Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Uterine NK cells

• NK tissue-residency

• Uterus transplant

Main Findings

Establishment of a pregnancy in mammals requires a delicate interplay of multiple immune cells at the maternal-fetal interface. Induction of tolerance towards the semi-allogeneic fetus is essential for maintaining a pregnancy. Conversely, embryo implantation and placentation require a pro-inflammatory immune environment. Uterine natural killer (NK) cells have specifically been linked to spiral artery remodelling and trophoblast differentiation and mice lacking these cells show abnormal placentation. Similarly, in humans abnormal placentation due to poor spiral artery remodelling has also been associated with pre-eclampsia and previous studies have suggested a link between a decrease in uterine NK cells and recurrent pregnancy loss. However, the exact contribution of these cells in early pregnancy remains elusive.

Asiimwe et al. use a cohort of patients having undergone uterus transplant (Utx) to study the distribution of uterine NK cells in these patients compared to healthy subjects. This patient group is known to be pre-disposed to pregnancy complications related to placentation, such as pre-eclampsia, making it a good human model to investigate the role of tissue-resident NK cells in human pregnancy and pregnancy related complications. Using high-throughput techniques such as single-cell RNA sequencing and CITE-seq the authors show that:

1. NK cells are depleted in patients having undergone a uterus transplant.

2. CD49a+CD103+ tissue-resident NK cells (trNK3) specifically are absent in Utx patients.

3. trNK3 lose the signature associated with TGF-b and subsequently downregulate their early tissue-residency program in Utx.

4. Inhibition of NFAT by a calcineurin inhibitor (e.g. tacrolimus) blocks induction of a trNK3 phenotype in healthy uterine NK cells.

In summary, the authors conclude that trNK3 development in Utx is absent due to inhibition of NFAT by calcineurin inhibitors commonly prescribed to these patients and subsequent failure to establish a tissue-residency phenotype in NK cells leads to the abnormal placentation observed in these patients.

Limitations

• Even though all CD45+ cells were sorted and sequenced for scRNA-seq, the focus on NK cells is immediate and the compositional difference for other cell types is not discussed. It would be essential to also show these in a supplemental figure to investigate whether the transcriptional changes observed are specific to NK cells or general to resident immune cells.

• As serial biopsies of Utx patients were taken, it would be valuable to show how/if the immune composition changes over time after transplantation and potentially see at what timepoint trNK3 are lost.

• While trNK3 are described in detail here, their role in a normal pregnancy should be addressed further if the authors want to claim they are an essential subset at homeostasis. This could be achieved by comparing placentas of healthy pregnancies to those that suffered from pre-eclampsia (and are not Utx) and stain for trNK3 in tissue sections.

• Using healthy donor biopsies (as shown in Fig. 8) could be used to characterise trNK3 functionally (e.g. ELISA for cytokines, more extensive flow cytometry staining) and perform more mechanistic experiments to convincingly show that NFAT signalling is responsible for their development/maintenance. This could be done by measuring NFAT activity with and without tacrolimus treatment using commercially available assay kits.

• The role of stromal cells in establishing trNK3 cells should be addressed as these cells are likely the main producers of IL-15 and TGF-b in the uterine environment. Stromal cells isolated form healthy donors could also be treated ex vivo with tacrolimus or other immunosuppressive agents used in patients to measure whether the production of these cytokines is inhibited or if trNKs do not react to these stimuli (e.g. downregulation of IL-15R, or NFAT inhibition downstream in NKs).

Significance/Novelty

This study characterises a novel subset of human uterine tissue-resident NK cells (trNK3). These cells could be essential for placentation and subsequently healthy pregnancies in humans and further investigation into the function of this cell type will open additional avenues in reproductive medicine. Further, the implication that calcineurin inhibitors could block the development of these cells is especially relevant in an organ transplant setting since patients receive these drugs as standard immunosuppression regime after transplantation. Therefore, clinicians should potentially reconsider dosing regimens and usage of calcineurin inhibitors in transplant patients with a desire to carry a successful pregnancy.

Credit

Reviewed by Teresa Neuwirth as part of a cross-institutional journal club between the Vanderbilt University Medical Center (VUMC), the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.