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

Abstract

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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“…(47) Recently, Tremante et al (60) tested the HLA-I binding of MEM-E/02 on Western blots using the purified heavy chains (NP40 lysates) from the cell lines that express HLA-A*11:01, HLA-B*35, HLA-Cw*04:01 (CJO), Cw*05:01 (221 C5), and HLA-Cw*07:01(221 C7). (60) Indeed, MEM-E/02 bound to A*11:01, B*35, Cw*04:01, Cw*05:01, and Cw*07:01 as shown in our report. (47) However, the authors contended that the MEM-E/02 binding to HLA-Ia antigens on Western blots is not as intense as the staining of HLA-E heavy chains.…”

“…The inhibition is further confirmed by dosimetic analysis as shown in Figure 4 of the report. (47) Recently, Tremante et al (60) tested the HLA-I binding of MEM-E/02 on Western blots using the purified heavy chains (NP40 lysates) from the cell lines that express HLA-A*11:01, HLA-B*35, HLA-Cw*04:01 (CJO), Cw*05:01 (221 C5), and HLA-Cw*07:01(221 C7). (60) Indeed, MEM-E/02 bound to A*11:01, B*35, Cw*04:01, Cw*05:01, and Cw*07:01 as shown in our report.…”