The ontogeny of naïve and regulatory CD4+ T-cell subsets during the first postnatal year: a cohort study

              OPEN
ORIGINAL ARTICLE
The ontogeny of naïve and regulatory CD4+ T-cell
subsets during the first postnatal year: a cohort study
Fiona M Collier1,2,3, Mimi LK Tang3,4,5, David Martino3,5, Richard Saffery3,5, John Carlin3,5, Kim Jachno3,
Sarath Ranganathan3,4,5, David Burgner3,5, Katrina J Allen3,4,5, Peter Vuillermin1,2,3,6 and
Anne-Louise Ponsonby3,4,5,6
As there is limited knowledge regarding the longitudinal development and early ontogeny of naı¨ve and regulatory CD4+ T-cell
subsets during the first postnatal year, we sought to evaluate the changes in proportion of naı¨ve (thymic and central) and
regulatory (resting and activated) CD4+ T-cell populations during the first postnatal year. Blood samples were collected and
analyzed at birth, 6 and 12 months of age from a population-derived sample of 130 infants. The proportion of naı¨ve and
regulatory CD4+ T-cell populations was determined by flow cytometry, and the thymic and central naı¨ve populations were sorted
and their phenotype confirmed by relative expression of T cell-receptor excision circle DNA (TREC). At birth, the majority (94%)
of CD4+ T cells were naı¨ve (CD45RA+), and of these, ~80% had a thymic naı¨ve phenotype (CD31+ and high TREC), with the
remainder already central naı¨ve cells (CD31− and low TREC). During the first year of life, the naı¨ve CD4+ T cells retained an
overall thymic phenotype but decreased steadily. From birth to 6 months of age, the proportion of both resting naı¨ve T regulatory
cells (rTreg; CD4+CD45RA+FoxP3+) and activated Treg (aTreg, CD4+CD45RA−FoxP3high) increased markedly. The ratio of thymic
to central naı¨ve CD4+ T cells was lower in males throughout the first postnatal year indicating early sexual dimorphism in
immune development. This longitudinal study defines proportions of CD4+ T-cell populations during the first year of postnatal
life that provide a better understanding of normal immune development.
Clinical & Translational Immunology (2015) 4, e34; doi:10.1038/cti.2015.2; published online 27 March 2015
Following birth and during the first postnatal year, the infant’s
immune system must rapidly develop in order to negotiate the
infectious milieu of the extrauterine environment as well as to
establish tolerance to abundant nonpathogenic antigens. The devel-opment
of immune phenotypes during this time may define the risk of
infective illnesses or program the subsequent risk of immune-related
disease.1,2 Several studies have described the proportions of basic
lymphocyte subsets at birth,3,4 over the first year of life,5,6 and during
childhood and adolescence,7 but there are limited data regarding the
longitudinal natural development of these subsets or potential gender
differences during the first postnatal year. At birth, thymic (T) cells,
derived from precursors that have undergone both positive and
negative selection and have a low affinity for self-peptides, comprise
approximately half of the circulating lymphocyte population.8 The
CD4 surface marker denotes two subsets: T helper cells that have a
central orchestrating role in the adaptive immune responses to various
infectious agents,9 and a smaller subset of T regulatory cells (Treg)
with a vital role in immune homeostasis.10
In cord blood, the phenotype of the neonatal CD4+ compartment is
primarily naïve (positive for CD45RA antigen), and thought to
comprise ‘recent thymic emigrants’ or ‘thymic naïve’ T cells.11,12
These thymic naïve T cells in cord blood can be characterized by
both high levels of T cell-receptor (TCR) excision circle DNA
(TREC)13 and the expression of the transmembrane marker,
CD31.11 CD31 is an immunoglobulin-like molecule with an important
role in modulation of T-cell responses,14,15 and stimulation of the
TCR leads to loss of CD31 expression.16 In adults and older children, a
proportion of naïve CD4+ T cells do not express CD31, have relatively
low levels of TREC, and are designated ‘central naïve’ T cells. These
factors indicate that they have undergone proliferation following
exodus from the thymus.12 It has been suggested that, as the thymic
output of naïve T cells reduces with age, the formation of central naïve
T cells in the periphery have a role in the maintenance of an adequate
naïve T-cell population.11,17 Although the central naïve CD4+ T cells
are present in significant proportions in adults and older children,
little is known about the levels at birth or during the first
postnatal year.
Treg cells that have a critical role in the control of self-tolerance
and the development of acquired immunity18 can be differentiated
according to a naïve phenotype and the transcription factor, forkhead
1Child Health Research Unit and Barwon Biomedical Research, University Hospital, Barwon Health, Geelong, Victoria, Australia; 2School of Medicine, Deakin University, Waurn
Ponds, Victoria, Australia; 3Murdoch Childrens Research Institute, Parkville, Victoria, Australia; 4The Royal Childrens Hospital, Parkville, Victoria, Australia and 5The University of
Melbourne, Parkville, Victoria, Australia
Correspondence: Dr FM Collier, Child Health Research Unit and Barwon Biomedical Research, University Hospital, Barwon Health, Geelong 3220, Victoria, Australia.
Email: fmcol@deakin.edu.au or fionac@barwonhealth.org.au
6These authors contributed equally to this work.
Received 20 January 2015; accepted 21 January 2015
Clinical & Translational Immunology (2015) 4, e34; doi:10.1038/cti.2015.2
& 2015 Australasian Society for Immunology Inc. All rights reserved 2050-0068/15
www.nature.com/cti
box P3 (FoxP3).19 Miyara et al.20 demonstrated that the resting Treg
(rTreg) and activated Treg (aTreg), two phenotypically and function-ally
distinct thymic-derived Treg subpopulations, could be discrimi-nated
on the basis of the degree of expression of FoxP3, and the
presence or absence of CD45RA. Their study and subsequent work
have shown age- and disease-related differences in the proportion of
rTreg and aTreg,21,22 which is consistent with the concept that they are
a useful measure of Treg maturation and function. As the overall
balance between Treg and T helper cells is linked to the development
of allergic disease and other noncommunicable diseases,23,24 measures
of Treg proportions early in life may be particularly informative.
Current studies of Treg at birth and during infancy are restricted to
flow cytometric measures defined by expression of CD4+/CD25+/
FoxP3+;25,26 however, this combination of markers does not clearly
differentiate the naïve rTreg from activation-induced FoxP3+ T cells.
The aim of this study, therefore, was to describe, in a population-derived
cohort of infants, the natural history of key CD4+ T-cell
subsets during the first postnatal year and to evaluate the cell
percentages according to gender. Specifically, we sought to evaluate
longitudinal age- and sex-related changes in naïve CD4+ T cells
(thymic and central), and naïve Treg subgroups.
RESULTS
Characterization of CD4+ T-cell subsets
All isolated mononuclear cells (MNCs) had a viability 499.5%. T-cell
and CD4+ T-cell subsets were identified using specific antibodies and
gating as shown in Figure 1.
Validation of thymic versus central naïve T-cell populations by TREC
PCR analysis. Anti-CD31 was used to discriminate two subpopula-tions
of naïve CD4+ T cells (Figure 2a). To confirm the thymic
(CD31+/CD45RA+) and central (CD31−/CD45RA+) naïve phenotypes,
target populations were flow sorted, genomic DNA extracted and
relative TREC quantitated. TREC content was highest in the thymic
naïve cells and significantly lower in the central naïve cells at both
birth and 6 months (Figure 2b). Cells that had a non-naïve (memory)
phenotype (CD31−/CD45RA−) were also sorted from the 6-month
samples. The TREC DNA in these cells was similar to the central naïve
cells (Figure 2b). TREC measures confirmed that the flow cytometric
analysis correctly delineated thymic naïve and proliferating central
naïve phenotypes.
Longitudinal analysis of T-cell subsets over the first postnatal year
The distribution of each T-cell population was examined, and the
mean percentages (with 95% confidence intervals (CIs)) at birth,
6 and 12 months are displayed in Table 1.
CD3+ and CD4+ T-cell subsets. Total T cells (CD3+) and CD4+
subsets (CD3+CD4+) were measured in whole blood as a percentage
of the total lymphocyte population. There was a minor increase in the
mean proportion of CD3+ T cells between birth and 6 months (5.4%
(95% CI 3.6, 7.1)), and no further change from 6 to 12 months
(Table 1). From birth to 6 months, there was a small increase in mean
percentage of CD4+ T cells (2.5% (95% CI 1.1, 3.9)), and then a
decrease from 6 to 12 months back to birth levels (3.7% (95% CI 2.4,
4.9)) (Table 1, Figures 3a and b).
100 103
103
1 102
BISC0660BirthWB3.4.
L s
s
Leukocytes
Lymphocytes
Monocyets
Granulocytes
800 100
CD45-PerCP
SSC-H
102 L2H
.4.45.001
l
ells
CD4+ ces
CD3neg
CD8+ T cells
CD4+ T cells
T cells
CD3-FITC
CD4-PE
2
Lymphocytes
CD4-FITC
CD5RA-PECy5
CD4+ Memory
T cells
10 10
101 102
FL1-H
CD45+Fvoex P3
FoxPh3ig h+ve
CD5RA-PECy5
CD4+T cells
CD45RA+/FoxP3low
=rTreg
FoxP3-AlexaFluor 488
CD45RA+
Naive
CD45RA-Memory
CD45RA-/
FoxP3low
=activation-induced
FoxP3 T cells
0 200 600
101
100
100
103 100
100 103
103
103
101
101
CD4+ Naïve
T cells
100 101
100 101 103
Lymphocytes
CD45RA-/FoxP3high
=aTreg
Figure 1 Gating for flow analysis of T-cell subsets. (a) CD3+ and CD4+ T cells. Whole blood cells were surface stained with fluorochrome-labeled monoclonal
antibodies to CD3, CD4 and CD45. Lymphocytes were gated on the basis of CD45 and granularity (side scatter). CD3+ and CD4+ T cells were selected on
the basis of CD3 and/or CD4 expression. (b) CD4+ naive T cells. MNCs were surface stained with fluorochrome-labeled monoclonal antibodies to CD4 and
CD45RA. CD4+ T cells were gated, and naive CD4+ T cells were selected on the basis of CD45RA expression. (c) CD4+ Treg. MNCs were stained with
fluorochrome-labeled monoclonal antibodies to CD4, CD45RA and FoxP3. Events were gated to the CD4+ T cells, and FoxP3+ subsets were selected on the
basis of CD45RA and FoxP3 expression. CD45RA+FoxP3low, thymus-derived rTreg; CD45RA−FoxP3high, aTreg; CD45RA−FoxP3low, activation-induced FoxP3+
T cells.
Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology
Naive CD4+ T-cell populations. At birth, the CD4+ T cells were
predominantly of a naïve (CD45RA+) phenotype. There was a stepwise
decrease in mean CD45RA expression over the 12-month period
(birth to 6 months, 9.4% (95% CI 8.7, 10.1), and 6–12 months, 5.7%
(95% CI 4.8, 6.6)) with some individual variation noted (Table 1,
Figure 3c).
Thymic and central naïve CD4+ T cells. We next investigated changes
in the percentages of the populations of thymic naïve, central naïve
and memory CD4+ T cells. There was a small decrease in the mean
proportion of thymic naïve CD4+ T cells between birth and 6 months
(3.0% (95% CI 2.1, 4.0)), followed by a more marked decrease
between 6 and 12 months (6.0% (95% CI 5.1, 7.0)). The percentage of
CD4+ T cells - Birth CD4+ T cells - 6MTH
(2)
Expression of TREC DNA - Birth Expression of TREC DNA - 6MTH
(2)
(3)
(1) (2) (1) (2) (3)
(1) (1)
103
103
Relative Expression
Relative Expression
100
CD45zRA-PECy5
100
CD45zRA-PECy5
100
∗
∗∗∗
∗∗∗
40
30
20
10
0
20
25
15
10
5
0
Flow Plot Quadrants Flow Plot Quadrants
101 103
CD31-FITC 100 101 103
CD31-FITC
Figure 2 Validation of thymic and central naive CD4+ T-cell subsets. (a) MNCs were stained with fluorochrome-labeled monoclonal antibodies to CD4, CD31
and CD45RA. Cells were gated to the CD4+ population, and CD45RA+ naive cells were divided according to quadrant (1) (CD31+/CD45RA+) and quadrant
(2) (CD31−/CD45RA+), with CD45RA− cells in quadrant (3) (CD31−/CD45RA−). Representative flow plots at birth and 6 months are shown. (b) Relative
expression of TREC DNA in sorted CD4+ T-cell populations from quadrants (1), (2) and (3). Thawed MNC samples from participants (n=5) at birth and
6 months were flow sorted and relative TREC expression was assessed. At birth, expression in quadrant (1) (CD31+/CD45RA+, thymic naïve) was greater than
quadrant (2) (CD31−/CD45RA, central naïve; *Po0.05 paired t-test). Similarly, at 6 months, TREC expression in cells from quadrant (1) were greater than
cells from either quadrant (2) or quadrant (3) (CD31−/CD45RA−, memory cells; ***Po0.0001, repeated measures analysis of variance). There was no
difference in TREC expression of cells in quadrant (2) versus quadrant (3) at the 6-month time point.
Table 1 Mean percentages (with 95% CIs) of T-cell subtypes during the first year of life (n=130 infants)
Cell populations
First year of life
Birth 6 months 12 months
Mean (%) 95% CI Mean (%) 95% CI Mean (%) 95% CI
As % of lymphocytes
CD3+ T cells 59.3 57.6, 61.1 64.7 63.5, 65.8 64.1 62.9, 65.3
CD4+ T cells 42.7 41.3, 44.2 45.2 44.1, 46.3 41.6 40.4, 42.7
As % of CD4+ T cells
CD45RA+ naïve 93.5 93.1, 93.8 84.1 83.3, 84.8 78.3 77.4, 79.3
CD31+ thymic naïve 76.1 75.2, 77.1 73.1 72.3, 74.0 67.1 66.0, 68.2
CD31− central naïve 17.4 16.5, 18.2 10.9 10.3, 11.5 11.2 10.5, 11.9
CD45RA−/CD31− memory 4.1 3.9, 4.4 10.7 10.2, 11.2 15.7 15.0, 16.5
rTreg, CD45RA+FoxP3low 4.1 3.9, 4.3 5.6 5.4, 5.8 5.3 5.0, 5.6
aTreg, CD45RA−FoxP3high 0.72 0.66, 0.79 1.46 1.32, 1.59 1.91 1.72, 2.10
Activation-induced FoxP3 T cells (CD45RA−FoxP3low) 0.53 0.53, 0.63 1.78 1.63, 1.92 2.65 2.48, 2.83
Abbreviations: aTreg, activated T regulatory cell; CI, confidence interval; rTreg, resting T regulatory cell.
Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology
central naïve CD4+ T cells (CD31−/CD45RA+) decreased from birth to
6 months (6.4% (95% CI 5.8, 7.0)), but there was no change in
percentage between 6 and 12 months. In contrast, the mean
percentage of CD4+ T cells with a memory phenotype
(CD31−/CD45RA−) increased from birth to 6 months (6.5% (95%
CI 6.0, 7.0)) and to 12 months (5.1% (95% CI 4.4, 5.8)) (Table 1,
Figure 4a).
FoxP3+ populations within the CD4+ T cells. We characterized the
CD4+ T cells according to the method of Miyara et al.,10 with the
CD45RA+ FoxP3low population defined as thymus-derived rTreg,
the CD45RA− FoxP3high cells designated as activated Treg (aTreg)
and CD45RA− FoxP3low as the activation-induced FoxP3+ CD4+
T cells (Figure 3a). There was a marked increase in mean rTreg from
birth to 6 months (1.5% (95% CI 1.2, 1.8)), and then a small
reduction in this population between 6 and 12 months (0.3% (95% CI
0.0, 0.6)). The aTreg, which are derived primarily from the rTreg,
increased more than twofold from birth to 6 months, and then
underwent a further increase between 6 and 12 months (0.45% (95%
CI 0.2, 0.7)). The activation-induced FoxP3+ CD4+ T cells also
increased in a linear fashion from birth to 6 months (1.25% (95%
CI 1.1, 1.4)) and from 6 to 12 months (0.90% (95% CI 0.7, 1.1);
Table 1, Figure 4b).
Investigation of CD4+ T-cell ontogeny over the first postnatal year by
gender. We evaluated the longitudinal changes in the CD4+ T-cell
population percentages according to gender. There was no difference
in the proportion of CD3+, CD4+
T cells, naïve CD4+ T cells or
FoxP3+ populations between males and females; however, for thymic
naïve CD4+ T cells, males had a lower percentage at all time points
compared with females (overall, −1.8% (95% CI − 3.4, 0.3)) and a
higher percentage of central naïve CD4+ T cells at all time points
(overall, 2.6% (95% CI 1.4, 3.7); Table 2).
Overall changes in composition of the CD4+ T-cell compartment
We combined measures of the various CD4+ phenotypes assessed in
this study into a composite view of the CD4+ T-cell compartment in
which changes in subset proportions are simply visualized (Figure 5).
DISCUSSION
This is the first study to report multiple longitudinal CD4+ T-cell
phenotypes from a large population of infants born at/near term,
sampled from a population-derived cohort, at three time points during
the first year of postnatal life. At birth, almost all CD4+ T cells are
naïve, but a significant proportion of these have undergone prolifera-tion
(that is, have reduced TREC and are CD31−). In the first
6 months, the naïve population decreases and the non-naïve
(memory) CD4+ T cells increase, in concert with a decrease of the
subset of central naïve CD4+ T cells. There are gender differences in
the birth thymic and central naïve CD4+ T-cell populations and these
persisted throughout the first postnatal year. At birth, both rTreg and
aTreg are present in the CD4+ T-cell compartment and their
proportions increase during the first year of life.
Despite some individual variation in percentages, a small mean
increase of both CD3+ and CD4+ T-cell subsets was observed in the
first 6 months of life. Previous studies that lack longitudinal data have
found similar T-cell population percentages at the three time points,6,7
whereas de Vries et al.5 showed, in a longitudinal study of 11 infants, a
comparable increase in the proportion of CD3+ but not CD4+ T cells.
Among those infants, the frequency of CD45RA+ CD4+ T cells at birth
was only 70%; however, we clearly show in a large population sample
that, consistent with other studies,28,29 the CD4+ T cells at birth are
primarily naïve (493%) and that over the subsequent 12 months,
there is a gradual reduction in the naïve phenotype, reflecting the
development of the infants’ adaptive immune response.30 A similar
reduction in the naïve CD4+ T-cell population has previously been
Birth 6MTH 12MTH
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*** # # #
CD3+ T cells (% of lymphocytes)
Birth 6MTH 12MTH
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CD4+ T cells (% of lymphocytes)
Birth 6MTH 12MTH
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*** # # #
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CD45RA+ Naive CD4+ T cells
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Figure 3 Longitudinal analysis of T-cell subsets. Longitudinal flow analysis of T-cell subsets at birth, 6 and 12 months (n=130 repeated measures) and
represented by a line graph (top panel), and box and whisker plot (5–95 percentile, lower panel) for: (a) CD3+ T cells expressed as a percentage of the
lymphocyte population. From birth to 6 months or 12 months, there was a mean increase (***Po0.0001), whereas from 6 to 12 months, there was no
change (P=0.72). (b) CD4+ T cells expressed as a percentage of the lymphocyte population. From birth to 6 months, there was a mean increase
(***Po0.0001), and then a mean decrease (###Po0.0001) from 6 to 12 months. (c) CD45RA+ naïve CD4+ T-cell subsets expressed as a percentage of the
CD4+ T cells. There is a stepwise decrease over the first year, with a reduction from birth to 6 months (***Po0.0001), and a further decrease from 6 to
12 months (###Po0.0001).
Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology
reported in cross-sectional studies;7 however, to further develop these
data, we analyzed the naïve CD4+ T cells subsets: CD31+, thymic
naïve; and CD31−, central naïve. The CD31+ population represented
the majority of naïve cells at all ages investigated and had the highest
relative expression of TREC; the signature marker of thymic naïve
cells.13,31 Interestingly, contrary to previous reports,31,32 we found that
at birth, the CD31− central naïve population represented a fifth of the
neonatal naïve CD4+ population, nearly double the level seen at 6 and
12 months, and similar to that observed in adults.33 Thus, it appears
that a substantial proportion of central naïve CD4+ T cells is formed
during gestation. The immunoglobulin-like molecule, CD31, is known
to be shed following TCR engagement with major histocompatibility
complex,16 but not following in vitro cytokine activation,34,35 which
suggests that further studies are required to determine which in utero
signals stimulate the proliferation of naïve CD4+ T cells. It has been
suggested that low-affinity self-peptide–major histocompatibility com-plex
interactions may be involved in the generation of the central naïve
cells, and that they have a role in the maintenance of the overall naïve
Birth 6MTH 12MTH
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60
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CD31+CD45RA+ Thymic Naive
CD31-CD45RA+ Central Naive
CD31-CD45RA- Memory
%
Naive and Memory CD4+ T cell Populations
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Thymic Naive
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Birth 6MTH 12MTH
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Memory
FoxP3+ CD4+ T-cell Populations
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CD45RA+FoxP3low rTreg
CD45RA-FoxP3high aTreg
CD45RA-FoxP3low
Act-induced FoxP3 Tcells
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%
Resting Treg (rTreg)
Activated Treg (aTreg)
0
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6
8
%
Activation-induced FoxP3 T-cells
Birth 6MTH 12MTH
0
2
4
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%
Figure 4 Longitudinal analysis of thymic and central naive CD4+ T cells and Treg subsets. (a) Longitudinal flow analysis of thymic, central naive and memory
CD4+ populations as a percentage of CD4+ T cells (n=130 repeated measures, line graph with mean±95% CI). There was a minor drop (Po0.0001) in
thymic naive CD4+ T cells between birth and 6 months, and a further decrease, from 6 to 12 months. There was a drop in central naive CD4+ T cells
(Po0.0001) between birth and 6 months, but no change in percentage between 6 and 12 months (P=0.18). The CD4+ T-cell memory population increased
(Po0.0001)) from birth to 6 months with a further increase to 12 months (Po0.0001). (b) Longitudinal analysis of FoxP3+ CD4+ T-cell subsets during the
first year of life expressed as a percentage of CD4+ T cells (n=130 repeated measures, line graph with mean±95% CI). There was a marked increase
(Po0.0001) in rTreg from birth to 6 months, and then a slight reduction between 6 and 12 months (Po0.032). There was a greater than twofold increase
(Po0.0001) in both aTreg and activation-induced FoxP3+ CD4+ T cells from birth to 6 months, and a further increase in both populations (Po0.0001)
between 6 and 12 months.
Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology
population.36 The central naïve CD4+ T cells we observed at birth may
at least in part explain the reported rapid proliferative responses of
cord blood MNCs to various antigens seen in other studies.32
Sexual dimorphism was observed with male newborns having an
increased proportion of CD31− central naïve CD4+ T cells (and
decreased thymic naïve) at birth, and this difference was maintained
throughout the postnatal year. This indicates that a greater number of
naïve CD4+ T cells in male infants are in a proliferative state and no
longer express the immature thymic phenotype.12 An age-related
reduction in TREC expressing thymic naïve T cells has previously been
reported in the elderly, and the rate of this reduction appears to be
greater in males.37 The gender differences reported here at birth are in
keeping with this observation, and these might be the earliest signs of
sexual dimorphism in immune function.38
The small but vital CD4+ subpopulation of Treg has important
suppressive functions that limit autoimmune and allergic responses.
We used specific cellular markers that provided longitudinal measures
of the thymic-derived and naïve rTreg.20 Within the Treg population,
rTreg predominated at birth and then increased during the first
6 months of life. This indicated accelerated Treg thymic activity in
contrast to the overall fall in the entire population of naïve CD4+
T cells. After 6 months, the rTreg population started to decrease, a
trend that presumably continues with increasing age. Indeed, by
adulthood, the proportion of rTreg has dropped to 0.5–1.0%20 from
an estimated 4.1% at birth observed in this study. The aTreg and
activation-induced FoxP3+ CD4+ T cells increased throughout the first
year of life. The population of aTreg is reported to be primarily
derived from vigorously proliferating rTreg cells and it displays
suppressive properties equal to that of rTreg,10 but are terminally
differentiated and undergo apoptosis following suppressive activity.20
We demonstrated that by 12 months, aTreg comprise (on average)
24% of the total Treg population. Their memory phenotype suggests
that they have undergone antigen recognition and may have a tailored
immune-suppressive response.39 Indicative of their clinical impor-tance,
the frequency of aTreg has been reported to be modulated in
aged donors,20 as well as disease states including active sarcoidosis,20
chronic obstructive pulmonary disease,21 type 1 diabetes40 and
systemic sclerosis.22 Our analysis is the first to report rTreg popula-tions
in an infant cohort and may define new measures for immune
outcomes related to infant disease.41
CONCLUSION
The results from this prospective cohort27 demonstrate the normal
ontogeny of CD4+ T-cell populations during the first 12 months of
life, a period when immune responses may be susceptible to
environmental influence. These findings provide insight into normal
Table 2 Mean percentages of thymic and central naive T-cell subtypes during the first year of life according to gender (n=62 males, n=68
females)
Birth 6 months 12 months Overall (longitudinal)a
Male Female Male Female Male Female Male Female
As % of CD4+ T cells
CD31+ thymic naive 74.9 77.3 72.1 74.1 66.6 67.6 − 1.8 (95% CI, − 3.4, 0.3)
P-value 0.018 0.024 0.37 0.021
CD31− central naive 18.5 16.3 12.4 9.5 12.5 10.1 2.6 (95% CI, 1.4, 3.7)
P-value 0.011 o0.001 o0.001 o0.001
Thymic/central naive ratio 4.6:1 5.2:1 6.5:1 8.7:1 6.0:1 7.7:1 − 1.2 (95% CI, − 1.9, 0.5)
P-value 0.056 o0.001 0.001 0.002
Abbreviation: CI, confidence interval.
aMean difference (male to female).
Activation-induced FoxP3+T cells
Memory
Central Naive
Activated Treg (aTreg)
Thymic Naive
Resting Treg (rTreg)
Birth 6MTH 12MTH
Naive Naive
Memory
Memory
CD4+ T cell Compartment in First Year of Life
The ratio of thymic:central
naïve CD4+ populations are
consistently lower
(p<0.002) in males
compared to females
Figure 5 Diagrammatic overview of proportional changes in naive and Treg subsets within the CD4+ T-cell compartment during the first year of life.
Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology
immune development and the ontogeny of naïve CD4+ during the first
year of life.
METHODS
Study design
The Barwon Infant Study is a population-derived birth cohort (n=1074) with
antenatal recruitment in Victoria (Australia) that has been established to
investigate the early life origins of a range of noncommunicable diseases in the
modern environment.27 Infants born before 32 completed weeks gestational age
were excluded, as were those with a serious illness, or major congenital
malformation and/or genetically determined disease. Blood samples were
collected from the participants at birth (cord blood), 6 and 12 months of
age. Here we present data from the first 130 infants (68 females and 62 males)
born 436 weeks gestation that had flow cytometric measures completed on
freshly collected blood samples at all three time points.
Blood sampling and processing
Umbilical cord blood was collected from the participants at the Geelong
Hospital (public) and at the St John of God Hospital (private), Geelong,
Victoria. Where possible, blood was collected before placenta delivery, by
syringe. Up to 20 ml of cord blood was added to a 50-ml Falcon tube that
already contained 20 ml of RPMI medium (Gibco, Life Technologies, Mulgrave,
VIC, Australia) and 200 IU preservative-free sodium heparin (Pfizer, Australia
Pty Ltd, West Ryde, NSW, Australia). At 6 and 12 months, venous peripheral
blood was added to a 10-ml Falcon tube containing 100 IU preservative-free
sodium heparin. All bloods were maintained at room temperature on a roller
and processed within 18 h of collection. An aliquot (100 μl) of the whole blood
was removed for measurement of the proportion of T cells and CD4+ T cells,
and the remaining blood sample was processed to isolate plasma and MNCs.
The MNCs were isolated from the whole blood sample using the density-gradient
centrifugation (Lymphoprep, AxisShield, Oslo, Norway), and cell
number and viability were assessed by Trypan Blue staining. Approximately
1–4×105 MNCs were used for analysis of the CD4+ T-cell subsets.
Characterization of CD4+ T-cell populations by flow cytometry
Antibodies were purchased from BD Biosciences (San Jose, CA, USA), and cells
were stained to evaluate the relative frequency of T cells and CD4+ T-cell
subpopulations. Isotype controls were used to set up the instrument and the
positive gating, and these settings maintained throughout. To determine the
percentage of CD3+ and CD4+ T cells, a sample of whole blood was stained
with antibodies to CD3-FITC, CD4-PE and CD45-PerCP for 15min before
lysis of the red blood cells (BD FACS Lysing Solution, BD, North Ryde, NSW,
Australia), phosphate-buffered saline wash and formalin fixation.
To measure specific subpopulations of CD4+ T cells, MNCs were stained
either with (a) antibodies to CD4-FITC, CD31-PE and CD45RA-PECy5 (naïve
CD4+ T cells and thymic and central naïve subsets); or (b) antibodies to
CD4-PE and CD45RA-PECy5 (Treg subsets); washed in phosphate-buffered
saline and formalin fixed. After overnight fixation, cells from (b) were
permeabilized (0.5% Tween) and stained with anti-FoxP3 Alexa Fluor 488.
All samples were analyzed by the 3-channel flow cytometry (FACSCalibur,
Becton Dickinson).
Sorting of naïve CD4+ cell populations
Cryopreserved MNCs from five participants (birth and 6 month samples from
each participant) were sorted for TREC analysis. In brief, MNCs were thawed
and washed in RPMI with 10% fetal calf serum, before being stained with
antibodies to CD4-FITC, CD31-PE and CD45RA-PECy5 to discriminate three
main populations using flow cytometric quadrant analysis. Cells were gated to
the CD4+ population of lymphocytes and divided into flow plot quadrant (1),
CD31+/CD45RA+; quadrant (2), CD31−/CD45RA+; and quadrant (3),
CD31−/CD45RA−. The cells from quadrants (1) and (2; birth) and from
quadrants (1), (2) and (3; 6 months) were sorted using a MoFlo (Beckman
Coulter, Lane Cove, NSW, Australia) and the relative TREC expression was
determined. A distinct population was not evident in remaining quadrant (
CD31+/CD45RA−).
Relative expression of TREC
Genomic DNA was extracted from each of the sorted CD4+ T-cell subsets
(Qiagen AllPrep Mini Kit, Qiagen Sciences, Germantown, MD, USA) and
measured using a NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA).
All the DNA samples were diluted to a concentration of 5.0 ng μl− 1, and a total
of 15 ng used in a real time PCR reaction (Applied Biosystems 7500 Fast Real
Time PCR System, Life Technologies) to amplify the following: (i) TCR delta
J3 section of excision circle DNA (forward primer: 5′-CTCAGGTCCTT
AGAAAGCCT-3′; and reverse primer: 5′-CTCTTGGGTCACAAGTACAG-3′),
and (ii) the single-copy 36B4 human sequence (forward primer: 5′-CAGCA
AGTGGGAAGGTGTAATCC-3′; and reverse primer: 5′-CCCATTCTATCAT
CAACGGGTACAA-3′) using Sybr Green chemistry. The CT value for 36B4
gene was subtracted from the CT value for the TCR delta J3 gene region (delta
CT), and an arbitrary value for relative expression of TREC DNA in each group
was calculated by measuring the power to base 2 of delta CT.
Statistical analysis
Flow cytometric data from 130 Barwon Infant Study infants at the three time
points were collected (birth, 6 and 12 months), with no missing values.
Estimates of differences in the mean percentages of various T-cell populations
over the first postnatal year were obtained using regression models with
generalized estimating equations employed to account for repeated measures at
the individual level. Results of the PCR comparative gene analysis of TREC
DNA (performed in a small subgroup of infant samples) were analyzed using a
paired t-test (birth) or repeated measures analysis of variance (6 months). All
statistical analyses were conducted using Stata/IC 13.0 for Mac (StataCorp LP,
College Station, TX, USA).
ACKNOWLEDGEMENTS
The funding for this work was totally supported by the National Health and
Medical Research Council (NHMRC), Australia, and Barwon Health. We
acknowledge scientists Danielle Kennedy and Carling Southall for their
consistent and meticulous work in processing all the blood and mononuclear
cell samples for flow cytometric analysis.
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Ontogeny of CD4+ T-cell subsets in the first postnatal year
FM Collier et al
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Clinical & Translational Immunology