Inhibitory NK Receptor Recognition of HLA-G: Regulation by Contact Residues and by Cell Specific Expression at the Fetal-Maternal Interface

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface

The triple HLA-G contact residues mutants affect peripheral NK clones killing activity.

<p>Various S³⁵-labeled cells were incubated with (A) LIR-1⁺, (B) KIR2DL1⁺ or (C) KIR2DL2⁺ peripheral NK clone in effector to target ratio (E∶T) of 4∶1. The expression of a particular NK receptor on each of the clones is presented in A–C. Shown is one representative experiment out of four performed.</p

The triple HLA-G contact residues mutants affect decidual NK clones killing activity.

<p>Various S³⁵-labeled 221 transfected cells were incubated with (A) KIR2DL1⁺ or (B) KIR2DL2⁺ decidual NK clone in effector to target ratio (E∶T) of 5∶1. Shown is one representative experiment out of two performed.</p

Binding of various fusion proteins to the mutated 221/HLA-G molecule is not affected by single or double mutations in the contact residues.

<p>221\HLA-G mutated in the KIR-HLA-C contact residues were stained with various fusion proteins followed by secondary antibody staining. Gray histograms represent background secondary antibody staining. The numbers shown in each histogram indicate the median fluorescence intensity, MFI. Expression levels, were monitored with anti-MHC class I mAb (left panel). Shown is a representative experiment of at least three independent experiments.</p

The conformed and FHC forms of HLA-G are differentially expressed in trophoblast cell columns.

<p>Double immunofluorescent staining of representative first trimester placental sections (week 10–11) with antibody MEM-G9 (A,A′) and 4H84 (B,B′) and the combined image (C,C′) (scale bar 50 µM). DAPI (blue), conformed HLA-G (green) and FHC HLA-G (red) staining is observed in the panels with the magnified inset (boxed area in image C) viewed in panels A′,B′,C′. Several areas where differential expression of conformed and FHC HLA-G are indicated by arrows. CC indicates a trophoblast cell column.</p