Natural Killer Cells in Pregnancy

National Institute Of Allergy And Infectious Diseases

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Abstract

Human natural killer (NK) cells express a family of polymorphic killer-cell Ig-like receptors (KIR), which bind HLA class I and regulate NK cell responses. The more conserved member KIR2DL4 signals from endosomes in response to soluble ligand or agonist antibody to reprogram NK cells towards a senescence-associated secretory phenotype. Its ligand HLA-G is expressed by fetal trophoblast cells at the NK-rich maternal-fetal interface in early pregnancy. Here, we show that primary NK cells stimulated with soluble HLA-G or a KIR2DL4-specific antibody increased transcription of mostly the same genes (R2 = 0.889) with the exception of type I interferon (IFN-I) stimulated gene (ISG) transcription induced by soluble HLA-G. Gene silencing in primary NK cells identified transcription factor IRF7 and kinase JAK1 as necessary for this ISG response. JAK substrate STAT2, which links IFN-I to ISG, and IRF7 underwent phosphorylation and nuclear translocation in HLA-G stimulated NK cells. We detected association of JAK1 with KIR2DL4, which relied upon a motif in KIR2DL4 analogous to conserved JAK1 binding sites in IFN receptors. Single-cell RNA sequencing of primary blood NK cells stimulated with HLA-G revealed a similar yet broader transcriptional response in CD56bright NK cells versus CD56dim NK cells, with a strong correlation in ISG expression. Analysis of scRNA-seq data from early pregnancy revealed a correlation of HLA-G-induced transcription with genes that distinguish decidual NK cells from maternal blood NK cells. Thus, ISGs inducible by HLA-G are detected in early pregnancy, which may provide intrinsic anti-pathogen protection of NK cells at this site. Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissues early in pregnancy and encounter decidual NK cells at the maternal fetal interface. The NK cell receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program in NK cells to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unknown. Random mutagenesis revealed that three cysteines in the first Ig domain (D0) regulate endocytosis and uptake of HLA-G. The Cys10-Cys28 bond in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a Cys28-Cys74 bond present in other KIR D0 domains. We have shown that only 2 out of the 3 possible disulfide bonds occur in human cells since the potential Cys10-Cys74 disulfide bond was not detected. KIR2DL4 in human cells was found in the two remaining disulfide-bonded states. The C10-C28 bond in a purified extracellular domain of KIR2DL4 was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI caused retention of KIR2DL4 at the plasma membrane and prevented uptake of HLA-G. A mutant in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, a mutant in the Cys28-Cys74 configuration remained at the plasma membrane and internalized in response to HLA-G. A structural change upon disulfide switching predicted by AlphaFold reorients the D0 domain into a conformation that binds HLA-G. Thus, we show how KIR2DL4 conversion from an inactive state to an HLA-G binding form can regulate NK cell function to promote fetal development.