Natural killer cell profiles in recurrent pregnancy loss: Increased expression and positive associations with TACTILE and LILRB1

Abstract

Problem: NK cells are important for healthy pregnancy and aberrant phenotypes or effector functions have been associated with RPL. We compared expression of a broad panel of NK cell receptors, including immune checkpoint receptors, and investigated their clinical association with RPL as this might improve patient stratification and prediction of RPL.

Method of study: Peripheral blood mononuclear cells were isolated from 52 women with RPL and from 2 women with an uncomplicated pregnancy for flowcytometric analysis and plasma was used to determine anti-CMV IgG antibodies.

Results: Between RPL and controls, we observed no difference in frequencies of T-, NKT or NK cells, in CD56dimCD16+ or CD56brightCD16- NK cell subsets or in the expression of KIRs, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46 or DNAM1. NK cells from women with RPL had a higher expression of LILRB1 and TACTILE and this was associated with the number of losses. The immune checkpoint receptors PD1, TIM3 and LAG3 were not expressed on peripheral blood NK cells. In RPL patients, there was a large variation in NKG2C expression and higher levels could be explained by CMV seropositivity.

Conclusion: Our study identified LILRB1 and TACTILE as NK cell receptors associated with RPL. Moreover, we provide first support for the potential role of CMV in RPL via its impact on the NK cell compartment. Thereby our study could guide future studies to confirm the clinical association of LILRB1, TACTILE and NKG2C with RPL in a larger cohort and to explore their functional relevance in reproductive success.

Keywords: cytomegalovirus; immune checkpoint; natural killer cell; recurrent pregnancy loss.

FIGURE 1

Frequencies of cell populations in women with a previous uncomplicated pregnancy (C n = 22) and women with recurrent pregnancy loss (RPL n = 52). Cell populations are presented as percentage of lymphocytes (NKT cells (CD3⁺CD56⁺), T cells (CD3⁺CD56^–) and NK cells (CD3^–CD56⁺)), as percentage of T cells (T helper cells (CD3⁺CD56^–CD4⁺) and cytotoxic T cells (CD3⁺CD56^–CD8⁺)) or as percentage of NK cells (CD56^dim NK cells (CD3^–CD56⁺CD16⁺) and CD56^bright NK cells (CD3^–CD56⁺⁺CD16^–)). All populations were measured by flow cytometry. Dots indicate individuals, lines indicate median and interquartile range

FIGURE 2

Expression of activating receptors in women with a previous uncomplicated pregnancy (C n = 22) and women with recurrent pregnancy loss (RPL n = 52). Data show percentage NKG2C positive cells of total NK cells (CD3^–CD56⁺) or corrected mean fluorescent intensity (MFI) of total NK cells (CD3^–CD56⁺) for NKG2D, DNAM1, NKp46 and NKp30. Correction was done by subtracting the MFI of the fluorescence minus one (FMO). All data were acquired by flow cytometry. Dots indicate individuals, lines indicate median and interquartile range. Bottom: representative histograms of activating receptors on peripheral NK cells, grey histograms represent the FMO (no receptor staining) and green histograms represent the receptor staining in RPL and C

FIGURE 3

Expression of inhibitory receptors in women with a previous uncomplicated pregnancy (C n = 22) and women with recurrent pregnancy loss (RPL n = 52). Data show percentage of KIR2DL2/3, KIR3DL1, KIR2DL1, NKG2A positive cells of total NK cells (CD3^–CD56⁺) or corrected mean fluorescent intensity (MFI) of total NK cells (CD3^–CD56⁺) for LILRB1 and TACTILE. Correction was done by subtracting the MFI of the fluorescent minus one (FMO). All data were acquired by flow cytometry. Dots indicate individuals, lines indicate median and interquartile range. Bottom: representative histograms of inhibitory receptors on peripheral NK cells, grey histograms represent the FMO (no receptor staining) and green histograms represent the receptor staining in RPL and C

FIGURE 4

Linear regression between logarithmically transformed LIRLB1 and TACTILE expression and number of pregnancy losses (n = 74), presented as regression line with correlation coefficient (r) and 95% confidence interval (dotted lines)

FIGURE 5

Frequencies of natural killer (NK) cells expressing NKG2C are shown for women with a previous healthy pregnancy (C n = 16) and women with recurrent pregnancy loss (RPL n = 30) being either positive or negative for the cytomegalovirus (CMV). Dots represent individuals, lines indicate median and interquartile range