Immunology of pregnancy: renewed interest

The long-standing question of pregnancy immunological paradox has been generating renewed interest. Recent insights have emerged from studies in pregnant mice and humans demonstrating a number of mechanisms that prevent potentially harmful effects of maternal anti-paternal allo-antibodies (complement inhibition, partial deletion of maternal B cells specific of paternal antigens), cytotoxic CD8+ T cells (lack of HLA-A and HLA-B expression on trophoblast, local immunosuppressive molecules, transient tolerance of paternal allo-antigens specific T cells) and uterine NK cells directed against fetal-derived trophoblast cells (limited NK cytotoxic potential, trophoblast resistance to NK killing). Interestingly, it appears that not only decidual NK cell/trophoblast interactions are not harmful for the fetus but are beneficial for the placental vascularization and its subsequent development. A recent report has indeed demonstrated that during pregnancy most of the combinations of uterine KIR (killer cell immunoglobulin-like receptor) NK cell receptors and fetal HLA-C molecules expressed by trophoblast led to normal pregnancies, whereas mothers lacking activating KIR of the AA genotype when the fetus possessed HLA-C of the C2 group were at a greatly increased risk of severe preeclampsia pathology.     

The role of the maternal immune system in the regulation of human birthweight

Human birthweight is subject to stabilizing selection. Large babies are at risk of obstetric complications such as obstructed labour, which endangers both mother and child. Small babies are also at risk with reduced survival. Fetal growth requires remodelling of maternal spiral arteries to provide an adequate maternal blood supply to the placenta. This arterial transformation is achieved by placental trophoblast cells, which invade into the uterine wall. Under-invasion is associated with fetal growth restriction; but if invasion is excessive large babies can result. A growing body of evidence suggests that this process is controlled by interactions between killer-cell immunoglobulin-like receptors (KIRs) expressed on maternal uterine natural killer cells (uNK) and their corresponding human leukocyte antigen-C (HLA-C) ligands on invading trophoblast. Mothers with the KIR AA genotype and a fetus with a paternal HLA-C2 allele tend to have small babies, because this combination inhibits cytokine secretion by uNK. Mothers with the activating KIR2DS1 gene and an HLA-C2 fetus are more likely to have large babies. When KIR2DS1 binds to HLA-C2 this increases secretion of cytokines that enhance trophoblast invasion. We conclude that specific combinations of the highly polymorphic gene systems, KIR and HLA-C, contribute to successful reproduction by maintaining birthweight between two extremes.     

Unique receptor repertoire in mouse uterine NK cells

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.     

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.     

Dynamic shift from CD85j/ILT-2 to NKG2D NK receptor expression pattern on human decidual NK during the first trimester of pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1⁺/KIR2DL2/L3/S2⁺ subset towards the double negative subset, coupled with a decrease of the CD85j⁺/NKG2D⁻ subset in favour of the CD85j⁻/NKG2D⁺ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14⁺ and CD3⁺ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14⁺ and CD3⁺ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14⁺ cells whereas it was not detected on CD3⁺ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.     

The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical alpha beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns

Killer cell Ig-like receptor (KIR) and CD94:NKG2A molecules were first defined as human NK cell receptors (NKR), but now are known to be expressed and to function on subpopulations of T cells. Here the repertoires of KIR and CD94:NKG2A expression by T cells from two donors were examined and compared with their previously defined NK cell repertoires. T cell clones generated from peripheral blood of both donors expressed multiple NKR in different combinations and used the range of receptors expressed by NK cells. In both donors alpha beta T cells less frequently expressed the inhibitory receptors CD94:NKG2A and KIR2DL1 than either gamma delta T cells or NK cells. In contrast to NK cells, not all NKR(+) T cells expressed an inhibitory receptor for autologous HLA class I. This lack of specific inhibitory NKR was especially apparent on alpha beta T cells of one donor. Overall, alpha beta T cells exhibited a distinct pattern of NKR expression different from that of gamma delta T and NK cells, which expressed highly similar NKR repertoires. In one donor, analysis of TCR rearrangement revealed a dominant subset of NKR(+) T cells sharing identical TCR alpha- and beta-chains. Remarkably, among 55 T cell clones sharing the same TCR alpha beta rearrangement 18 different KIR phenotypes were seen, suggesting that KIR expression was initiated subsequently to TCR rearrangement.     

HLA-C and HIV-1: friends or foes?

The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins.     

Spontaneous clustering and tyrosine phosphorylation of NK cell inhibitory receptor induced by ligand binding

Inhibition of NK cell cytotoxicity by killer cell Ig-like receptors (KIR) depends on phosphorylation of cytoplasmic tyrosines in KIR, which recruit tyrosine phosphatase Src homology protein tyrosine phosphatase 1. It is not clear how KIR, whose function lies downstream of a tyrosine kinase, succeeds in blocking proximal NK cell activation signals upon binding HLA class I on target cells. Here we show that mixing NK cells with insect cells expressing HLA-C was sufficient to induce clustering of KIR, and phosphorylation of KIR and SHP-1. Transient phosphorylation of KIR was detected in the presence of pervanadate, an inhibitor of protein tyrosine phosphatases, at suboptimal concentration. Phosphorylation of KIR was specifically induced by ligand binding because it was detected only when HLA-C was loaded with a peptide that permits KIR binding. KIR phosphorylation was not dependent on ICAM-1-mediated adhesion and was not blocked by inhibition of actin polymerization, but required Zn(2+). Fluorescence resonance energy transfer between HLA-C molecules revealed close molecular interactions induced by KIR binding. These results demonstrate tight clustering of KIR and rapid KIR phosphorylation induced simply by binding to HLA-C. The unique property of KIR to become phosphorylated in the absence of adhesion and of actin cytoskeleton rearrangement explains how KIR can efficiently block early activation signals during NK-target cell contacts.     

Association of HLA Class-I and Inhibitory KIR Genotypes in Gabonese Patients Infected by Chikungunya or Dengue Type-2 Viruses

Background

Natural killer (NK) cells provide defense in the early stages of the immune response against viral infections. Killer cell immunoglobulin-like receptors (KIR) expressed on the surface of NK cells play an important role in regulating NK cell response through recognition of human leukocyte antigen (HLA) class I molecules on target cells. Previous studies have shown that specific KIR/ligand combinations are associated with the outcome of several viral infectious diseases.

Methods

We investigated the impact of inhibitory and activating KIR and their HLA-class I ligand genotype on the susceptibility to Chikungunya virus (CHIKV) and Dengue virus (DENV2) infections. From April to July 2010 in Gabon, a large outbreak of CHIKV and DENV2 concomitantly occurred in two provinces of Gabon (Ogooué-Lolo and Haut-Ogooué). We performed the genotypic analysis of KIR in the combination with their cognate HLA-class I ligands in 73 CHIKV and 55 DENV2 adult cases, compared with 54 healthy individuals.

Results

We found in CHIV-infected patients that KIR2DL1 and KIR2DS5 are significantly increased and decreased respectively, as compared to DENV2⁺ patients and healthy donors. The combination of KIR2DL1 and its cognate HLA-C2 ligand was significantly associated with the susceptibility to CHIKV infection. In contrast, no other inhibitory KIR-HLA pairs showed an association with the two mosquito-borne arboviruses.

Conclusion

These observations are strongly suggestive that the NK cell repertoire shaped by the KIR2DL1:HLA-C2 interaction facilitate specific infection by CHIKV.     

Cutting edge: engagement of CD160 by its HLA-C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-gamma, TNF-alpha, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.     

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.     

Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10(-9) M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56(bright) blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.     

A novel trafficking signal within the HLA-C cytoplasmic tail allows regulated expression upon differentiation of macrophages

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.     

Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure.

We have previously shown that human cultured trophoblast cells are resistant to lysis by natural killer (NK) cells from both peripheral blood and decidua although cells are present in decidua which do exhibit NK activity against K562(1). Using a cold-target inhibition assay and a single-cell conjugate assay we have now examined whether these trophoblast cells have NK target structures on their surfaces. Our findings indicate that first-trimester human trophoblast cells do not express surface structures recognized by decidual Leu19+ (CD56+) large granular lymphocytes (LGLs) isolated from human decidua. Immunostaining of the conjugates formed between decidual NK effectors and K562 cells confirmed that these effector cells are CD56+ LGLs.     

Cutting edge: resistance to HIV-1 infection among African female sex workers is associated with inhibitory KIR in the absence of their HLA ligands

NK cells are regulated in part by killer Ig-like receptors (KIR) that interact with HLA molecules on potential target cells. KIR and HLA loci are highly polymorphic and certain KIR/HLA combinations were found to protect against HIV disease progression. We show in this study that KIR/HLA interactions also influence resistance to HIV transmission. HIV-exposed but seronegative female sex workers in Abidjan, C?te d'Ivoire, frequently possessed inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1 and KIR3DL1 homozygosity in the absence of HLA-Bw4. HIV-seropositive female sex workers were characterized by corresponding inhibitory KIR/HLA pairings: KIR2DL3 homozygosity together with HLA-C1 and a trend toward KIR3DL1/HLA-Bw4 homozygosity. Absence of ligands for inhibitory KIR could lower the threshold for NK cell activation. In addition, exposed seronegatives more frequently possessed AB KIR genotypes, which contain more activating KIR. The data support an important role for NK cells and KIR/HLA interactions in antiviral immunity.