Pregnancy imparts distinct systemic adaptive immune function

Abstract

Problem: Pregnancy represents a state of systemic immune activation that is primarily driven by alterations in circulating innate immune cells. Recent studies have suggested that cellular adaptive immune components, T cells and B cells, also undergo changes throughout gestation. However, the phenotypes and functions of such adaptive immune cells are poorly understood. Herein, we utilized high-dimensional flow cytometry and functional assays to characterize T-cell and B-cell responses in pregnant and non-pregnant women.

Methods: Peripheral blood mononuclear cells from pregnant (n = 20) and non-pregnant (n = 25) women were used for phenotyping of T-cell and B-cell subsets. T-cell proliferation and B-cell activation were assessed by flow cytometry after in vitro stimulation, and lymphocyte cytotoxicity was evaluated by using a cell-based assay. Statistical comparisons were performed with linear mixed-effects models.

Results: Pregnancy was associated with modestly enhanced basal activation of peripheral CD4⁺ T cells. Both CD4⁺ and CD8⁺ T cells from pregnant women showed increased activation-induced proliferation; yet, a reduced proportion of these cells expressed activation markers compared to non-pregnant women. There were no differences in peripheral lymphocyte cytotoxicity between study groups. A greater proportion of B cells from pregnant women displayed memory-like and activated phenotypes, and such cells exhibited higher activation following stimulation.

Conclusion: Maternal circulating T cells and B cells display distinct responses during pregnancy. The former may reflect the unique capacity of T cells to respond to potential threats without undergoing aberrant activation, thereby preventing systemic inflammatory responses that can lead to adverse perinatal consequences.

Keywords: B cell; T cell; adaptive immunity; cytotoxicity; flow cytometry; maternal circulation.

FIGURE 1

Comparison of basal T-cell subset composition between non-pregnant and pregnant women. (A) Peripheral blood samples were collected from non-pregnant (n = 25, indicated in blue) and pregnant (n = 18, indicated in red) women to isolate peripheral blood mononuclear cells (PBMCs) for T-cell phenotyping at baseline (day 0). (B) Heatmap representation showing the basal proportion of T cells with various immunophenotypes from non-pregnant (indicated in blue) and pregnant (indicated in red) women. The color key indicates the relative proportion of T cells with the various immunophenotypes considered, which were not compared among each other. (C) Proportion of CD4⁺ T cells expressing CD69 and (D) co-expressing CD69 and PD-1 at baseline from non-pregnant (blue circles) and pregnant (red circles) women. Data are presented as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum ranges. *p < .05

FIGURE 2

Comparison of CD4⁺ T-cell proliferation between non-pregnant and pregnant women. (A) Peripheral blood samples were collected from non-pregnant (n = 25, indicated in blue) and pregnant (n = 20, indicated in red) women to isolate peripheral blood mononuclear cells (PBMCs) for in vitro stimulation with anti-CD3/anti-CD28 and recombinant human IL-2. Cells were cultured for 6 days prior to phenotyping. Controls were cultured in parallel without stimulation. (B) Heatmap representation showing the proportion of CD4⁺ T cells with various immunophenotypes from non-pregnant (indicated in blue) and pregnant (indicated in red) women with (stimulated) or without (control) stimulation. The color key indicates the relative proportion of T cells with the various immunophenotypes considered, which were not compared among each other. (C) Absolute number of CD4⁺ T cells in control and proliferated samples from non-pregnant (blue symbols) and pregnant (red symbols) women. (D-F) Proportion of proliferated CD4⁺ T cells with the phenotype of (D) CD4⁺CD69⁺, (E) CD4⁺PD-1⁺, and (F) CD4⁺CD69⁺PD-1⁺. Data are presented as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum ranges. Each dot represents the mean of three biological replicates per sample. Grey lines and asterisks represent within-group differences between control and stimulated samples (i.e., pregnant control vs. pregnant stimulated), while black lines and asterisks represent significant differences between groups after stimulation (i.e., pregnant stimulated vs. non-pregnant stimulated). *p < .05; ***p < .001

FIGURE 3

Comparison of CD8⁺ T-cell proliferation between non-pregnant and pregnant women. (A) Peripheral blood samples were collected from non-pregnant (n = 25, indicated in blue) and pregnant (n = 20, indicated in red) women to isolate peripheral blood mononuclear cells (PBMCs) for in vitro stimulation with anti-CD3/anti-CD28 and recombinant human IL-2. Cells were cultured for 6 days prior to phenotyping. Controls were cultured in parallel without stimulation. (B) Heatmap representation showing the proportion of CD8⁺ T cells with various immunophenotypes from non-pregnant (indicated in blue) and pregnant (indicated in red) women with (stimulated) or without (control) stimulation. The color key indicates the relative proportion of T cells with the various immunophenotypes considered, which were not compared among each other. (C) Absolute number of CD8⁺ T cells in control and proliferated samples from non-pregnant (blue symbols) and pregnant (red symbols) women. (D-G) Proportion of proliferated CD8⁺ T cells with the phenotype of (D) CD8⁺CD69⁺, (E) CD8⁺PD-1⁺, (F) CD4⁺CD69⁺PD-1⁺, and (G) CD8⁺CD45RA⁺CCR7⁻ (terminal effector memory). Data are presented as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum ranges. Each dot represents the mean of three biological replicates per sample. Grey lines and asterisks represent within-group differences between control and stimulated samples (i.e., pregnant control vs. pregnant stimulated), while black lines and asterisks represent significant differences between groups after stimulation (i.e., pregnant stimulated vs. non-pregnant stimulated). *p < .05; **p < .01; ***p < .001

FIGURE 4

Comparison of lymphocyte cytotoxicity between non-pregnant and pregnant women. (A) Peripheral blood samples were collected from non-pregnant (n = 21, indicated in blue) and pregnant (n = 17–20, indicated in red) women to isolate peripheral blood mononuclear cells (PBMCs) for in vitro culturing with CFSE-labeled target cells. (B) Flow cytometry gating strategy used to identify killed target cells (CFSE⁺7AAD⁺), live target cells (CFSE⁺7AAD⁻), and live PBMCs (CFSE⁻7AAD⁻). (C) Percentage of target cells killed (calculated as [CFSE⁺7AAD⁺ / (CFSE⁺7AAD⁺ + CFSE⁺7AAD⁻) * 100]) among ratios of PBMCs:target cells ranging from 0:1 to 50:1 in non-pregnant (blue circles) and pregnant (red circles) women. Data are presented as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum ranges. Trend lines for each study group are included.

FIGURE 5

Comparison of B-cell subset composition and activation between non-pregnant and pregnant women. (A) Peripheral blood samples were collected from non-pregnant [n = 20 (phenotyping) - 25 (activation), indicated in blue] and pregnant (n = 19, indicated in red) women to evaluate B-cell phenotypes and activation following anti-human IgM/IgG stimulation. (B) Heatmap representation showing the basal proportion of B cells with various immunophenotypes from non-pregnant (indicated in blue) and pregnant (indicated in red) women. The color key indicates the relative proportion of B cells with the various immunophenotypes considered, which were not compared among each other. (C-E) Proportion of B cells with the phenotype (C) CD19⁺CD20⁺CD27⁺IgG⁺, (D) CD19⁺CD20⁺CD38⁺CD24⁻, and (E) CD19⁺CD20⁺CD40⁺CD138⁻ from non-pregnant (red circles) and pregnant (blue circles) women. (F) Representative flow cytometry gating strategy for B-cell activation assay: viable B cells were identified as CD19⁺, and then B-cell activation in control (open histograms) and stimulated (filled histograms) samples from non-pregnant (indicated in blue) and pregnant (indicated in red) women was determined as described in the Methods. (G) Fold change in B-cell activation in non-pregnant (blue triangles) and pregnant (red triangles) samples after anti-human IgM/IgG stimulation, calculated as the adjusted MFI of IgM/IgG-stimulated samples divided by the adjusted MFI of control samples. Fold changes < 1 were considered as “no change” and assigned a value of 1. Data are presented as box-and-whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum ranges. *p < .05; **p < .01