HLA Class I Allelic Sequence and Conformation Regulate Leukocyte Ig-Like Receptor Binding

Abstract

Leukocyte Ig-like receptors (LILRs) are a family of innate immune receptors predominantly expressed by myeloid cells that can alter the Ag presentation properties of macrophages and dendritic cells. Several LILRs bind HLA class I. Altered LILR recognition due to HLA allelic variation could be a contributing factor in disease. We comprehensively assessed LILR binding to >90 HLA class I alleles. The inhibitory receptors LILRB1 and LILRB2 varied in their level of binding to different HLA alleles, correlating in some cases with specific amino acid motifs. LILRB2 displayed the weakest binding to HLA-B*2705, an allele genetically associated with several autoimmune conditions and delayed progression of HIV infection. We also assessed the effect of HLA class I conformation on LILR binding. LILRB1 exclusively bound folded β2-microglobulin–associated class I, whereas LILRB2 bound both folded and free H chain forms. In contrast, the activating receptor LILRA1 and the soluble LILRA3 protein displayed a preference for binding to HLA-C free H chain. To our knowledge, this is the first study to identify the ligand of LILRA3. These findings support the hypothesis that LILR-mediated detection of unfolded versus folded MHC modulates immune responses during infection or inflammation.

Interaction of the LILRB1 inhibitory receptor with HLA class Ia dimers

“…The LILRB1 inhibitory receptor recognizes a variety of HLA-I molecules, albeit with different affinities [11,12]. Cytoplasmic Cys were reported to be required for HLA-I recognition by the inhibitory receptor [19], and HLA-B27 and -A2 molecules were shown to form β2m-associated dimers in exosomes through specific intracellular Cys [20,21].…”

“…Under these experimental conditions LILRB1 was confirmed to preferentially interact with HLA-I molecules able to dimerize. The formation of these conformers likely enhances the avidity of the LILRB1-HLA-I interaction, primarily determined by other structural features which modulate LILRB1 affinity [11,22].…”

“…Preparations of plasmid DNA were made using the Endo-free Maxi-prep Kit (QIAgen). LILRB1-Fc and LILRB2-Fc [11] were obtained by transfecting HEK293T cells with Signal pIgplus vector containing the coding sequence of each inhibitory receptors ectodomain using calcium phosphate, and 18 h post-transfection, the culture medium was replaced by fresh FBS-free medium (EX-cell ACF CHO Medium, Sigma-Aldrich) supplemented with 5 mM L-glutamine (Life Technologies). After 6 days, supernatants were harvested and fusion proteins purified by affinity chromatography using protein A-Sepharose CL-4B (GE Healthcare) and analyzed by SDS-PAGE and Coomassie blue staining.…”

“…LILRB1 was shown to recognize HLA-I, assessing the interaction of fusion proteins with transfected cell lines [1,2] or purified HLA-I-coated beads [11], as well as by surface plasmon resonance and structural analysis [12,13]. LILRB1 interaction with HLA-I molecules constitutively expressed in primary cells is relevant to understanding the physiological role of the inhibitory receptor, but information in this regard is limited.…”

“…LILRB1 interaction with HLA-I molecules constitutively expressed in primary cells is relevant to understanding the physiological role of the inhibitory receptor, but information in this regard is limited. In contrast to killer immunoglobulin-like receptors (KIR), LILRB1 interacts with the conserved α3 domain and β2m, potentially recognizing a wide variety of HLA-I molecules, albeit with different affinities determined by allelic variability and conformation [11,12,14]. At least three types of HLA-I conformers may be detected on the cell surface i.e.…”