Visualization of B10 regulatory cells in peripheral blood in physiological pregnancy

Cover Page

Cite item


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.

About the authors

M. M. Ziganshina

V. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology

Author for correspondence.

Marina M. Ziganshina - PhD (Biology), Laboratory of Clinical Immunology, V. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology.

117997, Moscow, Acad. Oparin str., 4.

Phone: 7 (903) 105-97-46.

Russian Federation

S. V. Khaidukov

V. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology; M. Shemyakin and Yu. Ovchinnikov Research Institute of Bioorganic Chemistry


Sergey V. Khaidukov - PhD, MD (Biology), Director's Advisor, V. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology; Senior Research Associate, Laboratory of Carbohydrates, M. Shemyakin and  Yu. Ovchinnikov Research Institute of Bioorganic Chemistry.


Russian Federation


  1. Alhabbab R.Y., Nova-Lamperti E., Aravena O., Burton H.M., Lechler R.I., Dorling A., Lombardi G. Regulatory B cells: Development, phenotypes, functions, and role in transplantation. Immunol. Rev., 2019, Vol. 292, no. 1, pp. 164-179.
  2. Candando K.M., Lykken J.M., Tedder T.F. B10 cell regulation of health and disease. Immunol. Rev., 2014, Vol. 259, no. 1, pp. 259-272.
  3. Iwata Y., Matsushita T., Horikawa M., Dilillo D.J., Yanaba K., Venturi G.M., Szabolcs P.M., Bernstein S.H., Magro C.M., Williams A.D., Hall R.P., St Clair E.W., Tedder T.F. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood, 2011, Vol. 117, no. 2, pp. 530-541.
  4. Jensen F., Muzzio D., Soldati R., Fest S., Zenclussen A.C. Regulatory B10 cells restore pregnancy tolerance in a mouse model. Biol. Reprod., 2013, Vol. 89, no. 4, 90. doi: 10.1095/biolreprod.113.110791.
  5. Lima J., Martins C., Leandro M.J., Nunes G., Sousa M.J., Branco J.C., Borrego L.M. Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study. BMC Pregnancy Childbirth, 2016, Vol. 16, no. 1, 139. doi: 10.1186/s12884-016-0927-7.
  6. Mizoguchi A., Mizoguchi E., Takedatsu H., Blumberg R.S., Bhan A.K. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity, 2002, Vol. 16, no. 2, pp. 219-230.
  7. Muzzio D., Zenclussen A.C., Jensen F. The role of B cells in pregnancy: the good and the bad. Am. J. Reprod Immunol., 2013, Vol. 69, no. 4, pp. 408-412.
  8. Rolle L., Memarzadeh Tehran M., Morell-Garda A., Raeva Y., Schumacher A., Hartig R., Costa S.D., Jensen F., Zenclussen A.C. Cutting edge: IL-10-producing regulatory B cells in early human pregnancy. Am. J. Reprod. Immunol., 2013, Vol. 70, no. 6, pp. 448-453.
  9. Tedder TF. B10 cells: a functionally defined regulatory B cell subset. J. Immunol., 2015, Vol. 194, no. 4, pp. 1395-1401.
  10. Wang L., Fu Y., Chu Y. Regulatory B Cells. Adv. Exp. Med. Biol., 2020, Vol. 1254, pp. 87-103.
  11. Wortel C.M., Heidt S. Regulatory B cells: Phenotype, function and role in transplantation. Transpl. Immunol., 2017, Vol. 41, pp. 1-9.
  12. Wu H., Su Z., Barnie P.A. The role of B regulatory (B10) cells in inflammatory disorders and their potential as therapeutic targets. Int. Immunopharmacol., 2020, Vol. 78, 106111. doi: 10.1016/j.intimp.2019.106111.
  13. Zhao H., Feng R., Peng A., Li G., Zhou L. The expanding family of noncanonical regulatory cell subsets. J. Leukoc. Biol., 2019, Vol. 106, no. 2, pp. 369-383.

Supplementary files

There are no supplementary files to display.

Copyright (c) 2021 Ziganshina M.M., Khaidukov S.V.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № 77 - 11525 от 04.01.2002.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies