Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules

Poláková, K; Bennink, Jack R.; Yewdell, Jonathan W.; Bystrická, M; Bandžuchová, E; Russ, G

doi:10.1016/s0198-8859(02)00777-2

Cited by 20 publications

(21 citation statements)

References 29 publications

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“…This suggests extreme caution when mAbs that strongly prefer linear epitopes are used to detect antigen under folding conditions that cannot be exactly controlled. A similar observation was made with a mAb to HLA-G [23].…”

Section: Cross-reactivity Of Mabs To Hla-e Depends On Foldingsupporting

confidence: 76%

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains

Tremante¹,

Monaco²,

Ingegnere³

et al. 2015

Eur J Immunol

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Since HLA-E heavy chains accumulate free of their light β 2 -microglobulin (β 2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β 2 m-associated and β 2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β 2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.Keywords: Conformation r HLA-E r HLA-A r -B r -C r mAbs r β 2 -microglobulin (β 2 m) Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionBeing the ligand of both the conserved CD94/NKG2A immune receptor and the variable TCR, the oligomorphic HLA-E molecule bridges classical and nonclassical HLA class I antigens [1]. Often classified among the latter, HLA-E may rather be considered an inbetweener. This definition also applies to HLA-C, since this is the least polymorphic among classical (HLA-A, -B, -C) Correspondence: Dr. Patrizio Giacomini e-mail: giacomini@ifo.it class I antigens, and shares with HLA-E several unorthodox properties, such as selective peptide binding, poor association with β 2 m [2-4], and massive accumulation in a β 2 m-free form [5,6].Although often unappreciated, unfolded and β 2 m-free heavy chains may share epitopes with fully folded HLA class I heterodimers. These "intermediate" folds may confound the assignment of antibody specificity. For instance, a mAb called Q1/28 binds HLA-B but not HLA-C when heavy chains are β 2 massociated, and HLA-C but not HLA-B when heavy chains are β 2 m-free [7]. Likewise, mAbs MEM-E/07 and MEM-E/08 bind β 2 m-free HLA-E, but cross-react with HLA-A, -B, -C heavy chainswww.eji-journal.eu Eur. J. Immunol. 2015. 45: 2356-2364 Molecular immunology 2357 associated with β 2 m [6]. Therefore, mAbs to inbetweeners require thorough characterization, including their systematic testing on both the β 2 m-associated and the β 2 m-free heavy chains specified by ...

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Section: Cross-reactivity Of Mabs To Hla-e Depends On Foldingsupporting

confidence: 76%

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains

Tremante¹,

Monaco²,

Ingegnere³

et al. 2015

Eur J Immunol

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“…Furthermore, the specificity of some Ab that are extensively used is questionable. For example, mild acid treatment of 4H84 (38) induces cross-reactivity with HLA class Ia H chains (40). Thus, 1-2C3 is entirely novel.…”

Section: Discussionmentioning

confidence: 99%

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

Morales¹,

Pace

Platt³

et al. 2003

The Journal of Immunology

106

101

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The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

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“…Our first goal was to determine whether CD85J/LIR-1 would interact with HLA-G FHC. Increased amounts of FHC proteins can be observed on the cell surface after acid treatment, which releases the peptide from the binding groove initiating ␤ 2 m dissociation (37). To assess the possible interaction of HLA-G FHC with the CD85J/LIR-1 receptor, we treated 721.221-transfected cells with wild-type or mutated HLA-G and HLA-A2 with glycine-HCl buffer as described in Materials and Methods.…”

Section: Cd85j/lir-1 Ig Does Not Bind ␤ 2 M-free Hla-g Molecules On Tmentioning

confidence: 99%

The CD85J/Leukocyte Inhibitory Receptor-1 Distinguishes between Conformed and β2-Microglobulin-Free HLA-G Molecules

Gonen‐Gross¹,

Achdout²,

Arnon³

et al. 2005

The Journal of Immunology

108

124

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For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with β2m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.

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Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules

Cited by 20 publications

References 29 publications

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

The CD85J/Leukocyte Inhibitory Receptor-1 Distinguishes between Conformed and β2-Microglobulin-Free HLA-G Molecules

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Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules

Cited by 20 publications

References 29 publications

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β2m‐free heavy chains

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β2m‐free heavy chains

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

The CD85J/Leukocyte Inhibitory Receptor-1 Distinguishes between Conformed and β2-Microglobulin-Free HLA-G Molecules

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Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains

Monoclonal antibodies to HLA‐E bind epitopes carried by unfolded β₂m‐free heavy chains