Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors

Abstract

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α1-α2-α3 non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α1-α3 structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α1-α3-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.

Fig. 1

HLA-G isoforms. Alternative splicing of HLA-G primary transcript yields seven isoforms. Excision of one or two exons encoding globular domain generates truncated isoforms, and translation of intron four or intron two yield secreted isoforms that lack the transmembrane domain

Fig. 2

HLA-G isoforms expression and dimerization. a Monomers of HLA-G isoforms were identified from transfected cell lysates by Western blotting in a reducing SDS-PAGE. b Homodimers of HLA-G isoforms were identified from the same lysates by Western blotting in a non-reducing SDS-PAGE. In both cases, 4H84, directed toward the HLA-G α₁ domain, was used as a blotting antibody

Fig. 3

Recognition of the HLA-G α₁–α₃ structure by LILRB1 and LILRB2. a Immunoprecipitation of HLA-G1 and HLA-G5 (α₁–α₂–α₃ domains), and HLA-G2 and HLA-G6 (α₁–α₃ domains) with LILRB2-Fc. b Differential direct binding of HLA-G6-GST recombinant protein to LILRB1-Fc and LILRB2-Fc-coated beads. c Differential binding of B2M-free HLA-G5-GST and HLA-G6-GST recombinant proteins to NKL-LILRB1⁺ and NKL-LILRB1⁺LILRB2⁺ cells by flow cytometry analysis, using GST recombinant protein as control and anti-GST antibody for detection

Fig. 4

Tolerogenic function of HLA-G α₁–α₃ structure (α₁–α₃-Fc) in vivo. C57BL/6 mice strongly recognize the MHC class II-disparate mutant bm12 mouse that carries the I-A^bm12 alloantigen. The capability of α₁–α₃-Fc-coated beads to delay rejection was evaluated with non-transgenic and LILRB2-transgenic recipient animals. Kaplan–Meier curves representing graft survival are shown for α₁–α₃-Fc (plain lines) and control treatment (dotted lines). Control treatment: beads coated with mAb but without α₁–α₃-Fc. Results are expressed as median of graft survival time. Associated values are indicated above the curves