of June 13, 2013.
This information is current as
the Recovery from Lymphopenia
during CD4+ T Cell Clonal Anergy Induction
CD25+Foxp3+ Regulatory T Cells Facilitate
and Daniel L. Mueller
Tracy L. Vanasek, Sarada L. Nandiwada, Marc K. Jenkins
2006; 176:5880-5889; ;
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The Journal of Immunology
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CD25?Foxp3?Regulatory T Cells Facilitate CD4?T Cell
Clonal Anergy Induction during the Recovery from
Tracy L. Vanasek,* Sarada L. Nandiwada,* Marc K. Jenkins,†and Daniel L. Mueller2*
T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen
instead induced aggressive CD4?cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-
deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a
CD25?Foxp3?CTLA-4?CD4?regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate
the induction of clonal anergy in nearby responder CD25?CD4?T cells. Thus, an early recovery of CD25?regulatory T cells
following a lymphopenic event can prevent exuberant Ag-stimulated CD4?cell cycle progression and promote the development
of clonal anergy. The Journal of Immunology, 2006, 176: 5880–5889.
in systemic inflammatory diseases such as systemic lupus erythem-
atosus (SLE), and those affected with rheumatoid arthritis or mul-
tiple sclerosis demonstrate a primary defect in either thymic output
or peripheral T cell homeostasis (2, 3). Mouse models of systemic
lupus erythematosus and diabetes demonstrate similar evidence of
premature thymic atrophy and/or peripheral lymphopenia (4, 5).
Finally, lymphopenia is associated with resistance to the develop-
ment of transplantation tolerance (6). Thus, clinical and experi-
mental data suggest an association between T cell lymphopenia
and defects in immune self-tolerance. Despite these data, immu-
nodepleting agents such as cyclophosphamide and antithymocyte
globulin remain the standard of care for the treatment of severe
necrotizing vasculitis and prevention of acute allograft rejection,
respectively (7, 8). Although effective to quickly suppress danger-
ous immunopathology, such treatments are not designed to achieve
durable Ag-specific tolerance and carry substantial risk of
Under nonlymphopenic conditions, naive CD4?T cells can be
induced into an unresponsive state termed clonal anergy by re-
peated systemic exposure to soluble Ag (9). Anergy is an inability
of CD4?T cells to produce IL-2 or to proliferate upon subsequent
Ag challenge, as a consequence of multiple intracellular signaling
defects (10–12). This contrasts with the aggressive priming of
ndividuals with primary immunodeficiency and reduced lym-
phocyte counts are at heightened risk for the development of
autoimmunity (1). Likewise, lymphopenia is not uncommon
CD4?T effector cells and generation of immunological memory
that follows the recognition of Ag in the presence of adjuvant,
tissue injury, and/or infection (9, 13, 14). Certain biochemical sig-
nals (e.g., activation of the mammalian target of rapamycin) that
occur as T cells move through cell cycle during a protective im-
mune response to Ag appear to durably increase the recall Ag
responsiveness of the T cells in vivo (15).
Within the lymphopenic immune system there is a strong ho-
meostatic drive to increase the total number of T cells, and this can
lead to cell cycle progression that is independent of exogenous
(foreign) Ag or infection (16, 17). Lymphopenia-induced prolifer-
ation is known to require the recognition of a self peptide/MHC
complex, and may continue until a sufficiently diverse repertoire
develops that can efficiently compete for every self peptide/MHC
complex (18–23). These observations raise the question of
whether this homeostatic drive can also promote a more intense
cell cycle progression in response to self Ag recognition that an-
tagonizes the development of clonal anergy. If true, any attempt by
the immune system to recover from lymphopenia through homeo-
static proliferation carries the risk of selecting for T cells with the
highest potential for clinical autoreactivity (4, 5, 24, 25). Never-
theless, CD4?regulatory T cells can also quickly expand during
the course of immune reconstitution, and such cells may act to
prevent the development of overt autoimmunity in individuals re-
covering from lymphopenia (26–28).
We have investigated the regulation of CD4?T cell clonal an-
ergy induction within the setting of lymphopenia. As described
below, we have found T cells to be resistant to clonal anergy in-
duction immediately following adoptive transfer into athymic
nu/nu (nude) mice as a consequence of unrestrained cell cycle
progression. This system thus afforded us the opportunity to ex-
amine CD4?regulatory T cells for their ability to influence the
development of anergy. We now confirm that the adoptive transfer
of mature CD4?T cells into lymphopenic mice leads to a spon-
taneous expansion of a large population of CD25?Foxp3?CTLA-
4?CD4?regulatory T cells, and demonstrate that these CD25?T
cells have the capacity to dampen the Ag-induced drive to prolif-
erate and facilitate the induction of clonal anergy after partial im-
*Department of Medicine and†Department of Microbiology, and Center for Immu-
nology, University of Minnesota Medical School, Minneapolis, MN 55455
Received for publication December 8, 2005. Accepted for publication February
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.
1This work was supported by National Institutes of Health Grants P01 AI35296, R01
GM54706, and P01 AI050162 (to D.L.M.). T.L.V. was also a recipient of a T32
Immunology Training Grant Predoctoral Fellowship.
2Address correspondence and reprint requests to Dr. Daniel L. Mueller, Center for
Immunology, Mayo Mail Code 334, 6-120 Nils Hasselmo Hall, 312 Church Street
S.E., Minneapolis, MN 55455. E-mail address: email@example.com
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
by guest on June 13, 2013
Materials and Methods
DO11.10 (DO11) TCR-transgenic (TCR-Tg)3were bred to homozygosity
and maintained in our animal facility (29). CD4?T cells in these mice are
uniformly reactive to chicken OVA peptide 323–339 (OVAp)/I-Adcom-
plexes and express a clonotypic TCR detectable with the mAb KJ1-26 (30).
Rag-2?/?DO11 TCR-Tg mice were purchased from Taconic Farms
through their Emerging Models Program. The HA TCR-Tg mice (31) were
obtained from A. Khoruts (University of Minnesota, Minneapolis, MN)
and were maintained in our animal facility. CD4?T cells in these mice are
specific for peptide 111–119 of influenza hemagglutinin (HAp) presented by
class II I-EdMHC molecules and can be detected with the anti-clonotypic
mAb 6.5 (31). Wild-type (WT) BALB/c and BALB/c nu/nu (nude) recipient
mice, 5–8 wk old, were purchased from Charles River Laboratories through a
contract with the National Cancer Institute at the National Institutes of Health
(Frederick, MD). All mice were housed under specific pathogen-free condi-
tions and used in accordance with National Institutes of Health guidelines and
the University of Minnesota Institutional Animal Care and Use Committee.
Mice were age and sex matched for all experiments.
Adoptive transfer and in vivo treatments of mice
Lymph nodes (axillary, brachial, inguinal, and mesenteric) and spleens of
TCR-Tg mice were harvested into complete media containing 10% FCS
(Atlas Biologicals), 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml
streptomycin, and 5 ? 10?5M 2-ME in RPMI 1640 (Mediatech), and
prepared for adoptive transfer as described previously (9). In some exper-
iments, cells were labeled with CFSE (Molecular Probes) before transfer,
using a modification of a technique previously described (32). Briefly,
lymph node and spleen cells in PBS at a concentration of 1 ? 107cells/ml
were incubated in 2.5 ?M CFSE for 5 min at 37°C. The labeling reaction
was stopped by the addition of complete media. The CFSE-labeled T cells
were washed twice with PBS before i.v. transfer of 2.5–5 ? 106cells to
recipient syngeneic mice. OVAp 323–339 was produced in our micro-
chemical facility (University of Minnesota), dissolved in PBS, and filter
sterilized for use. HAp 111–119 was kindly provided by A. Khoruts (Uni-
versity of Minnesota). OVAp and HAp were delivered i.v. at doses of 100
or 250 ?g. Rapamycin (RAPA) was obtained from S. N. Sehgal (Wyeth-
Ayerst Research, Princeton, NJ). A stock solution of 1 mg/ml RAPA in
100% ethanol was prepared. RAPA was then suspended in 0.2% car-
boxymethylcellulose, as previously described (33), and delivered i.p. at a
dose of 0.5 mg/kg/day for 4 days beginning on the day of adoptive transfer.
The rat anti-mouse CD25 mAb PC61 obtained from A. Khoruts (University
of Minnesota) was purified from hybridoma cell culture supernatants using
standard protein G-Sepharose chromatography techniques. Anti-CD25
mAb (400 ?g) was injected i.p. into mice every 3 days during the exper-
iment starting on the day of DO11 T cell adoptive transfer and throughout
the period of the CD4?cell immune reconstitution. Control mice given the
PC61 mAb starting only after the CD4?cell reconstitution and at the time
of OVAp administration demonstrated no effect of the Ab treatment on
either cell cycle progression or recall IL-2 production (data not shown).
Spleen cells were washed with staining buffer (PBS containing 2% FCS
and 0.2% azide), and then incubated with anti-Fc mAb 93 (eBioscience) to
block FcRs. Cells were then stained with the combination of PerCP-cyanin
5.5-labeled anti-CD4 (RM4-5) mAb (BD Pharmingen) and allophycocya-
nin-labeled DO11.10 TCR (KJ1-26) mAb (Caltag Laboratories) as well as
one or more of the following: biotin-labeled anti-CD25 (7D4), PE-labeled
anti-CTLA4 (4F10), (BD Pharmingen); PE-labeled anti-IL-2 (JES6-5H4),
FITC- or PE-labeled anti-CD25 (PC61), PE-labeled anti-CD69 (H1.2F3),
PE-labeled streptavidin, (eBioscience). Intracellular molecules were de-
tected essentially as previously described (34, 35). Briefly, washed and
Fc-blocked spleen cells were first incubated with PerCP-cyanin 5.5-labeled
anti-CD4 mAb and allophycocyanin-labeled KJ1-26 mAb. Cells were then
washed one time with PBS, fixed in 2% formaldehyde (Sigma-Aldrich) for
20 min atroom temperature,
(Sigma-Aldrich), and incubated at room temperature for 20 min with PE-
labeled anti-IL-2 mAb, PE-labeled anti-CTLA4 mAb, or PE-labeled irrel-
evant mAb. Finally, cells were washed once in 0.5% saponin and once in
staining buffer. For all experiments, at least 1000 KJ1-26?CD4?events
were collected using a FACSCalibur flow cytometer (BD Biosciences) and
analyzed using FlowJo (Tree Star) software.
permeabilizedin 0.5% saponin
Measurement of cell cycle progression in vivo
Cell cycle progression was monitored using the CFSE dye as a marker of
cell division (32). The average division of CFSE-labeled KJ1-26?CD4?
T cells at various times after stimulation was calculated as previously de-
scribed (15). Briefly, based on the peaks of CFSE fluorescence intensity
within the population, each T cell was assigned to a particular cell division
group d (with d ? 0 to n cell divisions), and the number of T cell events
(E) observed within each cell division group (Ed) was determined. Average
division was calculated using the following equation:
Average divisions ??
Cell purification and in vitro suppression assay
The suppressive properties of CD4?subpopulations isolated from intact
DO11 mice, or from DO11-reconstituted nude mice, were tested by their
addition to CD25?cell-depleted normal DO11 lymph node and spleen
cells. In some experiments, CD25?T cells from normal DO11 mice were
positively selected using CD25 mAb and MACS magnetic streptavidin
microbeads (Miltenyi Biotec), according to manufacturer’s instructions. To
obtain CD25?CD4?and CD25?CD4?subpopulations from DO11-recon-
stituted nude mice, CD4?cells were first isolated using CD4 Dynabeads
(Dynal Biotech) followed by CD4 Detachabead to remove the CD4 mag-
netic beads. The CD25?CD4?and CD25?CD4?subpopulations were then
separated using CD25 mAb and MACS-positive selection (Miltenyi Bio-
tec). To assay for in vitro suppression, the purified CD25?cells from intact
DO11 mice were compared with CD25?CD4?and CD25?CD4?subsets
from DO11-reconstituted nude mice following addition to CD25-depleted
DO11 lymph node and spleen cell cultures in the absence or presence of 10
?M OVAp. IL-2 secretion was measured in the 48 h supernatants by cap-
Real-time quantitative RT-PCR
CD25?CD4?and CD25?CD4?cell populations from intact DO11 mice
and DO11-reconstituted nude mice were purified as described above. One
million cells were lysed with TRIzol (Invitrogen Life Technologies) and
RNA was extracted according to the manufacturer’s instructions. RNA was
further purified using the RNA Easy Mini kit (Qiagen). Total RNA equiv-
alent to the cell number from each sample was reverse transcribed using the
Superscript II Platinum Two Step qRT-PCR kit (Invitrogen Life Technol-
ogies). PCR primers were synthesized in our microchemical facility (Uni-
versity of Minnesota) and real-time PCR was conducted using a Cepheid
Smart Thermocycler by adding SybrGreen (Molecular Probes) to the re-
action mixtures. Primers were designed to amplify the junction region of
exons 7 and 8 of the Foxp3 mRNA. The primers contained the following
sequences: Foxp3 (forward): 5?-AAA GGA GAA GCT GGG AGC TAT
G-3?; Foxp3 (reverse): 5?-CCT GAG TAC TGG CTA CGA T-3?. Hprt
mRNA was used as a positive control to normalize the Foxp3 data. The
Hprt primers were designed to amplify the junction of exons 7 and 8 and
contained the following sequences: Hprt (forward): 5?-TGA AGA GCT
ACT GTA ATG ATC AGT CA-3?; Hprt (reverse): 5?-AGC AAG CTT
GCA ACC TTA ACC A-3?. Data are expressed as the amount of Foxp3
mRNA present in a sample relative to Hprt.
Clonal anergy induction is defective in the setting of T cell
To explore clonal anergy induction in the setting of T cell lym-
phopenia, OVAp-reactive DO11 CD4?T cells were adoptively
transferred into athymic nu/nu (nude) BALB/c recipient mice and
then immediately exposed to repeated (three times) i.v. injections
of Ag (OVAp) in the absence of an adjuvant. In a parallel group of
WT recipient animals, this regimen of prolonged TCR stimulation
led to a state of unresponsiveness to Ag rechallenge by day 13 that
resulted in a defect in the in vivo production of IL-2 upon Ag
rechallenge (Fig. 1A). In contrast, the KJ1-26?CD4?T cells ex-
posed to the 3? OVAp regimen within the nude mice retained a
significantly higher capacity to synthesize IL-2 (p ? 0.001).
3Abbreviations used in this paper: TCR-Tg, TCR-transgenic; OVAp, OVA peptide;
HA, hemagglutinin; HAp, HA peptide; WT, wild type; RAPA, rapamycin.
5881The Journal of Immunology
by guest on June 13, 2013
Therefore, clonal anergy could not successfully be induced in the
Ag-stimulated cell cycle progression is stronger during T cell
Further analysis of the proportion of Ag-reactive KJ1-26?CD4?
T cells that remained within the spleens of the WT mice after the
3? OVAp infusion regimen revealed little change in their fre-
quency as compared with 3? PBS-treated control animals, con-
sistent with an ineffective clonal expansion response in the absence
of infection or adjuvant (Fig. 1B) (9). In contrast, a significant
increase in the percentage and total number of KJ1-26?CD4?T
cells was observed within the spleens of nude recipient mice
chronically exposed to Ag, as compared with nude mice exposed
to PBS alone (Fig. 1B and data not shown). This enhanced clonal
expansion response together with the persistent recall Ag respon-
siveness of the 3? OVAp-treated KJ1-26?CD4?T cells in lym-
phopenic mice thus was more reminiscent of a successful T cell
priming event than of an induction of immunological tolerance.
Previously, our work had indicated that aggressive in vivo cell
cycle progression during the primary response to Ag antagonizes
the development of clonal anergy (15). We, therefore, postulated
that the resistance to anergy induction observed in the nude mice
was caused by this lymphopenia-induced enhanced drive for cell
cycle progression. To test this, the intensity of cell cycle progres-
sion in lymphopenic mice was characterized based on the rate of
CFSE dye dilution in KJ1-26?CD4?T cells immediately exposed
to a single infusion of Ag (1? OVAp). KJ1-26?CD4?T cells in
both WT and nude recipients did demonstrate a reduced CFSE
fluorescence intensity indicative of multiple rounds of cell division
in response to Ag (Fig. 2A). However, the CFSE fluorescence of
the KJ1-26?CD4?T cells recovered from 1? OVAp-treated nude
mice was always much lower than in the WT mice, consistent with
a faster rate of cell division. A mathematical examination of the
flow cytometry data confirmed that KJ1-26?CD4?T cells in nude
mice had a significantly higher average cell division rate (5.58 ?
0.35 divisions/T cell over 5 days) than T cells stimulated within the
WT recipients (2.79 ? 0.05; p ? 0.016) (Fig. 2B). Such an in-
crease in the rate of cell division predicted a generation of daughter
cells in the lymphopenic mice that was nearly eight times greater
than in the WT case. Therefore, an enhanced cell cycle progression
may have accounted at least in part for the greater clonal expansion
originally observed in the 3? OVAp-treated lymphopenic animals.
A single i.v. infusion of Ag in the absence of adjuvant is a
relatively poor stimulus for T cell clonal anergy induction in WT
mice (15); nevertheless, it was apparent in these experiments that
the T cells exposed to 1? OVAp in nude mice achieved an even
greater level of recall Ag responsiveness as they underwent pro-
gressively more rounds of cell division (Fig. 2C). To determine
WT or nude mice were pretreated i.v. three times 3 days apart with 100 ?g
of OVAp (or PBS as a control) beginning 1 day after adoptive transfer of
4 ? 106DO11 T cells. Six days later, recipient mice were challenged i.v.
with 250 ?g of OVAp for 3 h. A, Intracellular IL-2 content among Ag-
specific KJ1-26?CD4?T cells, as a percentage of the 3? PBS-treated
controls. B, Percentage of KJ1-26?CD4?T cells accumulating within the
spleens of partially reconstituted nude mice on day 13 of the analysis. Error
bars represent the SEM for duplicate mice. The p value was determined
using the Student t test. This experiment was repeated with similar results.
Defective tolerance induction in the setting of lymphopenia.
following Ag-stimulated cell cycle progression in
lymphopenic recipients. WT or nude mice adoptively
transferred with 4 ? 106CFSE-labeled DO11 T cells
were treated with a single i.v. infusion of 100 ?g of
OVAp or PBS as a control. Some animals were treated
with simultaneous i.p. infusions of 0.5 mg/kg RAPA in
carboxymethylcellulose, whereas other mice received
the vehicle alone. Five days later, animals were rechal-
lenged i.v. with 250 ?g of OVAp for 3 h. A, CFSE dye
dilution (log FL1) of KJ1-26?CD4?T cells recovered
from the spleens of WT (filled histograms) or nude
(open histograms) recipient mice. B, Average division
calculation for the Ag-stimulated KJ1-26?CD4?T
cell groups as shown in A. C, Plot of the relationship
between cell division history and mean recall Ag-in-
duced IL-2 production in KJ1-26?CD4?T cells from
WT (filled symbol) or nude (open symbol) recipients
pretreated i.v. on day 1 with 100 ?g of OVAp in the
presence of RAPA (circles) or vehicle control
(squares). IL-2 production is calculated as the percent-
age of the T cell response observed in control animals
(diamond symbol) receiving a PBS pretreatment alone.
?, The mode number of cell divisions observed in the
KJ1-26?CD4?T cell population as a result of the
OVAp pretreatment. Error bars represent the SEM.
Data shown are representative of two independent
Increased recall Ag responsiveness
5882 HOMEOSTATIC REGULATION OF CLONAL ANERGY
by guest on June 13, 2013
whether the strength of cell cycle progression during primary Ag
challenge directly regulated the level of recall Ag responsiveness
in the nude mice, we examined the effects of the antiproliferative
agent RAPA on both the lymphopenia-enhanced cell cycle pro-
gression and the eventual level of Ag responsiveness achieved. As
previously described, treatment of WT mice with RAPA during a
primary Ag challenge significantly inhibited cell cycle progression
by the KJ1-26?CD4?T cells (Fig. 2, A and B) (15). Similarly,
treatment of nude mice with RAPA during the time of the primary
i.v. OVAp administration slowed the cell cycle progression and
reduced the average division rate of the KJ1-26?CD4?T cells by
36 ? 0.09%. Interestingly, RAPA also inhibited the lymphopenia-
induced proliferation of a fraction of the transferred KJ1-
26?CD4?T cell population in the absence of Ag. KJ1-26?CD4?
T cells in both WT and nude animals demonstrated reduced recall
IL-2 production in association with their blunted primary prolif-
erative response when RAPA was present at the time of priming
(Fig. 2C). Thus, these data showed that within the lymphopenic
host, a homeostatic drive toward immune reconstitution promotes
an overly aggressive cell cycle progression response during Ag
stimulation that prevents the development of clonal anergy.
Recovery from lymphopenia reduces the homeostatic drive for
excessive Ag-induced T cell proliferation
We previously demonstrated that KJ1-26?CD4?T cells will be-
come anergic even in nude recipients when a 3? OVAp infusion
regimen first begins at least 15 days after the T cell adoptive trans-
fer (35). An adaptive tolerance that resembles this peptide-induced
clonal anergy has also been observed to develop over extended
periods of time in 5C.C7 TCR-Tg CD4?T cells adoptively trans-
ferred into lymphopenic (CD3??/?) mice that express this T cell’s
specific Ag (pigeon cytochrome c) as a transgene (36). Therefore,
chronic TCR stimulation can induce T cell clonal anergy within
immunodeficient mice, but only after a partial reconstitution of the
lymphopenic immune system has taken place.
We directly compared Ag-stimulated cell cycle progression in
nonreconstituted or partially reconstituted nude recipients by the
adoptive transfer of a second, CFSE-labeled DO11 T cell popula-
tion. OVAp challenge was found to elicit significantly fewer av-
erage cell divisions by the CFSE-labeled cohort of KJ1-26?CD4?
T cells in the partially reconstituted nude recipients (2.65 ? 0.13)
as compared with nude mice that had not received an initial DO11
T cell adoptive transfer on day 0 (4.29 ? 0.31; p ? 0.001) (Fig.
3A). In fact, OVAp-induced cell cycle progression in the partially
reconstituted nude recipients closely resembled that observed in
WT mice (data not shown). Once again, those KJ1-26?CD4?T
cells that had divided the most during the primary Ag exposure in
the nonreconstituted nude mice also demonstrated a higher level of
recall Ag responsiveness than naive T cells, consistent with prim-
ing (Fig. 3B). In contrast, KJ1-26?CD4?T cells exposed to Ag
after partial reconstitution of the nude mice showed only poor
production of IL-2 in response to an OVAp rechallenge. Thus, the
results confirmed that a partial reconstitution of the lymphopenic
immune system can reduce the drive for aggressive cell cycle pro-
gression during primary Ag challenge and restore the ability to
induce clonal anergy.
Recovery from lymphopenia is associated with the expansion of
a large CD25?Foxp3?CTLA-4?CD4?regulatory T cell
Knoechel et al. (28) recently reported that following an adoptive
transfer of Rag?/?DO11 T cells into Rag-deficient and lym-
phopenic mice that constitutively expressed a soluble form of
OVA as a transgene, the T cells caused an early wasting disease
that resulted in the death of about half of the recipients. Nevertheless,
beyond 14 days after the T cell adoptive transfer (in surviving ani-
mals) a tolerance to OVA developed and these partially reconstituted
lymphopenic animals regained their health. In their studies, this late
immune tolerance was associated with a self Ag (OVA)-dependent
generation of a subpopulation of CD25?Foxp3?KJ1-26?CD4?
regulatory T cells. Both Rag-sufficient TCR-Tg and polyclonal
CD25?CD4?regulatory T cells have been previously shown to
undergo an MHC class II-dependent clonal expansion following
their adoptive transfer into Rag-deficient lymphopenic hosts, and
still retain their suppressive activity (27). Therefore, self Ag-spe-
cific CD25?CD4?regulatory T cells might also be expected to
arise over time following a partial reconstitution of lymphopenic
nude mice with DO11 T cells to promote the establishment of
Based on this information, we sought evidence that our nude
mice are resistant to clonal anergy induction because they lack
regulatory T cells. Freshly isolated Rag-sufficient DO11 T cells
were found to contain a small CD25?CD4?subpopulation (data
not shown), but these putative regulatory T cells did not become
enriched in response to an OVAp primary Ag challenge performed
immediately after T cell adoptive transfer (Fig. 3C). In contrast, 24
days after their adoptive transfer into nude recipients in the ab-
sence of OVAp, a sizable proportion of the reconstituting Rag-
sufficient KJ1-26?CD4?T cells appeared to have undergone mul-
tiple rounds of cell division (data not shown) and expressed a high
level of CD25 (Fig. 3C).
Consistent with a regulatory T cell phenotype, the CD25?KJ1-
26?CD4?T cells that reconstituted nude mice demonstrated a
high level of intracellular CTLA-4 and reduced expression of
CD45RB (Fig. 4A and data not shown). Furthermore, in response
to the infusion of OVAp this CD25?subpopulation demonstrated
little capacity to accumulate intracellular IL-2. Nevertheless, a par-
tial induction of CD69 expression was consistently observed fol-
lowing stimulation, suggesting that the CD25?KJ1-26?CD4?T
cells still retained some Ag reactivity (Fig. 4A and data not
shown). Foxp3 expression has been shown to be a very good
marker for the development of regulatory T cell function (37).
CD25?KJ1-26?CD4?T cells purified from nude mice after par-
tial immune reconstitution expressed high levels of Foxp3 mRNA,
relative to CD25?CD4?T cells found in either WT DO11 mice or
the same partially reconstituted nude mice (Fig. 4B). These same
CD25?KJ1-26?CD4?T cells also demonstrated a capacity to in-
hibit IL-2 production by activated CD25?KJ1-26?CD4?T cells
in an in vitro assay system (Fig. 4C). Therefore, the CD25?T cells
that arose during a partial reconstitution of lymphopenic mice
had a similar phenotype as the well-characterized natural
CD25?CD4?regulatory T cells (38–40).
Endogenous TCR gene recombination is necessary for optimal
CD25?CD4?regulatory T cell generation during partial
reconstitution of lymphopenic mice to fully dampen Ag-induced
cell cycle progression
An examination of TCR transgene and CD25 expression following
partial immune reconstitution of nude mice with Rag-sufficient
DO11 T cells did reveal a dimming of the clonotypic TCR staining
within the large CD25?KJ1-26?CD4?regulatory T cell popula-
tion, perhaps consistent with endogenous Tcra gene rearrangement
and the expression of a second TCR having self Ag specificity
(Fig. 5A). Such recognition of particular self peptide/MHC spec-
ificities appears in general to be important, because Rag?/?
TCR-Tg mice lacking in TCR diversity are often deficient in
CD25?CD4?regulatory T cells (41, 42). Thus, we reasoned that
5883The Journal of Immunology
by guest on June 13, 2013
a partial reconstitution of nude mice with Rag-deficient DO11 do-
nor T cells having limited TCR diversity would fail to give rise to
the CD25?KJ1-26?CD4?subset and would test whether the control
Ag-induced cell cycle progression depended on the presence of these
regulatory T cells. The generation of CD25?KJ1-26?CD4?regula-
tory T cells (in the absence of OVAp) was observed to be reduced
following reconstitution of the nude mice with Rag?/?DO11 donor
T cells (Fig. 5A). Furthermore, reconstitution with Rag?/?DO11 T
cells had a decreased capacity to suppress the proliferation of CFSE-
labeled OVAp-stimulated KJ1-26?CD4?T cells as compared with
Rag-sufficient donor cells (Fig. 5B). Thus, it appeared that a TCR-
diverse CD25?CD4?regulatory T cell subset that developed early on
during immune reconstitution acted to reduce the intensity of Ag-
induced cell cycle progression.
were adoptively transferred into nude mice that were either nonreconstituted or previously reconstituted with unlabeled Rag-2?/?DO11 T cells for
20 days. One day after transfer of the CFSE-labeled marker population, recipients were injected i.v. with OVAp (100 ?g) or with PBS as a control.
Three days later, a second infusion of OVAp (250 ?g) was given, and the splenic KJ1-26?CD4?T cells were analyzed 3 h later. A, OVAp-induced
CFSE dye dilution (log FL1) of splenic marker KJ1-26?CD4?T cells. Dotted lines to the right of the original Rag-2?/?DO11-reconstituting
population (shaded histograms) indicate the highest log FL1 autofluorescence of unlabeled T cells. Hatched lines indicate the cell division history
of the CFSE-labeled marker T cell population (filled histograms). Plots are representative of four individual mice within two independent exper-
iments. B, The relationship between cell division history and mean recall Ag-induced IL-2 accumulation within the marker KJ1-26?CD4?T cells
from nonreconstituted (E) or Rag-2?/?DO11 T cell-reconstituted (F) nude recipients was plotted. IL-2 production is calculated as the percentage
of the T cell response observed in control animals that were initially infused with PBS alone (?). Only those marker cells with CFSE fluorescence
greater than the autofluorescence of the reconstituting population were included in this analysis. ?, The mode number of cell divisions observed in
a marker T cell population as a result of the OVAp pretreatment. Error bars represent the SEM. Data shown in B are the average of two independent
experiments. C, Splenic KJ1-26?CD4?T cells were examined for surface CD25 expression as a function of CFSE dye content. Both unlabeled
reconstituting Rag-2?/?DO11 T cells (left quadrants) and the CFSE-labeled marker KJ1-26?CD4?T cell populations (right quadrants) are shown.
Horizontal lines in each plot indicate the maximum log FL2 fluorescence of isotype control Ab-stained cells. Plots are representative of duplicate
animals. This experiment was repeated more than three times with similar results.
Effect of partial immune reconstitution on Ag-induced cell cycle progression and clonal anergy induction. CFSE-labeled DO11 T cells
5884 HOMEOSTATIC REGULATION OF CLONAL ANERGY
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Suppression of OVAp-dependent cell cycle progression in
lymphopenic mice can occur following reconstitution with CD4?
T cells having no reactivity to OVAp
These findings indicated that a diverse TCR repertoire and broad self
peptide/MHC specificity within the population of reconstituting
CD4?T cells was more important than a high level of clonotypic
TCR expression to the counterregulation of OVAp-induced prolifer-
ation within nude mice. This predicted that suppression of the prolif-
eration of CFSE-labeled KJ1-26?CD4?T cells by the original
competition for OVAp/I-Adcomplexes (43, 44). Consistent with this,
a partial reconstitution of the nude mice with Rag?/?HAp-reactive
HA TCR-Tg CD4?T cells proved equally effective in dampening the
OVAp-induced KJ1-26?CD4?T cell proliferation, regardless of
whether these T cells were stimulated with HAp (Fig. 6). Note that
suppression was only found to occur when the DO11- or HA-recon-
stituting T cells expressed Rag proteins and were capable of endog-
enous Tcra gene rearrangements (Fig. 5 and data not shown).
tuting nude mice express high levels of intracellular
CTLA-4 and Foxp3, and are hyporesponsive to
OVAp stimulation. DO11 T cells were transferred
into either WT or nude mice. A, Twenty days after
adoptive transfer, recipient mice received an i.v. in-
fusion of OVAp (250 ?g). Later (2.5 h later), splenic
KJ1-26?and KJ1-26?CD4?T cells from WT re-
cipients and KJ1-26?CD4?T cells from nude re-
cipients were identified by flow cytometry and ana-
lyzed for surface CD25 expression and either
surface CD69, intracellular CTLA-4, or intracellular
IL-2, as indicated. Quadrant gates indicate the log
FL1 and log FL2 fluorescence of isotype control Ab-
stained cells. B, CD25?CD4?and CD25?CD4?T
cells were purified from intact Rag-2?/?DO11 Tg
animals (WT), or from Rag-2?/?DO11 T cell-re-
constituted nude (NU) mice as described in Materi-
als and Methods. Foxp3 mRNA levels in equivalent
numbers of T cells were then determined using real-
time quantitative RT-PCR. Foxp3 levels are ex-
pressed as arbitrary units, normalized to Hprt
CD25?CD4?and CD25?CD4?T cells were puri-
fied from Rag-2?/?DO11-reconstituted nude (NU)
mice, and examined for in vitro suppressive activity
as compared with CD25?T cells purified from in-
tact Rag-2?/?DO11 TCR-Tg (WT) animals. Sup-
pressor T cell and CD25?CD4?responder T cell
populations were mixed in the ratios shown and ex-
amined for the production of IL-2 (at 48 h) in re-
sponse to stimulation with 10 ?M OVAp using a
capture ELISA. Error bars indicate SEM. Data
shown are representative of two independent
CD25?CD4?DO11 T cells reconsti-
5885The Journal of Immunology
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CD25?CD4?T cells facilitate clonal anergy induction
Taken together, the data suggested that the development of a
CD25?Foxp3?CTLA-4?CD4?regulatory T cell population early
during the course of immune reconstitution of lymphopenic mice
was necessary to inhibit Ag-induced cell cycle progression in the
absence of adjuvant or infection, and this then led to an induction
of clonal anergy. To firmly establish that CD25?CD4?regulatory
T cells were responsible for suppressing the Ag-induced cell
cycle progression of CD25?CD4?responder T cells during re-
covery from lymphopenia, the proliferation of CFSE-labeled
KJ1-26?CD4?T cells was examined following adoptive transfer
into nude mice reconstituted in the presence of an anti-CD25 mAb
capable of inhibiting the development of this CD25?population.
Treatment of nude mice with anti-CD25 mAb PC61 throughout the
period of DO11 T cell immune reconstitution reduced the percent-
age of KJ1-26?CD4?T cells expressing CD25 (as detected using
the 7D4 anti-CD25 mAb) from 31 ? 2% to 12 ? 4% (Fig. 7A).
Although this Ab treatment never resulted in a complete elimina-
tion of the CD25?CD4?subpopulation, OVAp-stimulated cell di-
vision by marker KJ1-26?CD4?T cells was nevertheless signif-
icantly enhanced (p ? 0.008; Fig. 7, B and C). Remarkably, this
increased cell cycle progression was also associated with a resis-
tance to clonal anergy induction and increased recall Ag respon-
siveness, despitethe continued
CD25?CD4?DO11-reconstituting T cell population (Fig. 7D).
Thus, the rapid expansion of a CD25?Foxp3?CD4?regulatory T
cell population during partial immune reconstitution facilitated
clonal anergy induction in nearby CD25?CD4?T cells that rec-
ognized the presence of Ag.
development ofa large
CD25?Foxp3?CTLA-4?CD4?regulatory T cells were shown for
the first time to facilitate the induction of clonal anergy in vivo in
CD25?CD4?T cells that recognize Ag in the absence of an in-
fection or adjuvant. CD25?CD4?T cells accumulated in lym-
phopenic mice early during the course of immune reconstitution
and functioned to counterregulate the homeostatic drive for exces-
sive cell cycle progression during Ag encounter. Their inhibition
of Ag-stimulated cell cycle progression enabled them to promote
clonal anergy induction. Without such a partial T cell reconstitu-
tion of the nude mice, and in the absence of CD25?CD4?regu-
latory T cells, Ag-stimulated cell cycle progression was exuberant
and was invariably associated with the maintenance of a high level
of Ag responsiveness.
It is plausible that a resistance to T cell clonal anergy induction
in the absence of CD25?CD4?regulatory T cells also accounts for
the failure of transplantation tolerance-inducing regimens during
lymphopenia (6). Whether CD25?Foxp3?CD4?regulatory T
cells play a similar role in facilitating clonal anergy induction in
CD25?Foxp3?CD4?regulatory T cells generated in vivo in the
presence of low doses of Ag can be shown to interfere with the
priming of a second cohort of naive Ag-reactive CD25?CD4?T
cells and lead to their reduced capacity to produce IL-2 upon in
vitro rechallenge (45). Nonlymphopenic CD28?/?animals also
demonstrate reduced numbers of CD25?CD4?regulatory T cells,
and anergy induction by infusion of soluble peptide Ag can be
ineffective in these mice (46).
The CD25?CD4?regulatory T cell population observed here
during the recovery from lymphopenia is phenotypically and
deficient TCR-Tg T cells fails to coun-
terregulate Ag-stimulated cell cycle pro-
gression. Nude mice were partially
reconstituted with either Rag-2?/?or
Rag-2?/?DO11 T cells for 20 days. Fol-
lowing that time period, a CFSE-labeled
marker Rag-2?/?DO11 T cell popula-
tion was adoptively transferred, and 1
day later, 100 ?g of OVAp (or PBS as a
control) was infused i.v. to stimulate cell
cycle progression. A, CFSE?reconstitut-
ing Rag-2?/?(left) and Rag-2?/?(right)
DO11 CD4?T cell populations, stained
with anti-CD25 (log FL2) and anti-
clonotypic TCR KJ1-26 (log FL4). Gates
for CD25 expression are based on the log
cells. B, OVAp-induced CFSE dye dilu-
tion (log FL1) of splenic marker KJ1-
26?CD4?T cells in the Rag-2?/?(left)
and Rag-2?/?(right) DO11 T cell-recon-
stituted nude mice. Dotted lines separate
the reconstituting (gray tracings) and
marker (black histograms) T cell popula-
tions. The experiments shown were re-
peated twice with similar results.
Reconstitution with Rag-
5886HOMEOSTATIC REGULATION OF CLONAL ANERGY
by guest on June 13, 2013
functionally similar to naturally occurring CD25?CD4?regula-
tory T cells. CD25?CD4?T cells have previously been shown to
develop from purified CD25?CD4?T cells that have undergone
extensive homeostatic proliferation upon transfer into lym-
phopenic recipients (27). Purified CD25?T cells can also give
rise, through extensive lymphopenia-induced proliferation, to even
cells during immune reconstitution leads to
enhanced Ag-induced proliferation and resis-
tance to clonal anergy induction. Nude mice
were partially reconstituted with Rag-2?/?
DO11 T cells either in the absence or pres-
ence of the anti-CD25 mAb PC61. Infusions
of mAb (400 ?g) were initiated on day 0 (the
day of T cell transfer) and repeated every 3
days during the course of the experiment.
One group of mice was analyzed on day 8 for
the presence of CD25?KJ1-26?CD4?T
cells within the spleens following reconsti-
tution in the absence or presence of anti-
CD25 mAb, as indicated (A). Other recipient
mice were transferred with a CFSE-labeled
marker DO11 T cell population on the same
day (day 8) and 1 day later were infused with
OVAp i.v. (100 ?g). Three days into the
OVAp response (day 12), recipient mice re-
ceived a second i.v. infusion of OVAp (250
?g) and spleens were harvested 3 h later.
OVAp-induced CFSE dye dilution (B), aver-
age division (C), and IL-2 production (D)
were measured within the marker KJ1-
26?CD4?T cell population recovered from
recipients reconstituted in the absence or
presence of anti-CD25 mAb, as indicated.
Dotted and hatched lines in B are as indi-
cated in Fig. 3. The percentage of IL-2?KJ1–
26?CD4?T cells during recall Ag challenge
was determined based on staining with an
isotype control Ab (D). Data plotted in C and
D are the mean ? SEM of duplicate mice.
The p value was determined using the Stu-
dent t test. Results shown are representative
of two independent experiments.
Depletion of CD25?CD4?T
nude mice that were reconstituted for 40 days with Rag-2?/?DO11 or HA TCR-Tg cells, as indicated. One day after transfer of the DO11 marker cells
(day 41), recipient mice were infused i.v. with 100 ?g of OVAp alone and/or 100 ?g of HAp. Three days following the peptide infusion (day 44), splenic
KJ1-26?CD4?T cell were recovered and examined for CFSE dye dilution as in Fig. 3. Plots shown are representative of duplicate mice. This experiment
was repeated with similar results.
Inhibition of proliferation is not Ag specific and does not require acute activation. CFSE-labeled marker DO11 T cells were transferred into
5887 The Journal of Immunology
by guest on June 13, 2013
greater numbers of CD25?CD4?T cells that retain a capacity to
suppress in vitro proliferation (27, 47). Therefore, it cannot be
determined whether the CD25?CD4?T cells generated during the
course of immune reconstitution in these experiments arose from
pre-existing natural regulatory T cells, or developed from naive T
cells responding to self Ag in the lymphopenic environment. We
did observe that the formation of this CD25?CD4?subset was
significantly reduced when Rag?/?DO11 cells were used as the
reconstituting population, suggesting that endogenous TCR
?-chain-dependent recognition of self peptide/MHC regulates their
development during the immune reconstitution. Perhaps a broad-
ened TCR diversity allowed for a large expansion of the
CD25?CD4?subpopulation without too many cells competing
with each other for a single self peptide/MHC niche (43, 44). Nev-
ertheless, the capacity of HA-reconstituting CD4?T cells to fa-
cilitate the induction of anergy in DO11 T cells does not suggest
that competition for a single self peptide/MHC niche is their mech-
anism of immunoregulation (43, 44).
The observation that a reduction in homeostatic drive for intense
Ag-induced proliferation required reconstitution with Rag-suffi-
cient TCR-Tg T cells was perhaps surprising, because one might
have expected that an expanded population of DO11 Rag-2?/?CD4?
T cells would be fully competent to compete with newly trans-
ferred DO11 CD4?responder T cells for peptide/MHC complexes
and cause an inhibition of their proliferation (43). In fact, the shar-
ing of Ag specificity between the reconstituting population and the
responder CD4?T cells was not required to inhibit Ag-stimulated
cell cycle progression (Fig. 6). On the surface, this result appears
to be at odds with that of Moses et al. (43) who showed that only
TCR-Tg Rag?/?CD4?T cells that compete for the same self
peptide/MHC complex are capable of inhibiting the spontaneous
proliferation of a particular TCR-Tg CD4?T cell. It is important
to note that in our experiments, the proliferative response to ad-
ministered exogenous Ag given at high dose was examined rather
than self peptide-dependent lymphopenia-induced proliferation.
In vitro data have also indicated that suppression by
CD25?CD4?T cells can be Ag nonspecific (48, 49). Neverthe-
less, it has been reported that immune regulation in vivo can ap-
pear Ag specific (50). In those experiments, HA-specific regula-
tory T cells were not capable of inhibiting the proliferative
response of pigeon cytochrome c-specific CD4?T cells respond-
ing to peptide-loaded dendritic cells (pulsed with both peptides),
whereas the proliferative response of HA-specific responder T
cells to the same limited stimulus was significantly reduced when
HA-specific T regulatory cells were cotransferred. Perhaps in our
system, the recognition of numerous self peptide/MHC complexes
by the reconstituting Rag-sufficient CD25?CD4?regulatory T cell
population under lymphopenic conditions leads to a durable acti-
vation of this subset. This could then allow them to directly sup-
press either APCs or the effector CD4?T cells themselves in an
Ag-nonspecific manner, thus leading to an abortive cell cycle pro-
gression and the induction of clonal anergy in response to an Ag-
The molecular mechanism of in vivo suppression of this Ag-
induced cell cycle progression by these CD25?Foxp3 CD4?T
regulatory cells in the lymphopenic mice remains unknown. Both
in vitro and in vivo investigations have indicated a capacity of
CD25?CD4?regulatory T cells to inhibit the production of IL-2
in nearby CD25?CD4?T cells (Fig. 4C) (49, 51). In our study, the
CD25?regulatory cells themselves did appear anergic at the level
of the Il2 gene, but they did not suppress the production of IL-2 by
nearby Ag-stimulated CD25?T cells in vivo (Fig. 4A). Similarly,
a coexistence of anergic CD25?CD4?regulatory T cells and IL-
2-producing CD25?CD4?effector T cells has been demonstrated
in the lymph nodes of OVA-expressing lymphopenic mice that had
been reconstituted 30 days earlier by an adoptive transfer of
Rag?/?DO11 T cells (28). Finally, it is unclear whether IL-2 plays
any role in the cell division response observed in the setting of
lymphopenia (34). In our hands, the anti-CD25 mAb has demon-
strated no direct inhibitory effect on OVAp-induced cell cycle pro-
gression in the nude mice (Fig. 7B and data not shown). Therefore,
the mechanism of inhibition of cell cycle progression by these
CD25?CD4?regulatory T cells is likely independent of any ef-
fects on Il2 gene expression. Regardless of the molecular mecha-
nisms involved in this suppression, during the recovery from
lymphopenia CD25?CD4?regulatory T cells act to dampen Ag-
stimulated cell cycle progression and facilitate instead an induction
of clonal anergy.
We thank Dr. Alex Khoruts for valuable discussions related to T regulatory
cell development and function, and for the careful review of this
The authors have no financial conflict of interest.
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