The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 202, No. 6, September 19, 2005 771–781
Continuous control of autoimmune disease
by antigen-dependent polyclonal CD4
regulatory T cells in the regional lymph node
Eileen T. Samy,
Lucy A. Parker,
Colin P. Sharp,
and Kenneth S.K. Tung
Department of Pathology and
Department of Microbiology, University of Virginia, Charlottesville, VA 22908
This study investigated the unresolved issue of antigen-dependency and antigen-specificity of
autoimmune disease suppression by CD4CD25
disease (AOD) in day 3 thymectomized (d3tx) mice and polyclonal T regs expressing the Thy1.1
marker, we determined: (a) the location of recipient T cell suppression, (b) the distribution of
AOD-suppressing T regs, and (c) the relative efficacy of male versus female T regs. Expansion
of recipient CD4 T cells, activation/memory marker expression, and IFN-
inhibited persistently in the ovary-draining LNs but not elsewhere. The cellular changes were
reversed upon Thy1.1 T reg depletion, with emergence of potent pathogenic T cells and
severe AOD. Similar changes were detected in the regional LNs during autoimmune
dacryoadenitis and autoimmune prostatitis suppression. Although the infused Thy1.1
proliferated and were disseminated in peripheral lymphoid organs, only those retrieved from
ovary-draining LNs adoptively suppressed AOD at a suboptimal cell dose. By depriving d3tx
recipients of ovarian antigens, we unmasked the supremacy of ovarian antigen-exposed
female over male T regs in AOD suppression. Thus, disease suppression by polyclonal T regs
depends on endogenous antigen stimulation; this occurs in a location where potent antigen-
specific T regs accumulate and continuously negate pathogenic T cell response.
T cells (T regs). Based on autoimmune ovarian
There is now general acceptance that natural
CD25 T cells that express the functional
cellular component of the normal immune sys-
tem (1). They develop in the thymus and are
potent suppressors or regulators of effector and
helper T cell responses in the setting of organ-
specific autoimmunity, allergic responses, allo-
graft rejection, and microbial immunity (2). T
regs also modulate antitumor response to favor
tumor growth and metastasis; their elimination
has led to enhanced immunogenicity of tumor
vaccines (3). In studies on common human au-
toimmune disease, including multiple sclerosis
(4), rheumatoid arthritis (5), and autoimmune
polyendocrinopathy syndrome (6), the reduction
in T reg number or function has been reported.
These clinical findings prompted attempts in de-
ploying antigen-specific T regs for treatment of
autoimmune disease and allograft rejection (7).
Patients and mice deficient in T reg func-
tion that is due to mutation of the
develop severe and fatal autoimmunity (8); thus,
antigen-specific T regs is a likely mechanism
, are an important
for maintenance of peripheral tolerance that
normally prevents pathogenic autoimmune re-
sponse. In studies of T cells expressing transgenic
TCRs against nominal, transgenic, or allogeneic
antigens, peripheral T regs respond to cognate
antigen (9–12), proliferate extensively in normal
and lymphopenic hosts (13–15), and respond to
self-peptides presented by dendritic cells with
unique properties in tissue-draining LNs (15–
18). Naturally processed epitopes recognized
by T regs also have been identified (19, 20).
Therefore, T regs can recognize and respond
to specific antigenic peptides in vivo.
However, the more critical issue of the anti-
gen dependency of natural T regs with respect to
their functional acquisition and autoimmune
disease suppression has not been resolved. This
is a fundamental question because its clarification
will allow us to address more fully: (a) T regs as
significant participants in physiologic peripheral
tolerance, a process that depends on antigen-
specific immunoregulation; and (b) application
of T regs in antigen-specific immunotherapy.
The second question has been explored in
experiments that were based on T reg-expressing
transgenic TCRs. They showed that T regs
The online version of this article contains supplemental material.
Kenneth S.K. Tung:
Abbreviations used: AOD,
autoimmune ovarian disease;
d3tx, day 3 thymectomy; EAE,
experimental allergic encephalo-
myelitis; nOX, neonatal ovariec-
tomy; PLP, proteolipid protein;
T reg, CD4
ANTIGEN REQUIREMENT IN REGULATORY T CELL FUNCTION | Samy et al.
that had been expanded previously by the cognate antigen
had enhanced capacity in suppressing the relevant autoim-
mune disease (21–24). Transgenic T regs (BDC2.5) sup-
pressed diabetes that was transferred adoptively by the same
transgenic diabetogenic effector T cells (17). Similarly, pro-
teolipid protein (PLP)-specific T regs suppressed experimen-
tal allergic encephalomyelitis (EAE) that was induced by
PLP immunization (24), and thyroglobulin-specific T regs
suppressed thyroiditis that was induced by thyroglobulin im-
munization (23). Thus, T regs with known specificity sup-
press autoimmune disease that is induced by pathogenic T
cells of shared specificity. In EAE, PLP-specific T regs sup-
pressed EAE that was induced by PLP, but not by myelin
oligodendrocyte glycoprotein; this provides more definitive
support for antigen-specific suppression. However, these
studies also yielded data that implicated antigen-nonspecific
suppression. For example, the BDC2.5 transgenic T regs
suppressed disease that was transferred adoptively by whole
spleen cells from female nonobese diabetic donors (17), and
PLP-specific T regs suppressed EAE that was induced by im-
munization with brain homogenate (24). Moreover, when
the PLP-specific T regs were activated in vitro, they sup-
pressed EAE that was induced by PLP or by myelin oligo-
dendrocyte glycoprotein (24). The nonspecific suppression
observed could be due to cross-suppression of effector T
cells of one specificity by T regs of a second specificity,
when both cognate antigens are present in vivo (24, 25).
However, it also could be explained by the dual TCRs that
are expressed on the nonphysiologic transgenic T cells, due
to pairing of a random endogenous V
(26–28). Thus, T reg expansion may depend on engage-
ment of the transgenic TCR, whereas disease suppression
may depend on the second TCR of unknown specificity.
Studies with polyclonal T regs have addressed the ques-
tion of acquisition of T reg function in response to physio-
logic self-antigen. When polyclonal T cells from normal an-
tigen-positive donors were used to suppress autoimmune
thyroiditis in adult thymectomized and irradiated rats (29),
or autoimmune prostatitis of day 3 thymectomy (d3tx) mice
(30, 31), they suppressed disease with greater efficacy than
with the transgenic
the T cells from antigen-negative donors. Moreover, the T
regs from thyroid antigen-negative donors retained the abil-
ity to suppress autoimmune diabetes in the same animals.
However, when the same strategy was applied to autoim-
mune ovarian disease (AOD) suppression, the polyclonal T
regs from male or female donors suppressed the disease
To gain more insight into antigen-dependency and anti-
gen-specificity of physiologic polyclonal T regs, we studied
the classical AOD model of the d3tx mouse. D3tx renders
mice T lymphocytopenic and T reg-deficient (33, 34) and,
in this setting, the effector T cells spontaneously respond to
endogenous ovarian antigens and trigger AOD (35). The dis-
ease is inhibited completely by polyclonal T regs that are
transferred from normal adults soon after thymectomy (33).
Because it is not possible to analyze the function of antigen-
specific T regs within a polyclonal population, we deter-
mined disease specificity rather than antigen specificity of the
T reg function. In addition, the study is based on several
novel approaches that are built on current understanding
of antigen-specific T cell responses. First, we determined
whether T reg suppression, similar to the effector T cell re-
sponse, is triggered by organ-specific antigens that are pro-
cessed and presented uniquely in the regional LNs (36, 37).
Second, to explore the causal relationship among T reg pres-
ence, regional LN changes, and AOD suppression, we stud-
ied the effect of in vivo T reg depletion in d3tx mice that are
under suppression by T regs. Third, we investigated the lym-
phoid organ distribution of T regs and, most importantly, re-
isolated the input T regs from individual lymphoid organs
and studied for the first time the distribution of “memory” T
regs with enhanced capacity to suppress AOD. Fourth, we
revisited our earlier attempt to compare male and female T
regs in AOD suppression, but in a setting where the oppor-
tunity for the T reg to be educated by endogenous ovarian
antigens in the d3tx recipients was no longer permitted. The
results of our study uniformly and strongly support the con-
clusion that the acquisition of T reg function and AOD sup-
pression by polyclonal T regs are highly disease-dependent,
and, therefore, antigen-dependent.
suppression by T regs in d3tx B6AF1 mice. (A) AOD in d3tx mice of
Time course of AOD induction and dose response of
different ages. (B) AOD in 6-wk-old d3tx recipients of different numbers of
Thy1.1?CD25? T cells from normal female donors.
JEM VOL. 202, September 19, 2005
The ovary-draining LN is the site of T cell activation
required for AOD induction in d3tx mice
Following d3tx, ovarian inflammation appeared spontane-
ously in the B6AF1 mice at 3 wk of age, and, by 8 wk, 89%
developed severe inflammation and ovarian atrophy (Fig. 1
A). To investigate the requirement of endogenous antigenic
stimulation in AOD induction, we compared LN changes
between d3tx mice and d3tx mice with neonatal ovariec-
tomy (d3tx/nOX). The d3tx/nOX mice developed severe
lymphopenia without evidence of autoimmune response to
oocyte antigens or AOD (35), and were used as appropriate
control for this study and the subsequent studies on AOD
At 4 wk, there was a significant increase in the number of
total and CD4
T cells in the ovary-draining LNs of d3tx
mice, and the CD4 T cells expressed significantly higher lev-
els of CD69 and CD44
markers (Fig. 2; Table I). These
changes were not observed in the nondraining LNs. Therefore,
the autoimmune T cell response to endogenous antigen occurs
in the ovary-draining LNs, a finding that is consistent with
other models of spontaneous autoimmune disease (36, 38).
in ovarian and inguinal LNs at 4 wk (top), and from 3 to 8 wk
(bottom). Compared with d3tx/nOX control, the total cellularity and
CD4? T cell number in the d3tx mice were increased significantly in ovary-
The total cell number and recipient CD4? T cell number
draining LNs, but not inguinal LNs. Compared with d3tx mice, profound
reduction in the total cellularity and recipient CD4? T cell number occurred
in ovary-draining LNs but not inguinal LNs of the d3tx/suppressed mice.
This was detectable at 4 wk and persisted to 8 wk.
d3tx mice, d3tx/nOX mice, and d3tx/suppressed mice
Activation and memory T cell markers on recipient CD4
T cells from ovary-draining LNs and nondraining LNs from
) of recipient CD4
cells with marker
SEM (% positive)
Treatment of mice (
n) Inguinal LN
CD69 d3tx/nOX (6)
CD25d3tx (8) 28
2 (22)d3tx/supp (8)
D3tx/nOX, d3tx mice with neonatal ovarian ablation; D3tx/supp, d3tx mice suppressed with CD4
ANTIGEN REQUIREMENT IN REGULATORY T CELL FUNCTION | Samy et al.
Profound suppression of recipient T cell response occurs in
the ovary-draining LNs of d3tx mice with complete AOD
prevention by T regs
AOD was suppressed completely in d3tx mice that received
0.5 million Thy1.1 T regs that were pooled from the
LNs and the spleens of normal adult mice (d3tx/suppressed
mice). Partial suppression was noted in the recipients of 0.3
million cells; those that received 0.1 million cells showed no
evidence of disease suppression (Fig. 1 B). The ovaries of
d3tx/suppressed mice were free of pathology and devoid of
infiltrating T cells (Fig. 3, A and B). The mice did not have
oocyte autoantibodies (35), and their splenic T cells adop-
tively transferred only mild and infrequent AOD to neonatal
recipients. To locate where the autoimmune response was
suppressed, we compared the phenotype of the recipient
T cells in LNs and spleen between
d3tx mice and d3tx mice that had received 0.5 million
Thy1.1 T regs.
There was a profound reduction in the total cellularity in
the ovary-draining LNs at 4 wk (Fig. 2, top); this persisted
for at least 8 wk (Fig. 2, bottom). Morphologically, the
ovary-draining LNs of d3tx/suppressed mice were exceed-
ingly small, with empty-looking T cell zones and no germi-
nal centers (Fig. 3, C and D). These changes were not ob-
served in the spleen or the nondraining LNs, including the
inguinal and axillary LNs (unpublished data).
There also was significant reduction in the recipient
CD4 T cell numbers within the ovary-draining LNs (Fig.
2). The number in d3tx mice increased 400% from weeks 3
to 4, and remained elevated at week 8 (Fig. 2, bottom),
whereas the number of recipient CD4
draining LNs of the d3tx/suppressed mice remained very
low for the entire 8-wk period (Fig. 2, bottom). In parallel,
there was a profound reduction in recipient CD4
vision as documented by bromodeoxyuridine (BrdU) incor-
0.0001) (Fig. 4 A, left). This was not observed
in the nondraining LNs or the spleen (Figs. 4 A, right).
In the ovary-draining LNs of the d3tx/suppressed mice,
phenotypic changes that are characteristic of T cell activa-
tion and memory T cells (CD25
) were reduced greatly (Table I). In addition, signifi-
fewer recipient CD4
T cells in the ovary-draining
LNs produced IFN-
(Fig. 4 B). Again, these changes were
not detectable in the nondraining LNs or the spleen (Fig. 4
B). Thus, suppression of recipient T cell response of the d3tx
mice occurs exclusively in the ovary-draining LNs.
cells in the ovary-
T cell di-
Suppression of the recipient T cell response is not unique to
AOD or the ovary-draining LNs
To address whether regional LN suppression was unique to
the ovary-draining LNs in mice with AOD, we studied the
recipients’ T cell response in (a) the LNs draining the lacri-
mal glands in d3tx B6AF1 male mice with autoimmune
dacryoadenitis, and (b) the LNs draining the prostate in d3tx
B6AF1 male mice with autoimmune prostatitis. Similar re-
sults were found in both extraovarian autoimmune diseases.
As shown in Fig. S1 (available at http://www.jem.org/cgi/
content/full/jem.20041033/DC1), the recipient CD4
d3tx mice and the d3tx/suppressed mice. At 4 wk, the ovary of a d3tx
mouse has heavy T cell infiltration (A), whereas the ovary of a d3tx/suppressed
mouse is completely devoid of T cells (B) (Texas red–labeled anti-CD5 anti-
body plus DAPI). Findings are representative of 10 mice for each group.
The ovary-draining LN of the d3tx mice is enlarged, and contains a germinal
center and a broad T cell zone (C), whereas the ovary-draining LN of the
d3tx/suppressed mice has an empty T cell zone and is devoid of germinal
centers (D) (hematoxylin and eosin).
Ovarian immunohistology and ovarian LN histology of
negative recipient CD4? T cells in the ovary-draining LNs of d3tx
versus d3tx/suppressed mice. BrdU uptake in 12 h by cells in d3tx mice
greatly exceeds that of d3tx/suppressed mice; the difference is not found
in the inguinal LN. IFN-?–producing CD4? T cell numbers of d3tx mice exceed
those of d3tx/suppressed mice in ovarian LNs but not in inguinal LNs.
Proliferation (A) and IFN-? production (B) of Thy1.1-
JEM VOL. 202, September 19, 2005
cell number and the number of CD4
the activation/memory phenotypic markers (CD69, CD44
and CD62L ), were reduced significantly in the d3tx/sup-
pressed mice in which the extraovarian autoimmune disease
was suppressed completely by 0.5 million T regs.
T cells expressing
suppressing T regs are enriched in the ovary-draining LNs
To accrue additional evidence for the response of T regs to
endogenous antigens in regional LNs, we determined the
distribution of total and AOD-suppressing Thy1.1
in d3tx/suppressed mice. 5,6-carboxyfluorescein diacetate-
succinimidyl ester (CFSE)-labeled Thy1.1
regs proliferated extensively in the d3tx/suppressed mice. By
day 23, they made up
25% of CD4
and the spleen (Fig. 5; not depicted), and
at least eight times. We next reisolated the polyclonal Thy
T regs from the ovary or the nondraining LNs of d3tx/
suppressed mice, and studied the LN-specific input T regs
for suppression of AOD and dacryoadenitis in d3tx mice.
This approach was based on the assumption that if ovarian
antigen-specific memory T regs existed, they should have
enhanced regulatory capacity for suppression of AOD, but
0.1 million Thy1.1
T cells at
lated from the ovary-draining LNs from 10 d3tx/suppressed
donors (Fig. 5 C), and were transferred into a single d3tx
recipient. Remarkably, with the suboptimal 0.1 million
T regs from the ovary-draining LNs, a complete
suppression of AOD and oocyte autoantibody response was
T regs are distributed widely, but AOD-
T cells in the LNs
90% had divided
90% purity were reiso-
achieved (Fig. 5 D). In contrast, 0.1 million Thy1.1
that were reisolated from nondraining LNs did not affect the
development of AOD or autoantibody response, although
they completely suppressed dacryoadenitis (Fig. 5, C and D).
Also important was the failure of the T regs from ovary-
draining LNs to suppress dacryoadenitis (Fig. 5 D). Thus, in
the ovary-draining LN there is an accumulation of T regs
with enhanced AOD-suppressing capacity and a concomi-
tant reduction of dacryoadenitis-specific T regs.
If the T regs with enhanced AOD-suppressing property
that accumulated in the regional LNs were responsible for
AOD suppression and for negating host T cell responses in
this location, these changes should reverse following deple-
tion of the infused T regs from the d3tx/suppressed host.
ovary-draining LN suppression and emergence of severe AOD
Infusion of Thy1.1 antibody completely eliminated all
Thy1.1? T reg cells from the 3-wk-old, d3tx/suppressed
mice (Fig. 6 B). 4 wk later, the recipient CD4? T cells in the
ovary-draining LNs increased significantly in number as well
as in the expression of T cell activation and memory T cell
markers (Fig. 6 A). These changes were observed only in the
ovary-draining LNs (Fig. 6 A), and not in the nondraining
LNs or the spleen (not depicted). Remarkably, severe AOD
also emerged in essentially all Thy1.1? T cell-depleted mice;
disease incidence and severity matched that of 7-wk-old
d3tx female mice (Fig. 6 A).
Although suppression of the recipient CD4? T cell re-
sponse and AOD development depend on the persistent ac-
T reg depletion in vivo is followed by reversal of
reg cells in d3tx/suppressed mice. T regs proliferated and were distributed
equally in ovarian LNs and nondraining LNs (A, B). However, the Thy1.1?
T regs (C) that were reisolated from the ovarian LNs (OLNs) but not the
nondraining LNs (NDLNs) suppress AOD and oocyte antibody response in
d3tx mice at 0.1 million cells per recipient (D). Conversely, dacryoadenitis
was suppressed by T regs that were reisolated from NDLNs but not by T regs
Proliferation, distribution, and function of the polyclonal T
from OLNs (D). In D, closed circles denote mice with serum oocyte antibody.
The NDLNs included the inguinal, axillary, brachial, and cervical LNs. T reg
proliferation was determined by injecting 0.5 million CFSE-labeled Thy1.1?
T regs into 5-d-old d3tx B6AF1 mice, and studying them 23 d later. In A,
donor cells (gated on CD4?Thy1.1? cells) were analyzed for percentages
that are undivided, have divided one to six times, or more than six times. The
CFSE line graph represents data pooled from four to eight mice.
ANTIGEN REQUIREMENT IN REGULATORY T CELL FUNCTION | Samy et al.
tion of the Thy1.1? T regs in the ovary-draining LNs,
suppression was not evident in the spleen, despite the coex-
istence of polyclonal T regs and effector T cells. Therefore,
we investigated the function of regulatory and effector T
cells in the spleens of the d3tx suppressed mice by evaluating
their capacity to transfer AOD adoptively.
The spleen contains T regs and effector T cells relevant to
AOD but exhibits no evidence of T cell suppression in
the d3tx/suppressed mice
Compared with d3tx donors, the total splenocytes from the
d3tx/suppressed mice transferred much milder and less fre-
quent ovarian inflammation to young naive recipients (Fig. 6
C). This could be due to deficiency in effector T cells in the
spleen or to suppression of the donor effector T cells by the
Thy1.1? T regs in the neonatal recipients. To differentiate
the two possibilities, the Thy1.1? T cells were depleted from
the splenocytes ex vivo before cell transfer. As shown in Fig.
6 C, splenocytes depleted of Thy1.1? T regs transferred
AOD with comparable severity and frequency to the AOD
that was transferred by total splenocytes of d3tx donors.
Therefore, despite the colocalization of functional splenic ef-
fector T cells and T regs, T cell suppression was not evident
in the spleen, presumably because ovarian antigens are not
accessible to the spleen. However, when the cells were
cotransferred to the neonatal recipients where they are ac-
cessible to ovarian antigens, AOD suppression ensues.
AOD is suppressed more effectively by female T regs than
male T regs when their encounter with recipient ovarian
antigens is avoided for 2.5 wk after cell transfer
Our previous study showed that splenocytes from ovarian
antigen-positive females and ovarian antigen-negative males
suppressed d3tx-induced AOD with equal efficacy; this was
taken as evidence against ovarian antigen-dependency of
AOD suppression by T regs (32). However, the interpreta-
tion is not consistent with the cumulative evidence provided
by the present study. To reevaluate the old data, we rea-
soned that if T reg function was critically antigen-depen-
dent, then the male T regs would respond to ovarian anti-
gens of the neonatal d3tx recipients, gain AOD-suppressing
capacity, and suppress like female T regs. Therefore, we
studied AOD suppression in d3tx female recipients devoid of
ovarian antigens for the first 3 wk of life.
Neonatal mice were ovariectomized within 24 h of birth
and were thymectomized on day 3. On day 5–7, they re-
ceived 0.3 million CD4?CD25? T cells from male or female
donors. At 3 wk of age they were engrafted with an age-
matched ovary, which provided antigenic stimulus for effec-
tor T cell response and also served as the target of AOD. In
d3tx recipients that were devoid of neonatal ovarian antigen
expression, AOD was suppressed completely by T regs from
female donors (Fig. 7, A and C), whereas AOD was not sup-
pressed by T regs from male donors (Fig. 7, A and D). The
ovarian disease in the recipients of T regs from male donors
of Thy.1 T reg cells. (A) Reversibility of ovarian LN suppression and AOD.
(B) Thy1.1? T regs in d3tx/suppressed mice were eliminated by a single
injection of Thy1.1 mAb at 3 wk, and were studied at 7 wk. (C) To detect
Reversal of AOD suppression by in vivo and in vitro depletion
functional splenic effector T cells and T regs in the d3tx/suppressed mice,
spleen cells were transferred to 5 d-old recipients with or without ex vivo
depletion of the infused Thy1.1? T regs. AOD in cell recipients was determined
12 d later. AOD in recipients of spleen cells from d3tx mice served as control.
JEM VOL. 202, September 19, 2005
did not differ from the disease of the d3tx mice in frequency
and severity (Fig. 7 A). As an important tissue-specificity
control, the T regs from the same male and female donors
suppressed dacryoadenitis in the same d3tx/nOX recipients
with equal efficacy (Fig. 7 B). We conclude that T regs from
the ovarian antigen-positive female donors are intrinsically
more competent in AOD suppression than the T regs from
antigen-negative male donors. However, when the male T
regs have the opportunity to respond to ovarian antigens
from the ovary-intact female recipients, they gain AOD-
suppressing function and suppress AOD as efficiently as fe-
male T regs (32).
We have investigated the mechanism of AOD suppression in
d3tx mice by polyclonal Thy1.1? natural T regs from nor-
mal donors, and accrued results that strongly support the
conclusion that the polyclonal T reg action in this setting is
antigen-dependent and disease-specific. (a) Stimulation and
suppression of the pathogenic T cell response of d3tx mice
occurred exclusively in the ovary-draining LNs in AOD
suppression. (b) The LN changes were not unique to AOD
because the host T cell suppression was confined to the lacri-
mal gland–draining LNs in autoimmune dacryoadenitis sup-
pression, and to the prostate-draining LNs in autoimmune
prostatitis suppression. (c) Suppression of host T cell re-
sponse and AOD suppression were reversed upon depletion
of the infused Thy1.1? T regs. (d) In contrast, suppression of
the recipients’ T cell response was not detectable in the non-
draining LNs or the spleen even though effector T cells and
T regs relevant to AOD coexisted in these locations. (e) In
d3tx mice suppressed by Thy1.1? T regs, the ovary-draining
LN was enriched in Thy1.1? T regs that exhibited greatly
enhanced capacity to suppress AOD, but did not suppress
the autoimmune dacryoadenitis that also develops in d3tx
B6AF1 mice. (f) T regs from normal females had a greater
capacity than male T regs to suppress AOD; however this
was demonstrable only in d3tx recipients that were deprived
of ovarian antigens in the neonatal period. Thus, a short ex-
posure to ovarian antigens in neonatal recipients is sufficient
for male T regs to gain AOD-suppressing capacity to the
level of female T regs.
It is generally agreed that in spontaneous autoimmunity,
the regional LN is the location of autoimmune response in-
duction (36–39). The obligatory role of the regional LN re-
sponse in autoimmune diabetes was demonstrated by the ex-
periment wherein excision of the pancreatic LN of young
nonobese diabetic mice prevented development of diabetes
mellitus (36). In AOD of d3tx mice, neonatal ovarian abla-
tion also abrogated ovarian autoimmune response and pre-
vented AOD development in ovarian grafts at specific time
points (35); as shown here, ablation of ovarian antigens also
abrogated T cell activation in the d3tx/nOX mice. Thus,
changes in the regional LN in spontaneous autoimmune dis-
ease are indicative of cellular events of the antigen-specific
immune responses. We have now extended this paradigm
and demonstrated that the regional LN also is the site of sup-
pression of autoimmune diseases by polyclonal T regs. Even
AOD-specific T reg function. In this experiment, each neonatal recipient
received 0.1 million Thy1.1? cells retrieved from the LNs of d3tx/suppressed
mice. (A) AOD does not develop in the d3tx/nOX recipients of female
T regs, whereas the d3tx/nOX recipients of male T regs developed AOD
with the same incidence and severity as the AOD in control d3tx mice.
Ovarian antigen exposure in neonatal mice capacitates
(B) In contrast to AOD, dacryoadenitis in these d3tx/nOX recipients is
inhibited completely by male or female T regs. (C) The ovarian graft of
d3tx/nOX recipients of female T regs is normal histologically (arrow points
to normal oocyte). (D) The ovarian graft of d3tx/nOX recipients of male T
regs is infiltrated heavily by inflammatory cells, some replacing the oocyte
(arrow; hematoxylin and eosin).
ANTIGEN REQUIREMENT IN REGULATORY T CELL FUNCTION | Samy et al.
more remarkable was the finding that Thy1.1? T regs with
greatly enhanced capacity to suppress AOD also accumulate
in the ovary-draining LNs. Based on these observations, we
conclude that the regional LN is where polyclonal antigen-
specific T regs respond to self-antigens, gain disease-sup-
pressing function, suppress pathogenic T cell response, and
prevent autoimmune disease.
Suppression of the host CD4? T cells in the regional
LNs affected all levels of T cell response, including profound
reduction in DNA synthesis and proliferation of CD4? T
cells, changes in host CD4? T cell phenotype to indicate re-
duction of activated and memory T cells, and reduction in
proinflammatory IFN-? production. The regional LN re-
sponse was not unique to the ovary or to the ovary-draining
LNs, because similar changes were observed in autoimmune
dacryoadenitis and autoimmune prostatitis. This finding
strongly argues that the ovarian LN changes represent the re-
sponse to ovarian antigen, rather than the response to other
unique ovarian factors. The suppression of the host T cell re-
sponse in the regional LN is critically dependent on the con-
tinuous presence of the T reg. When the Thy1.1? T regs
were eliminated by antibody in vivo, host T cell suppression
in the regional LNs reversed and was followed by the emer-
gence of severe AOD. Thus, the presence of T regs in the
regional LN is linked causally to suppression of AOD. This
finding also indicates that T reg action is cytostatic, rather
than cytolytic (40–42); that the capacity of T regs to suppress
AOD has not been transferred to recipient T cells through
infectious tolerance (43–45); and that there has been no sig-
nificant conversion of recipient CD4?CD25? T cells to T
regs during disease suppression (46, 47). Conversely, we
consider the finding to be consistent with the requirement of
colocalization and cell–cell interaction of T regs and effector
T cells within the draining LNs where ovarian antigens are
presented to both T cell subsets (48). Depletion of the in-
fused Thy1.1? T regs allows the effector T cells to respond
to ovarian antigens presented in the regional LNs and medi-
ate AOD. The reversibility of T reg suppression was docu-
mented in EAE of Rag knockout mice that expressed trans-
genic T cell receptor for myelin basic peptide (49), allograft
rejection (45), and inflammatory bowel disease (50).
The new finding of the superactive Thy1.1? T regs in
ovary-draining LNs of the d3tx/suppressed mice is of consid-
erable interest. These cells are estimated to be at least five
times more potent than the T regs from nondraining LNs
when we take into consideration the dose response of AOD
suppression by T regs from normal B6AF1 donors, shown in
Fig. 1. The finding is reminiscent of the response of TCR
transgenic T regs to pancreas-specific cognate antigens (16,
17, 21, 51), and strongly supports the response of polyclonal
T regs to ovarian antigens in this location. It also should be
noted that although they exhibited superactive AOD-sup-
pressing capacity, the polyclonal T regs from ovary-draining
LNs are defective in dacryoadenitis-suppressing function
when compared with the T regs in nondraining LNs. Perhaps
the nonfunctioning T regs are excluded from the ovary-
draining LN when it is dedicated to AOD suppression.
In contrary to the ovary-draining LNs, the nondraining
LNs and the spleen showed no evidence of host T cell sup-
pression. This was not due to the absence of functional ef-
fector or regulatory T cells because both activities were de-
monstrable when the cells were transferred adoptively, singly
or together, into the naive neonatal recipients. As a more
tenable explanation, we believe that suppression did not oc-
cur in the nondraining LNs or the spleen of the d3tx/sup-
pressed host because ovarian antigens were not presented in
those locations; however, the function of the T cells was
manifested when they were stimulated by ovarian antigens of
the cell recipients after adoptive transfer. This possibility is
supported by the fact that immunogenic ovarian antigens are
present in neonatal mice and have been documented to
stimulate pathogenic T cells responsible to neonatal AOD
If endogenous antigens can prime antigen-specific T reg
function, then the polyclonal T regs from normal female
donors should suppress AOD more efficiently than the poly-
clonal T regs from normal male donors. However, our ear-
lier study showed that both populations suppressed d3tx-
induced AOD equally (32). We have now documented that
the equal AOD suppression by male and female T regs is ex-
plicable by the response of male T regs to recipient ovarian
antigens; this finding strongly supports, rather than refutes,
the antigen-dependency of polyclonal T reg function.
In conclusion, this study provides strong evidence that
endogenous antigens are major players in spontaneous au-
toimmunity. They stimulate pathogenic T cells in disease in-
duction. They also stimulate antigen-specific polyclonal T
regs to gain disease-suppressing capacity quickly and to accu-
mulate in the regional LNs, the site of ovarian antigenic
stimulation. In AOD, the ovarian antigen orchestrates im-
portant cellular events in the ovary-draining LNs during dis-
ease suppression, and allows the accumulation of highly
potent AOD-specific polyclonal T regs that continuously
negate pathogenic T cell response and inhibit AOD.
MATERIALS AND METHODS
Mice and surgery. C57BL/6 ? AJ (B6AF1) mice were produced from
C57BL/6 and AJ adults from the National Cancer Institute. B6.PL-Thy1a/
Cy (Thy1.1?) female mice came from The Jackson Laboratory. All mice
were kept in a pathogen-free facility; experiments were approved and per-
formed in accordance with the guidelines of the Animal Care and Use
Committee of University of Virginia. Thymectomy was performed by suc-
tion under hypothermia anesthesia (35); complete thymectomy was verified
histologically and mice with residual thymus excluded. Bilateral nOX was
performed through vertical posterior incision under hypothermia; mice
with residual ovaries were excluded. Ovary was implanted by insertion of a
3-wk-old ovary under kidney capsule through posterior incision.
Media, reagents, and antibodies. Cells were harvested in complete
RPMI 1640 medium (Biowhittaker) supplemented with 10% heat-inacti-
vated FCS, 2-mercaptoethanol (5 ? 10?5 M), L-glutamine (2 mM), peni-
cillin (100 U/ml), streptomycin (100 ?g/ml), Hepes (10 mM), nonessential
amino acids (0.1 mM), and sodium pyruvate (1 mM) (Invitrogen). The fol-
JEM VOL. 202, September 19, 2005
lowing antibodies and isotype controls (BD Biosciences) were used for flow
cytometric analysis and cell isolation: biotin-conjugated anti-CD25 (7D4),
anti-CD16/CD32 (2.4G2), anti-CD90.1 (HIS51), FITC-conjugated anti-
CD4 (H129.19), CyC-conjugated anti-CD4 (L3T4), PE-conjugated anti-
CD4 (L3T4), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CD25 (7D4),
anti-CD69 (H1.2F3), anti-CD90.1 (HIS51) rat IgG2a (R35-95), rat IgG2b
(R35-38), rat IgM (R4-22), hamster IgG (A19-3), anti–IFN-? (XMG1.2),
anti-CD25 (PC61), anti-CD90.1 (OX-7), rat IgG1 (R3-34), rat IgG2a
(R35-95), and rat IgG2a (R35-95).
LN dissection, cell purification, and CFSE labeling. LNs that drain
the ovary (renal), prostate (iliac or lumbar), and lacrimal gland (superficial
cervical), as well as the axillary, brachial, and inguinal (nondraining) LNs
were dissected from 8–10-wk-old Thy1.1? B6AF1 mice and dissociated
into single-cell suspension. CD4?CD25? T cells were purified as described
(55). Enriched on T cell enrichment columns (R&D Systems), T cells were
stained with biotin-conjugated anti-CD25 antibody (7D4) or anti-Thy1.1
antibody, followed by PE-conjugated streptavidin (Rockland Immu-
nochemicals) and anti-PE microbeads (Miltenyi Biotec), and isolated on the
autoMACS system (Miltenyi Biotec). Cell purity ranged from 90 to 95%.
To suppress disease, the CD4?CD25? T cells—in 50 ?l of HBSS—were
injected i.p. into 5-d-old d3tx pups. CD4?CD25? T cells were labeled
with CFSE (Invitrogen) by incubation for 10 min at 37?C in 10?M CFSE
in HBSS, washed in complete RPMI 1640 and then HBSS, and injected
i.p. in 5-d-old d3tx pups.
Histology and disease grading. Tissues were fixed in Bouin’s fixative,
embedded in paraffin, and 5-?m serial sections were stained with hematox-
ylin and eosin, and examined as unknown samples. Ovarian pathology was
evaluated in 50 step sections, and graded on a scale of 1–4 (35). Grade 1 in-
flammation consists of 1–2 foci of inflammatory cells, including inflamma-
tory infiltration restricted to the hilar region. Grades 2 and 3 include incre-
mental extent of ovarian inflammation, involving follicles and interstitial
space, but no ovarian atrophy. Grade 4 pathology has ovarian atrophy with
loss of mature, growing, and/or primordial oocytes. Dacryoadenitis and
prostatitis were graded from 1 to 4 as follows. Grade 1 disease was focal
mononuclear infiltration. Grade 4 lesions had diffuse inflammation with at-
rophy and loss of glandular and ductal structure. Grades 2 and 3 represent
increasing inflammation between grades 1 and 4.
Immunofluorescence microscopy. Antibody to ovarian oocytes was
detected by indirect immunofluorescence, and the intensity was graded
from 1–3. Serum diluted 1:50 in PBS, was incubated with frozen sections of
adult mouse ovaries prefixed in 95% ethanol and blocked by goat serum,
followed by incubation with FITC-conjugated goat anti–mouse IgG (Jack-
son ImmunoResearch Laboratories). Inflammatory cells in normal and dis-
eased ovaries were identified by immunofluorescence using the TSA Biotin
System (PerkinElmer), with antibody to CD5 (53–7.313), CD4 (GK1.5),
CD8 (53–6.7) T cells, macrophage (F4/80), B cell (B220), and MHC class
II (M5/114.15-2) for activated macrophages and dendritic cells, as described
Cell counting. LN cells, dissociated by syringe plunger, were incubated in
HBSS containing collagenase D (Roche Applied Sciences; 400 U/ml) and
collagenase VII (Sigma-Aldrich; 100 U/ml) for 20 min at 37?C. The cells
were filtered through nylon and washed in RPMI 1640. Cell number was
determined with hemacytometer or automatic counter (Hemavet 850,
CDC Technologies Inc.), with comparable results.
Flow cytometric analysis and intracellular cytokine staining. LN
cell suspension (106 per well) was incubated with anti-CD16/CD32 anti-
body to block IgG-Fc receptors, which was followed by 30-min incubation
with FITC-, PE-, and/or CyC-conjugated monoclonal antibodies. Isotype
control IgG was included. Cells were studied on a FACScan and analyzed
by Cell Quest software (BD Biosciences), the FlowJo software (Tree Star,
Inc.), or by WINMDI (http://facs.scripps.edu/software.html). For intracel-
lular cytokines, cells treated with phorbol-12-myristate-13-acetate, iono-
mycin, and brefeldin A (BD Biosciences) for 6 h were stained with anti-
CD4-CyC, fixed and permeabilized in formaldehyde/saponin (Fix/Perm
Buffer, BD Biosciences), washed, and stained with PE-conjugated antibody
to cytokine or control antibody. Cells were washed in buffer containing sa-
ponin before study.
BrdU labeling. D3tx B6AF1 mice were injected i.p. with 1 mg of BrdU
in PBS on day 25 and studied 12 h later, or they were fed BrdU, 0.8 mg/
ml, in drinking water starting on day 21 for 4 d. Cells from individual LNs
were analyzed for BrdU incorporation using BrdU Flow Kits (BD Bio-
sciences). In brief, LN cells, stained with PE-conjugated anti-CD4 anti-
body, fixed, and permeabilized, were treated with DNase (Sigma-Aldrich),
stained with FITC-labeled anti-BrdU antibody (BD Biosciences), and ana-
lyzed by flow cytometry.
In vivo and in vitro cell depletion. D3tx/suppressed B6AF1 mice re-
ceived one i.v. injection of 100 ?g mouse antibody to Thy1.1 [CD90.1
(HIS51)] at 3 wk, and the completeness of cell depletion was verified by
flow cytometry at 7 wk. For ex vivo Thy1.1? cell depletion, splenocyte sus-
pension was incubated with FITC-conjugated anti-Thy1.1 (CD90.1)
(HIS51) (BD Biosciences), followed by anti-FITC microbeads (Miltenyi
Biotec) and separation on the autoMACS system (Miltenyi Biotec).
Adoptive transfer of AOD. 107 total splenocytes, or those depleted of
Thy1.1 cells, were injected i.p. into 3–5-d-old naive pups. Ovarian pathol-
ogy of cell recipients was determined 12 d later.
Online supplemental material. Fig. S1 shows that the recipient CD4? T
cell number and the number of CD4? T cells expressing the activation/mem-
ory markers were reduced significantly in the prostate and lacrimal gland
draining but not the inguinal LNs. Online supplemental material is available at
We are grateful to S. Mangawang, V. Rubianes, Y.F. Sun, and J. Nash for expert
technical assistance. We also thank Dr. A. Thornton for her suggestions on CD25? T
cell purification; Dr. J. Goverman for suggestions on CFSE labeling; and Drs. M.S. Sy,
Y.-X. Fu, Z. Fehervari, and S. Sakaguchi for their helpful critique.
This study is supported by National Institutes of Health grants AI-41236, AI-
51420, AR45222, and HD-44415. This work benefitted from the Cancer Center
Support (P30 CA44579) Research Histology Core.
The authors have no conflicting financial interests.
Submitted: 26 May 2004
Accepted: 4 August 2005
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