Visualization of B10 regulatory cells in peripheral blood in physiological pregnancy

Abstract

The current trend in studies of the B-cell immunity is the study of small subpopulations of cells. It was found that a minor subpopulation of IL-10 producing B-cells (B10-reg cells) has the properties of limiting excessive reactions of the innate and adaptive immune response. Their regulatory and pathogenetic effect has been shown in various physiological and pathophysiological conditions, in particular in the pathological pregnancies.Due to the low content of B10-reg cells in the blood (up to 1%) and the difficulties of visualizing flow cytometry data, a previously developed method based on prolonged stimulation of peripheral blood cells with a combination of factors ((CD40L+CpG) and PMA) causing cell activation, proliferation and maturation, allows visualization of the enriched fraction of B10-reg cells (B10 cells + pro-B10 cells), the content of which exceeds 5%. The aim of this study was to obtain a stimulated ex vivo population of B10 cells + pro-B10 cells from the peripheral blood of patients with physiological pregnancy and to develop an optimal strategy for gating B10-reg cells for their visualization.Materials and methods: in patients with physiological pregnancy in the third trimester, peripheral blood was taken. The cells were stimulated according to two protocols. First protocol: short (5 hours) stimulation of whole blood cells under sterile conditions with mixture of PMA + ionomycin + brefeldin A. The second protocol: long-term (48 hours) stimulation of the isolated mononuclear fraction under sterile conditions with a mixture (CD40L + CpG) with the addition of PMA + ionomycin + brefeldin A during the last 5 hours. Cells were stained for surface markers (CD45, CD19, CD24, CD27, CD38) and the intracellular content of IL-10. Sample analysis was performed on a Navios™ flow cytometer.Results: a five-color cytometric analysis was performed and a sequential gating strategy was developed based on the isolation of the gate by lymphocytes (marker CD45); restriction of B-lymphocytes (marker CD19); isolating a subpopulation of B cells expressing the CD24 marker; limiting the two required subpopulations of B10-reg cells for CD27 and CD38: CD19+CD24hiCD27+IL-10+ and CD19+CD24hiCD38hiIL-10+. Stimulation of cells based on the first protocol allows visualizing up to 1% of both subpopulations, and based on the second protocol - about 10%. The method opens up prospects for fundamental research of B10-reg cells during pregnancy. The detectable amounts of an enriched population of B10-reg cells can be of diagnostic and prognostic value in the clinic for idiopathic obstetric complications.