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Early-life compartmentalization of human T cell
differentiation and regulatory function in mucosal and
lymphoid tissues
© 2015 Nature America, Inc. All rights reserved.
Joseph J C Thome1,2, Kara L Bickham1, Yoshiaki Ohmura3, Masaru Kubota3, Nobuhide Matsuoka3,
Claire Gordon1,4, Tomer Granot1, Adam Griesemer1, Harvey Lerner5, Tomoaki Kato3 & Donna L Farber1–3
It is unclear how the immune response in early life becomes
appropriately stimulated to provide protection while
also avoiding excessive activation as a result of diverse
new antigens. T cells are integral to adaptive immunity;
mouse studies indicate that tissue localization of T cell
subsets is important for both protective immunity1–4
and immunoregulation5,6. In humans, however, the early
development and function of T cells in tissues remain
unexplored. We present here an analysis of lymphoid and
mucosal tissue T cells derived from pediatric organ donors in
the first two years of life, as compared to adult organ donors,
revealing early compartmentalization of T cell differentiation
and regulation. Whereas adult tissues contain a predominance
of memory T cells7,8, in pediatric blood and tissues the main
subset consists of naive recent thymic emigrants, with effector
memory T cells (TEM) found only in the lungs and small
intestine. Additionally, regulatory T (Treg) cells comprise a
high proportion (30–40%) of CD4+ T cells in pediatric tissues
but are present at much lower frequencies (1–10%) in adult
tissues. Pediatric tissue Treg cells suppress endogenous T cell
activation, and early T cell functionality is confined to the
mucosal sites that have the lowest Treg:TEM cell ratios, which
suggests control in situ of immune responses in early life.
Knowledge of human immune responses during early life remains
sparse, owing to the difficulty and impracticality of obtaining blood
and tissue samples from infants. Our current view of normal infant
immune responses, including T cell differentiation and function, is
based mainly on the sampling of umbilical cord blood9–11 or fetal
tissue12,13, which reflect immune responses in utero but not responses
to the diverse antigens encountered in early life. Fundamental information about the establishment of adaptive immunity during infancy,
including how T cells respond, differentiate and populate tissue sites,
remains undefined. Through collaboration with organ procurement
agencies, we have established protocols for obtaining lymphoid and
mucosal tissues from individual organ donors for whom consent has
been given for the use of tissues for research 7,8. We showed previously that distinct subsets of T cells, including naive and previously
activated effector and memory T cell subsets, are differentially compartmentalized in the lymphoid and mucosal tissues of older children
and adults7,8,14,15. To investigate T cell immunity in tissues during
early life, we obtained multiple lymphoid and mucosal tissues (and
blood) from a cohort of infant and pediatric organ donors in the
first 2 years of life (n = 17, Supplementary Table 1). We compared
these to tissues from a cohort of adult donors stratified into young
adults, aged 15–25 years (n = 23), and adults, aged >25 years (n = 24)
(Supplementary Table 2). The tissues analyzed are representative of
circulatory, lymphoid and mucosal sites and included blood, spleen,
inguinal lymph nodes (ILNs), lung-draining lymph nodes (LLNs),
mesenteric lymph nodes (MLNs), lungs, jejunum, ileum and colon.
Samples from pediatric donors contained lower T cell:B cell ratios
than those from adult donors, in the blood, spleen, MLNs and intestinal sites, which indicated that T cell numbers in these tissues are
reduced in early life. The CD4+:CD8+ T cell composition, by contrast,
was similar in pediatric and young adult donor tissues, showing a
predominance of CD4+ T cells in lymphoid tissues (Supplementary
Fig. 1a). In pediatric tissues, however, major functional subsets of
T cells that are delineated by their expression of the cell surface CD45
isoform CD45RA and the lymph node homing/chemokine receptor
CCR7, including naive (CD45RA+CCR7+) and previously activated
T effector memory (CD45RA−CCR7−, TEM) cell subsets, exhibited
distinct frequencies and distributions in pediatric as compared to
young adult tissues, whereas central memory (CD45RA−CCR7+,
TCM) and terminal effector (CD45RA+CCR7−, TEMRA) cell subsets
were similar in both age groups (Fig. 1a,b). In pediatric donors (2
months to 2 years of age), naive T cells represented the predominant
population (70–95%) of CD4+ and CD8+ T cells in the blood, spleen,
lymph nodes and colon, whereas in young adults, naive T cells were
present only in blood and lymphoid sites and at frequencies of <50%
(Fig. 1a,b). CD4+ T cells in pediatric tissues also exhibited a high proportion of CD31+ cells, denoting recent thymic emigrants (RTEs)16
(>60% in lymphoid tissues; 25–40% in mucosal sites, Fig. 1c), which
1Columbia
Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA. 2Department of Microbiology and Immunology,
Columbia University Medical Center, New York, New York, USA. 3Department of Surgery, Columbia University Medical Center, New York, New York, USA.
4Department of Medicine, Columbia University Medical Center, New York, New York, USA. 5LiveOnNY, New York, New York, USA. Correspondence should be addressed
to D.L.F. ([email protected]).
Received 30 June; accepted 12 November; published online 14 December 2015; doi:10.1038/nm.4008
nature medicine advance online publication
letters
exceeded the proportion of CD4+ RTEs in young adult lymphoid
tissue (<30%) and at mucosal sites (<10%) (Fig. 1c). Pediatric and
young adult thymii exhibited similar percentages of CD4+CD8+ (double positive, DP) thymocytes (Fig. 1d), and naive T cells were largely
RTEs in both groups (Fig. 1c). Therefore, the reduction in tissue RTEs
in young adults does not correlate with differences in thymopoiesis;
CD4+ T cells
Blood
Jejunum
87
3
77
9 10
Spleen
10 41
Lung
78
9 27
12 19
MLN
6
9
11
1
90 5
7 3
Jejunum
93 15
2
Spleen
57
24
19
5
70 29
ILN
Blood
62
11
IIeum
59 11
16
Colon
89
2
95 1
95 3
7
1
2
1
95
**
**
*
*
40
*
20
60
40
*
* *
20
11
Jejunum
75 12
16
Spleen
27
32
25
26
Colon
70
58
12
10 9
21 20
22
22
1
86
Lung
IIeum
36 50
6 10
43
ILN
83
*
*
40
20
Bl
o
Sp od
le
en
IL
N
LL
N
Lu
ng
Je ML
ju N
nu
m
IIe
u
C m
ol
on
0
40
*
**
60
** *
**
**
20
100
100
*
80
* **
80
60
40
20
0
**
80
60
*
40
20
*
*
*
*
0
c
Bl
o
Sp od
le
en
IL
N
LL
N
Lu
ng
M
Je LN
ju
nu
m
IIe
u
C m
ol
on
*
*
0
CD8+ TEM cells (%)
60
80
Bl
o
Sp od
le
en
IL
N
LL
N
Lu
ng
M
Je LN
ju
nu
m
IIe
u
C m
ol
on
80
20 47
MLN
100
0
0
*
18
LLN
68 7
12
73
MLN
63 5
Colon
59 8
28
57
29
100
Pediatric
80
Young adult
60
40
20
*
0
*
100
Pediatric
Young adult
80
60
40
20
*
0
Bl
o
Sp od
le
en
IL
N
LL
N
Lu
ng
Je ML
ju N
nu
m
IIe
u
C m
ol
on
100
** **
60
100
18
CD4+ TEMRA cells (%)
**
CD4+ TEM cells (%)
**
CD4+ TCM cells (%)
80
6
12
IIeum
16
22 63
Blood
CCR7
CD8+ TCM cells (%)
CD4+ naive T cells (%)
100
13 69
Lung
ILN
CCR7
b
23 8
62
8
MLN
Jejunum
79
Spleen
76
CD8+ T cells
LLN
65
22
Lung
11
Colon
88
11 4
46
CD4+ T cells
LLN
CD8+ TEMRA cells (%)
CD45RA
83
Blood
IIeum
57
ILN
CD8+ naive T cells (%)
d
DP thymocytes (%)
Total CD4+CD31+
T cells (%)
Figure 1 Naive T cells predominate in
80
** ** ** ** ** **
infant tissues, with TEM cells confined
60
72
5
to mucosal sites. (a) Flow cytometry plots
** **
*
40
of CD45RA and CCR7 expression by CD4+
80
4 months
old
and CD8+ T cells in pediatric blood and
20
60
tissues from a representative infant (left,
0
20
donor 118, 2 months) and from an older baby
40
80
(right, donor 213, 2 years). (b) Mean
84
7
20
frequency ± s.e.m. of each T cell subset in
60
the order (left to right) naive; central
22 years
0
40
old
memory (TCM); effector memory (TEM);
Pediatric Young
Pediatric
terminal effector (TEMRA) expressed as a
adult
20
Young adult
8
+
+
percent of CD4 (top) or CD8 (bottom) T cells
CD4
0
in circulation, lymphoid and mucosal tissues
(ordered from circulation, red; to mucosal sites,
green) and separated by age group (pediatric
(0–2 years) 15 donors, red; young adult (15–25 years),
9 donors, black). (c) CD31 expression (mean frequency ± s.e.m.) by total CD4 T cells (top) and naive CD4+ T cells (bottom) from eight pediatric and nine
young adult donors as in b. (d) CD4 and CD8 expression by thymocyte populations in the thymic tissue of an infant (top, donor 107, 4 months) and a
young adult (bottom, donor 102, 22 years) and compiled frequencies of CD4 +CD8+ (double positive, DP) cells in the thymii of six pediatric and five young
adult donors (right). Statistical significance represents comparisons between the indicated frequencies in pediatric and young adult donors at the same
tissue sites, measured by multiple t-tests adjusted for multiple comparisons. *P < 0.05; **P < 0.001. Individual donors used, descriptive statistics and
individual P values are shown in Supplementary Tables 2–4. ILN, inguinal lymph nodes; LLN, lung-draining lymph nodes; MLN, mesenteric lymph nodes.
N
n
LL
N
Lu
ng
M
L
Je N
ju
nu
m
IIe
um
C
ol
on
IL
ee
Sp
l
Bl
oo
d
CD8
Naive CD4+CD31+
T cells (%)
© 2015 Nature America, Inc. All rights reserved.
11
CD8+ T cells
LLN
CD45RA
a
it could derive from differential seeding, retention or activation
at these sites.
In pediatric tissues, memory T cells (mainly TEM cells) were
found in frequencies >20% only in the lungs, jejunum and ileum,
and they were significantly less abundant (P < 0.05) in the blood,
spleen, lymph nodes and colon (Fig. 1a,b, Supplementary Fig. 1b and
advance online publication nature medicine
Supplementary Table 3). By contrast, in young adults, TEM cells predominated in all mucosal sites (>90%), and they comprised 30–40%
of lymphoid T cells (Fig. 1b). The compartmentalization of early
T cell memory to the lungs and small intestines, but not to the draining lymph nodes, suggests local in situ priming to inhaled and ingested
antigens, respectively. We examined whether infant tissue TEM cells
express markers of tissue resident memory T (TRM) cells1,3,14, including
the activation marker CD69 and the integrin CD103, which are both
involved in tissue retention17,18. CD69 was expressed by the majority of TEM cells in all pediatric tissues, similarly to TEM cells in adult
tissues, whereas blood TEM cells were CD69− (Fig. 2a,b)8. However,
in pediatric mucosal sites, a lower frequency of CD8 + TEM cells
coexpressed CD69 and CD103 compared to higher frequencies in
adult mucosal sites (Fig. 2c,d and Supplementary Table 3)8, suggesting that mucosal memory T cells in early life have not yet fully
differentiated into TRM cells.
To further establish the differentiation status of T cells in pediatric
tissues, we analyzed their capacity for effector cytokine production.
Lymph node T cells from adult tissues produced substantial levels
of interleukin 2 (IL-2), interferon-γ (IFN-γ) and IL-4 after stimulation mediated by the T cell receptor (TCR)-CD3 complex, whereas
a
Blood
Spleen
3
LLN
59
Lung
59
pediatric T cells produced lower levels of both IL-2 and IL-4 and
negligible levels of IFN-γ (Fig. 2e). Low-level IFN-γ production by
pediatric lymphoid T cells, compared to those of adults, was also
observed after TCR-independent stimulation of the cells with phorbol
12-myristate 13-acetate (PMA) and ionomycin (Fig. 2f), which
suggests that there are cell-intrinsic differences. Nor did pediatric
lymphoid T cells produce high levels of IL-8 (Supplementary Fig. 2),
as observed in T cells from newborn cord blood9. However, pediatric
memory T cells in the small intestines produced IFN-γ rapidly in
response to PMA stimulation, exceeding the low proportion of
IFN-γ producers in the LN and in the colon (Fig. 2g). Thus, memory
T cells that are generated during early life in mucosal sites acquire the
capacity to secrete proinflammatory cytokines.
We considered whether Treg cells have differential distribution
or function in pediatric, as opposed to adult, tissues. Treg cells are a
subset of CD4+ T cells that express the transcription factor forkhead
box P3 (FOXP3) and the IL-2Rα chain (CD25), and they are essential
for the establishment of self-tolerance and immune homeostasis (for
reviews, see refs. 19–21). In humans, there are few studies of Treg cells
in tissues22,23, and the role of Treg cells in early immune responses is
unknown. In infant tissues, a high proportion (20–40%) of CD4+
c
Jejunum
78
Lung
Jejunum
20
98
lleum
45
Colon
33
29
2 years
old
+
CD4 T
cells
Naive
TEM
13
65
68
86
% maximum
93
60
49
60
61
40
90
87
57
20 years
old
CD103
+
CD8 T
cells
31
8
11
35
CD69
d
100
80
CD8+CD69+
CD103+ TEM (%)
CD4+CD69+
T cells (%)
b
*
100
Pediatric
Young adult
100
**
80
CD8+CD69+
CD103– TEM (%)
CD69
80
on
um
ol
C
lle
um
ng
Lu
Je
ju
n
Bl
oo
d
Sp
le
en
M
LN
sp Ad
le ult
en
IFN-γ, IL-2 (pg/ml)
IL
N
LL
N
Lu
ng
M
Je LN
ju
nu
m
lle
um
C
ol
on
f
IL-4 (pg/ml)
Bl
oo
d
Sp
le
en
e
CD3+ lFN-γ+ T cells (%)
on
ol
C
um
lle
um
Je
j
un
Lu
ng
Figure 2 Distinct properties
60
**
**
*
60
**
60
of early tissue memory T cells
*
40
Pediatric
**
40
in lymphoid and mucosal sites.
40
Young adult
20
(a) Representative histograms
20
20
0
of CD69 expression by naive
0
0
+
100
(red) and TEM (blue) CD4 (top)
and CD8+ (bottom) T cells from
80
an infant donor (donor 127, 16
60
months). (b) Mean frequencies ±
40
50
+
+
s.e.m. of CD4 CD69 (top) or
20,000
Pediatric
80
20
of CD8+CD69+ (bottom) TEM cells
Young adult
40
0
15,000
60
from 15 pediatric donors (0–2 years,
30
red) or nine young adults (15–25 years,
10,000
40
black). (c) Representative flow cytometry
20
*
plots of CD69 and CD103 expression on
5,000
20
10
CD8+ TEM cells from a pediatric donor (top, donor 63, 26 months)
*
0
0
and a young adult donor (bottom, donor 105, 20 years). (d) Mean
0
IL-2
IL-4
IFN-γ
frequencies ± s.e.m. of CD8+CD69+CD103+ (left) and CD8+CD69+CD103−
(right) TEM in nine pediatric (white) donors and eight young adult
(black) donors. Statistical significance for each site is indicated as
Pediatric
*P < 0.05; **P < 0.001. (e) Cytokine content in culture supernatants
ILN
MLN
Jejunum
lleum
Colon
(pg per ml, mean ± s.e.m.) of infant (n = 4, white) and adult
5 22
5
3 58
3 9
2
3 5
(n = 3, black) mesenteric lymph node (MLN) T cells after stimulation
with anti-CD2/CD3/CD28–coupled beads for 4 days. Unstimulated
cells produced <1 pg per ml of cytokines (data not shown). Statistical
significance is indicated as *P < 0.05. (f) Average interferon-γ (IFN-γ)
32
59 29
3 52
46
59 35
22 47
production from indicated tissues of five pediatric donors and three
CD45RA
adult donors stimulated with phorbol 12-myristate 13-acetate (PMA)
and ionomycin for 4 h. (g) IFN-γ production versus CD45RA expression from CD3+ T cells of donor 213 (2 years) stimulated with PMA and ionomycin
for 4 h. Individual donors used, descriptive statistics and individual P values are shown in Supplementary Tables 2 and 3.
CD8+CD69+
T cells (%)
g
IFN-γ
© 2015 Nature America, Inc. All rights reserved.
letters
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7 Jejunum 2
–
CD25
+
CD127
+
CD4 T cells
3
34 lleum
27
Spleen
3 Lung
5 lleum
2
ILN
14 Colon
15
ILN
4 MLN
5 Colon
4
6 months
old
CD25
5
0
0
Spleen
MLN
0.2 <0.1
0.5
2
30
Lung
20
41
75
2
10
0
10
8 0.4
5 0.5
43
75
5
75
CD69
8
+
6
4
2
0
Bl
Sp ood
le
en
IL
LLN
Lu N
n
Je M g
ju LN
nu
lle m
C um
ol
on
Colon
lleum
100
80
60
40
20
0
CD69 CD103
–
CD69+CD103+
Bl
o
Sp od
le
en
IL
N
LL
N
Lu
ng
Je ML
ju N
nu
m
lle
u
C m
ol
on
*
*
*
20
<0.1 0.9
CD103
**
** ** **
**
*
*
40
Pediatric
Adult
40
+
**
60
Blood
1
Bl
Sp ood
le
en
IL
LLN
Lu N
n
Je M g
ju LN
nu
lle m
C um
ol
on
10
*
f
CD3
Bl
Sp ood
le
en
IL
LLN
Lu N
n
Je M g
ju LN
nu
lle m
C um
ol
on
20
15
**
Bl
o
Sp od
le
en
IL
N
LL
Lu N
ng
Je ML
ju N
nu
lle m
u
C m
ol
on
Figure 3 Elevated frequency and broad tissue distribution of Treg cells in pediatric, as compared to adult, tissues.
Treg
(a) Expression of CD25 and FOXP3 by CD4+ T cells in tissues from an infant (donor 118, 2 months, left) and from
30
Non-Treg
an adult (donor 105, 20 years, right) with frequency of CD25 +FOXP3+ cells indicated. (b) Gating strategy for
*
Treg cells from pediatric (top, donor 68, 6 months) and from adult (bottom, donor 110, 40 years) lung-draining
20
**
lymph node (LLN) tissue, showing an initial CD25+CD127− gate (left) subsequently gated on FOXP3 expression,
** ** ** **
**
** **
versus non-Treg (CD25−CD127+) cells as a control. (c) Treg cell frequency (mean ± s.e.m.) in tissues from
10
13 pediatric (red) and 24 adult (black) donors, expressed as the percent of CD25 +CD127−FOXP3+ cells from
0
total CD4+ T cells. *P < 0.05; **P < 0.001. (d) Treg cell tissue distribution in pediatric and adult donors.
Graphs show Treg cell frequency tissues from individual pediatric (top row, 2 months, 4 months, 1 year) and adult
(lower, 20 years, 38 years, 51 years) donors. (e) CD45RA expression by Treg cells from 13 pediatric (red) and 24 adult
(black) donors. *P < 0.05. (f) CD69 and CD103 expression on Treg cells, shown as representative flow cytometry plots (top)
and compiled percentages (mean ± s.e.m.) of CD69+CD103− (white) and CD69+CD103+ (gray) populations from 16 donors (bottom). (g) Percent Ki67
expression (mean ± s.e.m.) by Treg (purple) and by non-Treg (CD4+CD25−CD127+) cells (black) in blood and tissues from 12 pediatric and adult donors.
Statistical significance indicated as *P < 0.05; **P < 0.001. Tissues displayed are ordered from blood, red, to mucosal sites, green. Individual donors
used, descriptive statistics and P values are shown in Supplementary Tables 2, 4 and 5.
+
g
Bl
Sp ood
le
en
IL
LLN
Lu N
n
Je M g
ju LN
nu
lle m
C um
ol
on
+
% Ki67 CD4
Treg (%)
© 2015 Nature America, Inc. All rights reserved.
+
CD4 Treg (%)
25
Adult
+
CD4 Treg (%)
Pediatric
Adult
Pediatric
+
CD4 Treg (%)
d
40 years
old
FOXP3
CD25
c
39
16
CD127
FOXP3
2
18 MLN
80
77
44
Spleen 29 Lung
e
+
CD25
–
CD127
Bl
o
Sp od
le
en
IL
N
LL
Lu N
ng
Je M
ju LN
nu
lle m
u
C m
ol
on
5 LLN
+
Blood
CD4 Treg (%)
31 Jejunum 32
+
b
31 LLN
Blood
CD45RA CD4
Treg (%)
a
T cells were CD25+FOXP3+, whereas the frequency of these cells was
lower in young adult tissues (Fig. 3a). Quantitation of Treg cells on the
basis of gating of CD25+CD127−FOXP3+ CD4+ T cells24,25 (Fig. 3b
and Supplementary Fig. 3) revealed an elevated frequency of Treg
cells (10–30%) in all infant and pediatric tissues, which contrasted
with the 6- to 10-fold lower frequency of Treg cells (2–5% CD4+
T cells) seen in adult tissues (Fig. 3c, Supplementary Fig. 4a and
Supplementary Table 4). Treg cell distribution in tissues also differed
between pediatric and adult donors, with high frequencies of Treg cells
in pediatric mucosal and lymphoid tissues, whereas in adults, Treg cell
frequencies were highest in the lymphoid tissues (often in the LLN)
as compared to mucosal sites (Fig. 3c,d and Supplementary Fig. 4b).
A heat map of tissue Treg cell frequencies in individual donors ranging from infants to adults 63 years of age shows the sharp reduction
in Treg cell frequencies that occurs after childhood (17–20 years)
(Supplementary Fig. 4a).
We examined potential differences in Treg cell phenotypes and
turnover between tissues and the circulation. In pediatric and adult
tissues, Treg cells in mucosal tissues were predominantly CD45RA−,
whereas 40–60% Treg cells in lymphoid tissues and blood were
CD45RA+ (Fig. 3e and Supplementary Fig. 5a), suggesting that there
are tissue-intrinsic differences in the activation state26,27. Pediatric
and adult tissue Treg cells also expressed CD69 (similarly to TRM
cells) but not CD103, whereas blood Treg cells were CD69−CD103−
(Fig. 3f). All tissue and circulating Treg cells showed extensive proliferation compared to their non-Treg cell counterparts (Fig. 3g,
Supplementary Fig. 5b and Supplementary Table 5). Thus, Treg cells
in human tissues exhibit distinct properties that are independent of
age and are instead influenced by CD45RA− phenotypes28 and/or
tissue-specific signals.
Pediatric Treg cells expressed higher amounts of FOXP3 (which is
associated with increased suppressive capacity29) than did adult Treg
cells in lymphoid tissues, whereas levels of FOXP3 expression in adult
and pediatric blood Treg cells were similar (Fig. 4a,b). Functionally,
the depletion of CD25+CD4+ Treg cells from pediatric lymph node
T cell cultures resulted in increased T cell proliferation (Fig. 4c) and
greater cytokine production (Fig. 4d and Supplementary Table 6);
adult LN T cells, by contrast, were activated similarly whether or
not CD25 was depleted (Fig. 4c,d). A comparable increase in T cell
proliferation was observed when Treg cells were depleted from cultures derived from pediatric lungs and colon (Supplementary Fig. 6).
Together, these results suggest that early in life, Treg cells may be
suppressing tissue immune responses to a greater extent than they
do later in life. The findings are consistent with recent results in
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letters
Non-Treg
22 years old
c
2 months
∆MFl
2,064
∆MFl
3,819
4 months
2 years
16 months
22 years
CD4+
T cells
**
3,000
*
n.s.
n.s.
CD8+
T cells
CFSE
CD25-depleted
N
LL
M
LN
Sp
le
e
n
d
0
Total T cells
60
40
20
d
3
80
60
40
20
0
Pediatric
Adult
*
Fold change in
cytokine production
Unstimulated
1,000
Bl
oo
Undivided CD8+
T cells (%)
Pediatric
Adult
% max
FOXP3 MFI
b 4,000
e
**
2
4
16
Age (months)
2
22
Age (years)
4
3
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
This work was supported by the US National Institutes of Health (NIH) (grant no.
AI100119; D.L.F., AI106697: D.L.F., F31AG047003; J.J.C.T., AI083022; K.L.B.) and
a BD Bioscience Research Grant (J.J.C.T.). These studies were performed in the
Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core,
funded in part through an S10 Shared Instrumentation Grant from the NIH (grant
no. S10RR027050), with the excellent technical assistance of S.-H. Ho.
nature medicine advance online publication
on
ol
C
m
um
lle
LN
nu
M
ju
Je
N
ng
Lu
N
IL
LL
en
oo
Sp
Bl
-2
d
Methods
Methods and any associated references are available in the online
version of the paper.
IL
-γ
N
mice that show distinct roles for perinatal Treg cells in establishing
self-tolerance30.
Our results provide a new view of infant T cell distribution and
function throughout the body, with important implications for promoting immune health and protection in early life. We show that
naive and Treg cells populate the lymphoid and mucosal tissues during
infancy, and that differentiation of functional memory T cells occurs
in certain mucosal sites. We propose that the higher Treg:TEM cell
content seen in infant as compared to adult tissues (Fig. 4e) leads to
in situ regulation of T cell differentiation; this confines early T cell differentiation activation and memory formation to the lungs and small
intestines, both sites of continuous, high-level antigen exposure. Our
results suggest that immunization may be targeted during infancy to
sites with a low ratio of Treg:TEM cells for the promotion of immune
protection during the early years and beyond.
le
Treg / TEM
Figure 4 Pediatric tissue Treg cells suppress endogenous T cell proliferation
2
and function. (a) FOXP3 expression by pediatric and adult Treg cells.
2
Representative FOXP3 expression by mesenteric lymph node (MLN)
1
1
CD4+CD25+CD127− cells (red) compared to CD25−CD127+FOXP3− cells
(black dashed) from a pediatric (16 months, left) donor and an adult (22 years,
0
0
right) donor. ∆MFI (change in mean fluorescence intensity) = MFI(Treg
cells)–MFI(control non-Treg cells). (b) Normalized FOXP3 MFI (± s.e.m.) as in a of
Treg cells from the blood and lymphoid tissues of ten pediatric (white) and 17 adult
(black) donors. *P < 0.05; **P < 0.001; n.s., not significant. (c) Proliferation of unfractionated T cells (red dotted) or CD25-depleted T cells (black),
isolated from MLN tissue from pediatric and adult donors of the indicated ages after anti-CD3/CD28 stimulation. Left, CFSE dilution of activated
compared to unstimulated (gray shaded) CD4+ (top) and CD8+ (bottom) T cells. Right, percent of CFSE+ (undivided) CD4+ (top) or CD8+ (bottom)
T cells stimulated either with or without CD25-depletion for each individual infant and adult donor. (d) Cytokine production by spleen and MLN T cells
in the presence or absence of Treg cells. T cells from pediatric and adult spleen and MLN were activated as in c with or without CD25 depletion, and
interferon-γ (IFN-γ) and interleukin-2 (IL-2) in supernatants were measured after 48 h. Graph shows mean fold change (± s.e.m.) calculated by dividing
the cytokine level produced by the CD25-depleted by the level obtained from unfractionated pediatric (white, n = 4) and adult (black, n = 4) T cells.
*P < 0.05; **P < 0.001. (e) Average Treg:TEM cell ratios in tissues from pediatric donors (0–2 yrs, n = 17). Red dashed line indicates the maximum
Treg:TEM cell ratio in adult tissues. Tissues displayed are ordered from blood, red, to mucosal sites, green.
lF
© 2015 Nature America, Inc. All rights reserved.
CD25-depleted
80
0
100
FOXP3
2,000
Unfractionated
100
Undivided CD4+
T cells (%)
Treg
16 months old
% max
a
We gratefully acknowledge the generosity of the organ donor families and the
efforts of the LiveOnNY transplant coordinators and staff for making this study
possible. We also wish to thank S. Mickel and B. Kumar for their assistance with
tissue processing, B. Levin and N. Yudanin for their assistance with statistical
analyses and K. Zens, N. Yudanin and E. Lamouse-Smith for critical reading
of this manuscript.
AUTHOR CONTRIBUTIONS
J.J.C.T. designed experiments, generated and analyzed data from infant and adult
donor tissues, made figures and wrote and edited the paper; K.L.B. generated
phenotypic and functional data from infant donors, analyzed data and made
figures; Y.O., M.K., N.M. and A.G. obtained the tissues from organ donors for these
studies; C.G. and T.G. assisted with the processing of donor tissue; H.L. coordinated
the tissue donation and acquisition from adult donors; T.K. coordinated acquisition
for all infant donors; D.L.F. designed experiments, analyzed data, maintained
protocols for human tissue acquisition and wrote and edited the paper.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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advance online publication nature medicine
ONLINE METHODS
© 2015 Nature America, Inc. All rights reserved.
Acquisition of human tissue. Human tissues were obtained from deceased
(brain dead) organ donors at the time of organ acquisition for life-saving clinical transplantation. Infant donor tissue was obtained in collaboration with the
Columbia University/New York Presbyterian Hospital pediatric liver transplant
program. Adult donor tissues were obtained through an approved protocol and
material transfer agreement with LiveOnNY (formerly the New York Organ
Donor Network, NYODN). Organ donors were free of chronic disease and
cancer, negative for hepatitis B, hepatitis C and HIV and 79% male. The study
does not qualify as human subjects research, as confirmed by the Columbia
University IRB, because tissue samples were obtained from deceased individuals.
Cord blood was obtained as discarded samples from K. Liu, Columbia
University. Adult blood and pediatric thymus tissues were acquired through
the Columbia Center for Translational Immunology (CCTI) human studies
core, which operates in accordance with the Columbia University human
research protection office institutional review board.
Lymphocyte isolation from human lymphoid and non-lymphoid tissues.
Tissue samples were maintained in cold saline and brought to the laboratory
within 2–4 h of organ procurement. Samples were rapidly processed using
enzymatic and mechanical digestion to obtain lymphocyte populations with
high viability, as described in detail7,8. Tissues were minced and incubated
at 37 °C in enzymatic digestion media: RPMI (Thermo Fisher, Waltham,
MA) containing 10% FBS (Thermo Fisher), l-glutamate (Thermo Fisher),
sodium pyruvate (Thermo Fisher), nonessential amino acids (Thermo Fisher),
penicillin-streptomycin (Thermo Fisher), collagenase D (1 mg/ml, Roche,
Indianapolis, IN), trypsin inhibitor (1 mg/ml, Thermo Fisher) and DNase I
(0.1 mg/ml, Roche). Digested tissue was further disrupted using the gentleMACS tissue dissociator (Miltenyi Biotech, San Diego, CA); the resulting
suspension was passed through a tissue sieve (10–150 mesh size) and then
pelleted through centrifugation. Residual red blood cells (RBC) were lysed
using AKC lysis buffer (Corning Cellgro, Manassas, VA), and dead cells and
debris were removed via centrifugation through 30% Percoll (GE Healthcare
Life Sciences, Pittsburgh, PA). Lymphocytes were isolated from blood using
lymphocyte separation media (Cellgro) and AKC lysis buffer as described7.
Flow cytometry analysis. The following fluorochrome-conjugated antibodies
were used for surface staining: anti-human CD3 (Brilliant Violet 650, 1:100,
OKT3, BioLegend, San Diego, CA), CD4 (PeCy7, 1:100, SK3, BioLegend),
CD8 (APC-Cy7, 1:100, SK1, BD Biosciences), CD19 (PE Texas Red, 1:100,
SJ25-C1, Invitrogen or APC, HIB19, BioLegend), CD25 (FITC, 1:50, 2A3, BD
Biosciences, San Jose, CA), CD31 (APC, 1:100, WM59, eBioscience, San Diego,
CA), CD45RA (Brilliant Violet 605, 1:100, HI100, BioLegend), CD45RO
(PerCpFl710, 1:100, UCHL1, eBioscience), CD69 (Brilliant Violet 421, 1:100,
doi:10.1038/nm.4008
FN50, BioLegend or BUV395, 1:100, FN50, BD Biosciences), CD103, (Alexa
Fluor 647, 1:100, Ber-ACT8, BioLegend), CD127 (BV711, 1:100, A019D5,
BioLegend), CCR7 (Alexa Fluor 488, 1:100, TG8, BioLegend). For intracellular staining, surface stained cells were resuspended and incubated in fixation
buffer (eBioscience), washed, resuspended in 0.1 ml permeabilization buffer
(eBioscience) and stained with anti-FOXP3 antibodies (PE, 1:20, 236A/E7,
eBioscience) and Ki67 (α700, 1:100, Ki-67, BioLegend) for 30 min at room
temperature and washed twice with permeabilization buffer. Stained cells were
acquired on a 6-laser LSRII analytical flow cytometer (BD Biosciences) in
the CCTI flow cytometry core and analyzed using FlowJo software (Treestar,
Ashland, OR).
T cell proliferation and Treg cell depletion. T cells isolated from tissue suspensions as above were purified using a human T cell enrichment kit (STEMCELL
Technologies, Vancouver, BC). A portion of these cells was subjected to CD25
depletion using BD IMag CD25 Magnetic Particles (BD Biosciences), with
the CD25-depleted population being collected in the column flow-through.
Purified T cells (unfractionated and CD25-depleted) were labeled with 2.5 µM
CFSE for 15 min at 37 °C, washed with media + FBS to quench excess CFSE
and plated in 96-well round bottom plates (100,000 cells per well) +/− antiCD2/CD3/CD28-coupled beads (one bead/cell; T cell activation/expansion
kit, Miltenyi Biotech) for 4 days at 37 °C incubator with 5% CO2, after which
cells were harvested and analyzed by flow cytometry.
Cytokine analysis. Supernatants from T cell cultures treated as above were
analyzed for cytokine content by Luminex using a cytokine human 25-plex
panel (Life Technologies, Grand Island, NY), as performed by the Human
Immunology Core (University of Pennsylvania, Philadelphia, PA). For intracellular cytokine staining, isolated T cells were cultured for 4 h with PMA/
ionomycin in the presence of Brefeldin A as described7. Cells were then stained
for surface markers, fixed, permeabilized as above and stained with antibodies
for IL-8 (PerCP, 1:20, BH0814, BioLegend) and IFN-γ (Alexa Fluor 700, B27,
BD Biosciences). Cytokine concentrations were also measured in supernatants using cytometric bead array with the BD Biosciences Human Th1/Th2
cytokine kit II following stimulation with anti-CD2/CD3/CD28–coupled
beads, as indicated above, for 2 days.
Statistical analysis and data visualization. Descriptive statistics were calculated using Microsoft Excel. Statistical significance was calculated for each
subset using Student’s t tests or by two-way ANOVA assuming unequal variance and adjusted for multiple comparisons by Holm-Sidak using GraphPad
PRISM (GraphPad Software, Inc., La Jolla, CA). We did not use statistical
methods to predetermine sample size, nor were the investigators blinded to
sample identity or results.
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