Cutting Edge: Local Proliferation of Uterine Tissue-Resident NK Cells during Decidualization in Mice

Abstract

NK cells accumulate in adult murine and human uteri during decidualization induced physiologically, pathologically, or experimentally. Adoptive transfer studies indicate that uterine NK (uNK) cells arise from circulating progenitors. However, virgin uteri contain few circulating NK1.1⁺CD49a^- conventional NK cells, whereas NK1.1⁺CD49a⁺ tissue-resident NK (trNK) cells are abundant. In this study, we employed a novel, immune-competent NK cell-specific reporter mouse to track accumulation of uNK cells during unmanipulated pregnancies. We identified conventional NK and trNK cells accumulating in both decidua basalis and myometrium. Only trNK cells showed evidence of proliferation. In parabiosis studies using experimentally induced deciduomata, the accumulated uNK cells were proliferating trNK cells; migrating NK cells made no contribution. Together, these data suggest proliferating trNK cells are the source of uNK cells during endometrial decidualization.

Figure 1:. Emergence of GFP⁺ cells in Ncr1^iCre x Rosa^mT/mG dams in pregnancy.

A) Pregnant uterus (gd6.5) of Rosa^mT/mG control mouse and Ncr1^iCre x Rosa^mT/mG reporter mouse. A 500 um bar is shown. B) Pregnant Ncr1^iCre x Rosa^mT/mG reporter (gd11.5), with accumulation of GFP⁺ cells in the DB and myometrium. A 1mm bar is shown. Three independent experiments were analyzed at gd6.5 (n=4) and gd11.5 (n=8).

Figure 2:. Time course of GFP⁺ cells accumulating in Ncr1^iCre x Rosa^mT/mG dams during pregnancy.

A) The uteri of pregnant Ncr1^iCre x Rosa^mT/mG reporter mice were prepared for histology at the indicated gestational days (gd). B) The MLAp, DB and labyrinth were all physically separated. Each anatomical region was separately processed for histology. A 500um scale is shown on all images. C) At gd11.5 the MLAp and DB were separated and single cell suspensions prepared for flow cytometry, along with spleen from Ncr1^iCre x Rosa^mT/mG reporter mouse. The percentages in the gates represent CD45⁺CD19⁻ that express GFP or are GFP-negative that express NK1.1 and are CD3-negative. Three independent experiments were analyzed at each gd with 6–8 implantation sites each.

Figure 3:. The DB and MLAp both contain uNK cells that are phenotypically cNK and trNK cells.

The uterus of a pregnant B6 dam was processed at gd11.5. A) The MLAp and DB were physically separated and single cell suspensions were prepared for flow cytometry. The numbers in the gates represent the percentage of CD45⁺CD3⁻CD19⁻ NK1.1⁺ that expresses either CD49a or DX5. B) Cell numbers and percentages were plotted from 3 experiments with 6–8 implantation sites in each experiment. C) The MLAp and DB were prepared for flow cytometry. Histograms were gated on CD45⁺CD3⁻CD19NK1.1⁺ and overlays are of the DX5⁺ and CD49a⁺ from the distinct regions and DX5⁺ spleen. D) The tissues were prepared for flow cytometry and analyzed for BrdU incorporation and Ki67 expression. Representative histograms of 4 independent experiments are shown, (n=4).

Figure 4:. The trNK cells accumulate and proliferate in deciduomata.

A) A photograph of the decidualized and control horns. B) Ncr1^iCre x Rosa^mT/mG NK reporter mice were artificially decidualized and on d5 the decidualized and control horns were processed for histology. A 2.5mm scale is shown. C) The decidualized and control horns were processed for flow cytometry. The numbers in the gates represent the percentage of CD45⁺CD3⁻CD19⁻NK1.1⁺ that express CD49a or DX5. D) Cell number and percentage plotted of 2 independent experiments, n=10. E) Tissues were processed for flow cytometry and the uNK cells were analyzed for the expression of Ki67.

Figure 5:. CD49a⁺NK1.1⁺ uterine trNK cells are resident and proliferate.

Ncr1^iCre x Rosa^mT/mG NK reporter and B6 mice were artificially decidualized and parabiosed. A) A photograph of the decidualized and control horns of the parabionts. B) On d7 the decidualized and control horns from each parabiont were processed for histology. A 1mm scale is shown. C) The decidualized and control horns of each parabiont were processed for flow cytometry. The percentages in the gates represent CD45⁺CD3⁻CD19⁻NK1.1⁺ that express GFP or are GFP negative that express CD49a and DX5. D) Cell percentage of donor cells of 6 decidualized horns from 2 independent parabiosis experiments, (n=6). E) Tissues were processed as in C and the uNK cells were analyzed for the expression of Ki67.