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

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 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

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

221/HLA-G mutated in three contact residues are recognized by KIR-Ig fusion proteins.

<p>Wild-type, triple mutated 221/HLA-G, 221/HLA-Cw3 and 221/HLA-Cw6 were stained with various fusion proteins followed by secondary antibody staining. Gray histograms represent background secondary antibody staining. For confirmation of expression level, cells were stained with anti-MHC class I mAb (left panel). Black frames emphasize the unique KIR-Ig binding to the 221/HLA-G triple mutants. Shown is a representative experiment of at least three independent experiments.</p

HLA-G is markedly different from HLA-C and other selected MHC class I molecules in the contact residues between KIR and HLA-C.

<p>(A) Sequence alignment between representative MHC class I molecules of the HLA-A, B and C sub-classes and HLA-G in the binding interface with KIR inhibitory receptors. The HLA-G residues that were selected for site-directed mutagenesis are shown in bold and highlighted in yellow. The sequences are shown for two regions (positions 68–85 and 143–153). Conserved residues are indicated by dashes. (B) A ribbon diagram of the crystal structure of HLA-G with the contact residues superimposed. Cys 42 and Cys 147 which form disulfide bridges for the formation of HLA-G complexes and the contact residues that were mutated are indicated. Domains α1 and α2 are also indicated. This backbone modeling of the HLA-G molecule was generated using Swiss-PDB viewer v3.7 software. (C) A list of the single, double and triple mutations which were performed in the HLA-G molecule.</p