Neonates develop both protective and tolerogenic adaptive
immune responses in the first three weeks of life
Thirty-eight healthy mother-and-baby dyads were sampled in our study and
all deliveries were performed by caesarean section (male/female: 17/21,
gestational age: 39 [39-39] weeks, birth weight: 3530
[3298-3733] g). Maternal and umbilical cord blood samples were taken
at delivery and a further blood sample of up to 2 mL was taken from the
neonate at 3 weeks of age. A neonatal stool sample was collected at
birth and also at 3 weeks of age (n=29). Sixteen out of 38 babies (42%)
were exclusively breastfed for the duration of the study, while 9 babies
received mixed feeding and 13 babies were exclusively formula-fed.
Flow cytometric analysis was used to assess the profile of major
lymphocyte subsets in umbilical cord and neonatal blood samples at 3
weeks of age, as well as in adult blood samples. The proportion of CD3+
cells was lower in cord blood compared to maternal blood, while that of
CD4+ cells was higher. CD8+ cell percentages were lower both at birth
and at 3 weeks of age compared to maternal and non-pregnant adult
samples (Figure 1a).
The proportion of CD4+ FoxP3+ CD25hi Tregs increased from 6.4% in cord
blood to 8.0% within the first 3 weeks of life, comparable to the level
in non-pregnant adults but remained lower than third trimester maternal
samples (Figure 1b). Interestingly, within the different feeding groups
at 3 weeks of age, the frequency of Tregs was nearly two-fold higher
(9.3% vs 4.9%) in exclusively breastfed compared to exclusively
formula-fed neonates (Figure 1c). We also examined the expression of
selected cell surface markers on Treg cells at birth and at 3 weeks of
age. HLA-DR expression increased during the first 3 weeks of life,
potentially reflecting recent activation, however, no difference was
detected in the expression of CD45RA, CD31 or CD69 between the two time
points (Figure 1d). Of note, no difference was observed in the
expression of these markers between the different feeding groups.
We then went on to determine the functional activity of T cells through
analysis of intracellular cytokine and surface CD107a expression in
response to mitogenic stimulation. The proportion of IL-8+ CD4+ cells
was lower, whereas that of IFN-γ+ CD4+ cells was higher in maternal
compared to neonatal samples (Figure 2a). No differences were observed
in the profile of IFN-γ, IL-4, IL-6 or IL-8 expression by CD4+ and CD8+
cells between birth and 3 weeks of age. However, the number of IL-17+
CD8+ cells, as well as the mean fluorescence intensity of IL-17 in CD4+
and CD8+ cells, increased during the first three weeks (Figure 3a and
Supplementary Figure 1). Interestingly, at 3 weeks of age the mean
fluorescence intensity of IFN-γ in CD4+ and CD8+ cells was higher in
exclusively formula-fed neonates compared to those receiving breastmilk
(Figure 3b). No further differences were observed between feeding
groups.
A higher proportion of CD8+ cells expressed CD107a, a marker of
cytotoxic degranulation, in non-pregnant adult samples compared to
neonatal samples at birth and 3 weeks of age. The mean fluorescence
intensity of CD107a expression on CD8+ cells was higher in adult
compared to neonatal samples (Figures 2b and 3c). No differences were
observed between feeding groups.