Content uploaded by Rhodri Ceredig
Author content
All content in this area was uploaded by Rhodri Ceredig on Dec 17, 2013
Content may be subject to copyright.
Effects of Increasing IL-7 Availability on Lymphocytes during
and after Lymphopenia-Induced Proliferation
1
Nabil Bosco,* Fabien Agene`s,* and Rhodri Ceredig
2†
IL-7 is critically involved in regulating peripheral T cell homeostasis. To investigate the role of IL-7 on lymphopenia-induced
proliferation of polyclonal lymphocytes, we have transferred CFSE-labeled cells into a novel T-lymphopenic, IL-7-transgenic
mouse line. Results obtained indicate that T and B cells do not respond in the same way to IL-7-homeostatic signals. Overex-
pression of IL-7 enhances proliferation of both CD4
ⴙ
and CD8
ⴙ
T cells but with distinctly temporal effects. Expansion of naturally
arising CD4
ⴙ
-regulatory T cells was like that of conventional CD4
ⴙ
T cells. IL-7 had no effect on B cell proliferation. By
immunohistology, transferred T cells homed to T cell areas of spleen lymphoid follicles. Increasing IL-7 availability enhanced T
cell recovery by promoting cell proliferation and reducing apoptosis during early stages of lymphopenia-induced proliferation.
Taken together, these results provide new insights into the pleiotropic effects of IL-7 on lymphopenia-induced T cell
proliferation. The Journal of Immunology, 2005, 175: 162–170.
Despite declining thymic output with age, the peripheral T
cell pool of an adult animal remains remarkably stable
(1, 2). How the T cell pool is maintained remains a cen-
tral question in immunology. Compelling data have been provided
indicating that long term survival and homeostatic proliferation of
T lymphocytes is dependent on a combination of low level TCR
and cytokine stimulation. After transfer into a lymphopenic envi-
ronment, T cells sense the absence of T cells and proliferate
slowly, a process that has been termed lymphopenia-induced pro-
liferation (LIP)
3
(3). Cytokines, such as IL-7 and IL-15, have been
shown to play a major role in both LIP and T cell survival in mice
(1, 4). IL-7 is also a crucial cytokine for lymphocyte development
providing survival-promoting signals for immature and mature T
as well as for immature B cells (5).
Different experimental procedures have been designed to study
the role of IL-7 in LIP. IL-7 enhances survival of mature T cells in
vitro (6, 7), and exogenous IL-7 administration increases both the
pool size of peripheral T cells (8) and the rate of hemopoietic
reconstitution after bone marrow (BM) transplantation in vivo (9).
Nevertheless, when IL-7 is injected into normal mice, it has been
difficult to distinguish between its effects on thymic output and that
on peripheral T cells. Syngenic adoptive transfer experiments into
either IL-7-deficient or wild-type mice treated with anti-IL-7 or
anti-IL-7R
␣
mAb show that perturbation of IL-7 signals prevents
the expansion of transferred T cells (1, 10). Although several
groups have shown that IL-7 is crucial for T cell survival and LIP,
the mechanisms by which IL-7 levels regulate the size and diver-
sity of the peripheral T cell pool are still not well understood.
Differences in experimental systems used to investigate the role
of IL-7 in LIP may account for some apparently conflicting data.
These differences concern either the nature of the recipient mouse
or that of the transferred cells. Preconditioning the recipient mouse
by irradiation or other lymphocyte-depleting regimens probably
alters lymphoid organ architecture, Ag-presenting cell function,
and/or cytokine milieu (11, 12). For instance, after irradiation, IL-7
and TGF

levels may change, thereby affecting LIP (12, 13). Fur-
thermore, in sublethally irradiated recipients, residual bystander T
cells persist and compete with transferred T cells for cytokine and
or self peptide-MHC complexes, thereby influencing reconstitu-
tion of the T cell compartment (12). Transferred cells are fre-
quently derived from TCR-transgenic, recombinase-activating
gene-deficient (RAG
⫺/⫺
) mice. Such monoclonal T cell popula-
tions express a TCR of predefined specificity and affinity, two
parameters that correlate with CD5 expression and that may dictate
whether a T cell undergoes LIP (14 –16). Second, such monoclonal
T cell populations lack naturally arising CD4
⫹
CD25
⫹
-regulatory
T cells (17, 18) known to alter the behavior of cells during LIP (19,
20). In such experiments, long term survival is rarely monitored
because for various reasons, including the absence of
CD4
⫹
CD25
⫹
-regulatory T cells, recipients frequently develop au-
toimmune diseases (21, 22).
Under normal T cell-replete, nonlymphopenic conditions, IL-7
probably acts as a survival factor for T cells (4). In normal mice,
the net level of IL-7 availability is low, resulting both from limited
production by stromal cells and simultaneous consumption by T
cells (23). In T cell-lymphopenic conditions, the net IL-7 level
increases by a poorly defined mechanism, thereby allowing resid-
ual T cells to sense lymphopenia, augment TCR signaling, and
consequently triggering LIP. This notion is consistent with avail-
able data and particularly with the previously reported phenotype
of IL-7-transgenic (IL-7Tg) mice (24) that contain a stable ex-
panded (⬃20-fold) T cell pool (25, 26). It could be postulated that
net IL-7 availability provides the main clue whereby T cells sense
*Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 548, De´partement de
Re´ponse et Dynamique Cellulaire, Commissariat a` l’Energie Atomique-G, Grenoble,
France; and
†
Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 645,
Institut Federatif de Recherche 133, Etablissement Franc¸ais du Sang, Besanc¸on,
France
Received for publication January 4, 2005. Accepted for publication April 21, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by institutional grants from Institut National de la Sante´
et de la Recherche Me´dicale and the Commissariat a` l’Energie Atomique, a specific
grant “The´matiques Prioritaires de la Re´gion Rhoˆne-Alpes.” M.N.B. has a PhD schol-
arship from the Commissariat a` l’Energie Atomique.
2
Address correspondence and reprint requests to Dr. Rod Ceredig, Institut National
de la Sante´ et de la Recherche Me´dicale Unite´ 645, Institut Federatif de Recherche
133, Etablissement Franc¸ais du Sang, 1 Boulevard Alexander Fleming, 25020 Be-
sanc¸on, France. E-mail address: Rod.Ceredig@efs.sante.fr
3
Abbreviations used in this paper: LIP, lymphopenia-induced proliferation; LN,
lymph nodes; PALS, periarteriolar lymphocyte sheaths; RAG-2, recombinase-acti-
vating gene-2; Tg, transgenic; SP, CD4
⫹
or CD8
⫹
single-positive; BM, bone marrow;
CEA, Commissariat a` l’Energie Atomique; mEF1
␣
, mouse elongation factor 1
␣
.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
lymphopenia. Thus, in IL-7Tg mice, T cells increase in number
until capable of absorbing the increased available IL-7. Recently,
Li et al. (27) predicted that in lymphopenic recipients, where IL-7
availability was increased, T cell LIP would be triggered. How-
ever, to date, this hypothesis has not been directly demonstrated,
and no study has systematically examined the behavior of poly-
clonal T and B cells transferred together into mice differing in IL-7
availability.
For the studies reported herein, we have developed a novel T
cell lymphopenic mouse strain that overexpresses IL-7. The IL-7
transgene was introduced into C57BL/6.CD3
⑀
gene-deficient
(CD3
⑀
⫺/⫺
) mice (28). IL-7Tg.CD3
⑀
⫺/⫺
and nontransgenic
CD3
⑀
⫺/⫺
littermate controls provide a pair of T-lymphopenic, B
lymphocyte-containing mice differing only in net IL-7 availability
and not requiring further conditioning before use as recipients. To
investigate how IL-7 availability influences the behavior of cells
undergoing LIP in T-lymphopenic animals, we transferred CFSE-
labeled polyclonal T and B cells into recipient mice. Parameters
investigated included the kinetics of both CD4
⫹
and CD8
⫹
T cell
as well as B cell growth and division, anatomical localization, the
kinetics of Bcl-2 expression and cell loss by apoptosis, as well as
long term cell recovery and turnover. Taken together, our results
provide new insights into the dynamics of IL-7-dependent LIP and
the pleiotropic effects of IL-7 on T and B cell homeostasis.
Materials and Methods
Mice
IL-7-transgenic mice on a C57BL/6 (B6) genetic background (IL-7Tg.B6)
have been described previously (24); they were maintained in a heterozy-
gous state by breeding transgenic males with B6 females (Iffa-Credo). IL-
7Tg.CD3
⑀
⫺/⫺
(28) and IL-7Tg.RAG-2
⫺/⫺
mice (29) were generated in the
animal facility of the De´partement de Re´ponse et Dynamique Cellulaires,
Commissariat a` l’Energie Atomique (CEA)-Grenoble by intercrossing IL-
7Tg.B6 males with the respective knockout females, obtained from CDTA
France. Mice were screened for the IL-7 transgene by PCR on tail DNA
and FACS of PBL. Once obtained, IL-7Tg.CD3
⑀
⫺/⫺
or IL-7Tg.RAG-2
⫺/⫺
mice were maintained by intercrossing the respective IL-7Tg males with
nontransgenic knockout females. P14.RAG-2
⫺/⫺
mice (30) were a gift of
Dr. Jo¨rg Kirberg (Max Planck Institute for Immunobiology, Freiburg, Ger-
many). In most experiments, 6- to 8-wk-old mice were used and were
heterozygous for the IL-7 transgene. Animal care and experimental pro-
cedures conformed to those of the CEA-Grenoble animal care and users
committee.
Flow cytometry
The following mAbs, obtained from BD Pharmingen or e-Bioscience, were
used for staining: anti-CD4 (GK1.5); anti-CD8 (53-6.7); anti-CD19 (1D3);
anti-CD21 (7G6); anti-CD23 (B3B4); anti-CD45R (RA3-6B2); anti-CD25
(7D4); anti-CD44 (IM7); anti-CD62L (MEL-14); anti-CD69 (H1-2F3); anti-
CD117 or c-Kit (2B8); anti-CD122 (TM-

1); anti-CD127 (A7R34); anti-
CD132 (4G3); anti-NK1.1 (PK136); and anti-IgM (R6-60.2). Cells were
three- or four-color stained with appropriate combinations of FITC-, PE-,
Cy-, and biotin-labeled Abs, followed by streptavidin-APC (BD Pharmin-
gen). Dead cells were excluded from analysis by light scatter and when
possible propidium iodide staining. All analyses were performed using a
FACSCalibur flow cytometer and data analyzed using CellQuest (BD Bio-
sciences) or WinMDI (Joseph Trotter) software.
Adoptive transfer experiments
Single-cell suspensions of lymph nodes (LN) and spleen lymphocytes were
prepared and purified over a Ficoll gradient before counting viable cells by
trypan blue exclusion and labeling with 5
M CFSE (Molecular Probes) as
described previously (31). Labeled lymphocytes (10
7
) were administered
i.v. to unirradiated lymphopenic mice. Mice were sacrificed at different
indicated times after adoptive transfer. Then, spleen, LN, and thymus were
collected, and single-cell suspensions were prepared and counted before
being surface stained as described above. CFSE and surface staining were
then analyzed by FACS. The total number of each T cell subpopulation was
calculated as described above from the frequency determined by FACS,
and the total number of viable cells recovered in each organ determined by
trypan blue exclusion. For naive T cell transfer experiments, naive
CD4
⫹
CD45RB
high
and CD8
⫹
CD44
low
T cells were sorted from spleen of
8-wk-old B6 mice with a Moflo (Cytomation) with a purity of 97%, then
labeled with CFSE, and cotransferred as described above.
Apoptosis assay
Externalization of phosphatidylserine was detected by PE-conjugated an-
nexin V mAb using the apoptosis detection kit (BD Pharmingen) according
to the instructions of the manufacturer. In brief, 10
6
splenocytes from adop-
tively transferred mice at day 3 or 28 were surface stained and then washed
with binding buffer. Cells were incubated for 15 min at room temperature
in the dark with annexin V ⫾7-aminoactinomycin D in binding buffer.
Then, 10
6
cells were analyzed by FACS as described above.
In vivo BrdU labeling
At 23 days after T cell transfer, mice were given 1 mg of BrdU (Sigma-
Aldrich) by i.p. injection and 1 mg/ml BrdU in their drinking water for 5
days. Recipient mice were sacrificed at day 28 after T cell transfer, and
spleen cell suspensions were prepared, surface stained as described above,
washed, fixed, and permeabilized before labeling with anti-BrdU mAb as
described previously (32, 33) using the BrdU flow kit (BD Pharmingen).
Then, 10
6
cells were analyzed by FACS.
Intracellular staining for Bcl-2
As described above, 10
6
total spleen cells from unmanipulated B6 or adop-
tively transferred mice were prepared and surface stained. After unbound
mAb were washed, cells were subjected to intracellular staining for Bcl-2
using the Cytofix/Cytoperm kit (BD Pharmingen). For intracellular stain-
ing, FITC-conjugated hamster anti-mouse Bcl-2 mAb (clone 3F11) or an
isotype control mAb (hamster IgG) was used.
Quantitative RT- PCR for IL-7 expression
Quantification of IL-7 transcript was performed on 6- to 8-wk-old mice.
RNA was extracted from 2 to 5 ⫻10
6
thymus or spleen cells of IL-7
transgenic mice or their negative littermate controls. RNA was extracted
(Trizol; Invitrogen), DNase digested (DNase I; Invitrogen) and cDNA syn-
thesized using SuperScriptII reverse transcriptase (Invitrogen). Real time
quantitative PCR was performed using a Lightcycler Instrument using the
FastStart kit, 1U/reaction (Roche Diagnostics). The real time PCR condi-
tions were as follows: 95°C for 8 min; then 45 cycles with 95°C for 15 s
at 60°C for 1 min. Mouse elongation factor 1
␣
(mEF1
␣
) mRNA was used
as a positive control and to quantitate total amplified RNA. Analysis was
conducted with Light Cycler 3.5 software (Roche Diagnostics). Results
were expressed as the ratio of IL-7 mRNA to mEF1
␣
mRNA and arbi-
trarily set at 1 for reference mice. The primer (Eurogentec) sequences were
as follows: mIL7 sense, 5⬘-CAGACCATGTTCCATGTTTCTTTTA-3⬘;
mIL7 antisense, 5⬘-CTTTGTCTTTAATGTGGCACTCAGA-3⬘; mEF1
␣
sense, 5⬘-CTGAACCATCCAGGCCAAAT-3⬘; and mEF1
␣
antisense,
5⬘-TCTTTTCTTTAAGCTCAGCAAACTTG-3⬘.
Immunohistochemistry
Spleens were harvested from recipient mice at different times after adoptive
transfer, embedded in Cryo-M-Bed compound (Bright Instrument), and
frozen at ⫺80°C. Frozen sections 5–7
m thick were fixed for 10 min in
cold acetone and dried extensively. Sections were stained with PE-labeled
anti-B220 mAb (RA3-6B2) for B cells and, when CFSE staining had be-
come negative, FITC-labeled anti-CD90 mAb (T24) to detect T cells. Cells
were imaged using a fluorescent confocal microscope (Leica TCS-SP3).
Results
Characterization of lymphopenic recipient mice
To study the effect of increased IL-7 availability on LIP, two models
of lymphopenic mice overexpressing IL-7 were generated. For most
of the experiments reported herein, we used only IL-7Tg.CD3
⑀
⫺/⫺
and CD3
⑀
⫺/⫺
littermates. The expression of the mouse IL-7 cDNA
transgene is controlled by the mouse MHC class II promoter (24), and
MHC class II genes are actively transcribed in mouse B cells (34).
Because B cells are present in CD3
⑀
⫺/⫺
mice, we observed by quan-
titative RT-PCR that differences in IL-7 transcript levels were greater
in spleen and thymus between IL-7Tg.CD3
⑀
⫺/⫺
and CD3
⑀
⫺/⫺
litter-
mates (10- to 30-fold) than between IL-7Tg.RAG-2
⫺/⫺
and RAG-
2
⫺/⫺
littermates (3- to 5-fold) where B cells are absent (not shown).
163The Journal of Immunology
Overexpression of IL-7 perturbs B cell development, primarily
by expanding the progenitor pool in the BM (35, 36). To demon-
strate this in the IL-7Tg.CD3
⑀
⫺/⫺
mouse line and because no re-
liable assay exists to quantitate available IL-7 protein in organs, a
series of phenotypic analyses was performed. The total number of
CD19
⫹
B cells was increased ⬃6-fold in the BM of IL-
7Tg.CD3
⑀
⫺/⫺
mice compared with littermate controls (Fig. 1A).
Both CD117
⫹
pro-B/pre-BI and CD25
⫹
B220
⫹
IgM
⫺
pre-BII cells
were increased ⬃10-fold; the numbers of B220
⫹
IgM
low
immature
B and IgM
⫹⫹
or CD23
⫹
mature B cells remained unchanged.
Thus, the known IL-7-dependent stages of BM B cell development
were the most affected by MHC class II promoter-driven IL-7
overexpression (32, 35, 36).
Two major changes were noted in the spleens of adult IL-
7Tg.CD3
⑀
⫺/⫺
mice. First, the spleen contained large numbers of
IL-7-dependent CD117
⫹
pro/pre-BI cells (Fig. 1B, bar graph) and
B220
⫹
IgM
⫺
immature B cells (Fig. 1B, cytograms); such cells are
only normally found in neonatal mice (37). Second, although
CD23
⫹
CD21
⫹
follicular B cell numbers were similar, there were
fewer CD23
low
CD21
⫹
marginal zone B cells in IL-7Tg.CD3
⑀
⫺/⫺
mice (Fig. 1B, lower cytograms and bar graph). Importantly, con-
focal microscopy indicated that spleen architecture was largely
preserved (see below). Qualitatively, Ab responses to the T-inde-
pendent type II Ag trinitrophenyl-Ficoll were normal in IL-
7Tg.CD3
⑀
⫺/⫺
and CD3
⑀
⫺/⫺
littermate controls, but IgM and IgG
titers were 10-fold higher in transgenics (Ref. 32 and data not
shown). In the peritoneal cavity, CD23
⫹
CD5
⫺
B2 B cells were
increased 4-fold whereas in the peripheral blood, B220
⫹
IgM
⫺
pre-
BII and B220
⫹
IgM
low
immature cells were present (Ref. 32 and
data not shown).
Analysis of thymocytes as reviewed by Ceredig and Rolink
(33) indicated that the numbers of IL-7-responsive
CD117
⫹⫹
CD44
⫹
CD25
⫺
DN1 and CD44
⫹
, CD25
⫹
DN2 cells
were increased ⬃2-fold but that the number of CD44
⫺
CD25
⫹
DN3 thymocytes remained unchanged (Fig. 1C) (38). Signifi-
cantly, the proportion of total CD44
⫹
CD25
⫺
DN1 cells was in-
creased with many cells staining weakly for CD44 (Fig. 1C, upper
cytograms). As expected (37), additional analysis indicated that
these CD44
low
CD25
⫺
cells were CD19
⫹
B cells comprising a
mixture of B220
⫹
IgM
⫺
pre-BII and B220
⫹
IgM
low
immature B
cells. CD19
⫹
B cells in the thymus of CD3
⑀
⫺/⫺
mice were mature
IgM
⫹
cells (Fig. 1C, lower cytograms). Taken together, these phe-
notypic and quantitative PCR data indicate that in IL-
7Tg.CD3
⑀
⫺/⫺
mice, BM B cell development was drastically al-
tered, and immature B cells were present in the blood, spleen, and
even the thymus. These changes reflect the increased IL-7 avail-
ability in these mice.
Increase of IL-7 availability promotes polyclonal T cell recovery
in vivo
To carefully characterize the contribution of IL-7 to T cell ho-
meostasis, unirradiated IL-7Tg.CD3
⑀
⫺/⫺
and their CD3
⑀
⫺/⫺
lit-
termates received i.v. 10
7
pooled LN and spleen lymphocytes, con-
taining 2–3 ⫻10
6
CD4
⫹
and CD8
⫹
T cells. Recipients were
sacrificed between 1 and 28 days after transfer. Fig. 2Ashows that
after adoptive transfer, both CD4
⫹
and CD8
⫹
T cells persisted in
the spleen of CD3
⑀
⫺/⫺
recipient mice. Cell numbers increased
slowly up to day 3 and thereafter more rapidly. At day 28 post-
transfer, the CD3
⑀
⫺/⫺
spleen contained ⬃15 ⫻10
6
CD4
⫹
and 5 ⫻
10
6
CD8
⫹
T cells, respectively, or a ⬎10- to 15-fold expansion of
FIGURE 1. IL-7 overexpression perturbs B cell lym-
phopoiesis. A, BM cells were three-color stained as de-
scribed in Materials and Methods. Bars show the aver-
age (n⫽3) cell number (left ordinates) of the indicated
subpopulation. p, Ratio of each subpopulation in IL-
7Tg.CD3
⑀
⫺/⫺
vs CD3
⑀
⫺/⫺
mice (right ordinates). B,
Spleen cells were three-color stained with anti-CD19,
anti-B220, and anti-IgM or anti-CD19, anti-CD21, and
anti-CD23. Upper cytograms, B220 vs IgM; lower cy-
tograms, CD23 vs CD21 profiles of gated CD19
⫹
cells.
Right bar graph, Mean number and ratios of the indi-
cated subpopulations. Imm.B, Immature B cells; FB,
follicular B; MZB, marginal zone B. C, Thymus cells
were four-color stained with anti-CD25, anti-CD44, anti-
B220, and anti-IgM. Upper cytograms show CD25 vs
CD44 profiles of all live cells; lower cytograms show
the B220 vs IgM staining of cells gated for intermediate
CD44-expressing cells (round gate in upper cytograms).
Right bar graph shows mean number and ratios of the
indicated subpopulations. In each cytogram display,
numbers in quadrants represent the percentage of posi-
tive cells. Results are representative of at least five
experiments.
164 INCREASING IL-7 AVAILABILITY ALTERS T CELL HOMEOSTASIS
the transferred cells between days 1 and 28. Thus, numbers are
almost similar to those in the spleen of a normal B6 mouse.
Overexpression of IL-7 in CD3
⑀
⫺/⫺
mice had a significant effect
on the behavior of transferred T cells. Thus, we observed a greater
rate of expansion of both CD4
⫹
and CD8
⫹
T cells in IL-
7Tg.CD3
⑀
⫺/⫺
recipient mice and also an increase in long term cell
recovery. Therefore, from day 3 and up to day 28, IL-
7Tg.CD3
⑀
⫺/⫺
contained more CD4
⫹
and CD8
⫹
T cells than their
CD3
⑀
⫺/⫺
controls with lymphocyte numbers reaching a plateau at
about day 14. At day 28, the spleen of IL-7Tg.CD3
⑀
⫺/⫺
recipients
contained ⬃52 ⫻10
6
CD4
⫹
and 17 ⫻10
6
CD8
⫹
T cells, repre-
senting a 3- to 4-fold higher expansion compared with control
CD3
⑀
⫺/⫺
recipients (Fig. 2A). A similar pattern of growth and
recovery was obtained in the LN (not shown). Thus, increased
availability of IL-7 in vivo increases the yield of T cells after
adoptive transfer.
Next, we investigated in more detail the in vivo impact of IL-7
overexpression on transferred T cells. To rule out a possible pref-
erential and exacerbated monoclonal expansion of T cells, we an-
alyzed their TCR
␣
and TCR

repertoire by flow cytometry. The
repertoire of transferred T cells in both recipient mice was large,
polyclonal, and comparable with that of T cells from unmanipu-
lated B6 mice (not shown) with no obvious bias after expansion.
Then, we determined the phenotype of transferred T cells by
four-color flow cytometry. Analysis of T cells during early phases
of proliferation (days 3, 5, and 7 posttransfer) showed changes
similar to those in other published reports (3); namely, cells be-
came larger, as judged by forward light scatter, remained CD25
⫺
and CD69
⫺
and CD44
⫹
(not shown). One month after transfer,
CD4
⫹
and CD8
⫹
T cells had acquired a memory-like phenotype
(3, 39 – 42) (Fig. 2B, left and middle cytograms) with most CD4
⫹
T cells being CD44
high
CD62L
low
and many CD8
⫹
T cells being
CD44
high
CD122
high
. This phenotypic conversion that accompanies
expansion of polyclonal T cells in both recipient mice was only
slightly more pronounced in IL-7 Tg recipients. Additionally, we
observed that ⬃10% of the injected CD4
⫹
T cells spontaneously
express CD25. Therefore, among CD4
⫹
cells, the proportion of
CD25
⫹
cells in both types of recipient mice remained the same
with ⬃10% of CD4
⫹
T cells expressing CD25 at 28 days after
transfer (Fig. 2B, right cytograms).
Localization of transferred cells in vivo
It has been shown that transferred T cells migrate to the periarte-
riolar lymphocyte sheaths (PALS) of the spleen where they pre-
sumably receive division and survival signals (43). To exclude that
altered T cell recovery was due to a difference in spleen architec-
ture, a confocal immunohistology study was conducted. Despite
IL-7-dependent alteration of BM B cell development (Fig. 1B) and
the presence of immature B cells, no difference in B cell follicle
size or organization was observed in the spleen of IL-
7Tg.CD3
⑀
⫺/⫺
mice (Fig. 3). At day 1 posttransfer (Fig. 3, aand b),
CFSE
⫹
cells were found in PALS of both recipients, with slightly
more in the follicles of IL-7Tg.CD3
⑀
⫺/⫺
mice. At day 28 post-
transfer, T cells had lost CFSE labeling but were identified using
anti-CD90 (Thy-1) staining. Results indicated that transferred T
cells were localized to T cell areas of follicles but that restoration
of these areas in both recipients was only partial compared with B6
controls (Fig. 3, c– e). These findings suggest that T cells undergo
LIP within a dedicated area inside B cell follicles (43) that pre-
sumably provides specific factors, including IL-7, necessary for
maintaining T cell survival and proliferation.
IL-7 overexpression has differential proliferative effects on
CD8
⫹
and CD4
⫹
subpopulations
Using CFSE labeling, we compared the proliferation of T cells in
lymphopenic hosts differing in IL-7 availability. Three days after
transfer into CD3
⑀
⫺/⫺
recipients, few CD4
⫹
cells had divided (Fig.
4A, left panels) and the average number of divisions was below 1.
Over the same time period, CD8
⫹
T cells had proliferated more, with
an average of almost two divisions. In contrast, in IL-7Tg.CD3
⑀
⫺/⫺
recipients, a greater proportion of both CD4
⫹
and CD8
⫹
T cells had
reduced CFSE fluorescence and had undergone between one and four
rounds of division, respectively. That increasing IL-7 availability pro-
moted LIP of CD8
⫹
T cells was somewhat expected, but in contrast
to other published reports (6, 7), there was also a clear effect of IL-7
on CD4
⫹
T cells. At 5 days after transfer (Fig. 4A, right panels), T
cells had undergone more rounds of division. Again CD8
⫹
cells had
divided more than CD4
⫹
cells. This difference between CD8
⫹
and
CD4
⫹
cells was maintained regardless of differences in IL-7 avail-
ability. Both T cell subpopulations proliferated more in IL-
7Tg.CD3
⑀
⫺/⫺
vs CD3
⑀
⫺/⫺
littermates. Similar differences between
CD8
⫹
and CD4
⫹
cell division were also seen when the cells were
transferred to IL-7Tg.RAG-2
⫺/⫺
or RAG-2
⫺/⫺
littermates where the
difference in IL-7 transcript levels was 3- to 5-fold (not shown).
Again, because there was no up-regulation of CD69 or CD25 expres-
sion on either CD8
⫹
or CD4
⫹
T cells (not shown) this, together with
the relatively slow kinetics of proliferation, indicated that LIP rather
than activation-induced proliferation was being measured (39).
FIGURE 2. IL-7 enhances T cell recovery but not
memory phenotype conversion after adoptive transfer.
A, Upper graph, number (⫾SD, n⫽7) of recovered
CD4
⫹
cells (䡺,f)(⫻10
⫺6
); lower graph, number of
CD8
⫹
cells (‚,Œ)(⫻10
⫺6
) recovered at various times
after transfer into CD3
⑀
⫺/⫺
(f,Œ) or IL-7Tg.CD3
⑀
⫺/⫺
(䡺,⌬) recipients. B, Fresh spleen cells from B6 mice (i)
or 28 days after transfer into CD3
⑀
⫺/⫺
(ii)orIL-
7Tg.CD3
⑀
⫺/⫺
(iii) mice were stained with either anti-
CD4, anti-CD25, anti-CD44, and anti-CD62L (left and
right) or anti-CD8, anti-CD44, and anti-CD122 (mid-
dle). Left cytograms, CD44 vs CD62L on live-gated
CD4
⫹
spleen cells; middle cytograms, CD44 vs CD122
on live-gated CD8
⫹
;right cytograms, CD4 vs CD25
distribution on live-gated spleen cells. The percentages
of cells within the indicated region are shown in each
dot plot (left and middle). In the right dot plots, ⴱindi-
cates the percent CD25
⫹
among total CD4
⫹
cells. Three
mice were individually examined in each group, and a
representative result is shown. FL, Fluorescence.
165The Journal of Immunology
After 14 days, the number of transferred T cells had reached a
plateau, and they became mostly CFSE negative. BrdU labeling
experiments showed that after 28 days, the turnover of transferred
T cells was similar in both recipients with ⬃30 and 20% of CD4
⫹
and CD8
⫹
T cells, respectively, incorporating BrdU (Fig. 4B).
However, due to the overall increase in T cell recovery in IL-
7Tg.CD3
⑀
⫺/⫺
mice, (Fig. 2Aand data not shown), the total num-
ber of proliferating T cells was ⬃4- to 5-fold higher in IL-7
transgenics.
In most experiments, we transferred unseparated populations of
T cells. However, to see whether naive T cells were equally af-
fected by differences in IL-7 availability, sorted naive
CD4
⫹
CD45RB
high
and CD8
⫹
CD44
low
cells were CFSE-labeled
and cotransferred into pairs of CD3
⑀
⫺/⫺
and IL-7Tg.CD3
⑀
⫺/⫺
mice. As shown in Fig. 5A, the CFSE profiles of transferred naive
CD4
⫹
and CD8
⫹
T cells showed that both responded to increased
IL-7 availability by an increase in the proportion of cells engaged
in LIP and the mean number of divisions (about one more division
for both CD4
⫹
and CD8
⫹
T cells in IL-7Tg recipients 4 days
posttransfer). Simultaneous phenotypic analysis showed that inde-
pendent of the host, there was an increase in CD44 expression by
transferred CD4
⫹
and CD8
⫹
T cells (Fig. 5B).
Previous experiments had suggested that during LIP, naive T
cells transiently acquired a memory-like phenotype (40). However,
subsequent experiments clearly demonstrated that this transient
phenotypic conversion was due to the de novo production of naive
T cells by the freshly recolonized thymus of irradiated recipient
mice (3, 41, 42). As shown in Fig. 6, the thymus of both CD3
⑀
⫺/⫺
and IL-7Tg.CD3
⑀
⫺/⫺
murine recipients contained neither
CD4
⫹
CD8
⫹
double-positive cells (Fig. 6, left panels) nor
CD44
⫺
CD25
⫺
(DN4) thymocytes (Fig. 6, right panels). This
shows the absence of thymus reconstitution by T cell progenitors
in unirradiated recipients and that the observed BrdU incorporation
into peripheral T cells (Fig. 4C) was not the result of de novo
FIGURE 3. Transferred CFSE
⫹
cells homed to
PALS following transfer. Frozen spleen sections were
stained as described in Materials and Methods. Recipi-
ent mice were killed 1 day (aand b)or4wk(cand d)
after transfer of CFSE-labeled pooled LN ⫹spleen
cells. Sections were stained for B cells (red) with PE-
conjugated anti-B220 mAb (a– e). CFSE
⫹
donor cells
(green) are situated almost exclusively in the periarte-
riolar lymphocyte sheath (PALS) (aand b). Four weeks
after transfer (cand d), transferred T cells had lost CFSE
staining, and T cells were stained with FITC-conjugated
anti-CD90.2 (Thy1.2) mAb. The T area is not com-
pletely restored (cand d) compared with normal B6
mice (e). Sections aand care from CD3
⑀
⫺/⫺
recipients,
and sections band dare from IL-7Tg.CD3
⑀
⫺/⫺
recipi-
ents. Data are representative of one of three mice ana-
lyzed individually. Scale bar, 80
m.
FIGURE 4. IL-7 promotes proliferation of poly-
clonal CD4
⫹
and CD8
⫹
cells in lymphopenic mice. A,
CFSE profiles of gated CD4
⫹
or CD8
⫹
spleen cells 3
days (left panels) or 5 days (right panels) after transfer
of 10
7
pooled labeled LN and spleen cells into CD3
⑀
⫺/⫺
(upper panels) or IL-7Tg.CD3
⑀
⫺/⫺
(bottom panels)re
-
cipients. Similar results were obtained with LN cells
(not shown). B, Cytoplasmic BrdU profiles of gated
CD4
⫹
(left histograms)orCD8
⫹
(right histograms)
cells 28 days after transfer and labeled for the last 5 days
with BrdU as outlined in Materials and Methods.
Within each panel, the percent of BrdU
⫹
cells is indi-
cated. Negative controls were ⬍1% BrdU
⫹
. FL,
Fluorescence.
166 INCREASING IL-7 AVAILABILITY ALTERS T CELL HOMEOSTASIS
thymus T cell production. Surprisingly, more CD4
⫹
and CD8
⫹
single-positive (SP) cells were found in the thymus of IL-
7Tg.CD3
⑀
⫺/⫺
mice compared with CD3
⑀
⫺/⫺
recipients (Fig. 6,
left panels) and at early time points, these SP cells were CFSE
⫹
(not shown).
IL-7 decreases T cell apoptosis with a temporal effect on CD4
⫹
and CD8
⫹
T cells
Regulation of T cell survival is a critical feature for the mainte-
nance of peripheral T cell numbers. To determine whether increas-
ing IL-7 availability promoted cell survival during LIP, two pa-
rameters were measured, namely, annexin V staining and
cytoplasmic Bcl-2 expression. At 3 days after transfer, the propor-
tion of annexin V
⫹
CD4
⫹
T cells was significantly reduced in
spleens of IL-7Tg.CD3
⑀
⫺/⫺
mice, and CD4
⫹
T cell apoptosis was
reduced (20 – 40%) less for each round of division (Fig. 7A). At
this time, cytoplasmic Bcl-2 levels in CD4
⫹
T cells was already
elevated in transgenic recipients and this persisted until 28 days
posttransfer (Fig. 7C).
In contrast, the early antiapoptotic action of IL-7 on transferred
CD8
⫹
T cells was less pronounced (Fig. 7, Aand C). In IL-
7Tg.CD3
⑀
⫺/⫺
recipients, 3 days posttransfer, there was only a
5–10% reduction in apoptosis for each round of division (Fig. 7A)
and no increase in cytoplasmic Bcl-2 expression (Fig. 7C). How-
ever, by day 28, annexin V
⫹
cells had decreased (Fig. 7B), and
Bcl-2 levels had increased in CD8
⫹
T cells and were even higher
than in CD4
⫹
T cells (Fig. 7). Thus, the effects of increasing IL-7
availability have temporally independent effects on CD4
⫹
vs
CD8
⫹
T cells. All the changes described above in polyclonal pop-
ulations of CD8
⫹
lymphocytes were also observed with TCR-
transgenic cells. Thus, when CD8
⫹
T cells from P14.RAG-2
⫺/⫺
mice were transferred to IL-7Tg.CD3
⑀
⫺/⫺
or CD3
⑀
⫺/⫺
recipients,
proliferation was more rapid, apoptosis was decreased, expression
of cytoplasmic Bcl-2 was increased, and cell recovery at 28 days
increased 10-fold in IL-7Tg.CD3
⑀
⫺/⫺
compared with CD3
⑀
⫺/⫺
recipients (not shown).
The transferred CFSE-labeled cells contained B cells, but there
did not appear to be an effect of IL-7 overexpression on their
proliferation, survival, or Bcl-2 expression (Fig. 7Cand data not
shown) in either IL-7Tg.CD3
⑀
⫺/⫺
or CD3
⑀
⫺/⫺
recipients. In con-
trast, in completely lymphopenic RAG-2
⫺/⫺
or IL-7Tg.RAG-2
⫺/⫺
recipients, transferred B cells proliferated slowly but proliferation
was similar in IL-7 transgenic and nontransgenic recipients (N.
Bosco, unpublished observation).
Discussion
In this report, we have studied the behavior of polyclonal popula-
tions of lymphocytes transferred into novel T-lymphopenic IL-7-
transgenic recipient mice (Figs. 1 and 2). Results obtained indicate
that increasing IL-7 enhanced recovery of both CD4
⫹
and CD8
⫹
polyclonal T cells but had markedly different effects on prolifera-
tion (Figs. 4 and 5) and apoptosis (Fig. 7) of transferred cells
depending on whether cells were actively undergoing LIP or sur-
viving in a replenished peripheral compartment. The prediction
had been made that in lymphopenic recipients, increasing IL-7
availability in vivo should increase the rate of T cell proliferation
(27), but this hypothesis has not been directly addressed. Our in
vivo results clearly demonstrate that during LIP, increasing IL-7
availability increases the rate of division of both CD4
⫹
and CD8
⫹
T cells but not of B cells.
The rate of proliferation of CD8
⫹
T cells, as measured by CFSE
fluorescence, was enhanced by increasing IL-7 availability. This
was true for both polyclonal and P14 TCR-transgenic T cells. The
proliferation of CD4
⫹
cells, although intrinsically slower than that
FIGURE 6. Normal thymopoiesis is not restored after adoptive transfer
of polyclonal lymphocytes. One month after transfer, recipient thymuses
were recovered and stained with anti-CD4, anti-CD8, anti-CD25, and anti-
CD44. Shown are the CD4 vs CD8 cytograms on all (left) or CD25 vs
CD44 cytograms of gated CD4
⫺
/CD8
⫺
cells (right) from adult B6 (upper)
or adoptively transferred CD3
⑀
⫺/⫺
(middle) or IL-7Tg.CD3
⑀
⫺/⫺
(lower)
recipients. CD44
low
CD25
⫺
cells in recipient mice are B cells (see Fig. 1).
Left lower panels, Percentage of SP cells within the indicated regions.
Insets, Region used to define DN and SP cells.
FIGURE 5. Naive CD4
⫹
and CD8
⫹
T cells prolifer-
ate more rapidly in IL-7Tg.CD3
⑀
⫺/⫺
mice. A, CFSE
profiles of 4 ⫻10
6
sorted, pooled and labeled, naive
CD4
⫹
CD45RB
high
(left panels)orCD8
⫹
CD44
low
(right panels) T cells 4 days after transfer into CD3
⑀
⫺/⫺
(upper panels) or IL-7Tg.CD3
⑀
⫺/⫺
mice (lower panels).
Division numbers and percentages of nondivided cells
are indicated. B, CD44 expression level vs CFSE con-
tent of the indicated cells in the corresponding mice.
Data are representative of two independent experiments
with two mice analyzed individually. FL, Fluorescence.
167The Journal of Immunology
of CD8
⫹
cells, was also increased in IL-7Tg.CD3
⑀
⫺/⫺
recipients
(Figs. 4 and 5). Although initial results had indicated that prolif-
eration of CD4
⫹
SP neonatal thymocytes in vitro (44) could be
maintained in the presence of IL-7, subsequent reports had indi-
cated that IL-7 had no effect on CD4
⫹
T cell proliferation (6, 7),
but this notion has been recently revised (27, 45, 46).
In IL-7Tg.CD3
⑀
⫺/⫺
recipients, Bcl-2 was up-regulated in both
CD4
⫹
and CD8
⫹
T cells but with different kinetics (Fig. 7). Early
after transfer, there was a distinct effect of IL-7 availability on
Bcl-2 expression in CD4
⫹
cells whereas CD8
⫹
cells were not
affected. Later on, Bcl-2 expression and apoptosis resistance were
observed and were more pronounced in CD8
⫹
T cells. In contrast,
Bcl-2 levels in T cells transferred into CD3
⑀
⫺/⫺
recipients were
similar to those of unmanipulated B6 controls. It has been shown
that CD8
⫹
but not CD4
⫹
memory cells overexpress Bcl-2 protein
(47). Phenotypic analysis indicated that after LIP, in both trans-
genic and nontransgenic recipients, T cells were enriched in so-
called memory-like cells (3). Differences in the proportion of
memory-like cells within surviving cells were minor, yet there was
a clear increase in cytoplasmic Bcl-2 expression (Figs. 2 and 7).
This indicates that changes in Bcl-2 levels were IL-7 dependent
and not the result of an altered cellular composition of CD4
⫹
or
CD8
⫹
T cell subsets. Even so-called naive CD4
⫹
CD45RB
high
and
CD8
⫹
CD44
low
T cells respond to increased IL-7 availability (Fig.
5A) and undergo naive to memory-like phenotypic conversion af-
ter transfer into lymphopenic hosts (Fig. 5Band Refs. 3 and 48).
Although sharing certain properties with true memory cells, it is
generally agreed that the slower kinetics of induction of effector
functions by memory-like cells clearly distinguishes them from
true memory cells (48 –50). It could be either that IL-7 sustains
naive T cell differentiation into memory cells or that IL-7 promotes
selection of memory CD8
⫹
subset among the transferred cells that
then outcompete naive T cells in LIP. The latter hypothesis would
be in agreement with our previous report describing an increase of
naturally arising CD8
⫹
memory cells in nonimmunized IL-7Tg.B6
mice (26). Importantly, the combined effects of decreasing annexin
V staining and increasing cytoplasmic Bcl-2 expression could ac-
count for the increased T cell yield in IL-7-transgenic recipients.
This reproducible difference in the degree of apoptosis is important
if we consider the exponential growth of T cells during LIP. De-
creasing cell loss by 10 or 15% at each division can lead to a 50%
increase in cell recovery after eight or five divisions, respectively.
Indeed, cell recovery is a balance between the rate of cell prolif-
eration and cell loss. The inoculum used in most of our experi-
ments contained twice as many CD4 as CD8 T cells, and this
difference may partially explain the increased recovery of CD4
⫹
vs CD8
⫹
T cells 1 month after transfer. In addition, as shown in
Fig. 7, there is a major effect of IL-7 overexpression on Bcl-2
expression by CD4
⫹
but not CD8
⫹
T cells early after transfer.
This is likely to have major consequences on overall cell recovery.
This could explain why CD4
⫹
T cell recovery is higher than CD8
⫹
cells despite their slower proliferation rate.
One month after transfer, when the T cell compartment had
reached equilibrium, there was no difference in overall turnover of
CD4
⫹
or CD8
⫹
T cells, whereas the total number of T cells was
4 times higher in IL-7Tg.CD3
⑀
⫺/⫺
recipients (Figs. 2 and 4). That
increasing IL-7 availability per se does not increase turnover of the
established T cell pool is consistent with studies in IL-7Tg.B6
mice (26) (N. Bosco, unpublished observation) in which the pro-
portion of dividing CD4
⫹
or CD8
⫹
T cells is similar to that of
FIGURE 7. Kinetics of Bcl-2 up-regulation and survival by IL-7 differs between CD4
⫹
and CD8
⫹
cells. A, Bars show the mean percent (⫾SD, n⫽
3) of annexin V
⫹
CD4
⫹
(upper panels)orCD8
⫹
(lower panels) cells that had divided the indicated number of times 3 days posttransfer into CD3
⑀
⫺/⫺
(f)
or IL-7Tg.CD3
⑀
⫺/⫺
(䡺) recipients. Insets, CFSE vs annexin V cytograms of the corresponding cells in CD3
⑀
⫺/⫺
recipients. B, Bar graphs show the mean
(⫾SD, n⫽3) of apoptotic (annexin V
⫹
) gated CD4
⫹
(left)orCD8
⫹
(right) cells 28 days posttransfer into CD3
⑀
⫺/⫺
(f) or IL-7Tg.CD3
⑀
⫺/⫺
(䡺)
recipients. Cells were stained with anti-CD4, anti-CD8, anti-annexin V, and 7-aminoactinomycin D as described in Materials and Methods.C, Bar graphs
show the mean fluorescence intensity (MFI) of intracellular Bcl-2 expression in B220
⫹
, CD4
⫹
,orCD8
⫹
lymphocytes from WT (f) or in cells 3 days (upper
panels) or 28 days (lower panels) after transfer into CD3
⑀
⫺/⫺
(u) or IL-7Tg.CD3
⑀
⫺/⫺
(䡺) recipients. Four mice were individually examined in each group,
and the mean fluorescence intensity ⫾SD is represented. WT, Wild type.
168 INCREASING IL-7 AVAILABILITY ALTERS T CELL HOMEOSTASIS
nontransgenic controls. However, given the increased pool size of
T cells, the number of dividing cells is proportionately higher, and
equilibrium at this elevated cell number is maintained by balanced
proliferation and simultaneous cell loss.
The IL-7R signal transduction cascade has been shown to acti-
vate expression of Bcl-2 family genes (51). We showed a temporal
difference in Bcl-2 expression between CD4
⫹
and CD8
⫹
T cells in
response to increased availability of IL-7 (Fig. 7). As recently
reported (52), the signaling cascade downstream of the IL-7R may
be differentially regulated between CD4
⫹
and CD8
⫹
cells. IL-7R
␣
gene transcription is suppressed in response to IL-7 signaling, a
process that involves different molecular mechanisms in CD4
⫹
vs
CD8
⫹
T cells. CD8
⫹
T cells use the transcriptional repressor
GFI1; the transcriptional repressor used by CD4
⫹
T cells remains
to be identified. If major differences exist in the repertoire of tran-
scription activators and repressors used by different T cell subsets
after IL-7R engagement, these factors could constitute important
targets to control selectively T cell responses to various cytokines
and therefore T cell homeostasis.
T cells injected into lymphopenic animals migrated into lym-
phoid follicles, and reconstituted their T cell areas. Restricted
homing of transferred T cells into lymphoid organs suggests that
lymphocyte migratory properties are crucial for the initiation of
LIP. Pertussis toxin treatment of donor cells abrogates G protein-
dependent migration of T cells and reduces LIP in recipient mice
(43). IL-7 and additional resources, including chemokines, are
present inside lymphoid follicles, and the concentration of these
factors is presumably increased in lymphopenic conditions (23).
This may be how T cells sense lymphopenia and how LIP is trig-
gered. Experiments in vitro have shown that IL-7 decreases the
activation threshold of T cells, thereby serving as a “cofactor” for
activation (45). Our results confirm that a similar mechanism
might be operational in vivo, thereby promoting LIP. The cells that
deliver the signals promoting LIP remain to be defined. However,
dendritic cells are good candidates because some of them localize
inside lymphoid follicles, express MHC molecules, secrete cyto-
kines, and sustain LIP in vitro (53).
Where does T cell proliferation during LIP occur? Most trans-
ferred cells localize to the PALS of lymphoid follicles (Fig. 3), the
anatomical site where most proliferation occurs (43). However, it
is unclear whether cells remain fixed in the spleen during LIP.
Early after cell transfer, CFSE
⫹
CD4
⫹
and CD8
⫹
SP cells were
found in the thymus of recipients, and their CFSE profiles mirrored
those of peripheral T cells. Quantitative RT-PCR and analysis of
thymic B cell phenotype together indicated that the thymus was
enriched in IL-7 (Fig. 1Cand N. Bosco, unpublished observation).
That CFSE
⫹
T cells were present in the thymus indicated that their
survival could be maintained in anatomical sites rich in IL-7. Their
presence in the thymus could also indicate that cells circulate dur-
ing LIP. A homeostatic niche supporting T cell LIP could be sim-
ply defined as a location where the trophic factors are available.
These locations for T cells are somehow flexible, a situation anal-
ogous to that described for B cells by Agene`s and Freitas (54).
Transfer of B lymphocytes into IL-Tg.CD3
⑀
⫺/⫺
or IL-7Tg.Rag-
2
⫺/⫺
recipients indicated that IL-7 was not involved in B cell LIP
(not shown). This is consistent with our previous data using IL-
7Tg.B6 mice, where the increase in mature B cell compartments is
simply a consequence of increasing BM B lymphopoiesis and not
due to a role for IL-7 in mature B cell survival (32). Thus, as
previously reported, T and B cell homeostasis are independent of
one another and depend on different resources (55).
In conclusion, we describe for the first time the pleiotropic ef-
fects of IL-7 during and after lymphocyte polyclonal LIP. We
show that increasing IL-7 availability has effects on both CD4
⫹
and CD8
⫹
T cells but not on B cells. These results are relevant to
the clinical settings in which IL-7 is being used as an adjunct for
hemopoietic, in particular lymphocyte reconstitution, after BM
transfer (10). Much less information is available on the role of IL-7
in human T cell LIP, but recent advances, including the generation
of humanized RAG
⫺/⫺
.
␥
c
⫺/⫺
mice reconstituted with human cord
blood cells (56) could provide opportunities to explore the effect of
human IL-7 on adoptively transferred human lymphocytes.
Acknowledgments
We thank Patrice Marche and Institut National de la Sante´etdelaRe-
cherche Me´dicale for their support; Dr. Jo¨ rg Kirberg, Max Planck Institute
for Immunobiology, Freiburg for P14.RAG-2
⫺/⫺
mice; Dr. Ton Rolink for
invaluable help; and Drs. Simon Fillatreau and Didier Grunwald for con-
focal microscopy. We thank Eve Borel for mouse maintenance and
Ve´ronique Collin for cell sorting. We thank Drs. Serge Cande´ias and Chris-
tophe Viret for their comments and constructive criticisms of the
manuscript.
Disclosures
The authors have no financial conflict of interest.
References
1. Marrack, P., and J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immu-
nol. 22: 765–787.
2. Jameson, S. C. 2002. Maintaining the norm: T-cell homeostasis. Nat. Rev. Im-
munol. 2: 547–556.
3. Ge, Q., H. Hu, H. N. Eisen, and J. Chen. 2002. Naive to memory T-cell differ-
entiation during homeostasis-driven proliferation. Microbes Infect. 4: 555–558.
4. Sprent, J., and C. D. Surh. 2003. Cytokines and T cell homeostasis. Immunol.
Lett. 85: 145–149.
5. Ceredig, R. 2000. Interleukin-7, a non-redundant potent cytokine whose over-
expression massively perturbs B-lymphopoiesis. In Cytokines and Cytokine Re-
ceptors. C. A. Bona and J.-P. Revillard, eds. Harwood Academic Publishers, pp.
167–182.
6. Vella, A. T., S. Dow, T. A. Potter, J. Kappler, and P. Marrack. 1998. Cytokine-
induced survival of activated T cells in vitro and in vivo. Proc. Natl. Acad. Sci.
USA 95: 3810–3815.
7. Tan, J. T., E. Dudl, E. LeRoy, R. Murray, J. Sprent, K. I. Weinberg, and
C. D. Surh. 2001. IL-7 is critical for homeostatic proliferation and survival of
naive T cells. Proc. Natl. Acad. Sci. USA 98: 8732–8737.
8. Morrissey, P. J., P. Conlon, K. Charrier, S. Braddy, A. Alpert, D. Williams,
A. E. Namen, and D. Mochizuki. 1991. Administration of IL-7 to normal mice
stimulates B-lymphopoiesis and peripheral lymphadenopathy. J. Immunol. 147:
561–568.
9. Alpdogan, O., S. J. Muriglan, J. M. Eng, L. M. Willis, A. S. Greenberg,
B. J. Kappel, and M. R. Van Den Brink. 2003. IL-7 enhances peripheral T cell
reconstitution after allogeneic hematopoietic stem cell transplantation. J. Clin.
Invest. 112: 1095–1107.
10. Fry, T. J., and C. L. Mackall. 2002. Interleukin-7: from bench to clinic. Blood 99:
3892–3904.
11. Ferrara, J. L. 1993. Cytokine dysregulation as a mechanism of graft versus host
disease. Curr. Opin. Immunol. 5: 794–799.
12. Moses, C. T., K. M. Thorstenson, S. C. Jameson, and A. Khoruts. 2003. Com-
petition for self ligands restrains homeostatic proliferation of naive CD4 T cells.
Proc. Natl. Acad. Sci. USA 100: 1185–1190.
13. Chung, B., L. Barbara-Burnham, L. Barsky, and K. Weinberg. 2001. Radiosen-
sitivity of thymic interleukin-7 production and thymopoiesis after bone marrow
transplantation. Blood 98: 1601–1606.
14. Kieper, W. C., J. T. Burghardt, and C. D. Surh. 2004. A role for TCR affinity in
regulating naive T cell homeostasis. J. Immunol. 172: 40– 44.
15. Kassiotis, G., R. Zamoyska, and B. Stockinger. 2003. Involvement of avidity for
major histocompatibility complex in homeostasis of naive and memory T cells.
J. Exp. Med. 197: 1007–1016.
16. Ge, Q., A. Bai, B. Jones, H. N. Eisen, and J. Chen. 2004. Competition for self-
peptide-MHC complexes and cytokines between naive and memory CD8
⫹
T
cells expressing the same or different T cell receptors. Proc. Natl. Acad. Sci. USA
101: 3041–3046.
17. Hori, S., M. Haury, A. Coutinho, and J. Demengeot. 2002. Specificity require-
ments for selection and effector functions of CD25
⫹
4
⫹
regulatory T cells in
anti-myelin basic protein T cell receptor transgenic mice. Proc. Natl. Acad. Sci.
USA 99: 8213–8218.
18. Apostolou, I., A. Sarukhan, L. Klein, and H. von Boehmer. 2002. Origin of
regulatory T cells with known specificity for antigen. Nat. Immunol. 3: 756 –763.
19. Almeida, A. R., N. Legrand, M. Papiernik, and A. A. Freitas. 2002. Homeostasis
of peripheral CD4
⫹
T cells: IL-2R
␣
and IL-2 shape a population of regulatory
cells that controls CD4
⫹
T cell numbers. J. Immunol. 169: 4850– 4860.
20. Stockinger, B., G. Kassiotis, and C. Bourgeois. 2004. Homeostasis and T cell
regulation. Curr. Opin. Immunol. 16: 775–779.
169The Journal of Immunology
21. Annacker, O., O. Burlen-Defranoux, R. Pimenta-Araujo, A. Cumano, and
A. Bandeira. 2000. Regulatory CD4 T cells control the size of the peripheral
activated/memory CD4 T cell compartment. J. Immunol. 164: 3573–3580.
22. Banz, A., A. Peixoto, C. Pontoux, C. Cordier, B. Rocha, and M. Papiernik. 2003.
A unique subpopulation of CD4
⫹
regulatory T cells controls wasting disease,
IL-10 secretion and T cell homeostasis. Eur. J. Immunol. 33: 2419–2428.
23. Fry, T. J., and C. L. Mackall. 2001. Interleukin-7: master regulator of peripheral
T-cell homeostasis? Trends Immunol. 22: 564–571.
24. Fisher, A. G., C. Burdet, M. LeMeur, D. Haasner, P. Gerber, and R. Ceredig.
1993. Lymphoproliferative disorders in an IL-7 transgenic mouse line. Leukemia
7 (Suppl. 2): S66–S68.
25. Mertsching, E., C. Burdet, and R. Ceredig. 1995. IL-7 transgenic mice: analysis
of the role of IL-7 in the differentiation of thymocytes in vivo and in vitro. Int.
Immunol. 7: 401–414.
26. Kieper, W. C., J. T. Tan, B. Bondi-Boyd, L. Gapin, J. Sprent, R. Ceredig, and
C. D. Surh. 2002. Overexpression of interleukin (IL)-7 leads to IL-15-indepen-
dent generation of memory phenotype CD8
⫹
T cells. J. Exp. Med. 195:
1533–1539.
27. Li, J., G. Huston, and S. L. Swain. 2003. IL-7 promotes the transition of CD4
effectors to persistent memory cells. J. Exp. Med. 198: 1807–1815.
28. Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier,
and B. Malissen. 1995. Altered T cell development in mice with a targeted mu-
tation of the CD3-
⑀
gene. EMBO J. 14: 4641–4653.
29. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn,
J. Charron, M. Datta, F. Young, A. M. Stall, et al. 1992. RAG-2-deficient mice
lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell
68: 855–867.
30. Pircher, H., K. Burki, R. Lang, H. Hengartner, and R. M. Zinkernagel. 1989.
Tolerance induction in double specific T-cell receptor transgenic mice varies with
antigen. Nature 342: 559–561.
31. Agenes, F. 2003. B lymphocyte life span, rate of division and differentiation are
regulated by total cell number. Eur. J. Immunol. 33: 1063–1069.
32. Ceredig, R., N. Bosco, P. N. Maye, J. Andersson, and A. Rolink. 2003. In in-
terleukin-7-transgenic mice, increasing B lymphopoiesis increases follicular but
not marginal zone B cell numbers. Eur. J. Immunol. 33: 2567–2576.
33. Laurent, J., N. Bosco, P. N. Marche, and R. Ceredig. 2004. New insights into the
proliferation and differentiation of early mouse thymocytes. Int. Immunol. 16:
1069 –1080.
34. Hayakawa, K., D. Tarlinton, and R. R. Hardy. 1994. Absence of MHC class II
expression distinguishes fetal from adult B lymphopoiesis in mice. J. Immunol.
152: 4801–4807.
35. Mertsching, E., U. Grawunder, V. Meyer, T. Rolink, and R. Ceredig. 1996. Phe-
notypic and functional analysis of B lymphopoiesis in interleukin-7- transgenic
mice: expansion of pro/pre-B cell number and persistence of B lymphocyte de-
velopment in lymph nodes and spleen. Eur. J. Immunol. 26: 28–33.
36. Ceredig, R., J. Andersson, F. Melchers, and A. Rolink. 1999. Effect of deregu-
lated IL-7 transgene expression on B lymphocyte development in mice express-
ing mutated pre-B cell receptors. Eur. J. Immunol. 29: 2797–2780.
37. Ceredig, R. 2002. The ontogeny of B cells in the thymus of normal, CD3e knock-
out (KO), RAG-2 KO and IL-7 transgenic mice. Int. Immunol. 14: 87–99.
38. Ceredig, R., and T. Rolink. 2002. Opinion: a positive look at double-negative
thymocytes. Nat. Rev. Immunol. 2: 888– 897.
39. Surh, C. D., and J. Sprent. 2000. Homeostatic T cell proliferation: how far can T
cells be activated to self-ligands? J. Exp. Med. 192: F9–F14.
40. Goldrath, A. W., L. Y. Bogatzki, and M. J. Bevan. 2000. Naive T cells transiently
acquire a memory-like phenotype during homeostasis-driven proliferation.
J. Exp. Med. 192: 557–564.
41. Tanchot, C., A. Le Campion, B. Martin, S. Leaument, N. Dautigny, and B. Lucas.
2002. Conversion of naive T cells to a memory-like phenotype in lymphopenic
hosts is not related to a homeostatic mechanism that fills the peripheral naive T
cell pool. J. Immunol. 168: 5042–5046.
42. Ge, Q., H. Hu, H. N. Eisen, and J. Chen. 2002. Different contributions of thy-
mopoiesis and homeostasis-driven proliferation to the reconstitution of naive and
memory T cell compartments. Proc. Natl. Acad. Sci. USA 99: 2989–2994.
43. Dummer, W., B. Ernst, E. LeRoy, D. Lee, and C. Surh. 2001. Autologous reg-
ulation of naive T cell homeostasis within the T cell compartment. J. Immunol.
166: 2460–2468.
44. Ceredig, R., and C. Waltzinger. 1990. Neonatal mouse CD4
⫹
mature thymocytes
show responsiveness to interleukin 2 and interleukin 7: growth in vitro of neg-
atively selected V

6- and V

11-expressing CD4
⫹
cells from (C57BL/6 ⫻DBA/
2)F
1
mice. Int. Immunol. 2: 869– 877.
45. Seddon, B., and R. Zamoyska. 2003. Regulation of peripheral T-cell homeostasis
by receptor signalling. Curr. Opin. Immunol. 15: 321–324.
46. Kondrack, R. M., J. Harbertson, J. T. Tan, M. E. McBreen, C. D. Surh, and
L. M. Bradley. 2003. Interleukin 7 regulates the survival and generation of mem-
ory CD4 cells. J. Exp. Med. 198: 1797–1806.
47. Grayson, J. M., A. J. Zajac, J. D. Altman, and R. Ahmed. 2000. Cutting edge:
increased expression of Bcl-2 in antigen-specific memory CD8
⫹
T cells. J. Im-
munol. 164: 3950–3954.
48. Goldrath, A. W., C. J. Luckey, R. Park, C. Benoist, and D. Mathis. 2004. The
molecular program induced in T cells undergoing homeostatic proliferation.
Proc. Natl. Acad. Sci. USA 101: 16885–16890.
49. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and
R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies
effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4:
1191–1198.
50. Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4
⫹
and
CD8
⫹
effector and memory T cell generation. Nat. Immunol. 4: 835– 842.
51. Kovanen, P. E., and W. J. Leonard. 2004. Cytokines and immunodeficiency dis-
eases: critical roles of the
␥
-dependent cytokines interleukins 2, 4, 7, 9, 15, and
21, and their signaling pathways. Immunol. Rev. 202: 67–83.
52. Park, J. H., Q. Yu, B. Erman, J. S. Appelbaum, D. Montoya-Durango,
H. L. Grimes, and A. Singer. 2004. Suppression of IL7R
␣
transcription by IL-7
and other prosurvival cytokines; a novel mechanism for maximizing IL-7-depen-
dent T cell survival. Immunity 21: 289–302.
53. Ge, Q., D. Palliser, H. N. Eisen, and J. Chen. 2002. Homeostatic T cell prolif-
eration in a T cell-dendritic cell coculture system. Proc. Natl. Acad. Sci. USA 99:
2983–2988.
54. Agenes, F., and A. A. Freitas. 1999. Transfer of small resting B cells into im-
munodeficient hosts results in the selection of a self-renewing activated B cell
population. J. Exp. Med. 189: 319–330.
55. Freitas, A. A., and B. Rocha. 2000. Population biology of lymphocytes: the flight
for survival. Annu. Rev. Immunol. 18: 83–111.
56. Traggiai, E., L. Chicha, L. Mazzucchelli, L. Bronz, J. C. Piffaretti,
A. Lanzavecchia, and M. G. Manz. 2004. Development of a human adaptive
immune system in cord blood cell-transplanted mice. Science 304: 104–107.
170 INCREASING IL-7 AVAILABILITY ALTERS T CELL HOMEOSTASIS
