Host defense, self-tolerance, and tissue transplantation are major health issues that invoke both innate and adaptive immune responses. However, the immune defense mechanisms of the fetus have challenged the concepts embedded in the traditionally defined pathways of immunity. This is further complicated by “immunologically tolerant” phenotype of the predominant NK cell population in the pregnant uterus and their perceived role in placentation-associated endovascular activity (16
). In this study we describe novel mechanisms by which uNK cells maintain non-cytotoxic and pro-angiogenesis status.
Our results provide evidence for a central link between production of VEGF C, a pro-angiogenic factor, by uNK cells and their non-cytotoxic phenotype. Using primary human peripheral blood or endometrial NK cells and panel of clones derived from primary cells, we demonstrate that VEGF C production is a hallmark property of non-cytotoxic uNK cells. We further demonstrate that VEGF C, not VEGF A or PlGF, can protect target endothelial and trophoblast cells from killing by cytotoxic pNK cells. Lack of killing is directly associated with VEGF C-mediated induction of TAP-1 in target cells, a key molecule in the process of MHC class I assembly. This is directly demonstrated by siRNA-mediated knockdown of VEGF C-mediated TAP-1 expression which results in reversal of non-cytotoxic characteristics of uNK cells.
Using a three-dimensional culture system on matrigel to evaluate the endovascular activity of uNK cells, we demonstrate that there are differences in the ability of activated uNK cell clones to facilitate or disrupt the endothelial-trophoblast dual cell interactions mimicking vascular remodeling. While CD56bright
uNK cell clones consistently expressed NCRs, they differed in their cytotoxic profile. This is not surprising because uNK cell clones were propagated in vitro
in the presence of activating stimuli. In vivo
, most uNK cells are likely to be of non-cytotoxic phenotype. Despite in vitro
conditions, several uNK cell clones maintained their non-cytotoxic phenotype. In these clones, the non-cytotoxic phenotype correlated with the production of high amounts of VEGF C. Importantly, exogenous VEGF C could rescue trophoblasts and endothelial cells from traditionally cytotoxic NK cells. Dysregulation in VEGF C production at the maternal-fetal interface could be a signal for poor angiogenesis and pregnancy complications. Reduced expression of VEGF C has been reported in pregnancies experiencing intrauterine growth restriction (IUGR) and preeclampsia (43
). Involvement of VEGF C may thus explain non-killer phenotype of uNK cells despite possessing toxic granules and expressing cytotoxicity receptors.
Since our results show that uNK cells do not express receptors for VEGFs, it is tempting to argue that angiogenic factors are produced for a paracrine action on uterine endothelial cells and invading trophoblasts. Our results point to this activity of uNK cell derived VEGF C. VEGF C acts by binding to VEGF R2 and VEGF R3 and triggers intracellular survival signals in tumor cells and promote their invasion (44
). In addition to maintaining uNK cell non-cytotoxicity, VEGF C may support survival of endothelial cells (45
). Although during pregnancy, intrauterine hormones and factors that upregulate secretions of VEGF C in uNK cells are not known, in vitro
studies suggest involvement of cytokines like IL-15 (36
The non-cytotoxic capacity of NK cells is based on its ability to recognize surface major histocompatibility complex (MHC) class I molecules on target cells that deliver signals to suppress NK cell functions. A lack of engagement of such MHC-specific receptors leads to NK cell-mediated killing (8
). Interestingly, Bender and colleagues (39
) have shown that endothelial cells exhibited sensitivity to activated peripheral blood NK cells in the absence of expression of TAP-1. IFN-γ was shown to induce TAP-1 in endothelial cells and protect them from peripheral blood NK cell-mediated killing (39
). However, our data suggest that VEGF C is robustly produced by non-cytotoxic uNK cells compared to IFN-γ. It is thus proposed that VEGF C is the predominant regulator of TAP-1 expression in the uterus, although it is likely that VEGF C and IFN-γ act in tandem as supported by our observations (). Interestingly VEGF A or PLGF failed to induce TAP-1 expression (). TAP-1 is a key factor essential for peptide loading for MHC class I assembly and antigen presentation (33
). Silencing TAP-1 expression using siRNA in trophoblasts abolished the cyto-protective activity of VEGF C, confirming the mechanism of action of VEGF C.
Trophoblasts cells do not express classical MHC class I molecules but express HLA-C and unconventional HLA-G and HLA-E molecules (5
). By using HLA-E as a molecule of choice, we show that VEGF C enhanced surface expression of HLA-E on first trimester HTR8 trophoblasts. Surface expression of HLA-E normally depends on the recruitment and binding of TAP-dependent classical nonameric peptides with anchoring residues derived from HLA class I signal sequence (47
). Interestingly, TAP-1 expression has been shown to be significantly higher in extravillous trophoblasts that are positive for non-polymorphic HLA class I molecules (5
). We also detected significant presence of pan-MHC class I on both HTR8 trophoblast cells and HUVECs in response to VEGF C, suggesting that this angiogenic factor is a potent immunoregulator in the uterine microenvironment.
Taken together, these findings support a dual role of VEGF C in immune tolerance and promoting active angiogenesis by uNK cells. Our findings for the first time provide evidence that non-cytotoxicity of uNK cells is directly coupled to their angiogenic translational machinery. uNK cells apparently prepare trophoblasts and endothelial cells to express modest levels of MHC class I molecules to evade killing by a major cytotoxic cell population at the maternal-fetal interface.