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Endogenous Oocyte Antigens Are Required
for Rapid Induction and Progression of
Autoimmune Ovarian Disease Following
Day-3 Thymectomy
Pascale Alard, Claire Thompson, Sally S. Agersborg, Jayant
Thatte, Yulius Setiady, Eileen Samy and Kenneth S. K. Tung
J Immunol 2001; 166:4363-4369; ;
doi: 10.4049/jimmunol.166.7.4363
http://www.jimmunol.org/content/166/7/4363
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Copyright © 2001 by The American Association of
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References
Endogenous Oocyte Antigens Are Required for Rapid
Induction and Progression of Autoimmune Ovarian Disease
Following Day-3 Thymectomy1
Pascale Alard,2* Claire Thompson,* Sally S. Agersborg,† Jayant Thatte,* Yulius Setiady,*
Eileen Samy,† and Kenneth S. K. Tung3*†
T
he prevention of autoimmune disease in normal individuals and the events leading to autoimmune disease occurrence are likely dependent on multiple and complex
mechanisms. Recent studies on the spontaneous autoimmune diseases that follow perturbation of the normal immune system, such
as in mice undergoing thymectomy on day 3 after birth (d3tx),4
have begun to reveal the physiological role of T cell subsets that
guard against the pathogenic expression of autoreactive T cells in
the normal rodent (1–3). d3tx in different inbred mice results in a
high frequency of independent autoimmune diseases that target the
ovaries, stomach, thyroid, lacrimal gland, prostate, and testis, and
in the production of the respective organ-specific autoantibodies
and pathogenic T cell responses (4 –9). Importantly, the T cell
dependent autoimmune diseases occur in inbred mice that are not
genetically prone to development of spontaneous autoimmunity.
Therefore, the regulatory mechanism being disrupted by d3tx is
Departments of *Pathology and †Microbiology, University of Virginia, Charlottesville, VA 22908
Received for publication November 27, 2000. Accepted for publication January
24, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by National Institutes of Health Grants AI41236,
AR45222, and AI10298. The histological study was conducted in the Cell Science
Core of the Center for Research in Reproduction, supported by Grant U54 HD96-008.
2
Current address: Department of Microbiology and Immunology, University of Louisville Health Science Center, Louisville, KY 40292.
3
Address correspondence and reprint requests to Dr. Kenneth Tung, Department of
Pathology, University of Virginia, Box 214, Charlottesville, VA 22908. E-mail address: [email protected]
4
Abbreviations used in this paper: d3tx, thymectomy on day 3 after birth; AOD,
autoimmune ovarian disease; OX, ovariectomy/ovariectomized; B6AF1, (C57BL/6 ⫻
A/J)F1; HPRT, hypoxanthine guanine phosphoribosyl transferase; NOD, nonobese
diabetic.
Copyright © 2001 by The American Association of Immunologists
likely to be of physiological relevance and is generalizable to different self Ag present in many organs (10).
An imbalance of effector and regulatory T cells has been
proposed as the basis of the d3tx autoimmune diseases for several reasons. A regulatory T cell population has been found in
normal adult mice that expresses the IL-2 receptor ␣-chain
(CD25) (11–13). The infusion of CD4⫹ CD25⫹ spleen T cells
or CD4⫹CD8⫺CD25⫹ thymocytes from normal syngeneic
adults completely blocks d3tx diseases (11, 14). In addition, the
thymus derived CD25⫹ CD4 T cells are detected in the periphery after day 3 of life, thus this T cell population would be
depleted or reduced by d3tx (12). Finally, CD25⫺ T cells that
emigrate from the thymus before day 3 have pathogenic potentials in the absence of CD25⫹ T cells (12). For example, CD4⫹
T cells from 3-day-old, but not adult, euthymic BALB/c donors
were found to transfer autoimmune diseases into syngeneic
nu/nu recipients (12, 15).
In addition to elucidating the regulatory T cells and their mode
of action, it is important to define the parameters surrounding the
development of autoimmune diseases that follow regulatory T cell
depletion. One parameter is the source of antigenic stimulus responsible for the spontaneous autoimmune response. Because d3tx
disease induction does not follow deliberate Ag injection, a critical
question is whether the response is initiated by exogenous Ag
through molecular mimicry, or alternatively, represents the response of a dysregulated immune system driven by endogenous
Ag? One might also ask whether endogenous Ag are required for
the maintenance of the ongoing autoimmune response, and, would
the response regress upon removal of the endogenous Ag? These
questions are pertinent to the understanding of neonatal immune
responses, the capacity of Ag from normal organs to initiate pathogenic autoimmune responses, self tolerance mechanism, and eventually the regulatory T cell action.
0022-1767/01/$02.00
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Female (C57BL/6ⴛA/J)F1 mice undergoing thymectomy on day 3 after birth (d3tx) developed autoimmune ovarian disease (AOD)
and autoimmune disease of the lacrimal gland. As both were prevented by normal adult CD25ⴙ T cells, regulatory T cell depletion
is responsible for d3tx diseases. AOD began as oophoritis at 3 wk. By 4 wk, AOD progressed to ovarian atrophy with autoantibody
response against multiple oocyte Ag of early ontogeny. The requirement for immunogenic endogenous ovarian Ag was investigated
in d3tx female mice, d3tx male mice, and d3tx neonatally ovariectomized (OX) females. At 8 wk, all mice had comparable
lacrimalitis but only those with endogenous ovaries developed AOD in ovarian grafts. The duration of Ag exposure required to
initiate AOD was evaluated in d3tx mice OX at 2, 3, or 4 wk and engrafted with an ovary at 4, 5, or 6 wk, respectively. The mice
OX at 2 wk did not have oophoritis whereas ⬃80% of mice OX at 3 or 4 wk had maximal AOD, thus Ag stimulus for 2.5 wk
following d3tx is sufficient. AOD progression requires additional endogenous Ag stimulation from the ovarian graft. In mice OX
at 3 wk, ovaries engrafted at 5 wk had more severe oophoritis than ovaries engrafted at 6 or 12 wk; moreover, only mice engrafted
at 5 wk developed ovarian atrophy and oocyte autoantibodies. Similar results were obtained in mice OX at 4 wk. Thus endogenous
tissue Ag are critical in autoimmune disease induction and progression that occur spontaneously upon regulatory T cell
depletion. The Journal of Immunology, 2001, 166: 4363– 4369.
4364
ENDOGENOUS Ags INDUCE SPONTANEOUS AUTOIMMUNE DISEASE
For this study, we have taken advantage of autoimmune ovarian
disease (AOD) that occurs at high frequency in the d3tx (C57BL/
6⫻A/J)F1 (B6AF1) mice. Ovarian Ag is gender specific and its
expression can be manipulated experimentally. As an example,
ovarian Ag can be depleted surgically at any time of life. In mice
without endogenous ovaries, viable syngeneic ovarian grafts provide a potential source of ovarian Ag stimulus and can be the target
for AOD. Based on this model, we investigated the requirement
and the duration of endogenous ovarian Ag stimulation necessary
for the initiation and progression of AOD in d3tx mice. As an
integral part of this investigation, we defined the nature and ontogeny of ovarian Ag relevant to AOD in the d3tx mice.
Materials and Methods
Animals and surgery
Histologic evaluation of ovarian and lacrimal gland
inflammation
The ovaries and lacrimal glands were fixed in Bouin’s solution and embedded in paraffin, and 5-␮m sections were stained with hematoxylin and
eosin. The extent of ovarian pathology was determined as unknown samples. Ovarian inflammation and ovarian atrophy were graded separately,
each from 1 to 4, as described previously (16). Grade 1 inflammation
consisted of 1–2 foci of inflammatory cells, consisting of monocytes, lymphocytes, and a few granulocytes, usually at the ovarian hilum. Grade 4
represented diffuse inflammation throughout the ovary involving both follicles and interstitial space. Grades 2 and 3 represented intermediate inflammation of incremental severity. Atrophy was evidenced as loss of
growing or mature ovarian follicles, hypertrophy, leuteinization of interstitial cells, and eventually, loss of primordial oocytes. Lacrimal gland
inflammation was graded from 1 to 4. Grade 1 lacrimalitis contained 1–2
foci of periductal mononuclear inflammatory cells. Grade 4 disease exhibited atrophy of lacrimal acini and severe lymphoid infiltration with destruction of lacrimal ducts. Grades 2 and 3 lacrimalitis represented incremental intermediate degrees of inflammation between grades 1 and 4.
Detection of the 110-kDa oocyte Ag messenger RNA by RT-PCR
Total RNA was isolated from the ovaries and testes of B6AF1 mice at days
0, 1, 3, 7, and 21 after birth using RNAqueous-4PCR kit (Ambion, Austin,
TX). RNA concentration was quantified by absorbance at 260 nm. To
perform RT-PCR, ⬃2 ␮g of total RNA was reverse-transcribed into
cDNAs with 1 ␮g of oligo(dT)12–18 using SuperScript Preamplication System (Life Technologies, Rockville, MD). To compare the expression of the
mRNA of the oocyte 110-kDa Ag between samples, each sample was
coamplified with hypoxanthine guanine phosphoribosyl transferase
(HPRT) using PCR. To ensure the linearity of both the oocyte 110-kDa Ag
and HPRT, 10-␮l aliquots of PCR product at 22, 24, 26, 28, 30, 32, or 34
cycles were resolved onto 2% agarose gel using 1⫻ Tris-acetic acid-EDTA
(TAE) as a running buffer to ensure linearity in estimating the concentrations of primers and reverse transcription products for PCR. PCR was
performed by combining 1 ␮l of the reverse transcription product with 8
pmol specific primers of the oocyte 110-kDa Ag, known as OP1 or mater
(17) (sense primer, 5⬘-AGG ACT GTC TGC ATC AAG GAG AT-3⬘ and
antisense primer, 5⬘-AGT GTC GTC AGT TCT CTT CA-3⬘), and 40 pmol
each of the HPRT-specific primers (sense primer, 5⬘-GTT GGA TAC AGG
CCA GAC TTT GTT G-3⬘ and antisense primer, 5⬘-GAG GGT AGG CTG
GCC TAT AGG CT-3⬘) (18). The PCR mixture in a final volume of 50 ␮l
contained 5 ␮l of 10⫻ PCR buffer, 1.5 ␮l of 50 mM MgCl2, 1 ␮l of 10 mM
dNTPs, 2 U Taq DNA polymerase (Life Technologies), and double-distilled water. The cycling parameters for PCR were as follows: denaturation
at 95°C for 1 min, annealing at 57°C for 1 min and extension at 72°C for
1 min for a total of 28 cycles, which was followed by a 10-min extension
at 72°C in PTC-200 DNA Engine (MJ Research, Watertown, MA). The
identity of the PCR products was confirmed by the nucleotide sequences of
the 110-kDa oocyte Ag (18) and of HPRT (18).
Adoptive transfer of disease to young recipient
Spleen cells were dissociated and filtered through nylon mesh, and erythrocytes were lysed in hypotonic 0.1% ammonium chloride. After washing
in RPMI 1640, the cells were suspended in PBS. Each recipient (⬍7 days
old) was injected i.p. with 35 ⫻ 106 viable cells in a 100-␮l volume, and
their ovaries were studied histologically 10 days later.
Isolation and transfer of CD25⫹ CD4⫹ T cells to prevent AOD
in d3tx mice
Lymphoid cells were obtained from axillary, inguinal, brachial, and cervical lymph nodes, and from the spleen of 8-wk-old normal B6AF1 female
mice. They were enriched for CD4⫹ cells using the CD4 enrichment column (R&D Systems, Minneapolis, MN). CD4⫹ T cells were then labeled
with biotinylated anti-CD25 Ab (PharMingen, San Diego, CA), incubated
with streptavidin-iron beads (Miltenyi Biotec, Auburn, CA), and isolated
on a magnetic column. Seventy percent of the cell preparation were CD25⫹
T cells (data not shown). A total of 2 ⫻ 106 cells in 100 ␮l PBS were
injected i.p. into 7-day-old d3tx mice.
Detection of ovarian Ags and Abs by immunofluorescence and
immunoblot
Results
Frozen sections of ovaries from naive B6AF1 or BALB/c scid mice were
fixed in acetone, and processed for indirect immunofluorescence. All
mouse sera were tested at a 1:50 dilution, and FITC labeled goat Ab to
mouse IgG was used as the second Ab. The slides were viewed and photographed with an Olympus fluorescence microscope (New Hyde Park,
NY). Absence of endogenous IgG in young BALB/c scid mice eliminated
background staining and permitted detection of Ag in small ovarian oocytes in the neonatal ovaries.
Ovulated oocytes were used as Ag for immunoblot detection of Ab.
Four-week-old B6AF1 female mice were injected i.p. with 7.5 IU/mouse of
pregnant mare serum (Sigma, St. Louis, MO) and 48 h later with 7.5 IU/
mouse of human chorionic gonadotrophin (Sigma). Oocytes collected
12–13 h later from the ovarian bursa were treated with 0.1 mg/ml hyaluronidase to disperse granulose cells, and rinsed in medium 199 (Life
Technologies, Rockville, MD). The zona pellucida-intact oocytes were
added to the loading buffer that consists of 50 mM Tris-HCl (pH 6.8), 1%
␤ mercaptoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol,
and boiled for 5 min. Proteins from 10 oocytes per lane were electrophoresed in 8% polyacrylamide gel, transferred to a nitrocellulose membrane,
and blocked with 5% skim milk. The membrane was subsequently incubated for 1.5 h at room temperature with the antiserum, diluted 1:100, from
d3tx mice known to have positive fluorescent oocyte Ab. The antigenic
protein band was visualized by further reaction with HRP conjugated goat
We first established the prevalence and the time of onset of the
different components of AOD in female B6AF1 mice thymectomized at the University of Virginia facility (Charlottesville, VA).
Ovarian inflammation (oophoritis), detected in occasional mice at
2 wk, reached ⬃70% by 3 wk (Table I). At this time, ⬍10% of the
ovaries were atrophic. In parallel, the spleen cells from 3-wk-old
but not 2-wk-old d3tx donors transferred oophoritis to young recipients. The severity of AOD then escalated in the ensuing week.
Although oocyte autoantibodies were not detected at 3 wk, most
d3tx mice had oocyte autoantibodies in their sera by 4 wk. At the
same time, the ovaries of 40% of the animals became atrophic,
some with residual oophoritis. This sequence of events occurred in
d3tx mice from different B6AF1 colonies although the exact time
of onset could differ by 1–2 wk (8, 19, 20).
Progression of AOD in d3tx female B6AF1 mice
Endogenous ovarian Ag is required for AOD occurrence in
d3tx mice
To determine the requirement of endogenous ovaries as a source of
antigenic stimulus for ovarian autoimmune response, the responses
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A breeding colony B6AF1 mice was established by mating C57BL/6 female and A/J male mice purchased from National Cancer Institute (Frederick, MD). Mice were kept and handled in accordance of approved National Institutes of Health guidelines. Thymectomy was performed on
3-day-old B6AF1 pups using the suction technique (16). Completeness of
thymectomy was verified histologically, and mice with incomplete thymectomy were excluded from analysis. Ovariectomy (OX) was performed by
removing both ovaries through a posterior incision, under dissection microscopy. To implant ovaries, the ovary from a 6-wk-old B6AF1 mouse
was inserted under the kidney capsule through a posterior incision. Neonatal mice were anesthetized by hypothermia or metofane and adult mice
with tribromoethanol.
Ab to mouse IgG, followed by a chemiluminescent substrate (SuperSignal;
Pierce, Rockford, IL), and exposed on x-ray film.
The Journal of Immunology
4365
Table I. Induction of AOD in d3tx B6AF1 mice
Ovarian Pathology in Recipients of
Spleen Cells from d3tx Miceb
Ovarian Histopathologya
Age of d3tx Mice at
the Time of Study
(wk)
Incidence oophoritis
(%)
Oophoritis severity (1–4)
(means ⫾ SE)
Atrophy in mice with
oophoritis (%)
Serum Oocyte
Ab (%)a
Incidence (%)
Mean severity
(1–4)
2
3
4
1/22 (5)
13/18 (72)
17/20 (85)
1
2.0 ⫾ 0.19
2.7 ⫾ 0.26
0/1 (0)
0/13 (0)
7/17 (41)
0/6 (0)
1/7 (14)
11/16 (69)
0/7 (0)
5/6 (83)
8/8 (100)
0
1.8
2.4
a
b
Ovarian pathology and serum oocyte Ab in the d3tx mice.
Ovarian pathology in recipients of spleen cells from d3tx mice. Each mouse, at ⬍7 days of age, received 35 ⫻ 106 spleen cells i.p., and the ovaries were studied 10 days
later.
The nature and ontogeny of endogenous ovarian Ag
The majority of serum autoantibodies in the d3tx B6AF1 mice
reacted with the intracellular Ags of growing and mature oocytes
detected by immunofluorescence on ovarian sections (Fig. 2). By
immunoblot, the autoantibodies were found to react with three
distinct oocyte Ag with the apparent molecular mass of 110, 90,
and 75 kDa (Fig. 2B). Among 38 sera from B6AF1 mice with
oocyte Ab detected by immunofluorescence, 26 (69%) were also
detected by immunoblot, and among them, 50% reacted with the
110-kDa oocyte Ag, 37% reacted with the 75-kDa oocyte Ag, and
18% reacted with the 90-kDa oocyte Ag.
The sera of 10 d3tx mice were also evaluated weekly for oocyte
Ab from 6 –10 wk. In four mice, autoantibodies to oocyte was
detected only by immunofluorescence during the 4-wk period. In
the remaining six d3tx mice, the initial Abs were detected by immunofluorescence of which three also recognized the 110-kDa oocyte Ag by immunoblot. This was followed in the next 1–3 wk by
the emergence of Ab to the 110-kDa oocyte Ag in two additional
mice, and new Ab to the 90k-Da oocyte Ag and the 75-kDa oocyte
Ag in three and two mice, respectively (data not shown). Thus the
earliest autoantibodies in the sera of d3tx B6AF1 were directed to
native determinants of the oocyte cytoplasmic Ag including those
of the 110-kDa component.
We next determined the ontogeny of oocyte Ag to establish the
earliest age when endogenous antigenic stimulus could occur. As
shown in Fig. 2, D and E, oocyte Ag was detected in normal
BALB/c scid mice at 1 day (6 of 8 sera) or 5 days (11 of 12 sera)
of age by immunofluorescence. This finding parallels the detection
of mRNA for the major 110-kDa Ag by RT-PCR on the day of
birth (Fig. 2C). Thus ovarian autoAg that react with Abs, produced
by d3tx mice, are detectable close to the day of birth in
female pups.
To determine whether ovarian autoAg is recognized by pathogenic T cells from the d3tx mice, spleen cells from 6-wk-old d3tx
mice were injected i.p. into neonatal recipients on the day of birth.
Ovarian inflammation was detected 3 days later, whereas spleen
cells from sham thymectomized mice did not transfer disease (Fig.
2, F and G). Inflammation was not detected in organs outside the
ovaries. Thus, within the first 3 days of life, ovarian oocyte Ag was
processed and presented by ovarian APCs that can be recognized
by pathogenic oocyte-specific T cells. The expression of the oocyte
Ag early in life indicates that immunogenic ovarian Ag are available to trigger an autoimmune response in neonatal mice.
Endogenous ovarian antigenic stimulus for 3 wk is required for
induction of oophoritis
We next determined the duration of endogenous antigenic stimulation required for the induction of AOD and autoantibody response. By removing the ovaries from the d3tx female mice at 2,
Table II. Ovarian autoimmunity develops only in d3tx mice with endogenous ovariesa
Sex (tx)
Female (d3tx)
Female (d3tx)
Male (d3tx)
Female (d3tx)
Female (sham tx)
Ovarian
Ablation
Normal CD25⫹ T
Cell Transfer at
Day 5
2 ⫻ 106
Day 3
Ovarian Graft Pathology
Lacrimal Gland Pathology
Incidence
oophoritis (%)
Oophoritis severity (1–4)
(means ⫾ SE)
Atrophy in mice with
oophoritis (%)
14/14 (100)
0/6
0/18
0/14
0/13
3.6 ⫾ 0.23
11/14 (78)
Serum Oocyte
Ab (%)
12/13 (93)
0/6
0/18
0/8
0/13
Incidence
(%)
8/13 (61)
0/6
15/16 (94)
4/8 (50)
0/13
Mean severity
(1–4)
2.8
2.0
2.0
a
Male or female B6AF1 mice were subjected to d3tx or sham tx on day 3 after birth (day 0 ⫽ day of birth). At 6 wk, all mice received one ovarian graft from age-matched
B6AF1 female donors, and 2 wk later, the ovarian graft and lacrimal gland pathology was determined and the sera were studied for oocyte Abs by indirect immunofluorescence.
One group of d3tx mice also received i.p. 2 ⫻ 106 CD4⫹ CD25⫹ spleen T cells from normal 8-wk-old B6AF1 female donors.
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of female and male mice were compared (Table II). They were
thymectomized on day 3, engrafted with a 6-wk-old B6AF1 ovary
at 6 wk, and studied at 8 wk. Female mice developed AOD in the
ovarian graft and serum autoantibodies to oocytes (Fig. 1A). In
contrast, the ovarian grafts of male mice that lack endogenous
ovaries were normal and their sera were free of detectable oocyte
autoantibodies when they were studied at 2 wk (Fig. 1B), or at 6
wk after ovarian engraftment (one of nine had focal AOD). The
requirement of endogenous ovaries for AOD induction was confirmed by the absence of AOD in d3tx females that were OX at day
3 of age (Table II). In contrast to AOD, autoimmune lacrimalitis
occurred in d3tx mice regardless of whether they had endogenous
ovaries or not (Table II). Thus, loss of the endogenous ovaries does
not impact on pathogenic autoimmune responses in general.
As evidence that regulatory T cell depletion was the basis for the
autoimmune diseases, both AOD and autoimmune lacrimalitis were
found to be inhibited by the transfer of CD4⫹ CD25⫹-enriched T
cells from the normal B6AF1 adult females (Table II). In addition,
autoimmunity was absent in sham thymectomized mice. The findings
indicate that endogenous ovarian Ag is required for AOD development in the d3tx female mice. In the next study, we analyze the nature
of the ovarian autoantigens and the ontogeny of their expression.
4366
ENDOGENOUS Ags INDUCE SPONTANEOUS AUTOIMMUNE DISEASE
3, or 4 wk, and assuming that Ag stimulation occurs from the day
of thymectomy (day 3), we allowed endogenous ovarian antigenic
stimulation to persist for 1.5, 2.5, or 3.5 wk. Each mouse was then
engrafted with an adult ovary 2 wk after OX, and the ovarian graft
was studied 2 wk later at 6, 7, or 8 wk of age, respectively.
Of the d3tx mice OX at 2 wk, none developed AOD or autoantibody response although most animals in this group developed
autoimmune lacrimalitis (Table III). Therefore, consistent with the
time of onset of AOD in the d3tx mice, ovarian Ag stimulation
for ⬍1.5 wk is not sufficient to elicit AOD in the d3tx mice.
In contrast to mice OX at 2 wk, ⬎85% of the mice OX at 3 wk
developed severe oophoritis and ovarian atrophy by 5 wk (Table
III). Because these responses were indistinguishable from the responses of mice that were OX at 4 wk (and studied at 6 wk; Table
III), we concluded that Ag stimulation from endogenous ovaries
for 2.5 wk from d3tx was sufficient to elicit maximum autoimmune
response and AOD.
Regardless of the age when the OX was performed, all d3tx
mice developed high incidences of lacrimalitis (Table III). Thus
ovarian ablation impacted specifically on the ovarian immune
response.
Ovarian antigenic stimulus is required for progression from
oophoritis to ovarian atrophy and autoantibody response
Although d3tx mice OX at 3 wk and engrafted at 5 wk developed
severe AOD that progressed to atrophy and oocyte Ab production
(Table III), it was surprising that the ovarian disease of d3tx mice
studied at 3 wk had oophoritis without atrophy or autoantibodies
response (Table II). In addition, mice that were OX at 3 wk and
engrafted at 12 wk had significantly reduced incidences and severity of AOD, including oophoritis ( p ⬍ 0.05) and oocyte autoantibodies ( p ⬍ 0.01; Table IV). Both observations suggest that
endogenous ovarian Ag derived from ovarian grafts are required
for the maintenance and progression of the autoimmune response
associated with severe AOD. In the following experiments, we test
the hypothesis that endogenous ovarian Ag is also required for
disease progression.
Groups of d3tx mice were OX at 3, 4, or 5 wk. In each group,
we compared the immunopathology of ovaries that were engrafted
at 2 wk vs 3 wk after the mice were OX (Table IV). Indeed, the
ovarian inflammation was much more severe in ovaries engrafted
at 5 wk (Fig. 1C) than those engrafted at 6 wk (Fig. 1D) ( p ⫽
0.02). Moreover, the progression from oophoritis to atrophy and
induction of autoantibodies response were largely halted in mice
that were devoid of ovaries for 3 wk rather than 2 wk ( p ⬍ 0.01).
A similar finding was noted in d3tx mice OX at 4 wk, with a
higher incidence of atrophy in the ovaries engrafted at 6 wk rather
than 7 wk ( p ⬍ 0.05). The difference was less prominent in mice
OX at 5 wk. Although there was a significant reduction in autoantibodies response in mice engrafted at 8 wk instead of 7 wk
( p ⬍ 0.001), the incidence of oophoritis and atrophy in ovaries
engrafted at 7 or 8 wk were comparable. These data, together,
indicate that ovarian Ag from endogenous ovaries or ovarian graft
is required to stimulate full progression of AOD induced by d3tx.
Discussion
This study has investigated the requirement of endogenous Ag in
the initiation and propagation of autoimmune response and AOD
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FIGURE 1. Histopathology of ovarian grafts in d3tx
mice. A and B compare the changes in the ovarian graft
in a d3tx female mouse (A) with the graft in a d3tx male
mouse (B). Note that the ovary in the female mouse (A)
has heavy infiltration of lymphoid cells with loss of ovarian follicles, whereas the ovary in the male mouse (B) is
not inflamed and contains normal ovarian follicles (f). C
and D illustrate the different pathological changes in
ovarian grafts of d3tx mice OX at 3 wk. The ovary engrafted at 5 wk (C) is severely inflamed and atrophic,
whereas the ovary engrafted at 6 wk (D) has ovarian
follicles that are inflamed (arrows) or normal (f), without
evidence of atrophy. A, C, and D, Hematoxylin and eosin, magnification, ⫻100; B, magnification, ⫻200).
The Journal of Immunology
4367
in d3tx mice. Male mice and OX female mice both lack endogenous ovarian Ag and neither group developed oophoritis or produced detectable autoantibodies to oocyte Ag following d3tx. Thus
endogenous ovarian Ag is required for disease induction. The difference in AOD between d3tx male and female mice could be due
to gender difference or other OX effects. However, this is unlikely
because all d3tx animals developed a high prevalence of autoimmune lacrimalitis, a disease that is not gender specific. The present
finding is supported by the earlier studies wherein spleen cells
from d3tx mice without endogenous ovaries failed to transfer AOD
to young recipients (6, 20). The requirement of endogenous Ag has
also been demonstrated for spontaneous autoimmune thyroiditis in
obese strain chicken (21), and for autoimmune diabetes in the
nonobese diabetic (NOD) mice (22). In the latter, alloxan treatment destroyed the pancreatic islet ␤ cells of NOD mice, and their
T cells did not transfer diabetes to irradiated male NOD recipients.
A more recent study with a transgenic diabetogenic peptide-specific TCR indicated that T cell activation in the regional lymph
node was the first observable pathogenic event in NOD mice (23).
However, these studies did not investigate the duration of Ag stimulation required for disease induction, nor did they address the
impact of endogenous Ag on autoimmune disease progression.
The present study demonstrated clearly that Ag provided by an
ovarian graft is required for progression of AOD. As shown in
Tables III and IV, although Ag exposure for the first 2.5 wk in the
d3tx mice is sufficient for AOD development, including oocyte Ab
and atrophy, this maximum response was observed only when the
ovaries were engrafted 2 wk after removal of the endogenous ovaries. When ovarian engraftment was delayed by an extra week (i.e.,
ovarian engraftment at 3 instead of 2 wk after OX), oophoritis was
no longer accompanied by ovarian atrophy or autoantibodies response. Thus disease progression to atrophy and autoantibody response was halted when persistent Ag stimulation was interrupted
by 3 instead of 2 wk. This trend was also observed in mice OX at
4 wk; and in this case, more severe disease was observed in mice
engrafted at 6 rather than 7 wk. Indeed, the disease may regress
upon further withdrawal of Ag stimulus. For example, in d3tx mice
that were OX at 3 wk and studied at 12 wk, the incidence of
oophoritis was significantly reduced ( p ⬍ 0.05; Table IV). In mice
OX at 5 wk, reduction in subsequent Ag exposure from the ovarian
graft also reduced autoantibodies response but had little effect on
progression from oophoritis to atrophy. It is known that the number of oocyte progenitors is finite, and when they are lost upon
ovarian atrophy, recovery from AOD might not be possible. We
have recently obtained direct evidence for the capacity of an ovarian graft to confer antigenic stimulation. Male mice injected with
a T cell epitope of the ovarian zona pellucida Ag ZP3 (24) did not
produce Ab to distant B cell epitopes of ZP3 unless they were
engrafted with an ovary (P. Pramoonjago, C. Sharp, and K. S. K.
Tung, unpublished observation). Therefore, we conclude that endogenous or engrafted ovarian Ag is required for AOD induction
as well as for AOD progression.
The presence of the ovarian Ag for 3 wk from birth was required
for maximum disease induction, whereas Ag exposure for 2 wk
didnot induce autoimmune disease (Table III). Assuming that endogenous Ag stimulation occurs on day 3 when the mice were
thymectomized, the finding suggests that Ag stimulation for the
first 2.5 wk after thymectomy is sufficient to initiate a maximum T
cell response. This is consistent with AOD transfer by spleen cells
from d3tx mice at 3 wk but not at 2 wk of age (Table I). This
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FIGURE 2. Nature and ontogeny of oocyte
mRNA and Ag recognized by serum autoantibodies and splenocytes from d3tx B6AF1 mice. A and
B, Adult B6AF1 oocyte Ag recognized by serum
Ab of d3tx mice, detected by indirect immunofluorescence on B6AF1 frozen ovarian section (A)
and by immunoblot (B). B, The sera from four d3tx
B6AF1 female mice react with three oocyte Ags
with molecular mass of 110, 90, and 75 kDa. C–E,
The ontogeny of the 110-kDa oocyte expression.
C, The oocyte mRNA (592 bp) is detected by RTPCR in days 0, 1, 3, 7, and 21 in B6AF1 ovaries
(lanes 1–5, respectively); mRNA of 21-day-old
testis serves as negative control (lane 6). In all
lanes, the HPRT transcript (352 bp) is shown. The
110-kDa oocyte Ag is detected by indirect immunofluorescence in the oocyte cytoplasm of BALB/
c.scid ovaries at 1 day (D) and 5 days (E) of age.
F and G, Ontogeny of B6AF1 ovarian Ag recognized by effector T cells from d3tx B6AF1. Spleen
cells (35 ⫻ 106) from 6-wk-old sham OX (F) and
d3tx (G) B6AF1 mice were injected i.p., to each
B6AF1 female mouse on the day of birth, and the
recipients’ ovaries were studied histologically 3
days later. Note inflammatory cells surround small
oocytes in G and not in F. A, Magnification, ⫻200;
D and E, magnification, ⫻100; F and G, magnification, ⫻200; F and G, hematoxylin and eosin.
4368
ENDOGENOUS Ags INDUCE SPONTANEOUS AUTOIMMUNE DISEASE
Table III. Duration of endogenous antigenic stimulus required for induction of AOD and pathogenic T cell and autoantibody responses in the d3tx
micea
Findings at 2 wk After Ovarian Implant
Ovarian graft pathology
Age (wk)
a
Lacrimal gland pathology
Bilateral
OX
Ovarian
implantation
Incidence
oophoritis
(%)
Oophoritis severity
(1–4) (means ⫾ SE)
Atrophy in mice
with oophoritis
(%)
Serum oocyte
Ab (%)
Incidence
(%)
Mean
severity
(1–4)
2
3
4
4
5
6
0/7
7/8 (88)
9/10 (90)
3.7 ⫾ 0.29
3.5 ⫾ 0.29
0/7
6/7 (86)
7/9 (77)
0/7
6/8 (75)
9/12 (75)
5/7 (71)
5/8 (63)
5/11 (45)
2.1
1.7
2.0
All B6AF1 female mice were subjected to d3tx and later OX. Two weeks after OX, each mouse received an ovarian graft from 6-wk-old B6AF1 female mice.
Table IV. Ovarian antigenic stimulus is required for the progression from oophoritis to ovarian atrophy and oocyte autoantibody response in the
d3tx micea
Findings at 2 wk After Ovarian Implantation
Ovarian graft pathology
Age of Mice (wk)
Bilateral
OX
Ovarian
implantation
Incidence
oophoritis
(%)
Oophoritis severity
index (1–4)
(means ⫾ SE)
Atrophy in mice
with oophoritis
(%)
Serum oocyte
Abs (%)
Incidence
(%)
Mean
severity
(1–4)
3
5
6
12
6
7
7
8
7/8 (88)
9/10 (90)
8/17 (47)
9/10 (90)
8/11 (73)
7/8 (88)
7/8 (88)
3.7 ⫾ 0.29
2.7 ⫾ 0.29
3.1 ⫾ 0.23
3.6 ⫾ 0.29
3.0 ⫾ 0.27
3.9 ⫾ 0.14
3.3 ⫾ 0.42
6/7 (86)
1/9 (11)
3/8 (38)
7/9 (77)
2/8 (25)
6/7 (86)
5/7 (71)
6/8 (75)
1/10 (10)
2/17 (12)
9/12 (75)
4/11 (36)
6/8 (75)
2/8 (25)
5/8 (63)
8/10 (80)
16/17 (94)
5/11 (46)
9/11 (82)
7/8 (88)
7/8 (88)
1.7
2.6
2.6
2.0
2.4
3.0
2.0
4
5
a
Lacrimal gland pathology
See footnote in Table III. See text for statistical differences in data between experimental groups.
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ative oocytes in the prepubertal ovaries are eliminated by apoptosis
(26) and, therefore, are available to invoke the autoimmune responses observed in the d3tx mice.
With regard to the availability of autoreactive effector T cells, it
has been shown that neonatal T cells are immunocompetent (28,
29). Moreover, when transferred to nude recipients, the spleen
cells from normal 3-day-old BALB/c mice induce ovarian and gastric autoimmune diseases spontaneously (12, 15). Therefore, the
accessibility of endogenous Ag and the competence of pathogenic
T cells are not limiting for induction of spontaneous autoimmune
disease in the neonatal mice. A more likely limiting factor is the
immaturity of the neonatal APC.
Macrophages from neonatal mice have minimal MHC II expression (30). A recent study indicated that B cells and plastic adherent
cells from normal neonatal spleens presented Ag to memory T
cells at ⬃30% of the adult level in the first week (31). However,
it has also been shown that the immature neonatal APC function
might be modified, for example, after adjuvant or CD40 agonist
Ab treatment. Thus autoimmune responses and autoimmune diseases were elicited in the neonatal mice by injection of self peptides in complete or incomplete adjuvant (32–34); and CD40 Ab
was found to abrogate induction of neonatal tolerance to alloantigen (35). In the d3tx mice, because the self Ag-driven autoimmune
disease is inhibitable by CD25⫹ T cells, it is possible that deletion
of the regulatory T cell population by d3tx somehow capacitates
neonatal APC to present ovarian self Ag to naive autoreactive T
cells. Recent literature supports this contention. For example,
CD4⫹ CD25⫹ T cells (36) and anergic regulatory T cells (37) have
been reported to down-regulate expression of costimulatory molecules on APC and to reduce APC function. In addition, cytokines
produced by T cells, including IL-10 (38, 39) and TGF␤ (40), also
duration is comparable to the time required for induction of autoimmune diseases by immunization with tissue peptides in complete adjuvant. The rapid induction of autoimmune response by
endogenous Ag in the neonatal mice without the use of adjuvant is
quite surprising and has several interesting and important implications. It suggests that endogenous ovarian Ag are available and
are presentable in immunogenic form by APC capable of activating neonatal T cells that are presumably naive. It also suggests that
neonatal T cells in the d3tx mice have full capacity to mount a
pathogenic autoimmune response. Thus tight regulation must normally guard against spontaneous autoimmune disease in the neonatal mice, and this begs the question as to how the regulation is
abrogated by CD25⫹ regulatory T cell depletion.
The lack of autoimmune response in the d3tx mice studied at 2
wk, or in the d3tx mice OX at 2 wk and engrafted at 4 wk, is not
due to a late ontogeny of oocyte Ag. In this study, we have documented the multiple oocyte Ag to which the autoimmune responses of d3tx B6AF1 mice are directed. The 110-kDa oocyte
Ag, which may elicit the earliest autoimmune response, has been
characterized by Tong and Nelson (17) as a prefertilization oocyte
protein required for blastocyst formation after fertilization (25). In
this study, we have shown clearly that the transcript for this oocyte
autoantigen is detectable in B6AF1 ovaries on the day of birth, and
that oocyte Ag are recognized by autoantibodies 1 day later, and by
CD4 T cells as early as 3 days later. In the neonatal week, a large
fraction of oocytes are normally eliminated by apoptosis from normal ovaries. For example, an C57BL/6 ovary contains 60,000 oocytes on day 0, and the number is reduced to 20,000 by day 3 (26)
(J.L. Tilly, unpublished observation). This rate of oocyte loss is
further accelerated by d3tx, before detectable oophoritis (27). Although the fraction of the apoptotic oocytes that contains relevant
autoAg is currently unknown, 100% of Ag positive and Ag neg-
The Journal of Immunology
Acknowledgments
We are grateful to Dr. Kristina Garza and Dr. Michelle Kosiewicz for their
expert advice during the investigation. We thank Jason Borillo, Alice
Chang and Melissa Bevard for technical assistance.
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inhibit APC function. Powrie and colleagues (41, 42) have provided in vivo evidence that these two cytokines are involved in the
control of T cell-mediated colitis by regulatory T cells that express
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In summary, although there is considerable evidence that autoimmune disease can be elicited by exogenous Ag through molecular mimicry at the level of T or B cell response (43, 44), research
based on the AOD and other spontaneous autoimmune models
emphasize a more critical role for endogenous Ag in the prevention, induction, and maintenance of pathogenic autoimmune responses. In a recent study, we documented the requirement of endogenous Ag for the maintenance of physiological tolerance to
ovarian self Ag (45). Now, in the d3tx context, we have demonstrated that ovarian endogenous Ag can also trigger and sustain
AOD progression. To accommodate this apparent paradox, we
propose that endogenous Ag stimulation occurs normally, and that
the outcome is dependent on the integrity and the continuous operation of the immune regulatory mechanisms.
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