. 2016 Dec 1;197(11):4482-4492.
Epub 2016 Oct 28.
Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy
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Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy
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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.
Copyright © 2016 by The American Association of Immunologists, Inc.
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 (
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
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 (
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
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
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.
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Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, P.H.S.
Research Support, Non-U.S. Gov't
CD4-Positive T-Lymphocytes / immunology*
CD8-Positive T-Lymphocytes / immunology*
Extracellular Signal-Regulated MAP Kinases / immunology
Immunity, Innate / physiology*
Killer Cells, Natural / immunology*
Pregnancy Proteins / immunology
STAT1 Transcription Factor / immunology
STAT1 Transcription Factor
Extracellular Signal-Regulated MAP Kinases
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