Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2

Abstract

Many common disorders of pregnancy are attributed to insufficient invasion of the uterine lining by trophoblast, fetal cells that are the major cell type of the placenta. Interactions between fetal trophoblast and maternal uterine NK (uNK) cells--specifically interactions between HLA-C molecules expressed by the fetal trophoblast cells and killer Ig-like receptors (KIRs) on the maternal uNK cells--influence placentation in human pregnancy. Consistent with this, pregnancies are at increased risk of preeclampsia in mothers homozygous for KIR haplotype A (KIR AA). In this study, we have demonstrated that trophoblast expresses both paternally and maternally inherited HLA-C surface proteins and that maternal KIR AA frequencies are increased in affected pregnancies only when the fetus has more group 2 HLA-C genes (C2) than the mother. These data raise the possibility that there is a deleterious allogeneic effect stemming from paternal C2. We found that this effect also occurred in other pregnancy disorders (fetal growth restriction and recurrent miscarriage), indicating a role early in gestation for these receptor/ligand pairs in the pathogenesis of reproductive failure. Notably, pregnancy disorders were less frequent in mothers that possessed the telomeric end of the KIR B haplotype, which contains activating KIR2DS1. In addition, uNK cells expressed KIR2DS1, which bound specifically to C2+ trophoblast cells. These findings highlight the complexity and central importance of specific combinations of activating KIR and HLA-C in maternal-fetal immune interactions that determine reproductive success.

Figure 1. Representative KIR A and B haplotypes illustrate centromeric (Cen-A and Cen-B) and telomeric (Tel-A and Tel-B) gene regions, with the HLA-C ligands (C1 and C2) depicted above their cognate receptors.

In addition, KIR2DL2 also binds subsets of C2 allotypes (54), and KIR2DS4 binds some C1 and C2 allotypes (63). A KIR2DS5 locus may also be found in the Cen-B region, but this is seen infrequently in the white British population. Similarly, KIR2DS3 is most often associated with the KIR2DL5B locus in the Cen-B region, as shown here, but may occasionally occur in the Tel-B region (18).

Figure 2. HLA-C is expressed predominately on the EVT in the decidua basalis.

(A and B) Immunofluorescence staining of frozen sections of human first-trimester implantation site showed infiltrating EVTs in sections of decidua basalis identified by staining for HLA-G. EVTs were observed mingling and in close apposition with CD56⁺ NK cells. (C) Cytokeratin staining identified EVTs and glandular epithelial cells. (D and E) Only EVTs were strongly labelled with the mAb DT9, specific for HLA-C allotypes. There was weak staining of other maternal cells in the decidua identified as CD45⁺ leukocytes (not shown). Scale bars: 200 μm (A); 30 μm (B); 60 μm (C–E).

Figure 3. Fetal EVTs express both maternally and paternally inherited HLA-C allotypes.

EVTs isolated from the genotyped placental samples shown in Table 1 were identified by flow cytometric staining for HLA-G. Double staining with the mAb WK4C11 showed that HLA-G⁺ EVTs expressed both paternal and maternal C1 allotypes. Examples are shown in which the C1 allele detected by mAb WK4C11 was inherited maternally or paternally (samples 8 and 9, respectively, in Table 1). No staining was seen on EVTs with WK4C11 when the trophoblast had the C2/C2 genotype.

Figure 4. KIR2DS1 and KIR2DL1 bind specifically to C2 molecules on EVTs.

(A–H) Binding of KIR-Fc fusion proteins was measured by flow cytometry to peripheral blood CD3⁺ T cells (A–D) or HLA-G⁺ EVTs isolated from placentae of healthy first-trimester pregnancies (E–H). KIR2DL1 and 2DS1 bind cells from C2-positive (A, B, E, and F) but not C2-negative (C, D, G, and H) donors. The indicated KIR-Fc fusion protein histograms are outlined in black and secondary antibody histograms in gray. Pre-incubating with an anti-KIR mAb (HP-3E4) blocked KIR2DS1-Fc and KIR2DL1-Fc binding to both PBLs and trophoblasts (histograms with dotted line).

Figure 5. Categorization of pregnancy types according to the relative number of C2 genes or by parental origin of the fetal C2.

(A) Pregnancies were separated into 3 groups: those in which the fetus had fewer C2 genes than the mother (i.e., B and E), those in which the fetus and mother had the same number of C2 genes (i.e., A, D, and G), and those in which the fetus had more C2 genes than the mother (i.e., C and F). (B) Alternatively, pregnancies were selected in which the origin of the single fetal C2 gene could be definitively identified as being derived from either the father (i.e., C and D1) or the mother (i.e., D2 and E). HLA-C allele typing to 4 places was used to identify the parental origin of the fetal C2, distinguishing D1 from D2. See Table 3 for comparison of the groups.