Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study

Jorge Lima, Catarina Martins, Maria J Leandro, Glória Nunes, Maria-José Sousa, Jorge C Branco, Luís-Miguel Borrego, Jorge Lima, Catarina Martins, Maria J Leandro, Glória Nunes, Maria-José Sousa, Jorge C Branco, Luís-Miguel Borrego

Abstract

Background: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women.

Methods: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation.

Results: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women).

Conclusion: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Keywords: B cell subsets; Flow cytometry; Human pregnancy; Obstetrics.

Figures

Fig. 1
Fig. 1
Identification of B cell subsets according to Bm1-5 classification system. a and b Gating strategy for CD19+ B cells using an initial CD19/SSC plot and refinement of the gate using a plot of FSC vs SSC. c Bm1-5 classification from double staining for IgD and CD38 (unswitched memory Bm1: IgD+CD38−; naïve Bm2: IgD+CD38+; transitional Bm2′: IgD+CD38hi; plasmablasts Bm3 + Bm4: IgD−CD38hi; post-germinal memory/early eBm5: IgD−CD38+; and resting memory/late Bm5: IgD−CD38−)
Fig. 2
Fig. 2
Identification of regulatory B cell subsets. a and b Gating strategy for CD24hiCD27+ (a) and CD24hiCD38hi (b) Bregs; c and d IL-10-producing CD19+ B cells (CD19+ B cells were analyzed for the expression of IL-10 after a 5-h incubation period without stimulation (c) and with stimulation (d) with phorbol 12-myristate 13-acetate, calcium ionophore and lipopolysaccharide)
Fig. 3
Fig. 3
B cell population in peripheral blood samples. Delivery, within 15 min after placental expulsion; Post-partum, at least 6 weeks after delivery; The bottom line represents the 25th percentile, the top line represents the 75th percentile, and the middle line represents the median. * p < 0.05; ** p < 0.001
Fig. 4
Fig. 4
Maturational stages of B cells (absolute counts) in peripheral blood samples according to Bm1-5 classification. Bm1-5 classification, IgD/CD38 cell surface markers; Delivery, within 15 min after placental expulsion; Post-partum, at least 6 weeks after delivery; Non-pregnant women. The bottom line represents the 25th percentile, the top line represents the 75th percentile, and the middle line represents the median. * p < 0.05; ** p < 0.001
Fig. 5
Fig. 5
Maturational stages of B cells (percentages) in peripheral blood samples according to Bm1-5 classification. Bm1-5 classification, IgD/CD38 cell surface markers; Delivery, within 15 min after placental expulsion; Post-partum, at least 6 weeks after delivery; Non-pregnant women. The bottom line represents the 25th percentile, the top line represents the 75th percentile, and the middle line represents the median. * p < 0.05; ** p < 0.001
Fig. 6
Fig. 6
Regulatory B cells in peripheral blood samples (absolute counts and percentages). Delivery, within 15 min after placental expulsion; Post-partum, at least 6 weeks after delivery; Non-pregnant women. The bottom line represents the 25th percentile, the top line represents the 75th percentile, and the middle line represents the median. * p < 0.05; ** p < 0.001

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