HLA-E: Strong Association with β2-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides

Abstract

), all of the MEM Abs unexpectedly reacted with ␤ 2 -microglobulin (␤ 2 m)-free and denatured (but not ␤ 2 m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas ␤ 2 m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to ␤ 2 m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, ␤ 2 m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.

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

“…Substitutions at the dimorphic S 57 position reduced mAb MEM-E/02 binding in all cases, but only the nonconservative substitution with the naturally occurring P 57 residue (carried by all class I alleles except HLA-E) virtually abolished mAb binding. In complete agreement with substitution scan, retrospective analysis of our own IEF blotting studies [6] showed that under denaturing conditions MEM-E/02 does not detectably react with HLA-F, HLA-G, and 37 common HLA-A, -B, -C alleles from HLAhomozygous cell lines (listed in Supplemental Results). These alleles sample all the possible variations at each position of the epitope (compare with Supporting Information Fig.…”

“…The MEM mAbs were characterized by at least three groups [6,[8][9][10][11]. We found that they selectively bind unfolded HLA-E heavy chains free of β 2 m. Whereas mAb MEM-E/02 was particularly restricted to HLA-E in IEF/Western blotting, mAbs MEM-E/07 and MEM-E/08 cross-reacted to different extents with detergent-soluble HLA-A, -B, -C heavy chains, suggesting recognition of three distinct, although unmapped, nonconformational epitopes [6,12,13]. In contrast, using microbeads carrying HLA-E and HLA-A, -B, -C molecules, Ravindranath et al mapped mAbs MEM-E/02, MEM-E/07 and 3D12 to the same discontinuous, conformational, widely HLA-B/-C-cross-reactive epitope [8][9][10], and concluded that all these mAbs (particularly mAb MEM-E/08) are suitable to bind "intact HLA-β 2 m complexes."…”

“…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 the more than 6000 HLA-A, -B, -C, -E, -F, and -G alleles known to date (http://www.ebi.ac.uk/ipd/imgt/hla/).…”

“…Likewise, mAbs MEM-E/07 and MEM-E/08 did not detectably coimmunoprecipitate β 2 m (not shown, and see ref. [6] densitometry. When the mAb W6/32 ratio was normalized to 100, the ratios of mAbs 3D12, MEM-E/02, and DT9 were 6.5, <1, and <1, respectively, demonstrating a >100-fold difference in β 2 m association.…”

“…Precise epitope mapping may help to settle several controversial issues regarding the specificity of 4 HLA-E-restricted mAbs, namely MEM-E/02, MEM-E/07, MEM-E/08, and 3D12. The MEM mAbs were characterized by at least three groups [6,[8][9][10][11]. We found that they selectively bind unfolded HLA-E heavy chains free of β 2 m. Whereas mAb MEM-E/02 was particularly restricted to HLA-E in IEF/Western blotting, mAbs MEM-E/07 and MEM-E/08 cross-reacted to different extents with detergent-soluble HLA-A, -B, -C heavy chains, suggesting recognition of three distinct, although unmapped, nonconformational epitopes [6,12,13].…”