Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy

Abstract

Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4⁺ and CD8⁺ T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet⁺CD4⁺ T cells, CD8⁺ T cells, B cells, and CD56^loCD16⁺ NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.

FIGURE 1. Scaffold maps provide a reference for the cellular organization of the normal fetal and maternal peripheral immune systems at term gestation

Scaffold maps of immune cells in maternal peripheral (A) and fetal cord (B) blood. A representative example from pairs of maternal and fetal samples is shown. Black nodes denote landmark cell populations that define the basic structure of the Scaffold map (i.e., manually-gated granulocytes, Gr; classical monocytes, cMCs; classical dendritic cells, cDCs; natural killer cells, NK cells; B cells; and CD4⁺ and CD8⁺ T cells, Supplemental Fig. 1). Landmark populations are the critical anchors for organizing cell clusters derived from unsupervised clustering analyses. Cell clusters are connected to landmark populations of similar phenotypes and are sized based on the frequency of cells contained in each cluster. In this example, the color scale (grey to red) varies proportionally to the median CD45RA expression of cells contained within each cluster. Rectangles highlight noticeable neonatal-maternal differences for naïve CD4⁺ T cells (CD45RA⁺ cells), IgM⁺ B cells, and CD56^loCD16⁺ NK cells (see also Supplemental Fig. 2A–D). (C) Statistically significant differences in immune cell frequencies between maternal and fetal samples. The analyses comprised 19 manually gated cell populations (see gating hierarchy, Supplemental Fig. 1) that represented the majority of displayed cell clusters (FDR < 0.01, SAM two-class paired, n = 20). Cell populations that differed significantly are plotted on a log10 scale. The relative distribution of naïve and memory CD4⁺ and CD8⁺ T-cell subsets were further quantified as a percentage of total CD4⁺ and CD8⁺ T cells and highlighted the preponderance of naïve T cells in fetal compared to maternal samples (Supplemental Fig. 2E).

FIGURE 2. Scaffold maps provide a reference for the functional organization of the normal fetal and maternal peripheral immune systems at term gestation

Scaffold maps of immune cell functions in maternal peripheral (A, C, E) and fetal cord (B, D, F) blood samples. Representative examples from one pair of maternal and fetal samples are shown. Individual cell clusters are colored based on indicated intracellular signaling activities. Median endogenous p-ERK1/2 signal (Arcsinh transform) in unstimulated maternal (A) and fetal (B) samples. Increased fetal p-ERK1/2 signaling in clusters within the CD4⁺ T, CD8⁺ T, and B cell landmark populations is highlighted in blue rectangles. Median p-STAT1 signaling response to cytokine stimulation with IL-2, IL-6, GM-CSF, and IFNα2A in maternal (C) and fetal (D) samples. Decreased fetal p-STAT1 signaling response in clusters within the classical monocyte (cMC), classical dendritic cell (cDC), and natural killer (NK) cell landmark populations is highlighted in blue rectangles. Median p-P38 signaling response to TLR4 stimulation with LPS. Decreased fetal p-P38 signaling response in clusters within the cMC, cDC, and granulocyte landmark cell populations in maternal (E) and fetal (F) samples are highlighted in blue rectangles.

FIGURE 3. Paired analysis of related maternal and fetal samples enables detection of unique pair-specific differences in endogenous signaling activity

(A) Principal component analysis (PCA) perfectly separated signaling activities (functional immune features; total of 570 per subject) in fetal and maternal immune cells. Dot plots represent values along the first PC-axis for neonatal cord blood (teal circles) or maternal blood samples (orange circles). (B, C) Dot plots represent p-values for fetal-maternal differences between functional immune features when incorporating paired information (paired t-test, y-axis) or excluding paired information (unpaired t-test, x-axis). In (B), colors indicate whether signaling features were statistically significant when using a paired analysis only (purple circles, SAM two-class paired), an unpaired analysis only (orange circles, SAM two-class unpaired), or both, i.e., a paired and unpaired analysis (teal circles). In (C), colors indicate stimulation conditions. All features that were significant only in a paired analysis corresponded to endogenous signaling activities, i.e., the signaling tone of immune cell subsets close to the in vivo condition.

FIGURE 4. Endogenous signaling activity of fetal and maternal immune cells at term gestation

(A) Significant endogenous immune features ordered by effect size (mean difference between values in paired maternal and fetal samples, FDR < 0.01, SAM two-class paired). Dashed line indicates noticeable change in slope of the ranked FDR values, which separates the 16 most significant immune features (red circles) from all other significant immune features. These 16 immune features are plotted in panels (B) (higher in fetal than in maternal samples) and (C) (higher in maternal than in fetal samples). All features that were higher in maternal samples were identified in innate immune cell subsets, while 10 out of 11 features that were higher in fetal samples were identified in adaptive immune cell subsets.

FIGURE 5. Signaling activity of fetal and maternal immune cells in response to cytokine stimulation (IL-2, IL-6, IFNα2A, and GM-CSF)

(A) Significant “evoked immune features” ordered by effect size (mean signal induction from unstimulated condition, FDR < 0.01, SAM two-class paired). The dashed line indicates change in the slope of the ranked data. The 32 most significant evoked immune features are shown in red. (B) Evoked immune features that are higher in fetal samples are shown. (C) Evoked immune features that are lower in fetal samples are shown. About 70% of evoked immune features that were lower in fetal samples are identified in innate immune cell subsets, while 100% of evoked immune features that were higher are identified in adaptive immune cells.

FIGURE 6. Signaling activity of fetal and maternal immune cells in response to TLR4 agonist (LPS)

(A) Significant evoked immune features in response to LPS stimulation ordered by effect size (mean signal induction from unstimulated condition, FDR < 0.01, SAM two-class paired). The dashed line indicates change in the slope of ranked data. The 22 most significant evoked immune features (red circles) are plotted in panel (B). All significant evoked immune features were lower in the neonatal samples. (C) Scaffold map summarizing all functional immune features that differed significantly between fetal and maternal immune cells (endogenous and LPS and cytokine stimulations). Immune cell clusters containing functional immune features characterized by decreased signaling activity in maternal cell clusters relative to fetal cell clusters are shown as blue circles, while increased signaling activity in maternal cell clusters relative to fetal cell clusters are shown as red circles.