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The Journal of Experimental Medicine
J. Exp. Med.
The Rockefeller University Press • 0022-1007/2003/12/1797/10 $8.00
Volume 198, Number 12, December 15, 2003 1797–1806
http://www.jem.org/cgi/doi/10.1084/jem.20030735
1797
Interleukin 7 Regulates the Survival and Generation of
Memory CD4 Cells
Robyn M. Kondrack,
1
Judith Harbertson,
1
Joyce T. Tan,
2
Meghan E. McBreen,
1
Charles D. Surh,
2
and Linda M. Bradley
1
1
Department of Immunology, The Sidney Kimmel Cancer Center, San Diego, CA 92121
2
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
Abstract
Cytokines, particularly those of the common
chain receptor family, provide extrinsic signals
that regulate naive CD4 cell survival. Whether these cytokines are required for the maintenance
of memory CD4 cells has not been rigorously assessed. In this paper, we examined the contri-
bution of interleukin (IL) 7, a constitutively produced common
chain receptor cytokine, to
the survival of resting T cell receptor transgenic memory CD4 cells that were generated in
vivo. IL-7 mediated the survival and up-regulation of Bcl-2 by resting memory CD4 cells in
vitro in the absence of proliferation. Memory CD4 cells persisted for extended periods upon
adoptive transfer into intact or lymphopenic recipients, but not in IL-7
mice or in recipients
that were rendered deficient in IL-7 by antibody blocking. Both central (CD62L
) and effector
(CD62L
) memory phenotype CD4 cells required IL-7 for survival and, in vivo, memory cells
were comparable to naive CD4 cells in this regard. Although the generation of primary effector
cells from naive CD4 cells and their dissemination to nonlymphoid tissues were not affected by
IL-7 deficiency, memory cells failed to subsequently develop in either the lymphoid or non-
lymphoid compartments. The results demonstrate that IL-7 can have previously unrecognized
roles in the maintenance of memory in the CD4 cell population and in the survival of CD4
cells with a capacity to become memory cells.
Key words: CD4 memory • cell subsets • T cell survival • homeostasis
Introduction
Prolonged protective immunity depends on the persistence
of memory T cells that arise during the primary immune
response. As with naive T cells, the survival and the overall
size of the memory T cell pool is tightly regulated (1),
even though naive and memory T cell populations are
thought to be regulated in an independent manner (2, 3).
For naive T cells, recent advances have shown that their
homeostasis is largely controlled by signals from contact
with self-MHC–peptide ligands and the cytokine IL-7 (4,
5). In contrast to naive T cells, homeostatic control of
memory CD4 and CD8 cell populations appears to occur
independently of TCR engagement (6, 7), although repeated
exposures to the priming or cross-reactive epitopes can
improve the longevity and functionality of memory T cells
(1, 8). In terms of cytokines, homeostasis of memory CD8
cells appears to be largely controlled by the common
chain (
c) receptor family cytokines, IL-15 and IL-7,
whereas these cytokines have appeared to be dispensable
for memory CD4 cells.
Clues to the cytokine requirements for homeostasis of
memory T cells were initially gained from the profiles of
receptor expression. The receptor for IL-15, as assessed by
expression of the
chain (CD122), is expressed at signifi-
cantly higher levels on memory phenotype CD8 cells than
on memory phenotype CD4 cells (9). This difference pre-
sumably explains why IL-15 has a profound role on the
homeostasis of memory CD8 cells, but not on memory
CD4 cells (9–13). In contrast to the IL-15 receptor, the
receptor for IL-7 is expressed at comparable high levels on
all populations of T cells, including both memory CD4 and
CD8 cells (14, 15). The role for IL-7 in memory CD8 cell
homeostasis has been recently realized by the finding that
IL-7 can synergize with, or act in place of IL-15 to support
survival and homeostatic division of memory CD8 cells
Address correspondence to Linda M. Bradley, Dept. of Immunology, The
Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121.
Phone: (858) 410-4213; Fax: (858) 450-3251; email: lbradley@skcc.org
Abbreviations used in this paper:
APC, allophycocyanin; BrdU, bromode-
oxyuridine;
c, common
chain; ICS, intracellular staining; mIgG,
mouse IgG; PCC, pigeon cytochrome
c
; rIgG, rat IgG.
Survival of Memory CD4 Cells In Vivo
1798
(11, 13, 16). In contrast to other subsets of T cells, the in-
volvement of IL-7 and IL-15 on homeostasis of memory
CD4 cells has yet to be fully explored, even though this is a
distinct possibility as human memory CD4 cells were
found to proliferate in vitro in response to IL-7 and IL-15
(17). In this regard, a recent analysis in the mouse showed
that CD4 cells deficient in the
c chain, a crucial compo-
nent of receptors for IL-7 and related cytokines, have a
normal lifespan once they are activated and become mem-
ory cells, even though they have a short lifespan as naive
cells (18). In addition, we have found previously that mu-
rine memory phenotype CD4 cells undergo efficient ho-
meostatic proliferation in IL-7–deficient mice (13). Al-
though these findings together suggest that agents other
than IL-7 and IL-15 regulate homeostasis of memory CD4
cells, it is nevertheless possible that these cytokines may
have roles, but that memory CD4 cells are more flexible in
their homeostatic regulation than memory CD8 cells.
Consistent with this hypothesis, a recent work suggests that
TCR signals may mask a contribution of IL-7 to the ho-
meostasis of memory CD4 cells (19). Thus, it is conceiv-
able that multiple factors may be involved in controlling
homeostasis of memory CD4 cells depending on the his-
tory of antigen exposure, stage of differentiation, or ana-
tomic location (20).
In light of the increasing appreciation of the role of IL-7
in homeostasis of resting T cells, we sought to carefully ad-
dress whether IL-7 can also contribute to the homeostasis of
memory CD4 cells. To this end, we have studied the sur-
vival of defined populations of antigen-specific memory
CD4 cells generated under in vivo conditions. This ap-
proach was taken to avoid potential problems associated
with using CD4 cells that have developed in the absence of
cytokine signaling (18, 21) and spontaneously generated
memory phenotype cells of unknown history (13). We
show that memory CD4 cells express high levels of IL-7R
that are comparable to naive T cells and respond to IL-7 by
prolonged in vitro survival. Strikingly, under in vivo condi-
tions, the absence of IL-7 results in the dramatic disappear-
ance of resting memory CD4 cells, and a failure to generate
memory cells in both lymphoid and nonlymphoid tissues.
We conclude that IL-7 regulates the survival of memory
CD4 cells, playing critical roles in the initial development as
well as long term maintenance of memory cells.
Materials and Methods
Mice.
C57BL/6, B10.BR, B6.PL Thy1
a
/Cy, C57BL/
6.SCID, and IL-7
mice were purchased from The Jackson Lab-
oratories. C57BL/6 Rag2
mice were obtained from Taconic.
B10.BR.SCID mice were provided by P. Linton (Sidney Kim-
mel Cancer Center, San Diego, CA) and bred in our facility to
C57BL/6.SCID mice. IL-7
mice (22) were bred at The Scripps
Research Institute. OT-II TCR (v
2 and v
5) CD4 transgenic
mice that respond to peptide 323-339 of OVA were from W.
Heath (Walter and Eliza Hall, Melbourne, Australia; reference
23). AND TCR (v
11 and v
3) CD4 transgenic B10.BR mice
that are specific for peptide 88-103 of pigeon cytochrome
c
(PCC; reference 24) were bred in our vivarium. The TCR trans-
genic mice were bred to B6.PL Thy1
a
/Cy mice to facilitate
tracking by Thy 1.1 in syngeneic Thy 1.2 recipients.
Antibodies for Flow Cytometry and In Vivo Blocking.
mAbs
specific for IL-7 (M25; reference 25) and IL-7R (A7R34 and
CD127; reference 14) were generated from the hybridoma lines
and purified by Protein G separation for blocking studies. Mouse
IgG (mIgG) and rat IgG (rIgG) obtained from Jackson Immu-
noResearch Laboratories were used as the respective controls.
TCR-specific reagents (v
2 [B20.1], v
11 [RR8.1], v
3 [KJ25],
and v
5 [MR9-4]), anti–IL-7R (SB/14/CD127), anti–L-selectin
(Mel-14/CD62L), anti-CD44 (IM7), anti-CD4 (GK1.5), Thy
1.1 (CD90/OX7), anti–IFN-
(R46A2 and XMG1.2), IL-4
(11B11 and BVD6–24G2), anti–Bcl-2 (3F11), and anti-BrdU
(B44) were obtained from BD Biosciences. Reagents for cell per-
meabilization and intracellular staining (ICS) with mAbs specific
for IFN-
, Bcl-2, and bromodeoxyuridine (BrdU) were obtained
from BD Biosciences, and staining was performed according to
the manufacturer’s instructions. Polyclonal anti–IL-13 (goat anti–
mouse) antibodies were obtained from R&D Systems. Isotype
control (rIgG
1
, rIgG2a, and rIgG2b) mAb and Thy 1.1 (CD90.1/
HIS51) were obtained from eBioscience.
Memory T Cell Priming.
Naive TCR transgenic CD4 cells
were primed in adoptive hosts. CD4 cells were isolated from the
spleens and pooled LNs (inguinal, axillary, brachial, cervical, and
mesenteric) of OT-II Thy 1.1 or AND Thy 1.1 mice by negative
selection using magnetic sorting with an enrichment cocktail ob-
tained from Stem Cell Technologies, Inc. according to the manu-
facturer’s instructions. v
2, v
5
OT-II cells, or v
11, v
3
AND cells were quantitated by flow cytometry. 5
10
6
trans-
genic OT-II cells were injected i.v. into C57BL/6 or C57BL/6
Rag 2
recipients. These mice were immunized by i.p. injection
of 100
g alum-precipitated OVA (Sigma-Aldrich) with 10
9
Bor-
detella pertussis
vaccine organisms (26). 5
10
6
AND (B10.BR
B6 PL Thy 1.1) F
1
cells were injected i.v. into (C57BL/6
B10.BR) F
1
SCID recipients. These mice were immunized by
s.c. injection of 50
g PCC peptide in CFA (BD Diagnostic Sys-
tems) distributed on the back and at the base of the tail. At 4–8
wk after priming, donor CD4 cells were enriched by magnetic
sorting using negative selection as described in this paragraph, and
when isolated from intact mice, anti-Thy 1.2 magnetic beads
(Miltenyi Biotec) were also used. In some experiments, resting
CD4 cells were isolated by Percoll density separation as described
previously (27). CD44
hi
CD4 cells were sorted by positive selec-
tion using a FACSVantage™ flow cytometer (Becton Dickinson).
In some experiments, CD62L
and CD62L
memory CD4 cells
were sorted magnetically after labeling with biotinylated anti-
CD62L and streptavidin beads (Imag; BD Biosciences). To evalu-
ate the contribution of IL-7 to the maintenance of memory,
0.5–5
10
6
in vivo–primed transgenic CD4 cells were injected
i.v. into a second set of recipients that included intact normal
mice, SCID mice, IL-7
mice, or IL-7R
mice.
Analysis of Memory CD4 Cell Cytokine Responses.
In vivo–
primed memory CD4 cells were tested for frequency of IFN-
producers by ICS 12 h after restimulation of 2
10
6
cells plated in
24-well plates with 2
10
6
mitomycin-treated splenic APCs and
8
g/ml OVA peptide in 2-ml cultures (28). For ELISPOT anal-
ysis, donor CD4 cells were isolated by positive selection with bio-
tinylated-anti-Thy 1.1 and streptavidin magnetic beads (Imag;
BD Biosciences), and 1.5
10
4
cultured together with 2
10
5
APCs and 8
g/ml OVA peptide for 24 h in Multiscreen-IP plates
(Millipore) that were coated with 2
g/ml anti–IL-4 or 4
g/ml
anti–IL-13 capture antibodies. ELISPOTs were developed by se-
Kondrack et al.
1799
quential incubation of plates with the appropriate detecting anti-
bodies (biotin-anti–IL-4 or -anti–IL-13, respectively), streptavi-
din-alkaline phosphatase, and nitroblue tetrazolium/5-bromo-
4-chloro-3-indolyl phosphate substrate (Kirkegaard & Perry
Laboratories) as described previously (29). ELISPOTs were counted
with an ImmunoSpot Analyzer (Cellular Technology Limited).
In Vitro Analysis of CD4 Cell Responses to IL-7.
Naive or
memory CD4 cells were cultured at 10
6
/ml in 2-ml volumes for
2 wk with or without 10 ng/ml murine rIL-7 (R&D Systems) in
RPMI 1640 media (Irvine Scientific) containing 7% FCS (Irvine
Scientific), 200
g/ml penicillin, 200 U/ml streptomycin, 4 mM
l
-glutamine, 10 mM Hepes, and 5
10
5
M 2-ME. Viable cell
recovery was determined by harvesting and counting cells by try-
pan blue exclusion at various times after incubation. Expression
of IL-7R, CD44, CD62L, and Bcl-2 was assessed by staining with
PE-conjugated mAb using a FACSCalibur™ flow cytometer with
CELLQuest™ software (Becton Dickinson). For blocking stud-
ies, mAbs to IL-7 and IL-7R were each added at 10
g/ml at the
initiation of culture.
In Vivo Analysis of CD4 Cell Responses to IL-7.
Donor naive
or memory CD4 cells were identified in the spleens, pooled pe-
ripheral LNs (cervical, inguinal, axillary, and brachial), mesenteric
LNs, livers, and lungs of recipient mice at various times after trans-
fer as indicated in the text for individual experiments by staining
with PerCP (peridiunin chlorophyll-a protein)-labeled anti-Thy
1.1, allophycocyanin-labeled CD4, and FITC and PE-labeled
anti-TCR
and
reagents specific for the OT-II (v
2 and v
5)
or AND (v
11 and v
3) transgenic receptors. Each experimental
design was repeated a minimum of twice using at least 2 recipients
per group. PBS containing 10 U heparin was used to perfuse the
lungs via the atrium of the heart, and to perfuse the liver through
the hepatic vein before excision of these tissues. After disruption
to obtain single cell suspensions, lymphocytes were isolated at the
interface of 40 and 80% layers of Percoll density gradients.
For antibody blocking studies, anti–IL-7 was administered i.p.
to adoptive recipients in 1-mg doses on the day of cell transfer,
and every other day thereafter for the duration of the experi-
ments, up to 2 wk. BrdU (Sigma-Aldrich) was administered by
i.p. injection of 800
g, followed by treatment in the drinking
water at 800
g/ml for 5–9 d before analysis by flow cytometry.
To evaluate memory generated in vivo, recipients of naive OT-II
cells were primed on the day of cell transfer by i.p. injection of
100
g alum-precipitated OVA with 10
9
B. pertussis
organisms
and boosted 3 wk later by i.p. administration of the same dose of
Ag and adjuvant.
Results
IL-7 Promotes Survival of Resting Memory Cells In Vitro.
Ag-specific memory CD4 cells were generated by transfer-
ring purified naive CD4 OT-II Thy 1.1 TCR transgenic
cells into Rag-2
recipients and immunizing the hosts with
alum-precipitated OVA protein plus
B. pertussis
vaccine or-
ganisms. Approximately 10% of the injected OT-II cells
Figure 1.
In vivo–primed TCR transgenic memory CD4 cells survive
in response to rIL-7 in vitro. Purified naive OT-II Thy 1.1 CD4 cells
were transferred into C57BL/6 Rag2
mice (5
10
6
cells/recipient) and
immunized with OVA protein and adjuvant as indicated in Materials and
Methods. 1 mo later, resting memory OT-II cells were isolated and com-
pared with freshly isolated naive OT-II cells. (A) Phenotype of memory
OT-II cells. Naive and memory OT-II cells were stained for expression
of CD62L, CD44, and IL-7R
and analyzed by flow cytometry (shaded
histograms; unshaded histograms denote background staining). (B) Fre-
quencies of effector cytokine producers among memory OT-II cells.
Memory OT-II Thy 1.1 cells were restimulated with OVA peptide in the
presence of splenic APC and tested for secretion of IFN-
at 12 h by ICS,
and after enrichment of Thy 1.1 cells, for production of IL-4 or IL-13 at
24 h by ELISPOT analysis. (C) IL-7 promotes survival of OT-II cells.
Naive and memory OT-II Thy 1.1 cells were cultured at 10
6
/ml for the
indicated number of days without or with rIL-7 at 10 ng/ml (left and
right, respectively). (D) Blocking IL-7 prevents up-regulation of Bcl-2.
Naive (top left) and memory (top right) OT-II Thy 1.1 CD4 cells were
stained for expression of Bcl-2 (light gray histograms). The cells were cultured
as in C for 6 d with 10 ng/ml rIL-7 and stained for BcL-2 (dark gray
histograms). Memory cell cultures were also treated with either 10
g/ml
each anti–IL-7 and anti–IL-7R mAb or with an equivalent amount of rat
and mouse IgG (bottom, rIgG and mIgG, respectively). The cells were
stained for Bcl-2 on day 6 after culture with blocking mAb (shaded histo-
gram) or with control mAb (unshaded histogram).
Survival of Memory CD4 Cells In Vivo
1800
were recovered from the hosts at day 1, before immuniza-
tion, and nearly all of these cells were found to have under-
gone vigorous proliferation when analyzed 5 d after immu-
nization by BrdU uptake or by dilution of carboxy
fluorescein diacetate succinimidyl ester (unpublished data).
Typically, OT-II cells underwent four- to fivefold expan-
sion during this time, and
50% of these cells survived as
memory cells 1 mo later (unpublished data). At this time,
OT-II cells in the spleen were resting and possessed an
effector memory phenotype (Fig. 1 A, CD44
hi
and
CD62L
lo
). The memory OT-II cells expressed similar lev-
els of IL-7R
as naive OT-II cells (Fig. 1 A), and 67% re-
sponded to OVA peptide–loaded APC by producing IFN-
,
as measured by ICS (Fig. 1 B). Although the frequency
of IL-4 producers was too low to be reliably assessed by
ICS, using ELISPOT analysis, we determined that 3% of
the cells secreted this cytokine and that a similar number
secreted the coordinately regulated cytokine, IL-13 (Fig. 1
B). Consistent with our previous studies, naive OT-II cells
secreted only IL-2 under the same conditions (28). As de-
scribed previously (4), spontaneous homeostatic prolifera-
tion of OT-II cells driven by lymphopenia was not a con-
founding problem as we found that both naive and
memory OT-II cells did not undergo homeostatic prolifer-
ation in Rag-2
hosts (unpublished data).
To study the in vitro effects of IL-7 on CD4 cells, naive
and resting memory OT-II cells were purified by magnetic
sorting and Percoll density separation and cultured with
murine rIL-7 (Fig. 1 C). Without rIL-7 (Fig. 1 C, left), na-
ive OT-II cells died precipitously within 24 h. A more
gradual decline was observed for memory OT-II cells, but
by day 6, few cells survived. Notably, adding a single dose
of rIL-7 (10 ng/ml) rescued survival of both naive and
memory OT-II cells for 2 wk (Fig. 1 C, right). In other ex-
periments, memory OT-II cells were kept alive as long as 4
wk without significant cell loss (unpublished data). These
results are in agreement with previous data showing that
resting murine T cells can use IL-7 for survival in vitro
(30). Consistent with papers suggesting that IL-7 does not
induce T cell division in vitro in the absence of TCR en-
gagement or costimulation (31, 32), both naive and mem-
ory OT-II cells survived with no surface phenotypic
change in a resting state with low forward scatter and with-
out cycling. Comparable results were obtained using naive
and memory CD4 AND TCR transgenic cells specific for
PCC, with memory cells generated in vivo from naive cells
in an analogous fashion by priming in SCID recipients with
PCC peptide and CFA.
As expected, the efficacy of exogenous rIL-7 on memory
OT-II cell survival was abolished in the presence of block-
Figure 2. Survival of memory CD4 cells in IL-7–
deficient recipients. (A) Comparison with intact recipi-
ents. Resting OT-II Thy 1.1 memory CD4 cells were
generated and isolated as for Fig. 1 and sorted by flow
cytometry to obtain CD44
hi
cells. These cells were
transferred into normal C57BL/6 or IL-7
mice (2
10
6
cells/recipient). 1 wk later, donor cell recoveries
were quantitated in the host spleens and LNs from the
total nucleated cell counts and fractions of transgenic
donor (v2, v5, and Thy 1.1
) CD4 cells. (B) Com-
parison with IL-7R
recipients. OT-II Thy 1.1 memory
CD4 cells were isolated and transferred as in A into
IL-7R
and IL-7
mice, and analyzed for transgenic
donor cell recovery after 1 wk. (C) Susceptibility of
CD62L
memory cells to IL-7 deprivation. OT-II
Thy 1.1 memory cells were isolated from the LNs of
OVA-primed Rag2
that were primed as for Fig. 1.
The cells were stained for CD62L before (top) and after
magnetic selection for positively expressing cells (bottom,
overlay of sorted CD62L
and CD62L
subsets).
CD62L
cells were transferred into either IL-7R or
IL-7 hosts (2 10
6
cells/recipient), and the recovery
of donor memory CD4 cells was assessed after 10 d.
(D) Naive OT-II Thy 1.1 CD4 cells were primed by
transfer into normal C57BL/6 mice (5 10
6
each) and
immunization of the recipients with OVA and adjuvant.
4 wk later, donor memory CD4 cells isolated from the
spleens were transferred into IL-7R
and IL-7
mice
(0.5 10
6
cells/recipient). These mice were evaluated
1 wk after cell transfer for the presence of transgenic
donor cells in the LNs and spleens.
Kondrack et al.
1801
ing antibodies to IL-7 and IL-7R (unpublished data). Be-
cause induction of the antiapoptotic protein, Bcl-2, is a
hallmark of responses to IL-7 (32), we determined if IL-7
also has such an effect on memory CD4 cells. Naive and
memory OT-II cells expressed similar basal levels of Bcl-2
before rIL-7 addition and up-regulated Bcl-2 to compara-
ble levels in response to rIL-7 during the 6-d culture period
(Fig. 1 D, top). Blocking IL-7 and its binding to the IL-7R
inhibited Bcl-2 induction in the residual surviving CD4
memory cells (Fig. 1 D, bottom). These finding indicate
that IL-7 can serve as a survival factor for memory CD4
cells under in vitro conditions.
IL-7 Promotes Survival of Resting Memory Cells In Vivo.
To assess the contribution of IL-7 to the survival of mem-
ory CD4 cells under in vivo conditions, resting memory
OT-II Thy-1.1 cells were purified from OVA-primed
Rag-2
recipients by sorting CD44
hi
, v2
CD4 cells and
transferred into intact C57BL/6 or IL-7
recipients (both
Thy-1.2
). Analysis of the recipients 1 wk later revealed
that the recoveries of Thy-1.1
CD4 donor OT-II cells
were markedly lower in IL-7
mice compared with con-
trol C57BL/6 hosts (Fig. 2 A). One reason that memory
OT-II cells failed to survive efficiently in IL-7
mice may
be that these knockouts possess atrophied lymphoid tissues
due to defective T and B cell lymphopoiesis. To rule out
this possibility, two approaches were taken. First, survival
of purified OT-II memory cells in IL-7
mice was com-
pared with that in IL-7R
mice, which also possess small
lymphoid tissues, but produce IL-7 (33). As shown in Fig.
2 B, although the recoveries of memory OT-II cells at 1
wk after transfer were somewhat lower in IL-7R
mice
than in normal C57BL/6 hosts, a profound loss of OT-II
memory cells was observed only in the IL-7
mice.
Second, dependence on IL-7 for survival of memory OT-
II cells was measured under normal T-sufficient conditions
by depleting circulating IL-7 using mAb specific for IL-7
(25). Thus, purified OT-II Thy 1.1 memory cells generated
in Rag-2
recipients were transferred into normal C57BL/6
Figure 3. Effects blocking IL-7 on transgenic and
polyclonal memory CD4 cells. (A) Blocking of trans-
genic memory cell survival in normal recipients. OT-II
Thy 1.1 memory cells were primed in vivo and isolated
as for Fig. 1. The cells were transferred into normal C57
BL/6 mice (2 10
6
cells/recipient). On the day of cell
transfer, separate groups of recipients were injected with
1 mg of either anti–IL-7 or mIgG. Additional doses
were given every other day through day 12. On days 1,
7, and 14, animals from each group were evaluated for
transgenic donor cell recovery from the spleens and
LNs. (B) Lack of division of donor transgenic memory
cells but expansion of host polyclonal memory cells in
anti–IL-7–treated recipients. Recipients were adminis-
tered BrdU as described in Materials and Methods to
assess proliferation of OT-II Thy 1.1 memory cells and
in a separate experiment, division of host memory phe-
notype (CD44
hi
) CD4 cells. BrdU uptake was assessed
by ICS of splenic lymphocytes. Histograms gated on
donor Thy 1.1, v2, and v5
cells (top) and on host
CD44
hi
and CD4 cells (bottom) are shown. (C) Dimin-
ished recovery of host naive and memory CD4 cells.
The effects of anti–IL-7 treatment on the recovery of
naive phenotype (CD44
lo
) and memory phenotype
(CD44
hi
) CD4 cells were assessed by quantitating Thy
1.2
host CD4 cells from the spleens and LNs of the
recipients from A on day 14. Comparable results were
obtained using high versus low expression of 4 integrin
to distinguish memory versus naive phenotype host
CD4 cells (not depicted). (D) Recovery of naive and
resting memory CD4 cells after IL-7 treatment of immu-
nodeficient mice. Naive CD4 cells were isolated from
(AND B10.BR B6PL.Thy)F
1
mice and were trans-
ferred into (B10.BR C57BL/6) F
1
SCID mice (5
10
6
cells/recipient). One set of recipients was treated
with anti–IL-7 or mIgG as for A without immunization.
A second set of recipients was primed with PCC peptide
as described in Materials and Methods. 1 mo later,
when AND CD4 cells from the spleen were uniformly
CD62L
and CD44
hi
, recipients were treated either
with anti–IL-7 or with mIgG as for A. Recovery of
transgenic donor (v11, v3, and Thy 1.1
) from the
spleens and LNs was determined on day 14 after treat-
ment for naive and primed recipients (left and right,
respectively).
Survival of Memory CD4 Cells In Vivo
1802
mice, and the hosts were injected with 1 mg anti–IL-7 or
mIgG on an every other day basis. Analysis of spleen and of
hosts 1, 7, and 14 d later revealed that a progressive loss of
memory OT-II cells occurred in recipients treated with
anti–IL-7 mAb, whereas the numbers of these cells stabilized
in hosts injected with control Ab (Fig. 3 A). The difference
in the recoveries was not due to variability in cell division as
OT-II memory cells failed to undergo proliferation in both
types of hosts (Fig. 3 B, top). In addition to reducing the
lifespan of donor memory OT-II cells, anti–IL-7 treatment
also decreased survivability of host polyclonal memory CD4
cells without altering their homeostatic turnover (Fig. 3 B,
bottom). Thus, the recovery of host memory phenotype
(CD44
hi
) CD4 cells at the day 14 time point was signifi-
cantly lower in mice injected with anti–IL-7 mAb compared
with control mice (Fig. 3 C). These data suggest that depen-
dence on IL-7 for survival may be a general property of
memory CD4 cells. However, memory T cells are hetero-
geneous and central memory cells have been distinguished
on the basis of their expression of CD62L (L-selectin) that,
together with CCR7, regulates the capacity to localize in
LNs (34, 35). Therefore, it was of interest to determine if
the survival of transgenic memory CD4 cells that reside in
LNs was affected by withdrawal of IL-7.
Although memory OT-II cells induced by immunization
of Rag-2
recipients were predominantly in the spleen
(90%) and were CD62L
(Fig. 1), transgenic memory
cells were detected in LNs, where donor cells were exclu-
sively CD44
hi
and comprised of 28% CD62L
cells (Fig. 2
C, top left). To evaluate the IL-7 dependence of this popu-
lation, CD62L
cells were purified to 90% (Fig. 2 C, bot-
tom left) from enriched CD4 cells using positive selection
and transferred into either IL-7R or IL-7–deficient recipi-
ents. In the absence of IL-7, OT-II cells with a central
memory phenotype survived poorly in either the spleen or
LNs of recipients (Fig. 2 C, middle and right, respectively).
In light of our finding that CD4 cells with both central
and effector memory phenotypes require IL-7 for persis-
tence, it was of interest to compare the relative dependency
of memory CD4 cells on IL-7 for survival with that of na-
ive CD4 cells. A potentially similar requirement was sug-
gested by our observation that anti–IL-7 treatment reduced
the recovery of polyclonal naive phenotype (CD44
lo
) as
well as memory phenotype (CD44
hi
) CD4 cells from oth-
erwise intact recipients (Fig. 3 C). Thus, we measured the
decay of naive and memory AND TCR transgenic cells in
SCID hosts where CD4 cell survival could be assessed
independently of the presence of peripheral T cells. As
shown in Fig. 3 D, naive or memory AND cells residing
in SCID hosts treated for 14 d with anti–IL-7 or control
Figure 4. Depletion of primed CD4 cells in the absence of IL-7. (A) Cell recovery from lymphoid and nonlymphoid tissues after primary immunization.
Naive OT-II Thy CD4 cells were transferred into IL-7
or IL-7R
mice (5 10
6
cells/recipient). The mice were immunized with OVA as described
in Materials and Methods. On day 5, donor cell recovery was assessed in the spleen, LNs, lung, and liver as depicted in Fig. 2. (B) Recovery of primed
CD4 cells after rest and challenge of IL-7
and IL-7R
recipients. Mice from the same experiment shown in A were evaluated for the presence of donor
CD4 cells at 3 wk after OT-II Thy 1.1 CD4 cell transfer and immunization (left), and 5 d later after a secondary response was induced by challenge with
OVA (right). Thy 1.1 and v5 staining of the total CD4 cells recovered from each tissue is shown. (C) Summary. Total donor OT-II cell recovery from
each site before and after boosting with OVA, as determined from the cell counts as for Fig. 2.
Kondrack et al.
1803
IgG decayed to similar extents when IL-7 was blocked. To
measure survival of naive cells, purified AND cells from
(B10.BR B6 PL Thy 1.1) F
1
mice were injected into
(B10.BR C57BL/6) F
1
SCID mice and the recipients
were treated immediately with anti–IL-7 or control IgG
Abs (Fig. 3 D, left). For memory cell analysis, SCID mice
were injected with naive AND cells and immunized with
PCC peptide with CFA; these mice were rested for 1 mo
and treated with anti–IL-7 or control antibody for 14 d
(Fig. 3 D, right). Importantly, we found that naive AND
cells failed to undergo homeostatic proliferation in the
SCID hosts (unpublished data).
Despite the clarity of the data in Figs. 2 (A–C) and 3, a
potential caveat is the fact that OT-II memory cells were
generated in the presence of elevated basal levels of IL-7 in
lymphopenic Rag-2
hosts. Therefore, it is possible that
such conditions favored production of memory cells de-
pendent on IL-7. To address this issue, OT-II memory
cells were generated under conditions of normal IL-7 levels
by transferring naive OT-II cells into intact T-sufficient
C57BL/6 hosts and priming the mice with OVA protein
and adjuvant. Although many fewer OT-II memory cells
were recovered from normal mice than from Rag-2
mice
1 mo later, both types of memory OT-II cells displayed
similar dependence for IL-7. Thus, memory OT-II cells
from C57BL/6 hosts survived with significantly reduced
efficiency in IL-7
hosts as compared with IL-7R
hosts
at 1 wk (Fig. 2 D).
IL-7 Regulates the Generation of Memory CD4 Cells in Both
Lymphoid and Nonlymphoid Tissues. The aforementioned
results support the conclusion that IL-7 can promote the
survival of memory CD4 cells that reside in the lymphoid
compartment. Because memory CD4 cells are thought to
be generated in situ in multiple tissues throughout the body
from the widely disseminated effector cells (36), next, we
sought to determine whether IL-7 also affects the develop-
ment of memory CD4 cells. To this end, purified naive
OT-II Thy 1.1 cells were transferred into groups of IL-7
and IL-7R
mice immunized with OVA protein and ad-
juvant, and the fate of OT-II cells followed in both lym-
phoid and peripheral tissues. Examination of one group of
recipients on day 5 revealed that similar numbers of acti-
vated OT-II cells were recovered in the spleens, LNs,
lungs, and livers from both types of mice (Fig. 4 A). As
seen with Rag-2
hosts, prominent proliferation of OT-II
cells was observed in the draining mesenteric LNs at this
time by BrdU incorporation (unpublished data). Strikingly,
analysis of the recipients on day 21 showed that OT-II cells
were no longer detectable in the lymphoid or nonlym-
phoid tissues of IL-7
recipients, whereas these cells were
clearly present in all the tissues examined in IL-7R
hosts
(Fig. 4 B).
To better ascertain whether memory OT-II cells gener-
ated in IL-7
hosts disappeared completely from various
tissues, IL-7
and IL-7R
hosts from the aforementioned
experiment that were rested for 3 wk after priming were
challenged with OVA protein and adjuvant. As depicted in
Fig. 4 (B and C), at 5 d after boosting, OT-II cells were
very sparse in all the tissues we examined from IL-7
hosts,
whereas control IL-7R
mice showed significant expan-
sion of OT-II cells in both lymphoid and nonlymphoid tis-
sues. We conclude that IL-7 deprivation can lead to im-
paired development of memory CD4 cells in both the
lymphoid and nonlymphoid compartments.
Discussion
In this paper, we investigated the potential of IL-7 to
function in the homeostasis of memory CD4 cells in vivo.
Our results suggest that IL-7 is a key regulator of memory
CD4 cell survival not only long term, but also during ear-
lier stages when resting memory cells develop from primary
cells that have undergone response to antigen. The notion
that IL-7 might be a cytokine candidate for control of
memory CD4 cell homeostasis was initially revealed by our
in vitro assessment of the effects of rIL-7 on the survival of
resting memory CD4 cells compared with naive CD4 cells
(Fig. 1). By using highly purified resting TCR transgenic
CD4 cells, we ensured greater uniformity of memory pop-
ulations than in previous analyses of memory phenotype
cells from normal animals. Importantly, we observed naive
and memory CD4 cells express equivalent amounts of IL-
7R and Bcl-2 and up-regulate Bcl-2 to comparable levels
after exposure to IL-7, supporting the possibility that both
populations have the potential to be similarly regulated by
this cytokine. IL-7–mediated survival by memory CD4
cells was not accompanied by division, as shown previously
for naive CD4 cells (32). The data suggest that survival and
homeostatic division can be separately regulated in both
naive and memory CD4 cells.
In vivo, IL-7 deprivation resulted in the disappearance of
transferred memory CD4 cells in both intact and immuno-
deficient recipients (Fig. 2). Although deficiency of IL-7
also disrupts both T and B cell lymphopoeisis (37), it is
striking that IL-7– and IL-7R–deficient animals, which dis-
play a similar phenotype with regard to a lack of mature
lymphocytes, show a profound difference in their ability to
maintain resting memory CD4 cells. Few memory CD4
cells were recoverable from either the lymphoid or non-
lymphoid compartments in the absence of IL-7. The po-
tential for IL-7 to regulate memory CD4 cell survival in
vivo is further supported by the results of IL-7 blocking
studies in normal recipients, where both donor and host
memory CD4 cells decayed in recipients rendered deficient
in IL-7 (Fig. 3). These data provide strong support for a
conclusion that IL-7 is at least one factor that contributes to
maintaining memory CD4 cells in vivo.
Although several previous studies have not detected a re-
quirement for IL-7 in regulating the homeostasis of mem-
ory CD4 cells, it is important to bear in mind that a pri-
mary focus has been proliferative capacity in lymphopenic
recipients (13). Our data suggest that IL-7 may not affect
memory CD4 cell expansion. In addition, we find that un-
der lymphopenic conditions where heterogeneous popula-
tions of normal memory CD4 cells undergo homeostatic
division, reduced recovery is observed in the absence of IL-7
Survival of Memory CD4 Cells In Vivo
1804
(unpublished data), in line with our current results from
IL-7 blocking studies in normal recipients. The use of irra-
diated hosts for analysis of cytokine dependence in some
studies may further complicate analysis of requirements for
individual cytokines due to the rapid induction of multiple
cytokines, including TGF- (38) and IL-6 (39), that have
the potential to mediate T cell survival.
Analyses of c cytokine receptor–deficient mice indicate
that some T cells can be generated in the absence of a ca-
pacity to respond to this cytokine family (21). In addition,
when TCR transgenic mice are crossed to c receptor
knockout mice, CD4 cells with an activated phenotype
that are highly susceptible to apoptosis arise. These findings
suggest that, whereas other mediators can support expan-
sion of the few naive CD4 cells that are generated in the
absence of a c cytokine response, most of c-deficient na-
ive CD4 cells fail to survive. However, TCR transgenic
CD4 cells from c receptor–deficient mice can survive
upon activation and differentiation into memory CD4 cells
in c receptor–deficient recipients (18). Although this find-
ing was interpreted to indicate that CD4 memory cell ho-
meostasis is regulated independently of c family cytokines,
it is also possible that the finding either applies to only a
minor subset of memory CD4 cells or is a reflection of a
capacity to engage aberrant compensatory survival mecha-
nisms that arise when responses to c cytokines are geneti-
cally impaired (18). In either case, c-deficient T cells
might utilize other cytokines that become available, and
these cytokines may promote homeostatic division under
conditions of lymphopenia to sustain long-term memory.
Consistent with previous conclusions that CD4 cells do
not depend solely on c family cytokines for proliferation
during an Ag-induced response (18), we found that naive
TCR transgenic CD4 cells could be comparably expanded
by cognate Ag and that the resulting effector cells distrib-
uted normally in both IL-7–sufficient and IL-7–deficient
recipients. Our results suggest that IL-7 is not critical for
survival of CD4 cells during Ag-induced activation or divi-
sion, or for their survival during dissemination to nonlym-
phoid sites. However, the primed population disappeared
from both lymphoid and nonlymphoid tissues only under
conditions of IL-7 deprivation. Thus, we envision that IL-7
is necessary for the survival of developing memory cells.
Because our previous analyses demonstrate that cytokines
are not required for activated CD4 cells to return to rest
and acquire properties of memory cells (28), it is less likely
that IL-7 also regulates the differentiation of memory cells.
IL-7 is not only produced by stromal cells in lymphoid tis-
sues but also by epithelium, liver, and skin (14, 37, 40).
Thus, it is possible that local availability of IL-7 can support
the survival of memory cells in many different tissues, and
that the decay we observed in nonlymphoid sites is not due
to T cell migration/recirculation that results in IL-7 with-
drawal only in the lymphoid compartment.
Although it could be argued that memory CD4 cells
primed in lymphopenic animals might be atypical because
of their induction and maintenance in an environment
where survival factors may be elevated due to the lack of
normal consumption, our finding that transgenic CD4 cells
induced in normal recipients showed a similar dependence
on IL-7 for survival suggests that this is an unfounded con-
cern. In addition, when polarized Th1 and Th2 cells were
induced under conditions where fully differentiated effec-
tors are generated, and rested to induce a memory-like
population (28), we observed similar susceptibility to IL-7
withdrawal in vivo (unpublished data). Thus, recently
primed CD4 cells also appear to be highly susceptible to
sudden withdrawal of IL-7. Consistent with the antiapop-
totic effects of IL-7, our data suggest that IL-7 may play a
key role in preventing the demise of effectors by activated
T cell autonomous death (41), thereby promoting CD4 cell
survival during the effector to memory transition. Thus,
IL-7 may be an important survival factor for early memory
CD4 cells as well as for maintaining long term memory
CD4 cells.
Because we studied TCR transgenic memory CD4 cells
generated by specific immunization protocols, it is possible
that the findings may not be generally typical for memory
CD4 cells. However, our observations that transgenic
memory CD4 cells with either central or effector memory
phenotypes can utilize IL-7 for persistence and that trans-
genic and polyclonal naive and memory CD4 cells have
comparable IL-7 dependence are striking (Figs. 2 and 3).
Together with previous papers on naive and memory CD8
cells (11, 13, 15, 16, 30, 42–44), our results support the hy-
pothesis IL-7 is a key cytokine for the physiologic survival
of resting peripheral T cells and in this capacity contributes
to regulation of the pool sizes of naive and memory cells
for both the CD4 and CD8 subsets. Nevertheless, alter-
ations in cytokine receptor expression during an immune
response and the relative abundance of various cytokines in
the local milieu may determine changes in the preferential
usage of growth and/or survival factors.
It remains unclear why IL-7 appears to function to medi-
ate survival but not homeostatic proliferation of memory
CD4 cells. However, this finding is reminiscent of the fact
that IL-2 and IL-4, in addition to IL-7, transduce signals
that promote survival of resting T cells without inducing di-
vision (45). Furthermore, a recent analysis of polyclonal
memory phenotype CD4 cells suggests that TCR-mediated
signals can support homeostatic cycling of memory CD4
cells in the absence of IL-7 but that IL-7 is required in addi-
tion for optimal survival (19). Our results also suggest that
survival and homeostatic turnover of T cells need not be
linked in vivo. Mediators that might exclusively promote
turnover of memory CD4 cells have yet to be identified,
and it is possible that multiple cytokines could exhibit re-
dundancy in this regard. Importantly, our data suggest that
an essential step in the maintenance of CD4 cell memory is
the provision of survival signals from cytokines, and that IL-7
may play a prominent role in this process.
This work was supported by National Institutes of Health grants
AI32978, AI46530, and DK59438 to L.M. Bradley and AI41079,
AI45809, and AG20186 to C.D. Surh. C.D. Surh is a Scholar of
the Leukemia and Lymphoma Society. J.T. Tan is supported by
Kondrack et al.
1805
postdoctoral fellowship grant PF-02-121-01LIB from the American
Cancer Society.
Submitted: 5 May 2003
Accepted: 15 October 2003
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