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doi:10.1182/blood.V97.6.1525
2001 97: 1525-1533
Terry J. Fry, Barbara L. Christensen, Kristin L. Komschlies, Ronald E. Gress and Crystal L. Mackall
depleted hosts−Interleukin-7 restores immunity in athymic T-cell
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Plenary paper
Interleukin-7 restores immunity in athymic T-cell–depleted hosts
Terry J. Fry, Barbara L. Christensen, Kristin L. Komschlies, Ronald E. Gress, and Crystal L. Mackall
Thymic-deficient hosts rely primarily on
antigen-driven expansion to restore the
peripheral T-cell compartment following
T-cell depletion (TCD). The degree to
which this thymic-independent pathway
can restore immune competence remains
poorly understood but has important im-
plications for a number of clinical condi-
tions including stem cell transplantation
and human immunodeficiency virus (HIV)
infection. A model of HY-mediated skin
graft rejection by athymic, TCD mice was
used to show that restoration of naive
and recall responses via peripheral expan-
sion requires transfer of only 25 ⴛ106
lymph node (LN) cells representing ap-
proximately 10% of the T-cell repertoire.
Consitutive expression of bcl-2 in the
expanding inocula restored recall re-
sponses to HY at a substantially lower LN
cell dose (1 ⴛ106), which is normally
insufficient to induce HY-mediated graft
rejection in athymic hosts. Interestingly,
bcl-2 had no effect on primary responses.
Interleukin-7 (IL-7) potently enhanced thy-
mic-independent peripheral expansion
and led to HY graft rejection using an LN
cell dose of 1 ⴛ106in both primary and
recall models. The restoration of immune
competence by IL-7 appeared to be medi-
ated through a combination of pro-
grammed cell death inhibition, improved
costimulation, and modulation of antigen-
presenting cell (APC) function. These re-
sults show that immune competence for
even stringent antigens such as HY can
be restored in the absence of thymic
function and identify IL-7 as a potent
modulator of thymic-independent T-cell
regeneration. (Blood. 2001;97:1525-1533)
©2001 by The American Society of Hematology
Introduction
Acute depletion of the T-cell compartment occurs in a number of
clinical situations including stem cell transplantation, human
immunodeficiency virus (HIV) infection, and after intensive cyto-
toxic therapy for cancer. Restoration of immune competence
following T-cell depletion (TCD) requires the regeneration of a
functionally intact, diverse T-cell pool that is capable of responding
to foreign antigens and, potentially, altered self-antigens expressed
by tumors. Previous studies have shown that there are 2 main
pathways capable of substantial peripheral T-cell regeneration:
thymic-dependent regeneration from bone marrow progenitors and
thymic-independent peripheral expansion of mature T-cell popula-
tions.1The relative contribution of these 2 pathways is dynamic and
dependent on the degree of thymic function. Current concepts hold
that the capacity of the host to restore immune competence depends
primarily on the extent to which thymic pathways contribute to
T-cell regeneration. However, due to disease, therapy-related
toxicity, and age-related changes, thymic function is frequently
limiting, resulting in a relative reliance on thymic-independent
peripheral expansion in many clinical situations associated with
TCD.2-8 The degree to which the peripheral expansion of mature
T-cell populations can restore host immune competence following
TCD remains poorly understood and is the focus of this report.
Peripheral expansion is predicted to give rise to limited immune
competence for a variety of reasons. First, although dramatic
expansions in cell number can occur,9-12 hosts reconstituted in this
manner display chronically reduced T-cell numbers.10,13 Second,
because the expansion of peripheral T cells via this process
proceeds from limited numbers of cells and is heavily influenced by
interactions with cognate antigen,14 the regenerated repertoire is
susceptible to dramatic skewing15 and shows limited T-cell receptor
diversity.16-19 Third, the process of peripheral expansion leads to an
accumulation of T cells that display a highly activated pheno-
type.13,20 Continuous or repeated exposure of these activated T cells
to antigens that contribute to the process of expansion could also
provide a signal for programmed cell death.21,22 This is suggested
by studies in patients with TCD due to HIV infection showing
increased susceptibility of T cells to apoptosis with restimulation in
vitro23-27 and by direct labeling studies in vivo.28-30 Although these
observations could potentially relate to direct or indirect effects of
HIV, similar results have been observed in T-cell–depleted hosts
after intensive chemotherapeutic regimens20 and after stem cell
transplantation,31 suggesting that increased susceptibility to cell
death is a common phenomenon in regenerating T-cell populations.
Thus, a propensity for programmed cell death in peripherally
expanding T-cell populations may also limit the capacity of
peripheral expansion to restore host immune competence.
Interleukin-7 (IL-7) exerts potent effects on T- and B-cell
progenitors and is absolutely required for early T-cell develop-
ment.32-34 Because T-cell development can be largely restored in
IL-7 knockout mice by the concurrent expression of a bcl-2
From the Molecular Oncology Section, Pediatric Branch, National Cancer
Institute, National Institutes of Heath, Bethesda, Maryland; Intramural Research
Support Program, SAIC Frederick, National Cancer Institute-Frederick, Frederick,
Maryland; and Experimental Immunology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland.
Submitted June 20, 2000; accepted November 7, 2000.
The contents of this publication do not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does mention of trade
names, commercial products, or organizations imply endorsement by the U.S.
Government. This project was funded in whole or part with funds from the
National Cancer Institute, National Institutes of Health, under contract no. NO1-
CO-56000. By acceptance of this article, the publisher or recipient
acknowledges right of the U.S. Government to retain a nonexclusive, royalty-
free license in and to any copyright covering the article.
Reprints: Terry J. Fry, Bldg 10, Rm 13N240, MSC 1928, 10 Center Dr,
Bethesda, MD, 20892-1928; e-mail: tf60y@nih.gov.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
1525BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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transgene,35,36 current models suggest that IL-7 acts primarily by
inhibiting programmed cell death of immature thymocytes during
development. Indeed, the administration of IL-7 after cyclophospha-
mide or bone marrow transplantation in mice leads to enhanced
T-cell reconstitution, presumably via enhanced thymopoiesis.37-41
Recent studies have shown that IL-7 is also capable of rescuing
mature T cells from apoptosis induced by glucocorticoids, cytokine
withdrawal, and radiation.42-47 These effects are correlated with
alterations in intracellular levels of bcl-2, bcl-xL, and caspase 3
activity, suggesting modulation of intracellular pathways of pro-
grammed cell death. In this report, we sought to examine the
capacity of peripheral expansion to restore immune competence
following TCD and to investigate the role of programmed cell
death in limiting immune competence following reconstitution by
peripheral expansion. Our results show that programmed cell death
plays an important role in limiting immune competence in thymic-
deficient hosts undergoing T-cell regeneration and, in addition, we
have identified IL-7 as a cytokine that dramatically enhances
immune competence in thymic-deficient hosts reconstituted via
this pathway.
Materials and methods
Mice and thymectomies
C57BL/6 (B6/Thy-1.2, Ly-5.1), B6 PLThy-1a (B6/Thy 1.1), and B6/Ly-5.2
mice were purchased from the Animal Production Unit, National Cancer
Institute (NCI; Frederick, MD) and housed in a specific pathogen-free
environment at the National Institutes of Health (NIH). At 4 to 6 weeks of
age, mice were anesthetized and suction thymectomy was performed
through a sternal incision according to approved protocol. Completeness of
the thymectomies was confirmed by visual inspection at the completion of
the experiments. C57BL/6-TgN(BCL2)36Wehi mice48 were originally
purchased from Jackson Laboratories (Bar Harbor, ME) and bred at the
National Institutes of Health. Mice were screened for human bcl-2 (hbcl-2)
transgene expression at 8 weeks of age. A small aliquot of blood was
obtained from the tail followed by separation of mononuclear cells over
lymphocyte separation media (Biowhitaker, Walkersville, MD). The pres-
ence of hbcl-2 protein was determined by flow cytometry using intracellular
labeling with a human-specific, monoclonal antibody (clone 6C8, Pharmin-
gen, San Diego, CA) and detected with goat antihamster IgG fluorescein
isothiocyanate (FITC; Southern Biotechnology Associates, Birmingham,
AL). The validation of this screening technique was confirmed by
polymerase chain reaction (PCR) analysis. Transgene-negative littermates
were used as control lymph node donors. C57BL/6 IL-7R␣⫺⫺ mice were
purchased from Jackson Laboratories and bred at the NCI-Frederick.
TCD and skin grafting
Mice were depleted of T cells using rat antimouse anti-CD4 (clone GK1.5)
and anti-CD8 (clone 2.43) purchased from the Biological Resources
Branch, NCI (Frederick, MD). Injections were given intraperitoneally with
schedules and doses determined for individual lots by in vivo experiments
to achieve more than 98% depletion of CD4 and CD8 cells in spleen and
lymph node (LN) at 4 days (data not shown). After 2 weeks, to allow
clearance of the monoclonal antibodies, mice were injected with LN
populations as indicated for the individual experiments. Skin grafting was
performed using a modification of a protocol described elsewhere49 with
a 0.5-cm patch of male tail skin grafted over the thorax and covered with a
pressure dressing for 7 days. After 1 week, bandages were removed and a
blinded observer monitored graft rejection. Complete rejection was defined
as more than 80% of the graft surface area involved. Animal care was
provided in accordance with procedures outlined in the “Guide for the Care
and Use of Laboratory Animals” (NIH Publication no. 86-23, 1996) and all
protocols were approved by the animal care and use committee at the NCI.
Cell injections
Axillary and inguinal LNs were harvested from syngeneic female mice and
placed in iced complete media (RPMI, with 10% heat-inactivated fetal
bovine serum, penicillin, streptomycin, L-glutamine, Hepes buffer, nones-
sential amino acids, sodium pyruvate (all from Gibco Life Technologies,
Gaithersburg, MD), and -mercaptoethanol (Sigma, St. Louis, MO). LNs
were teased apart with fine forceps, gently minced with the plunger of a
syringe, and passed through a nylon mesh. The cells were washed twice in
iced complete media, viable cell number was determined with trypan
blue exclusion, and cells were resuspended at the appropriate concentration
for injection via tail vein in RPMI without fetal calf serum at the
doses described.
Male dendritic cells were purified from splenocytes following plate
adherence and incubation with IL-4 and granulocyte-macrophage colony-
stimulating factor (GM-CSF). Briefly, male spleens were minced with the
plunger of a syringe in iced complete media and passed through a nylon
mesh. Red blood cells (RBCs) were lysed with ammonium hydroxide
lysing buffer, washed twice in complete media, resuspended in iced
Dulbecco phosphate-buffered saline (DPBS; Gibco Life Technologies) and
separated over a 50% Percoll gradient by centrifugation at 1800gfor 12
minutes. The cell layer was removed, washed twice in complete media,
resuspended, and counted using trypan blue exclusion. Viable cells
(100 ⫻106) were loaded onto a 150 ⫻25-mm tissue culture dish (Falcon
3025, Becton Dickinson, Lincoln Park, NJ), incubated at room temperature
for 20 minutes followed by incubation at 37°C/5% CO2for 2 hours.
Nonadherent cells were removed by washing with 50 mL warm complete
media, the media was replaced with complete media containing 1 ng/mL
GM-CSF and 0.1 g/mL IL-4 (Peprotech, Rocky Hill, NJ), and the plate
was incubated overnight at 37°C/5% CO2. On day 2, the nonadherent
fraction was removed by gentle pipetting, washed twice in complete media,
resuspended, and counted. A fraction of the cell suspension was analyzed
with flow cytometry and the remainder was injected in RPMI intraperitone-
ally at a dose of 1 ⫻105cells per mouse.
In vitro studies
Single-cell suspensions of RBC-depleted splenocytes were made as de-
scribed followed by passage over a negative selection T-cell enrichment
column (R&D Systems, Minneapolis, MN) according to the manufacturer’s
instructions. The enriched fraction was washed, counted, and resuspended
in complete media. The 24-well plates were coated for 18 hours with 0.1
g/mL rat antimouse CD3 (clone 145-2C11) in DPBS or with DPBS alone.
This concentration of 2C11 is insufficient to induce maximal T-cell
proliferation (data not shown). T-cell–enriched splenocytes were plated at
4⫻106cells/well in 2 mL complete media. Recombinant human IL-7
(Peprotech) was added at a concentration of 10 ng/mL. At the time points
indicated, wells were washed, pooled, and analyzed with flow cytometry.
For proliferation studies, T-cell–enriched splenocytes were labeled with
CFSE (5-[and 6]-carboxyfluorescein diacetate succinimidyl ester; Molecu-
lar Probes, Eugene, OR) according to the manufacturer’s instructions.
Labeled cells were incubated on a 96-well plate coated with anti-CD3 as
described above.
Flow cytometric analysis
Cell suspensions were prepared in staining buffer (Hanks balanced salt
solution without phenol red with 0.2% human serum albumin [Sigma] and
0.1% sodium azide [Sigma]). For intracellular labeling, cell suspensions
were washed in staining buffer containing 0.03% saponin (Sigma). Prior to
antibody labeling, FC␥III/II receptors were blocked with monoclonal antibody
(clone 2.4G2). The monoclonal antibodies used were CD4 phycoerythrin (PE)
(clone CT-CD4) and CD8a PE (clone CT-CD8a) (Caltag Laboratories, Burlin-
game, CA), TCR chain PE (clone H57-597), CD45R/B220PE (clone RA3-
6B2), CD11b PE (clone M1/70), CD11c FITC (clone HL3), CD45.1/Ly5.2 FITC
(clone A20), CD45.2/Ly5.1 FITC (clone 104), CD54/ICAM-1 FITC (clone 3E2),
CD80/B7-1 FITC (clone 1G10), CD86/B7-2 FITC (clone GL-1), CD90.1/Thy
1.1 FITC (clone HIS51), and CD90.2/Thy1.2 FITC (clone OX-7) (Pharmingen).
Intracellular murine bcl-2 was detected with purified hamster antimouse bcl-2
1526 FRY et al BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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(clone 3F11; Pharmingen) and detected with goat antihamster IgG FITC
(Southern Biotechnology Associates).Appropriate isotype controls were used for
all experiments. Annexin V labeling was performed in phosphate-buffered saline
(PBS) containing 10 mM NaOH, 140 mM NaCl, and 2.5 mM CaCl2. Cells were
then analyzed in buffer containing 2 g/mL propidium iodide (Sigma).
Flow cytometric analysis was performed on a single laser FACScan or
dual laser FACSCalibur (Becton Dickinson). Fluorescence data were collected
and analyzed using CELLQuest software. Viable lymphocyte populations were
gated based on forward-scatter and side-scatter characteristics.
Statistical analysis
Survival analysis was performed on the HY skin graft rejection experiments
considering the time of complete rejection in days as the end-point. A
dichotomous variable of 1 was assigned if a rejection occurred during the
period of observation (100 days) and value of 0 was assigned if rejection did
not occur and the event was censored. For each comparison, a log-rank test
was performed and the 2-sided Pvalue is reported.
Results
Restoration of immune competence in athymic hosts
reconstituted via peripheral expansion is critically dependent
on the size of the expanding inocula
To assess the capability of hosts reconstituted via peripheral
expansion to respond to recall antigen, thymectomized, T-cell–
depleted C57BL/6 females were grafted with male tail skin and
injected intravenously with 1 ⫻106primed LN cells as a source of
mature T cells. In the recall model, primed cells refer to LN cells
taken from syngeneic female mice within 3 weeks after complete
male skin graft rejection. Thymus-bearing mice reject HY-disparate
skin grafts within 8 weeks. In contrast, thymectomized, T-cell–
depleted mice were unable to reject HY-disparate skin grafts
despite administration of primed LN cells as a source for peripheral
expansion. Importantly, these mice are capable of rejecting alloge-
neic skin grafts within 8 to 10 days (data not shown). Therefore, the
impairment in T-cell immune competence following reconstitution
via thymic-independent pathways is limited to responses to antigen
with a low precursor frequency suggesting that a simple limitation
in the number of cells bearing antigen-specific T-cell receptor
specificities may play a central role.
To determine the degree to which the deficiency in HY graft
rejection observed following thymic-independent T-cell regenera-
tion was related to an inadequate starting T-cell inocula, and, if so,
to establish the size of the inocula required to restore responses to
recall antigen, thymectomized T-cell–depleted mice were reconsti-
tuted using progressively larger primed mature LN inocula. As the
dose of primed LN cells was increased, there is an increase in the
rate of graft rejection (Figure 1A). Indeed, T-cell–depleted mice
reconstituted from 25 ⫻106primed LN cells were able to reject
HY grafts at a rate analogous to that observed in control groups.
To examine the induction of primary immune responses after
thymic-independent regeneration, thymectomized T-cell–depleted
mice were injected with LN cells from syngeneic female mice
naive to male antigen at the same time as enriched male dendritic
cells as a sensitizing population. In this model sensitization must
occur following transfer into T-cell–depleted hosts. In T-cell–
replete mice, sensitization with enriched male dendritic cells
accelerated the rate of graft rejection by approximately 2 weeks,
confirming the ability of these cells to sensitize to HY antigen
(Figure 1B). In contrast, as seen in the recall experiments,
thymectomized T-cell–depleted mice reconstituted with 1 ⫻106
naive LN cells failed to reject male grafts despite sensitization with
enriched male dendritic cells. Importantly, titration of the lymph
node dose to 25 ⫻106LN cells led to complete restoration of
responses to HY antigen.
Therefore, in both the primary response and the recall model,
limitations in the capacity of athymic hosts to respond to nominal
antigen could be overcome by supplying increased numbers of T
cells for peripheral expansion. These results confirm that the
impairment in T-cell responses observed in thymic-deficient hosts
is not absolute and can be induced in this setting if sufficient T-cell
numbers are provided.
Inhibition of apoptosis during reconstitution from a limited
T-cell inocula restores responses to recall antigen
but not to naive antigen
To study the mechanisms that contribute to the limitation in T-cell
responses following immune reconstitution via peripheral expan-
sion, subsequent experiments attempted to identify factors limiting
peripheral expansion in hosts reconstituted from suboptimal num-
bers of T cells. Based on observations made in clinical settings
associated with TCD,20,31 we hypothesized that increased apoptosis
Figure 1. Rejection of HY-disparate skin grafts via peripherally expanded
lymph node cells is critically dependent on the size of the starting inocula.
(A) Adult thymectomized, C57BL/6 females were T cell depleted and grafted with
male tail skin as described in “Materials and methods.” Primed cells were
collected from the draining LN of T-cell–replete syngeneic female mice 3 weeks
after successful male skin graft rejection, teased into a single-cell suspension,
and injected via the tail vein 24 hours after skin grafting. Unprimed cells were
collected from LN of T-cell–replete females naive to male antigen. Percent
surviving grafts are shown as measured by visual inspection as described in
“Materials and methods.” Thymectomized, T-cell–depleted mice (TXY/TCD mice)
receiving 5 ⫻106primed LN cells are partially able to reject HY-disparate skin
grafts (f,n⫽4) when compared to mice receiving no LN inocula (F,n⫽5).
When the LN cell dose is increased to 10 ⫻106, there is rejection in all mice but
the rate of graft rejection remains delayed (䉬,n⫽5). At a dose of 25 ⫻106LN
cells (Œ, solid line, n ⫽5), graft rejection occurs at a rate analogous to
thymus-bearing control animals (, dashed line, n ⫽6) also receiving primed
inocula. Note the modest effect of the primed inocula when this group is compared
to thymus-bearing animals receiving an unprimed inocula (ƒ, dashed line, n ⫽5).
(B) To assess primary immune responses, TXY/TCD mice were given graded
numbers of LN cells from syngeneic females naive to male antigen via tail vein.
The mice were then sensitized by intraperitoneal injection of 1 ⫻105enriched
male dendritic cells from male splenocytes as described in “Materials and
methods.” These cells express B7-1, B7-2, CD11c, and major histocompatibility
complex (MHC) class II and represent 50% to 60% of the cells injected in all
experiments. Twenty-one days after LN cell transfer and enriched male dendritic
cell sensitization, the mice were grafted with male tail skin as before and observed
for rejection. As with the recall responses, mice receiving 1 ⫻106naive cells were
unable to reject HY-disparate skin grafts (F,n⫽5). Administration of 10 ⫻106
naive cells led to graft rejection in all of the mice but at a delayed rate (f,n⫽7).
Transfer of 25 ⫻106LN cells (},n⫽7) completely restored responses so that
rejection occurred at similar rate to thymus-bearing control groups receiving
enriched dendritic cells (Œ, dashed line, n ⫽6). Thymus-bearing control groups
receiving no enriched dendritic cells (, dashed line, n ⫽5) required approxi-
mately 2 weeks longer to reject HY grafts.
RESTORATION OF IMMUNITYBY IL-7 1527BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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of peripherally expanding T cells could limit immune competence
in this setting. To determine whether expanding populations are
susceptible to programmed cell death in our model, T cells from
thymectomized, T-cell–depleted C57BL/6 (Thy-1.2) mice reconsti-
tuted from 10 ⫻106congenic B6 PL Thy-1a(Thy-1.1) LN cells
were analyzed for apoptosis at various time points after transfer.
When analyzed on the day of collection there was no evidence for
increased T-cell apoptosis in peripherally expanding cells when
compared to normal, nonexpanding populations. However, as
shown in Figure 2, peripherally expanding congenic T cells from
thymectomized, T-cell–depleted animals collected on day 15 after
transfer and restimulated with concanavalin A for 18 hours show
increased annexin V staining when compared to nonexpanding
controls. This provides evidence that T cells undergoing peripheral
expansion in thymectomized T-cell–depleted hosts are susceptible
to apoptotic cell death on restimulation and suggested that a loss of
T cells to apoptosis during peripheral expansion from an insuffi-
cient inocula could potentially play a role in limiting T-cell
responses in these hosts.
Although the finding of increased annexin V staining of
restimulated, peripherally expanding T cells, along with the
findings of other investigators,20,31 suggests that these cells are
prone to apoptosis, it is unclear whether this process contributes
to the limitation in immune responses observed in hosts
reconstituted in this manner. If the loss of peripherally expand-
ing T cells to apoptosis in our skin graft rejection model
contributes to the inability to induce responses to HY antigen in
animals reconstituted from a suboptimal T-cell number, we
hypothesized that inhibition of apoptosis in vivo could restore
HY-mediated graft rejection. Thymectomized T-cell–depleted
mice were injected with 1 ⫻106primed LN cells from female
mice expressing an hbcl-2 transgene in lymphocytes. T cells
from these animals are resistant to apoptotic cell death induced
by a number of mechanisms including radiation and cytokine
withdrawal.50,51 As shown in Figure 3A, mice reconstituted via
peripheral expansion of 1 ⫻106primed, hbcl-2 transgenic LN
cells were able to rapidly reject HY-disparate skin grafts,
whereas mice reconstituted from the same size inocula of LN
cells from nontransgenic littermate controls were unable to
reject these grafts by 100 days. Therefore, inhibition of apopto-
sis via constitutive bcl-2 expression during peripheral expansion
is sufficient to restore recall responses to nominal antigen,
thereby confirming the role of apoptosis in limiting immune
responses in this setting. Interestingly, no significant increase in
the rate of graft rejection was observed in T-cell–replete hosts
suggesting that programmed cell death is uniquely limiting in
the setting of TCD.
Given the similar results obtained in the LN cell titration
experiments between recall and primary responses detailed above,
we predicted that the constitutive expression of bcl-2 transgene in
peripherally expanding T cells would also lead to restoration of
primary immune responses to HY. Surprisingly, however, 1 ⫻106
naive hbcl-2 transgenic LN cells sensitized with enriched male
dendritic cells failed to restore responses to HY antigen in athymic,
T-cell–depleted hosts (Figure 3B). Indeed, the same size inocula of
both transgenic and nontransgenic LN cells (25 ⫻106) was re-
quired to fully restore HY responses in this model. These results
indicate that, unlike responses to recall antigen, constitutive bcl-2
expression in T cells undergoing thymic-independent regeneration
from a limited inocula is insufficient to induce primary responses to
HY antigens in thymic-deficient hosts and suggests that additional
factors beyond the susceptibility to programmed cell death contrib-
ute to limitations in induction of primary T-cell responses in
thymic-deficient hosts.
IL-7 has diverse effects on mature T cells in vitro, and in vivo
administration leads to enhanced peripheral expansion
The T cells undergoing peripheral expansion display an activated
phenotype regardless of activation status prior to injection into
Figure 2. Increased annexin V staining of peripherally expanding T cells.
TXY/TCD C57BL/6 (Thy 1.2) mice were injected with 10 ⫻106congenic LN cells
from B6 PL Thy-1a females (Thy 1.1). At 5, 11, and 21 days, mice were killed for flow
cytometric analysis of apoptosis in Thy 1.1⫹T cells. This figure is a representative
histogram showing annexin V staining on Thy 1.1⫹propidium iodide–negative
splenocytes (gray line) at 11 days after an 18 hours incubation with concanavalin A
compared to Thy 1.2⫹splenocytes from thymus-bearing, T-cell–replete controls
(black line) also incubated with con A for 18 hours. In a separate experiment there
was no increased annexin V staining on Thy 1.2⫹splenocytes from thymectomized
T-cell–replete females when compared to sham thymectomized animals treated in
the same manner (data not shown).
Figure 3. Inhibition of programmed cell death by constitutive expression of
hbcl-2 in peripherally expanding T cells restores recall responses to HY-
disparate skin grafts at suboptimal inocula but fails to restore primary
responses. (A) TXY/TCD mice were reconstituted with 1 ⫻106primed LN cells
constitutively expressing hbcl-2 (F,n⫽9) 24 hours after placement of a male tail skin
graft. This resulted in restoration of HY-disparate skin graft rejection when compared
to mice receiving unprimed hbcl-2 transgenic inocula (f,n⫽6) or primed cells from
nontransgenic littermate controls (䉬,n⫽9). Primed hbcl-2 transgenic inocula did not
enhance graft rejection in thymus-bearing controls (, dashed line, n ⫽5) when
compared to primed inocula from nontransgenic littermate controls (Œ, dashed line,
n⫽5,
P
⫽.49). (B) Reconstitution of TXY/TCD mice with 1 ⫻106naive hbcl-2
transgenic LN cells followed by sensitization with enriched male dendritic cells does
not restore rejection of HY-disparate skin grafts placed 3 weeks after cell transfers
(F,n⫽5). Titration of the inocula to 10 ⫻106(f,n⫽5) and 25 ⫻106(䉬,n⫽5)
leads to HY-disparate graft rejection at a rate comparable to thymus-bearing controls
(Œ, dashed line, n ⫽5) analogous to nontransgenic inocula (Figure 2B).
1528 FRY et al BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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T-cell–depleted hosts.15 Therefore, modulation of T-cell activation
could potentially enhance the process of peripheral expansion. To
examine the effects of IL-7 on the activation of mature T cells in
vitro, T-cell–enriched splenocytes were stimulated with plate-
bound anti-CD3 antibody at a suboptimal concentration with and
without recombinant human IL-7 (rhIL-7). By day 5, T cells
incubated with rhIL-7 plus anti-CD3 displayed increased bcl-2
protein (Figure 4A) consistent with the antiapoptotic effect of this
cytokine. In addition, prelabeling of T cells with CFSE indicated
that rhIL-7 enhanced proliferation (Figure 4B). By day 7, the
number of viable T cells recovered after incubation with rhIL-7
plus anti-CD3 was significantly increased when compared to
unstimulated groups or to groups incubated with anti-CD3 alone
(Figure 4C). Consistent with prior reports,42-46 these results indicate
that IL-7 can have multiple effects on mature T cells including an
antiapoptotic effect and a costimulatory effect and, therefore, may
be particularly capable of modulating peripheral expansion in vivo.
To test this hypothesis, rhIL-7 was administered at a dose of
5g/d intraperitoneally for 28 days to thymectomized, T-cell–
depleted C57Bl/6 Ly5.2 mice during peripheral expansion of
10 ⫻106Ly5.1⫹LN cells. In addition, in the same experiment,
the potential for peripheral expansion of hbcl-2 transgenic (Ly
5.1) inocula was determined. As seen in Figure 4D, in vivo
administration of rhIL-7 led to a significant increase in Ly5.1⫹
T-cell number. A similar result was observed in mice receiving
hbcl-2 transgenic inocula, further confirming the role for
apoptosis in limiting peripheral expansion. Therefore, both
rhIL-7 treatment and constitutive expression of bcl-2 numeri-
cally enhance the peripheral expansion of mature T cells in
athymic T-cell–depleted hosts.
IL-7 treatment restores recall and primary immune responses
in athymic, T-cell–depleted hosts undergoing immune
reconstitution from a limited T-cell inocula
Due to the in vitro effects of IL-7 and the potent enhancement of
peripheral expansion observed in vivo, we tested whether IL-7
could restore recall responses during reconstitution from subop-
timal inocula. As shown in Figure 5A, administration of rhIL-7
during peripheral expansion from a limited primed LN inocula
(1 ⫻106) leads to rapid rejection of HY-disparate skin grafts.
Importantly, rejection of male skin grafts in all animals required
administration of rhIL-7 along with a primed LN inocula,
providing evidence that the effect of IL-7 in this model was
dependent on the presence of a population of mature T cells
undergoing peripheral expansion. We also tested whether the
ability of IL-7 to restore immune responses extended to primary
responses. As with recall responses, administration of rhIL-7
also led to rapid rejection of HY-disparate skin grafts in mice
reconstituted from a limited naive LN inocula and sensitized
with enriched male dendritic cells (Figure 5B). The rhIL-7
appeared to have no effect on responses in T-cell–replete hosts
in both recall and primary models. Therefore, IL-7 potently
restores immune competence in thymic-deficient hosts undergo-
ing T-cell reconstitution via peripheral expansion from a limited
Figure 4. IL-7 has diverse effects on mature T cells in vitro and leads to
enhanced peripheral expansion in vivo. Female C57BL/6 splenocytes were
enriched for T cells using a positive selection column as described in “Materials
and methods.” T-cell–enriched splenocytes were incubated in 24-well plates
precoated with anti-CD3 at a suboptimal concentration of 0.1 g/mL with or
without rhIL-7 at 10 ng/mL. At days 2, 5, and 7, wells were harvested. Viable cells
were counted with trypan blue exclusion and analyzed with flow cytometry. (A) By
day 5 of culture, there is increased bcl-2 fluorescence intensity in cells incubated
with rhIL-7 and stimulated by suboptimal anti-CD3 (heavy solid line) when
compared to suboptimal ␣CD3 alone (gray dotted line) or media (light solid line).
Isotype control shown as dark dotted line. (B) T-cell–enriched splenocytes were
prelabeled with CFSE according to the manufacturer’s instructions. At day 5,
there is decreased CFSE intensity in a subset of T cells from wells incubated with
suboptimal anti-CD3 in combination with rhIL-7 (solid line) when compared to
suboptimal anti-CD3 alone or media only (dotted lines) indicating increased
proliferation. Analogous results were obtained with H3incorporation and the
difference was not seen at optimal anti-CD3 stimulation (data not shown). (C)
Incubation with suboptimal anti-CD3 and rhIL-7 (f) results in increased viable
T-cell number per well by days 5 and 7 compared to controls with suboptimal
anti-CD3 (F) or media alone (䉬). (D) TXY/TCD, C57BL/6 Ly 5.2 mice were
injected intravenously with an inocula of 10 ⫻106LN cells from normal or hbcl-2
transgenic donors (both Ly 5.1). One group receiving nontransgenic LN cells was
treated daily with rhIL-7 at a dose of 5 g/d intraperitoneally for 28 days. By day
28, there was a significant increase in the number of Ly 5.1⫹T cells in the
rhIL-7–treated group (n ⫽7,
P
⫽.004) or the group receiving hbcl-2 transgenic
inocula (n ⫽7,
P
⫽.007) compared to control animals (n ⫽7). Data were
analyzed using an unpaired
t
test.
Figure 5. IL-7 potently restores both recall and primary responses to
HY-disparate skin grafts in T-cell–depletedhosts receiving suboptimal T cell
inocula. (A) TXY/TCD mice were grafted with male tail skin and injected with
1⫻106primed LN cells via tail vein 24 hours after grafting. Then rhIL-7 was
injected intraperitoneally at a dose of 5 g/d beginning on the day of primed cell
injection and continuing for 28 days. Animals receiving rhIL-7 and 1 ⫻106primed
LN cells, shown in previous experiments to be a suboptimal inocula (f,n⫽8),
rapidly rejected HY-disparate skin grafts at nearly the same rate as thymus-
bearing animals (Œ, dashed line, n⫽9,
P
⫽.2) and significantly faster than animals
injected with vehicle only plus primed LN cells (F,n⫽9,
P
⫽.0002) or rhIL-7 without
primed LN cells (䉬,n⫽6,
P
⫽.019). Note the lack of effect in thymus-bearing
animals injected with rhIL-7 (, dashed line, n ⫽5) compared to vehicle (Œ, dashed
line, n ⫽5). (B) TXY/TCD mice were injected with 1 ⫻106naive LN cells and
enriched male dendritic cells. The rhIL-7 at 5 g/d intraperitoneally was initiated on
the day of cell injection and continued for 28 days. Male skin grafting was performed
on day 21. Similar to recall responses, administration of rhIL-7 in the primary model
led to rapid rejection of HY-disparate skin grafts in TCD mice (F,n⫽8), whereas
mice receiving the same inocula and injected with vehicle were completely unable to
reject these grafts (f,n⫽7,
P
⫽.0002). Again, there was no difference between
thymus-bearing mice injected with rhIL-7 (Œ, dashed line, n ⫽6) or vehicle (䉬, dashed
line, n ⫽6).
RESTORATION OF IMMUNITYBY IL-7 1529BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
For personal use only. by guest on June 2, 2013. bloodjournal.hematologylibrary.orgFrom
inocula. These findings contrast with the experiments using
T-cell inocula constitutively expressing the bcl-2 transgene
where effects were only seen with recall responses. Thus, we
postulated that the ability of IL-7 to restore immune competence
in this setting involves other effects on peripherally expanding T
cells beyond a modulation of programmed cell death via
up-regulation of bcl-2. In addition, we could not exclude the
possibility that IL-7 may also enhance other cell populations
critical to the generation of immune responses.
Sensitization with IL-7R␣ⴚ/ⴚantigen-presenting cells partially
abrogates the effect of IL-7 on naive T-cell responses
To dissect possible effects of IL-7 on antigen-presenting cells
(APCs) from those on expanding T cells, mice were injected with a
suboptimal naive T-cell inocula as before, along with a sensitizing
population of enriched dendritic cells from IL7R␣⫺/⫺males. Thus,
the peripherally expanding T cells would still respond to IL-7, and
the APCs used for sensitization would be unresponsive due to a
lack of a functional receptor. Although the dendritic cell yield per
animal was lower from the IL-7R␣⫺/⫺mice than from IL-7R␣⫹/⫹
animals, the phenotype was identical (Figure 6). Further, as seen in
Figure 7, IL-7R␣⫺/⫺APCs are able to induce HY graft rejection in
animals receiving a sufficient LN inocula (25 ⫻106) at essentially
the same rate as thymus-bearing animals, thus establishing function-
ality of these APCs. However, when an insufficient LN inocula was
sensitized with enriched IL-7R␣⫺/⫺dendritic cells, administration
of rhIL-7 resulted in significantly delayed rejection relative to mice
receiving enriched dendritic cells capable of responding to IL-7
(P⫽.006). Therefore, a partial loss of the effect of IL-7 on
restoration of naive responses was seen, demonstrating that the
effects of IL-7 in this model are not limited to peripherally
expanding T cells but also include modulation ofAPCs.
Discussion
Depletion of T cells is a hallmark of a number of disease
processes and is an anticipated side effect of therapeutic
modalities such as cancer chemotherapy and stem cell transplan-
tation. Although much has been learned about the pathways that
lead to T-cell immune reconstitution, including the substantial
degree to which thymic-independent pathways can contribute to
this process, it remains unclear to what extent thymic-
independent pathways can restore host immune competence. In
this report, we describe a model that illustrates functional
deficits in immune responses to nominal antigen after thymic-
independent T-cell regeneration. Using this model, we have
shown that immune competence can be restored in the absence
of thymic pathways by modulation of the process of peripheral
expansion by increasing T-cell inocula, by inhibiting pro-
grammed cell death, and by cytokine administration.
A number of interesting findings emerge from these experi-
ments. First, in this murine model, the number of T cells
required to restore host immune competence (25 ⫻106LN cells
or approximately 20 ⫻106T cells) is approximately equal to
10% of the total T-cell number estimated for an intact mouse.52
Although the number required would be likely to vary depend-
ing on the antigen and the precursor frequency of the antigen-
specific T cells within the inocula, these results illustrate the
degree of redundancy that exists within the immune system.
What remains unclear is whether this number is relative and
must be increased proportionally to restore immune competence
in larger hosts (such as humans) or whether this represents an
absolute number of T-cell receptor specificities required for
response to diverse environmental antigens. Second, the finding
Figure 6. Identical phenotype of enriched dendritic cells from
normal and IL-7R␣ⴚ/ⴚmales. Dendritic cells were enriched from
male splenocytes following plate adherence and incubation with
IL-4 and GM-CSF as described in “Materials and methods.”
Before injection, portions of the cells were analyzed by flow
cytometry. Staining with FITC-conjugated antibodies expressed
by dendritic cells were analyzed against a cocktail of lineage-
specific antibodies conjugated to phycoerythrin (PE). The dashed
circle in each graph delineates the dendritic cells. Staining for PE
in the indicated cells represents intermediate labeling with anti-CD11b
with no expression of B220 or CD3 (data not shown), a finding
consistent with the observations of other investigators.65 The pheno-
type of dendritic cells from normal (top panels) and IL-7R␣⫺/⫺mice
(bottom panels), as characterized by this set of markers, was identical.
MHC indicates major histocompatibility complex.
Figure 7. Lack of IL-7 signaling in APCs leads to a partial abrogation of the
effect of IL-7 in restoring primary responses to HY skin grafts. TXY/TCD mice
were injected with naive LN cells and 1 ⫻105enriched dendritic cells from the
spleens of male IL-7R␣⫺/⫺mice or normal, IL-7R␣⫹/⫹males. The rhIL-7 at 5 g/d or
vehicle was initiated on the day of cell injections and continued for 28 days. Male skin
grafting was performed on day 21. IL-7R␣⫺/⫺APCs are functionally able to induce HY
graft rejection in animals receiving a sufficient T-cell inocula (25 ⫻106) with vehicle
(f,n⫽5) at a rate analogous to thymus-bearing animals receiving enriched
IL-7R␣⫹/⫹dendritic cells (,n⫽5). Mice receiving an insufficient LN inocula
(1 ⫻106) with vehicle are completely unable to reject these grafts (䉬,n⫽6). Mice
injected with a suboptimal T-cell inocula, sensitized with IL-7R⫺/⫺APCs and given
rhIL-7 (F,n⫽11) rejected HY-disparate skin grafts but significantly slower than mice
receiving IL-7R␣⫹/⫹dendritic cells (Œ,n⫽10,
P
⫽.006).
1530 FRY et al BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
For personal use only. by guest on June 2, 2013. bloodjournal.hematologylibrary.orgFrom
that restoration of immune responses can occur in the absence of
newly developed T-cell receptor specificities via thymic path-
ways emphasizes that the deficits in T-cell receptor repertoire
that result from peripheral expansion are relative rather than
absolute. Thus, by improving the efficiency of peripheral
expansion by inhibiting cell death and, potentially, by increasing
APC capacity, substantially lower numbers of antigen-specific T
cells may be adequate for successful induction of immune
responses. Third, the data show a clear role for programmed cell
death in limiting immune responses after thymic-independent
peripheral expansion. Although this is suggested by the finding
of increased annexin V staining on peripherally expanded T
cells manipulated ex vivo, it is more definitively shown by the
increased number of T cells derived from hbcl-2 transgenic
inocula and, more importantly, by restoration of immune
responses to nominal antigen by constitutive bcl-2 transgene
expression. Thus, peripherally expanding T cells that would
otherwise be lost to apoptotic cell death are rescued and,
therefore, contribute to the reconstitution of the T-cell pool.
However, restoration of immune responses by inhibition of
apoptosis appears to be confined to the setting of recall
responses indicating important differences in the requirements
for induction of responses to recall and naive antigens after
thymic-independent T-cell reconstitution. This phenomenon
may partially explain the restoration of recall responses and
protection from opportunistic infections, which has been ob-
served during immune reconstitution of HIV-infected pa-
tients53-56 and cannot be clearly attributed to restoration of T-cell
repertoire via thymic-dependent pathways.57
The widely accepted model for T-cell death holds that there
are 2 primary pathways that ultimately end in apoptosis.58,59 The
active process requires T-cell receptor stimulation, is mediated
by Fas ligand and other tumor necrosis factor (TNF) family
molecules, occurs at effective cytokine concentrations, and is
inefficiently inhibited by bcl-2. The passive pathway, such as
occurs with insufficient cytokine support, is strongly inhibited
by bcl-2. Based on this model, our results present a potential
paradox. The degree of activation of peripherally expanded T
cells and the requirement for antigen would suggest that the
limitations in immune responses observed in our experiments
are the result of active apoptosis of peripherally expanding T
cells. These models would predict that bcl-2 would be unable to
restore immune responses in this setting. Apotential explanation
for this finding may be that there is some degree of passive cell
death occurring during peripheral expansion due to competition
for limiting growth factors. Alternatively, it has been reported
that up-regulation of bcl-2 family members can lead to inhibi-
tion of Fas-mediated cell death in some situations.60,61 Whether
inhibition of the processes involved in active cell death would
have a more potent effect on immune responses in these models
is currently under study.
Interleukin-7 is a cytokine with extremely diverse and potent
effects on T- and B- lineage cells. It is uniquely suited to restore
immune competence after TCD because of its effects on
multiple pathways of T-cell regeneration. The effects of IL-7 on
thymopoiesis have been well characterized. Emerging data
suggest that IL-7 may also have effects on extrathymic T-cell
differentiation (K. Weinberg, written communication, 2000)
potentially providing a partial explanation for the restoration of
responses to HY antigen observed in some animals when rhIL-7
is administered without primed T cells. In addition, IL-7
has been shown to induce proliferation of naive T cells with-
out altering the phenotype.62 However, the effects of IL-7 on
mature T-cell populations in vivo are less well characterized
and the ability for this cytokine to enhance immune reconstitu-
tion via thymic-independent peripheral expansion has not been
reported previously. In this report we show that IL-7 potently
modulates the peripheral expansion pathway to T-cell regen-
eration. This action appears to be multifactorial and due to
effects on expanding T cells as well as effects on APC
populations. Modulation of APCs by IL-7 has not been reported
and the mechanisms of this effect are currently being investi-
gated in more detail (K. Komschlies, in preparation). Impor-
tantly, we have found IL-7 to be unique among the T-cell–active
cytokines tested in inducing peripheral expansion.63 This finding
raises the possibility that endogenously produced IL-7 could
also play a role in regulating this process during T-cell
regeneration and thus contribute to T-cell homeostasis. Indeed,
it is well known that TCD leads to enhanced expansion of
mature T cells and preliminary results from our laboratory (Fry
et al, manuscript submitted) in patients with HIV and studies by
others64 have shown elevated serum IL-7 levels in patients
with TCD.
Interleukin-7 may have potential application as a biologic
modifier in a number of clinical situations. First, after stem
cell transplantation, prolonged lymphodepletion results in sus-
ceptibility to infection that contributes to transplant-related
morbidity, especially in allogeneic transplants. Administration
of IL-7 during this period would be predicted to lead to more
rapid lymphocyte recovery, improved responses to foreign
antigens, and a reduction in mortality from infections in the
posttransplant period. Although there are clearly inherent dif-
ferences between our model, which uses antibody depletion
of T cells, and a stem cell transplantation model that uses
cytotoxic conditioning regimens, there are many similarities
including the reliance on peripheral expansion and the contribu-
tion of residual T cells to immune reconstitution. Thus, these
findings are likely to be relevant to clinical transplantation.
Second, although there has been success in substantially reduc-
ing viral load in patients with HIV infection, a proportion of
patients fail to show complete immunologic recovery and
discontinuation of antiretroviral agents results in eventual
rebound in the viral load. Therefore, one of the current
challenges in the treatment of HIV lies in restoring immune
responses to the virus. IL-7, with its ability to restore both recall
and naive responses after TCD, has the potential to be an
excellent adjuvant in this setting. Whether activation of resting
T cells will contribute to infection of new cells is not known.
Lastly, immunotherapeutic approaches to cancer treatment will
probably be most effective in the eradication of minimal
residual disease remaining after standard cytotoxic therapy.
However, as indicated by these findings, the TCD that results
will likely provide a major barrier to the success of these
approaches. Immune adjuvants such as IL-7 might vastly
improve the results of cancer immunotherapy trials that are
attempted in the setting of TCD.
Acknowledgment
We would like to thank Dr Scott Durum for his careful review of
this manuscript.
RESTORATION OF IMMUNITYBY IL-7 1531BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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Erratum
In the letter by Stachowitz et al entitled “Variable course of patients with
plaque psoriasis: lack of transformation into tumorous mycosis fungoides,”
which appeared in the June 1, 2000, issue of
Blood
(Volume 95:3635-3636),
the term “psoriasis” should have been “parapsoriasis” throughout.
RESTORATION OF IMMUNITYBY IL-7 1533BLOOD, 15 MARCH 2001 䡠VOLUME 97, NUMBER 6
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