E, one each of HLA-F and HLA-G) alleles. A striking diversity in the HLA-Ia and/or HLA-Ib reactivity of mAbs was observed. The number of the mAbs reactive to (1) only HLA-E (n = 25); (2) all HLA-Ib isomers (n = 8); (3) HLA-E and HLA-B (n = 5); (4) HLA-E, HLA-B, and HLA-C (n = 30); (5) HLA-E, HLA-A*1101, HLA-B, and HLA-C (n = 83); (6) HLA-E, HLA-A, HLA-B, and HLA-C (n = 54); and (7) HLA-Ib and HLA-Ia (n = 8), in addition to four other minor groups. Monospecificity and polyreactivity were corroborated by HLA-E monospecific and HLA-I shared sequences. The diverse HLA-I reactivity of the mAbs are compared with the pattern of HLA-I reactivity of serum-IgG in non-alloimmunized males, cancer patients, and ESKD patients. The findings unravel the diagnostic potential of the HLA-E monospecific-mAbs and immunomodulatory potentials of IVIg highly mimicking HLA-I polyreactive-mAbs.
Highlights
human leukocyte antigens (HLA) class-I expressed on human cell surfaces are heterodimers formed by the non-covalent association of a polymorphic, glycosylated 42-kDa heavy chain (HC) with β2-microglobulin (β2m), a 12-kDa light chain
Heavy chains (HCs) of two different alleles of HLA-E, which differ in their amino acid residue at position 107, were used for immunization
The number of monoclonal antibody (mAb) were higher in HLA-ER than in HLA-EG
Summary
HLA class-I expressed on human cell surfaces are heterodimers formed by the non-covalent association of a polymorphic, glycosylated 42-kDa heavy chain (HC) with β2-microglobulin (β2m), a 12-kDa light chain. HLA-I molecules are polymorphic consisting of several alleles of classical HLA-Ia (HLA-A 6921 alleles, HLA-B 8181 alleles, and HLA-C 6779 alleles) and non-classical HLA-Ib (HLA-E 271 alleles, HLA-F 45 alleles, and HLA-G 88 alleles). Intact HLA-I molecules bind to peptides that are derived from proteolytic degradation of cytoplasmic proteins, viruses, and pathogens. After presenting or losing the peptide, HC dissociates from β2m and is released by membrane matrix metalloproteinases (MMPs) [2] from the cell surface into the microenvironment and subsequently the circulation, where it undergoes proteolysis. The HC shedding determines the half-life of HLA-I, which may vary with cells, depending on glycosylation and sialylation [3]. The intact HLA-I molecules are identified with the monoclonal antibody (mAb) W6/32 that recognizes an epitope on β2m (residues 3 and 89) and on the HC residue 121 of all isoforms of HLA-I molecules [4]
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