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

MiRNA-Mediated Control of HLA-G Expression and Function

HLA-G is a non-classical HLA class-Ib molecule expressed mainly by the extravillous cytotrophoblasts (EVT) of the placenta. The expression of HLA-G on these fetal cells protects the EVT cells from immune rejection and is therefore important for a healthy pregnancy. The mechanisms controlling HLA-G expression are largely unknown. Here we demonstrate that miR-148a and miR-152 down-regulate HLA-G expression by binding its 3′UTR and that this down-regulation of HLA-G affects LILRB1 recognition and consequently, abolishes the LILRB1-mediated inhibition of NK cell killing. We further demonstrate that the C/G polymorphism at position +3142 of HLA-G 3′UTR has no effect on the miRNA targeting of HLA-G. We show that in the placenta both miR-148a and miR-152 miRNAs are expressed at relatively low levels, compared to other healthy tissues, and that the mRNA levels of HLA-G are particularly high and we therefore suggest that this might enable the tissue specific expression of HLA-G

The miRNA-mediate reduction of HLA-G expression enhances NK cell cytotoxicity.

<p>(A–B) 721.221 and 721.221/HLA-G cells, expressing either the G variant (A) or the C variant (B), transduced with control miRNA, miR-148a or miR-152, were used as target cells in killing assays against NK clones expressing LILRB1. The effector: target (E∶T) ratio was 1∶1. Values are mean ± s.d. for triplicate samples. * <i>P</i><0.02 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown.</p

MiR-148a and miR-152 specifically target the 3′UTR of HLA-G.

<p>(A) Luciferase activity in RKO cells transduced with control miRNA (black columns), miR-148a (white columns) or miR-152 (grey columns). Results are presented relative to control reporter activity. G/C variant, reporter containing the WT sequence of the 3′UTR of HLA-G, which contains guanine or cytosine at position +3142, as indicated in the figure. Mut, reporter mutated at the seed sequence of HLA-G 3′UTR. Values are mean ± s.d. of triplicate samples. * <i>P</i><0.05 (two-tailed Student's <i>t</i>-test). One out of three representative experiments is shown. (B) FACS histogram showing GFP levels (indicative for miRNA expression) of the miRNA-infected 721.221 cells. (C–D) Quantitative real-time PCR analysis of miR-148a (C) or miR-152 (D) in 721.221/HLA-G cells expressing the G variant transduced with the relevant miRNA or a control miRNA, as indicated in the figure. Results presented relative to U6. One out of three representative experiments is shown.</p