Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy

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Abstract

The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.

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E. G. Orlova

Institute of Ecology and Genetics of Microorganisms, UB RAS – branch of Perm Federal Research Center UB RAS

Author for correspondence.
Email: orlova_katy@mail.ru
Russian Federation, Perm

О. А. Loginova

Institute of Ecology and Genetics of Microorganisms, UB RAS – branch of Perm Federal Research Center UB RAS

Email: orlova_katy@mail.ru
Russian Federation, Perm

О. L. Gorbunova

Institute of Ecology and Genetics of Microorganisms, UB RAS – branch of Perm Federal Research Center UB RAS

Email: orlova_katy@mail.ru
Russian Federation, Perm

S. V. Shirshev

Institute of Ecology and Genetics of Microorganisms, UB RAS – branch of Perm Federal Research Center UB RAS

Email: orlova_katy@mail.ru
Russian Federation, Perm

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2. Fig. 1. Gating strategy used to analyze different subpopulations of peripheral blood NK cells and expression of Glut-1, CD94, CD107a molecules, and mitochondrial masses (a) – isolation of the lymphocyte gate according to the parameters of the area of direct (FSC-A) and the height of lateral (SSC-H) light scattering; discrimination of clumped cells (duplets) according to the parameters of the area and height of direct light scattering (FSC-A/FSC-H); determination of living cells (living cells) negative for staining ZombieUV™ (ZombieUV™ Fixable Viability Kit, Bio Legend); isolation of the negative population by CD3/CD14/CD19 markers in the gate of living PBMC; determination of the regulatory subpopulation of NK cells as a percentage of CD16–CD56bright and cytotoxic subpopulations of NK cells as a percentage of CD16+CD56dim and CD16hiCD56– – The CD3/CD14/CD19 gate has negative PBMC. Figure 1 shows histograms of one representative experiment. (b) Isotypic control and evaluation of the expression of the Glut-1 molecule in the studied NK cell subpopulations. (c

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3. Fig. 2. Percentage of cells positive for the expression of Glut-1, CD107a, CD94, and inclusion of the MitoSpy Green FM mitochondrial probe in different subpopulations of peripheral blood NK cells in non-pregnant (NP), pregnant women in the I and III trimesters of physiological pregnancy. CD16–CD56brightNK – regulatory NK cells; CD16+CD56dimNK – cytotoxic NK cells; CD16hiCD56NK – – minor subpopulation of cytotoxic NK cells; data are presented in the form of median and interquartile range, Me (Q1 – Q3); Figure 2b shows the median fluorescence brightness along the ordinate axis (MFI, Median Fluorescence Intensity) in cells containing MitoSpy Green FM; # – p < 0.05 relative to the group of non–pregnant (NP) according to the Kruskal - Wallis criterion; * – p < 0.05, ** – p < 0.01, *** – p < 0.001, **** – p < 0.0001 between the percentage different types of NK cells in the same group according to the Kruskal–Wallis criterion.

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