Interleukin 7 and T cell receptor signals regulate homeostasis of CD4 memory cells

Abstract

Immunological memory depends on the long-term maintenance of memory T cells. Although the factors that maintain CD8 T cell memory are well understood, those responsible for CD4 memory are not well defined. We have shown here that interleukin 7 (IL-7) was an important survival factor for CD4 memory T cells that together with T cell receptor (TCR) signals regulated homeostasis of the CD4 memory population in lymphopenic conditions and in the intact immune system. Thus, IL-7 contributes to the maintenance of all naive and memory T cell subsets, and therefore controls the overall size of the T cell pool.

Full text

ARTICLES
680 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY
The size of the peripheral T cell pool is notably constant despite
thymic production of new T cells and expansion of existing cells after
their stimulation by cognate antigen during immune responses
1
. This
is achieved through specific homeostatic mechanisms that regulate
both cell survival and proliferation. The precise nature of the environ-
mental cues that regulate these responses vary depending on the T cell
subset and on whether the cell is a naive or memory cell. It has been
proposed that these different subsets occupy independently regulated
niches
1
that are maintained by differences in the survival and prolifer-
ative requirements between compartments in both replete and lym-
phopenic conditions.
The homeostatic resources required by naive T cells and CD8
+
memory cells are well defined. The survival of CD4
+
and CD8
+
naive T
cells depends on the cytokine interleukin 7 (IL-7)
2–5
and also requires
contact with self-peptide major histocompatibility complexes
(MHCs)
6–11
. In conditions of lymphopenia, these same ligands drive
naive T cells into homeostatic proliferation
9,12–14
. Memory CD8
+
cells
persist in the absence of class I MHC ligands
15
but require IL-15 for
homeostatic proliferation in replete hosts
16–18
. Although there is no
absolute requirement for IL-7 to maintain CD8 memory cells in
replete mice, IL-7 does contribute to their homeostatic proliferation in
lymphopenic hosts
19,20
and can compensate for the absence of IL-15
(ref. 21). In addition, IL-7 and IL-15 promote survival of CD8 mem-
ory cells in vitro
21
. In contrast, the factors that regulate the survival
and proliferation of memory CD4
+
cells are less well defined and are
instead elucidated by intermittent reports of what is not required.
CD4
+
T cell memory can be generated from effector cells and persist in
the absence of MHC antigens
22
and does not require expression of
either TCR
23
or the Src-family kinases p56
lck
(Lck) and p59
fyn
(Fyn)
for long-term survival
5
. Homeostatic proliferation of CD4 memory
cells in response to lymphopenia occurs in the absence of IL-7 (ref.
24). Furthermore, a study of class II MHC–restricted TCR transgenic
mice lacking expression of the common cytokine receptor γ-chain (γ
c
)
indicates that although naive T cell survival is fundamentally
impaired, activation and memory cell formation is essentially normal,
potentially excluding involvement of all the cytokines whose receptors
require the γ
c
component: IL-2, IL-4, IL-7, IL-9 and IL-15 (ref. 25). It
has therefore been suggested that CD4 memory cells do not require
any specific signals for their homeostatic maintenance
26
.
Despite the burgeoning interest in the signals that influence home-
ostasis of the different T cell subsets, little is known about how CD4
+
memory T cells are regulated. As CD4 memory cells express IL-7
receptor (IL-7R) and receive the same MHC-derived TCR signals that
regulate naive T cell survival in vivo
27
, we sought to determine whether
these pathways were involved in their homeostasis. Using mice
expressing an inducible Lck transgene that permit the manipulation of
signals through the TCR, we were able to dissect the influence of TCR
from IL-7R signals. Our data showed that both of these signals regu-
lated memory CD4 T cell homeostasis. Moreover, we found an overlap
between these signals, such that the influence of one was able to mask
the effects of the other. Therefore, unlike CD8 T cells, naive and mem-
ory CD4 cells were regulated by overlapping signals, challenging the
idea that all T cell subsets necessarily occupy independent niches.
RESULTS
CD4 memory persists without TCR signals
Initially we examined CD4 memory homeostasis in the absence of
TCR stimulation. We used a system that generated resting memory
cells in which TCR signaling could be manipulated. Mice expressing a
tetracycline-regulated inducible Lck transgene (Lck1
ind
; Methods) on
either an Lck
–/–
or Lck
–/–
Fyn
–/–
background reconstitute the T cell
compartment
28
and generate a polyclonal repertoire of naive and
memory (CD44
lo
and CD44
hi
) T cells, similar to that found in wild-
type mice. The wild-type mice and doxycycline-fed Lck1 transgenic
mice (Lck
ind
) used in this study were not deliberately immunized with
antigen, but they nevertheless had T cells with the characteristics of
Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Correspondence should be addressed to
R.Z. (rzamoys@nimr.mrc.ac.uk).
Interleukin 7 and T cell receptor signals regulate
homeostasis of CD4 memory cells
Benedict Seddon, Peter Tomlinson & Rose Zamoyska
Immunological memory depends on the long-term maintenance of memory T cells. Although the factors that maintain CD8 T cell
memory are well understood, those responsible for CD4 memory are not well defined. We have shown here that interleukin 7 (IL-
7) was an important survival factor for CD4 memory T cells that together with T cell receptor (TCR) signals regulated homeostasis
of the CD4 memory population in lymphopenic conditions and in the intact immune system. Thus, IL-7 contributes to the
maintenance of all naive and memory T cell subsets, and therefore controls the overall size of the T cell pool.
© 2003 Nature Publishing Group http://www.nature.com/natureimmunology

ARTICLES
NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY 2003 681
memory T cells
29
likely to be specific for environmental antigens
found in gut flora and food. These naturally occurring memory cells in
both wild-type and Lck1
ind
transgenic mice bore all the hallmarks of
memory cells that distinguished them from naive T cells
30,31
: pheno-
typically they were CD44
hi
and CD62L
lo
(Fig. 1a); their survival was
independent of TCR signaling (Fig. 1a)
5
; they had the ability to ‘home’
to tissue sites (Supplementary Fig. 1 online); and, like wild-type mem-
ory T cells, they had the ability to rapidly secrete interferon-γ after
short-term restimulation in vitro (Supplementary Fig. 1 online). We
studied these cells rather than monoclonal TCR transgenic memory
cells because they not only represented a diverse memory pool, but
they also provided the most physiologically relevant setting to examine
signals that regulated homeostasis of CD4 memory cells.
When Lck expression was switched off in adult mice by removal of
doxycycline (Lck1
off
Lck
–/–
Fyn
–/–
), the T cells lacked expression of both
Lck and Fyn and were incapable of being stimulated through the TCR
(data not shown). Thymic development is completely aborted after
removal of doxycycline, so no new naive T cells are produced, and in
the absence of Lck and Fyn, existing naive T cells fail to survive
5
. In
contrast, the CD4
+
CD44
hi
compartment undergoes a brief contrac-
tion (this was approximately twofold in both Lck1
ind
Lck
–/–
and
Lck1
ind
Lck
–/–
Fyn
–/–
mice; data not shown). Although loss of some
memory CD4
+
cells may contribute to this reduction, the absence of
Lck will also cause loss of effector cells, absence of new input from the
naive pool and reduced turnover of existing memory cells. The
remaining population of CD4
+
CD44
hi
T cells in these mice persists in
the absence of TCR signaling
5
. Within several weeks, their CD4
+
com-
partment contained almost exclusively CD44
hi
CD62L
lo
memory phe-
notype cells (Fig. 1a), in contrast to that of either wild-type or
doxycycline-fed Lck1
ind
Lck
–/–
Fyn
–/–
mice, in which both naive and
memory T cells were supported. The range of TCR V
β
expression
among the persisting CD4
+
CD44
hi
T cells was similar to that found in
wild-type and doxycycline-fed mice, confirming the polyclonality of
this memory population (Supplementary Fig. 1 online). Because we
wanted to examine the influence of IL-7R signals on CD4 memory cell
behavior in these mice, we confirmed that IL-7R expression was nor-
mal. CD4
+
CD44
hi
-phenotype cells both from wild-type and the vari-
ous Lck1 mice, on or off doxycycline, expressed large amounts of
IL-7R (Fig. 1b). Expression of IL-7R was slightly increased on the
memory population from Lck1
off
Lck
–/–
Fyn
–/–
mice, perhaps because
of the loss of TCR signaling capacity, as signals through the TCR
down-modulate IL-7R expression
2
. These data confirmed that TCR
signals were not required for long-term survival of polyclonal memory
CD4 T cells.
Influence of IL-7 on CD4 memory
Because CD4 memory can persist in the absence of antigen, MHC lig-
ands and Src kinases, we investigated whether IL-7 was involved in
their homeostasis. We obtained CD4
+
CD44
hi
T cells from
Lck1
off
Lck
–/–
Fyn
–/–
mice off doxycycline for between 1 and 3 months,
labeled the cells with carboxyfluorescein diacetate succinimidyl ester
(CFSE) and transferred them to recipient mice deficient in recombina-
tion activating gene 1 (Rag1
–/–
): Il7
+/+
Rag1
–/–
, Il7
+/–
Rag1
–/–
or
Il7
–/–
Rag1
–/–
. On days 1, 21 and 35 after transfer, we assessed T cell
recoveries from lymph nodes and spleens of recipient mice and exam-
ined CFSE labeling of donor cells. After being transferred to
Il7
+/+
Rag1
–/–
recipients, Lck1
off
Lck
–/–
Fyn
–/–
memory CD4
+
cells
underwent a number of divisions that increased over time (Fig. 2a).
Moreover, cell proliferation was also accompanied by an increase in
cell recovery. Cells transferred to Il7
–/–
Rag1
–/–
recipients failed to
divide. Although recoveries from Il7
+/+
Rag1
–/–
and Il7
–/–
Rag1
–/–
mice
were identical at day 1 after cell transfer (Fig. 2b), indicating that the
initial seeding of transferred cells was similar between the recipient
strains, on days 21 and 35 after transfer, cell recoveries were far greater
from mice expressing IL-7 than from Il7
–/–
Rag1
–/–
recipients (Fig. 2b).
To determine whether IL-7 was influencing both expansion and
Figure 1 Persistence of memory CD4 T cells in the absence of TCR signaling. Lck1
ind
Lck
–/–
Fyn
–/–
mice were switched from doxycycline-containing food
to conventional diets to turn off Lck expression (Lck1
off
Lck
–/–
Fyn
–/–
). Lymph node cells from these mice or Lck1
ind
Lck
–/–
Fyn
–/–
mice fed doxycycline
continuously and from wild-type controls were stained for CD4, CD44, CD62L and TCR and were analyzed by FACS. (a) CD4 versus CD8 expression on live-
gated lymph node cells (top row) and CD62L versus CD44 expression on gated CD4
+
TCR
hi
lymph node cells (bottom row) from wild-type controls (left
column), doxycycline-fed Lck1
ind
Lck
–/–
Fyn
–/–
mice (middle column) and Lck1
off
Lck
–/–
Fyn
–/–
mice (off doxycycline for 6 weeks; right column). Percentages
indicate frequencies of CD4
+
and CD8
+
cells (top row) or frequencies of CD62L
hi
CD44
lo
and CD62L
lo
CD44
hi
populations (bottom row). (b) IL-7R expression
by either CD4
+
CD44
hi
or CD4
+
CD44
lo
cells from age-matched wild-type, Lck1
ind
Lck
–/–
or Lck1
ind
Lck
–/–
Fyn
–/–
mice either maintained on or taken off the
doxycycline-containing diet for at least 2 weeks. Vertical markers were set against negative staining controls and indicate the threshold of positive staining.
+ Dox, with doxycycline; – Dox, without doxycycline.
ab
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ARTICLES
682 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY
survival of the transferred cells, we adjusted results for cell recoveries
to compensate for the effects of cell division (Fig. 2c). Even with this
adjustment, cell recoveries were approximately three times greater
from Il7
+/+
Rag1
–/–
mice than from Il7
–/–
Rag1
–/–
recipients at day 21
(Fig. 2c). By day 35, all cells transferred to Il7
+/+
Rag1
–/–
mice had lost
their CFSE labeling, so those results could not be adjusted for division
number; however, the difference in cell recoveries between
Il7
+/–
Rag1
–/–
and Il7
–/–
Rag1
–/–
became greater over time (Fig. 2c).
Examination of peritoneal exudate cells showed that cell recoveries
from the tissues at day 35 (Fig. 2d) mirrored those from lymph node
and spleen, indicating that loss of cells from Il7
–/–
mice was not simply
a reflection of differences in ‘homing’. Inclusion of Il7
+/–
Rag1
–/–
recip-
ients showed a distinct gene-dose effect for IL-7. Cells transferred to
these hosts proliferated less (Fig. 2a), accumulated less (Fig. 2b) and
survived less well (Fig. 2c) than did cells in Il7
+/+
Rag1
–/–
hosts, con-
firming that the availability of IL-7 directly influenced both expansion
and survival of CD4
+
memory cells.
CD4 memory maintenance without IL-7R signals
Our data demonstrated involvement of IL-7 in the homeostasis of
CD4 memory, but previous studies have shown that mice with
impaired cytokine signaling generate CD4 memory cells that per-
sist
32–34
. Because we showed that in the absence of both TCR and IL-
7R signals, CD4 memory T cells could not proliferate and failed to
survive, we asked what function TCR signals have in the homeostasis
of CD4 memory.
We first examined the phenotype of IL-7R-deficient mice, which
are profoundly lymphopenic due to a developmental block in T and
B cell development caused by the absence of this receptor
35,36
.
However, analysis of splenocytes from these mice showed that
although naive phenotype CD4
+
CD44
lo
CD62L
hi
cells were scarce,
memory phenotype CD4
+
CD44
hi
CD62L
lo
cells were unexpectedly
abundant (Fig. 3a), as in other mice with defective cytokine signal-
ing
32–34
. Therefore, CD4
+
CD44
hi
memory phenotype cells were
produced in response to TCR signals in these mice, in numbers simi-
lar to those of wild-type mice (Fig. 3b).
ab
c
d
Figure 3 IL-7R signals were not required for the generation of CD4
+
CD44
hi
cells. Spleen cells from Il7r
–/–
and wild-type control mice (n = 5) were stained
for CD4, CD8, CD44, CD62L and TCR expression and analyzed by FACS.
(a) CD4 versus CD8 expression on splenocytes gated on live cells (top row),
and CD62L versus CD44 expression by gated CD4
+
TCR
+
splenocytes from
wild-type and Il7r
–/–
mice (bottom row). Percentages indicate frequencies of
CD4
+
cells (top row) or CD62L
hi
CD44
lo
and CD62L
lo
CD44
hi
populations
(bottom row). (b) Total numbers of CD4
+
CD44
lo
and CD4
+
CD44
hi
cells
recovered from the mice in a.
ab
Figure 2 IL-7R signals promoted proliferation and survival of CD4
+
CD44
hi
cells in the absence of TCR signaling. CD4
+
CD44
hi
cells, purified from
Lck1
ind
Lck
–/–
Fyn
–/–
mice taken off doxycycline 4–12 weeks previously,
were labeled with CFSE and transferred to Il7
+/+
Rag1
–/–
, Il7
+/–
Rag1
–/–
or
Il7
–/–
Rag1
–/–
recipients (n =3 per group). (a) CFSE profiles of gated
CD4
+
TCR
hi
CD44
hi
cells from blood (day 6) or spleen (days 21 and 35).
(b,c) Total numbers of CD4
+
CD44
hi
TCR
+
cells recovered from lymph nodes
and spleens from different recipient mice (b) or adjusted figures that
compensated for the effects of cell division on cell numbers (c). The expansion
index was calculated from CFSE profiles (Methods) as 3.9 and 1.75 for cells from Il7
+/+
Rag1
–/–
and Il7
+/–
Rag1
–/–
hosts, respectively, on day 21, and 6.7 for cells
from Il7
+/–
Rag1
–/–
hosts on day 35. N.D., not determined. (d) CD4 and TCR expression by lymphocytes recovered from the peritoneum of recipient mice 35 days
after cell transfer. Percentages indicate frequencies of CD4
+
TCR
hi
cells. Data were pooled from three independent experiments in which mice received either
1 × 10
6
(day 21) or 4 × 10
6
(days 1 and 35) memory cells per mouse and were representative of two further experiments.
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ARTICLES
NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY 2003 683
The fact that thymic selection in IL-7R-deficient mice and other mice
lacking essential cytokine signaling components
25,32–34
is profoundly
blocked may favor selection of T cells that are relatively cytokine-
independent. Therefore, we also examined the ability of wild-type
CD4
+
CD44
hi
cells to undergo homeostatic responses after their trans-
fer to lymphopenic hosts in the absence of IL-7. CFSE labeled
CD4
+
CD44
hi
cells from wild-type mice were transferred into
Il7
+/+
Rag1
–/–
, Il7
+/–
Rag
–/–
, Il7
–/–
Rag1
–/–
recipient mice or Rag2
–/–
recipient mice deficient in the IL-2 receptor common γ-chain
(Il2rg
–/–
Rag2
–/–
). Il2rg
–/–
Rag2
–/–
mice have the same defects in lym-
phoid architecture as do Il7
–/–
Rag1
–/–
mice
37
, lacking almost all lymph
nodes
33
, but still express IL-7. As with Lck1
ind
Lck
–/–
Fyn
–/–
T cells trans-
ferred to Il7
+/+
Rag1
–/–
or Il7
–/–
Rag1
–/–
hosts (Fig. 2), recoveries of
wild-type T cells from all these hosts were similar at day 1 (data not
shown). By day 7, analysis of peripheral blood showed that transferred
cells had undergone extensive proliferation, losing CFSE dye, regardless
of whether hosts had IL-7 or not (Fig. 4a), as reported before
19
.
Similarly, transfer of these cells to Rag1
–/–
hosts treated with antibody
to IL-7R had little effect on the proliferative response of the cells
(Fig. 4b). However, donor cell numbers at day 14 showed that in the
absence of IL-7, or in the presence of IL-7R-blocking antibodies, there
was a profound reduction in cell recovery (Fig. 4c). This reduction
could not be attributed to the altered state of the lymphoid compart-
ment in Il7
–/–
Rag1
–/–
mice, as recoveries from Il2rg
–/–
Rag2
–/–
hosts,
which have a similar lymphoid defect, were normal. In addition, we
found a similar reduction after treatment of normal Rag1
–/–
recipients
Figure 4 Proliferation but no accumulation of
wild-type CD4
+
CD44
hi
T cells in the absence
of IL-7. CD4
+
CD44
hi
cells were purified from
spleens of 6- to 8-week-old wild-type mice,
labeled with CFSE and transferred (4 × 10
6
cells
per mouse) to Rag1
–/–
recipient mice that were
Il7
+/+
, Il7
+/–
or Il7
–/–
, and to Il2rg
–/–
Rag2
–/–
recipient mice. (a) On days 7 and 14, blood
was obtained from mice, and CFSE labeling
of CD4
+
CD44
hi
cells in peripheral blood was
determined by staining of cells for CD4, TCR
and CD44 expression and analysis by FACS.
(b) Using a protocol similar to that in a.
CD4
+
CD44
hi
cells from wild-type mice were
transferred to Rag1
–/–
recipients (4 × 10
6
cells
per mouse) treated with monoclonal antibody to
IL-7R or with PBS as a control. CFSE labeling in
peripheral blood was measured on days 7 and 14.
(c) Total numbers of CD4
+
TCR
+
cells recovered
from lymph nodes and spleens of at least three
recipient mice per group described in a and b.
Data were representative of two independent
experiments.
ab
c
ab
c
de
Figure 5 Maintenance of CD4 memory in full mice by TCR signals and IL-7.
(a) CD44 versus antibody-to-BrdU staining by CD4
+
TCR
+
peripheral blood
lymphocytes from wild-type mice and doxycycline-fed Lck1
ind
Lck
–/–
and
Lck1
off
Lck
–/–
Fyn
–/–
mice (n =3 per group) fed BrdU-containing water for
1 week. Percentages indicate the mean frequency ± s.d. of CD4
+
CD44
hi
cells
staining for BrdU incorporation. (b–e) Lck1
ind
Lck
–/–
mice 6–8 weeks of age
were taken off doxycycline and treated either with monoclonal antibody to
IL-7R or with PBS as control. At 0, 2 and 4 weeks after treatment, total
numbers of CD8
+
CD44
hi
and CD4
+
CD44
hi
cells from groups of mice (n = 3)
were determined by counting of total cells in lymph nodes and analysis of the
expression of CD8, CD4, TCR and CD44 by FACS. Data represent average
numbers ± s.d. of recovered CD4
+
CD44
lo
(b), CD4
+
CD44
hi
(c), CD8
+
CD44
lo
(d) and CD8
+
CD44
hi
(e) cells in mice treated with antibody to IL-7R (open
symbols) or control mice (filled symbols). Data were representative of three
(a) or four (b–e) independent experiments.
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ARTICLES
684 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY
with antibody to IL-7R. These data confirmed that TCR-mediated sig-
nals were able to induce proliferation of CD4
+
memory T cells and,
therefore, contributed to their persistence in the absence of IL-7. The
presence of CD4 memory cells in Il7r
–/–
mice and the observation that
some wild-type CD4 memory cells were recovered after transfer to
Il7
–/–
Rag1
–/–
hosts indicated limited involvement of TCR signals in
promoting survival of these cells. However, the decrease in recovery of
cells in the absence of IL-7 showed an important function for this
cytokine in CD4 memory cell survival.
CD4 memory cells in replete hosts
Having established the signals that regulated CD4 memory T cell
homeostasis in lymphopenic conditions, we asked how these signals
contributed to the maintenance of these cells in normal mice in
steady-state conditions. Memory cells undergo nonexpansive cell divi-
sions in replete hosts
38
and this slow turnover may contribute to their
ability to mount more rapid responses than naive cells, by allowing
quicker entry into cell cycle. Although TCR signals do not contribute
much to the long-term survival of CD4 memory cells, we asked
whether these signals were responsible for the memory cell turnover in
replete mice. We fed bromodeoxyuridine (BrdU) to wild-type mice or
control doxycycline-fed Lck1
ind
Lck
–/–
and Lck1
off
Lck
–/–
Fyn
–/–
mice for
1 week and analyzed T cells for BrdU incorporation. Although naive T
cells did not incorporate any BrdU, just over 50% of CD4
+
CD44
hi
cells
from Lck1
ind
Lck
–/–
mice and wild-type controls had undergone cell
division in 1 week (Fig. 5a). In contrast, less than 10% of CD4
+
CD44
hi
cells from Lck1
off
Lck
–/–
Fyn
–/–
mice had incorporated BrdU during this
time (Fig. 5a), indicating that TCR signals were important in stimulat-
ing proliferation of the CD4 memory compartment of replete mice.
Although we were able to show that TCR signals induced prolifer-
ation of CD4 memory T cells in replete mice, neither proliferation
(Fig. 5a) nor TCR signals
5
were essential for the long-term survival
of these cells. We asked, therefore, whether IL-7 was important for
the maintenance of CD4 memory in unmanipulated replete hosts.
We examined the survival of memory T cells in mice treated with a
blocking antibody to IL-7R. In these experiments, we used
Lck1
ind
Lck
–/–
mice taken off doxycycline, to maintain a static T cell
pool. In the absence of Lck expression not only does thymic atrophy
prevent export of newly differentiated T cells, but also the incum-
bent peripheral T cells are unable to expand to TCR-mediated
homeostatic signals
39
. However, both naive and memory cells still
survive in the absence of Lck alone. We treated groups of
Lck1
off
Lck
–/–
mice with antibody to IL-7R or with PBS for 4 weeks.
We determined naive and memory T cell subset numbers in lymph
nodes obtained from mice 0, 2 and 4 weeks after treatment was
begun. As reported before
5
, blockade of IL-7R resulted in loss of
both naive CD4
+
CD44
lo
(Fig. 5b) and CD8
+
CD44
lo
(Fig. 5d) T cells.
In the presence of IL-7R blockade, CD4
+
CD44
hi
cells (Fig. 5c)
showed a decrease in number similar to that of their naive counter-
parts. There was also an initial smaller decrease in CD4
+
CD44
hi
cells
in the PBS-treated control mice, which indicated that there was a
subset of these cells that relied on TCR-mediated signals for their
persistence. Consistent with published results
20
, CD8
+
CD44
hi
cell
numbers were completely unaffected by treatment with antibody to
IL-7R (Fig. 5e), indicating that the antibody did not eliminate T
cells by antibody-dependent cell-mediated cytotoxicity in vivo, as
CD8 memory cells also expressed large amounts of IL-7R
(Supplementary Fig. 1 online). These results confirmed that
although TCR-mediated signals could contribute to turnover of
CD4 memory cells in an intact mouse, the maintenance of CD4
memory cell numbers depended on the availability of IL-7.
DISCUSSION
The signals that regulate CD4 memory cell homeostasis are ill-defined.
In this study, we examined whether signaling by the IL-7R and TCR
contributed to the survival and homeostatic proliferation of CD4
memory cells in normal mice. Our data showed that in conditions of
lymphopenia and in the intact immune system, CD4 memory cell
homeostasis was regulated through the combined effects of TCR and
IL-7R signaling. In the absence of both these signals, CD4 memory
cells were unable to divide and failed to survive.
Our data seemed to contradict published reports indicating no
involvement of the TCR
22,23
or γ
c
cytokines
19,25
in the maintenance
of CD4 memory cells. One possible explanation for this discrepancy
is that TCR and IL-7R signals can have similar outcomes with
respect to the maintenance of CD4 memory cell numbers. We
showed here that in the absence of TCR signals, a polyclonal popula-
tion of CD4 memory cells survived long term and turned over
slowly in response to IL-7. However, mice with defective cytokine
signaling maintained a population of memory phenotype cells sim-
ilar in size to that of wild-type mice. CD4 memory cell persistence in
mice deficient in cytokine signaling will be influenced by extensive
lymphopenia and/or environmental antigen-induced cell division
which may mask defects in cell survival. Indeed, polyclonal Jak3
–/–
(ref. 40) and Il2rg
–/–
(ref. 34) mice have more extensive proliferation
(assessed by BrdU uptake) of CD4
+
CD44
hi
cells than do wild-type
mice, and have excessive cell death. Furthermore, spectratype analy-
sis of memory cells from Jak3
–/–
mice shows a skewed TCR reper-
toire
40
, indicating the selection of an autoantigen-reactive,
TCR-driven population, and these mice ultimately develop colitis.
These data also indicate that TCR and IL-7 signals are not acting
redundantly, as they have different outcomes on the composition of
the memory population. TCR signals may preferentially promote
survival of cells proliferating in response to persistent antigen,
whereas IL-7 may promote survival of memory cells that do not
require TCR signals and therefore do not require the presence of
antigen. In normal mice with full T cell compartments, there is
clearly extensive TCR-driven cell division in the memory pool, but
no accompanying expansion. In such situations, competition for IL-
7 most likely regulates the overall pool size. It is possible that cell
division induced by TCR stimulation fulfills a different function,
perhaps being more important for the functional competence of the
cell. Indeed, although memory cells may persist in the absence of
MHC ligands, they do show considerable functional defects
41
.
Our findings also indicated that the signals required for homeostasis
of CD4 memory were essentially the same as those that regulated naive
CD4
+
T cells: IL-7 and TCR signals. The fact that naive and memory
cells shared common resources seemed initially at odds with the view
that these populations should occupy non-overlapping and non-com-
peting niches
1
. Although the behavior of the naive and memory com-
partments may be operationally independent, there are examples of
CD4 memory phenotype cells interfering with the homeostasis of
naive T cells. For example, the few T cells present in Lck
–/–
Fyn
+/–
mice,
all of which are of a memory phenotype, can prevent almost all IL-7-
driven proliferation of naive T cells
42
, presumably by competing for
available IL-7. However, the key difference between the homeostasis of
naive and memory CD4 cells may be the degree of redundancy
between the signals involved, rather than the nature of the specific sur-
vival factors. Thus, memory CD4 cells are substantially influenced by
either IL-7R or TCR signaling, whereas naive CD4 cells require the co-
operative activity of both of these receptor signaling pathways, and this
operational difference in the use of common resources allows their rel-
atively independent regulation.
© 2003 Nature Publishing Group http://www.nature.com/natureimmunology

ARTICLES
NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY 2003 685
Our finding that IL-7 was important in the maintenance of CD4
memory identifies a single factor that regulates the homeostasis of all
naive and memory peripheral T cell subsets. Although there are subtle
differences in the specific requirements and use of resources by these
subsets that may afford a degree of independence, the universal
requirement of IL-7 strongly indicates it is the master regulator that
controls the overall size of the T cell compartment.
METHODS
Mice. The generation of inducible Lck transgenic Lck1
ind
mice has been
described
28
. Mice expressing the reverse tetracycline-responsive transactivator
domain (rtTA) under control of the human CD2 promotor on an endogenous
Lck
–/–
background (rtTA-C Lck
–/–
) were intercrossed with mice bearing the
mouse Lck transgene under control of a tetO-CMVmin promotor, also on an
endogenous Lck
–/–
background (Lck1Lck
–/–
). Mice were given doxycycline in
food (1 mg per g body weight) throughout pregnancy and before weaning.
After being weaned, Lck1 rtTA-C Lck
–/–
F1 progeny were given food containing
doxycycline to maintain Lck1 transgene expression (Lck1
ind
Lck
–/–
) or were
switched to conventional diets to shut off transgene expression (Lck1
off
Lck
–/–
).
Lck1
ind
Lck
–/–
Fyn
–/–
mice were similarly derived by intercrossing rtTA-C
Lck
–/–
Fyn
–/–
and Lck1Lck
–/–
Fyn
–/–
strains. C57BL/10 wild-type, Rag1
–/–
, Il7r
–/–
,
Il7
–/–
Rag1
–/–
and Il2rg
–/–
Rag2
–/–
mice were bred in a conventional colony free of
pathogens at National Institute for Medical Research (London, UK). All lines
used were of H-2
b
haplotype. Animal experiments were done according to
institutional guidelines and Home Office regulations.
Flow cytometry and cellularity determinations. Flow cytometry used 2 × 10
6
to 5 × 10
6
lymph node, spleen cells or peritoneal exudate cells. Lymph node
cell suspensions were prepared from superficial cervical, brachial, axillary,
inguinal and mesenteric lymph nodes of individual mice, whereas peritoneal
exudate cells were collected after injection of 5 ml media into the peritoneal
cavities of mice. Cell concentrations were determined using a Scharf
Instruments Casy Counter. Cells were incubated for 1 h at 4 °C with saturating
concentrations of antibodies in 100 µl PBS in BSA (0.1%) plus azide (1 mM),
followed by a three washes in the buffer of PBS, BSA and azide. Biotin-
conjugated antibody labeling was developed with streptavidin–peridinin
chlorophyll protein (PharMingen). Monoclonal antibodies used in this study
were: allophycocyanin- and phycoerythrin-conjugated CD4 (GK1.5;
PharMingen); fluorescein isothiocyanate–conjugated TCR (H57-597;
PharMingen); allophycocyanin-conjugated CD44 (Leinco Technologies);
phycoerythrin-conjugated CD44 (PharMingen); Mel14 (antibody to CD62L);
allophycocyanin-conjugated CD8 (PharMingen); and IL-7 receptor α-chain
(A7R34). Four-color cytometric staining was analyzed on a FACSCalibur
Instrument (Becton Dickinson) and data were analyzed with Cell Quest soft-
ware (Becton Dickinson). To determine cell proliferation in vivo by BrdU
incorporation, mice were first injected intraperitoneally with 1 mg BrdU, then
were fed BrdU in drinking water (0.8 mg/ml) protected from light. BrdU water
was renewed every 2 d. Intracellular staining for BrdU incorporation and
cytokines was done using Fastimmune Anti-BrdU with DNase (Becton
Dickinson) and antibody to interferon-g (PharMingen), respectively, accord-
ing to manufacturers’ instructions.
Purification, labeling and adoptive transfer of T cells. Lymphocytes were
‘teased’ from lymph nodes and spleens of donor mice, and single-cell suspen-
sions were prepared. For experiments requiring purified CD4
+
CD44
hi
T cell
preparations, lymphocytes were labeled for 30 min at 4 °C with a mixture of
antibodies to CD62L-biotin and DX5-biotin (PharMingen), CD8-biotin and
B220-biotin (PharMingen) and I-A
b
-biotin (PharMingen) at a concentration
of 1 µg/ml (100 µl per 1 × 10
7
cells). After being washed, cells were incubated
for 30 min with M280-streptavidin Dynal beads (Dynal) , after which cells were
separated by application of a magnet. Contaminating cells were consistently
<1%. Cells were labeled for 10 min at 37 °C with 2 µM CFSE (Molecular
Probes) in Dulbecco’s PBS (Life Technologies) and washed twice. Cells were
transferred into various recipient mice through tail vein injections. Treatment
of recipient mice with purified monoclonal antibody to IL-7R (A7R34; a gift
from S. Nishikawa, Kyoto University, Kyoto, Japan) in PBS was done on a 7-day
cycle of intraperitoneal injections of 500 µg/mouse of monoclonal antibody on
days 1, 3 and 5 for the duration of the experiment. Control mice received injec-
tions of PBS alone. Cellular expansion of CFSE-labeled cells was calculated by
determining the frequency (F) of donor cells that had undergone different
numbers of divisions (d). Adjusted frequencies (AdF) were calculated by divid-
ing F by 2
d
. The predicted expansion was determined by the calculation
ΣF/ΣAdF.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTS
We thank T. Norton, K. Williams and the Biological Services staff for assistance with
mouse breeding and typing. We also thank B. Stockinger and G. Kassiotis, as well as
other members of the Division of Molecular Immunology, for discussions. This
work was supported by the Medical Research Council and the Leukaemia Research
Fund, UK.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 13 March; accepted 28 May 2003
Published online 15 June 2003; doi:10.1038/ni946
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