How do KIR on uterine Natural Killer cells (uNK) regulate human pregnancy success?

Several major disorders that women face during pregnancy, result from failure of the placenta to implant correctly into the mother's uterus or womb. During early pregnancy, fetal cells from the placenta, known as trophoblast cells, invade into the uterus and tap into her blood supply to sustain the growing baby. Failure of this process can lead to insufficient blood supply to the placenta and low birth weight babies. The 'starved' placenta can also cause a life-threatening syndrome (pre-eclampsia), which is characterised by a high maternal blood pressure and possible damage to her kidneys and other organs.

We are investigating how maternal immune cells in the lining of the uterus, known as uterine Natural Killer (uNK) cells, regulate the extent of trophoblast invasion. uNK cells have receptors called KIR that bind like a lock and key to HLA molecules on the invading trophoblast cells from the placenta. Both lock and key can vary. Our genetic studies show that when certain KIR receptors (present only in some mothers) bind to trophoblast HLA, this causes abnormal placental development and increases the risk of pre-eclampsia or a small baby. However binding of other KIR receptors to HLA does not cause this problem. Our question is: how do the mother's NK cells respond when these two different types of KIR recognise and bind the HLA molecules on the fetal trophoblast cells? We also want to ask why are responses triggered by some types of KIR detrimental while others enhance placental development? We will identify 'beneficial' and 'detrimental' responses by uNK cells and examine how they affect trophoblast invasion. Understanding the different responses of uNK will help us to pinpoint which pregnancies are at greatest risk and is an essential first step if we are to develop ways to treat these common disorders of pregnancy.

NK cells like those in the uterus are found in many tissues in the body, but show significant differences between organs. These 'tissue NK cells' have been very little studied compared with NK cells in blood. Also, NK cells even within the same tissue show tremendous variation in their responses. This will be one of the first studies of tissue NK cells, using techniques that can see how the responses of the many different types of NK cells in the tissue are controlled. Our findings will have wider implications, since therapeutic interventions targeting tissue NK cells are being developed for a range of diseases including cancer and in transplantation.

Our aim is to understand how the complex system of interactions between variable maternal KIR receptors on uterine NK cells (uNK) and fetal HLA-C ligands regulates trophoblast behaviour during placental development.

Our genetic studies show that binding of the inhibitory receptor KIR2DL1 on uNK to the cognate HLA-C group 2 ligands (C2) on trophoblast increases risk of abnormal placentation, whereas binding of activating KIR2DS1 to C2 is protective. Both KIR and HLA-C are polymorphic and we will use mass cytometry (CyTOF) to determine how this variation affects the phenotype and responsiveness of primary uNK cells from the decidua compared with matched NK cells from peripheral blood (pbNK). This will be one of the first studies using high resolution CyTOF to directly compare matched uNK and pbNK responses from the same donor.
Using in vitro assays that mimic different maternal KIR/fetal HLA-C combinations, CyTOF will enable us to study how functional responses of uNK depend upon KIR genotype and maternal or fetal HLA-C status across the full range of uNK subsets. We will employ degranluation assays (CD107), cytokine production and microarrays, to analyse how KIR2DL1+ or KIR2DS1+ uNK subsets respond to targets expressing C1 or C2 and to primary trophoblast cells expressing these epitopes. This will allow us to define novel responses mediated by these KIR in uNK and determine how responses differ between KIR2DL1 alleles that confer different risks to pregnancy. Finally we will examine how these KIR responses regulate trophoblast invasion and differentiation.

These studies will lead to an understanding of how the functions of tissue NK cells are regulated compared to pbNK and how uNK regulate trophoblast behaviour during placentation in normal and abnormal pregnancies.

Scientific Impact.
This research will initially benefit scientists and obstetricians because it will generate new knowledge and understanding of how maternal immune cells regulate placentation. Our findings will also have implications for immunologists more broadly, since no study of NK cells from solid tissue has previously been performed using CyTOF and therapeutic interventions targeting NKRs on tissue NK cells are being developed for a range of diseases including cancer and in transplantation. The way they will benefit is elaborated in the "academic beneficiaries" section.

Potential societal and economic impact of this research.
If we show how specific KIR/HLA-C combinations or KIR alleles increase risk of abnormal placentation this could lead to KIR and HLA-C typing before or during pregnancy to inform clinical management of high-risk pregnancies. Reducing the burden of pregnancy diseases such as pre-eclampsia, FGR or miscarriage would represent a long-term goal. This work could therefore benefit clinicians and other health care professionals in Obstetrics as well as the patients and their families.

Identified beneficiaries could also include those in industry and the commercial sector who wish to develop relevant screening tests in a clinical setting for genotyping maternal KIR at the allele level and maternal and fetal HLA-C typing of specific KIR and HLA-C alleles that increase pregnancy risk. Understanding the mechanisms by which KIR/HLA interactions increase the risk of pregnancy disorders will open up the possibility to develop methods to manipulate uNK/trophoblast interactions therapeutically. The project therefore has significant translational potential.

Development of new skills.
A key project goal involves the establishment of protocols and techniques for analysing NK cell function using mass cytometry (CyTOF) in Cambridge. Although this newly developed approach holds considerable promise for both research and clinical use, expertise is very limited in the UK. Cambridge only opened a CyTOF facility in December 2015. Both the Research Associate and a PhD student at Cambridge University (funded by a CASE studentship, from MedImmune) will receive training in these techniques through my collaboration with Peter Parham at Stanford. This will enhance capability and training here in Cambridge, which is essential if the technique is to be developed as a research or clinical tool. Through this project, both RA and PhD student will expand their skills in advanced cytometry, and in using complex bioinformatics tools for multiparameter data analysis such as ViSNE.
We aim to share this CyTOF antibody panel and expertise with others to enhance studies of NK biology and its clinical applications in the UK.

Engagement with the public about normal and abnormal pregnancy is clearly important.
My laboratory is actively involved in the Science Festival, which is held in Cambridge every year for two weeks in March. The general public come in large numbers to discuss with us the practical and ethical aspects of our work. During this project, we plan to continue to be actively involved in the Science Festival in the future. All the applicants frequently give public lectures about their research to specialist and non-specialist audiences and discuss their work with journalists to highlight potential translational findings to clinicians and to the public. The primary means of dissemination to the scientific and clinical communities will be results through publications and reviews in journals and at international meetings. The Cambridge University PR Department has well-established procedures for communication of important research findings to the public through newsletters, press-releases and websites. If translational findings emerge from this research, for example by identifying how KIR/HLA-C combinations significantly increase pregnancy risk, these will be rapidly communicated to clinicians and scientists.