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*
Division of Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, and Department of Pathology, and
Sackler Institute of Graduate Biomedical Sciences, New York University Medical Center, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 TCR chain or
cells lacking N nucleotides. We also investigated whether coexpression
of the myelin basic protein-specific TCR with another TCR in a single
cell would alter either pathogenesis or regulation. This was not the
case, as myelin basic protein-specific/OVA-specific recombinase
activating gene-1-/- double TCR transgenic mice still
developed experimental autoimmune encephalomyelitis spontaneously even
after immunization with OVA. Based on this evidence, we conclude that
CD4+ T regulatory cells do not express canonical TCRs and
that the altered signaling properties brought about by coexpression of
two TCRs are not sufficient for the generation of regulatory T cells.
Instead, our results indicate that regulatory T cells belong to a
population displaying wide TCR diversity, but in which TCR specificity
is central to their protective function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The protective effect of regulatory T cells has been observed in
several experimental disease models. For instance, colitis induced by
adoptive transfer of
CD4+CD45RBhigh T cells into
SCID mice can be prevented by cotransfer of
CD4+CD45RBlow T cells
(9) or in vitro-generated CD4+ Tr1
cells (10). A variety of organ-specific autoimmune
diseases that arise in day 3-thymectomized animals can be prevented by
administration of CD4+CD25+
T cells (11, 12). In thymectomized and irradiated adult
PVG.RT1u rats, the onset of autoimmune diabetes
can be prevented by transfer of
CD4+CD45RClowRT6+
T cells (3, 13). The role of regulatory T cells has also
been established in spontaneous autoimmune disease models. For
instance, in diabetes-prone BB/W rats and nonobese diabetic mice,
disease can be prevented by transfer of CD4+ T
cells from normal donors (14, 15). We and others have
shown that CD4+ ß T cells confer protection
from spontaneous experimental autoimmune encephalomyelitis
(EAE)3 (16, 17) (see below). Finally, regulatory T cells have also been
shown to play a major role in transplantation tolerance
(18, 19, 20). These results demonstrate the importance of the
regulatory T cell populations in preventing disease in normal
individuals and highlight their potential as targets for therapeutic
intervention.
Despite all the studies on different aspects of regulatory T cells, little is known about their TCR usage. In myelin basic protein (MBP)1–11-induced EAE in H-2u/u mice, Vß14-expressing T cells and, to a lesser extent, Vß3-expressing T cells have been implicated in the recovery from disease. These Vß14 and Vß3-expressing cells recognize epitopes in the TCR Vß8.2 chain expressed by the vast majority of MBP-specific T cells (21, 22). However, we have previously shown that spontaneous EAE can be prevented in a fraction of animals harboring T cells that exclusively express Vß8.2 chains (16). The fact that disease can be prevented by T cells expressing neither Vß14 nor Vß3 argues against a major role for anti-MBP TCR T cell responses in the protection against spontaneous EAE in our experimental system.
NK T cells express canonical V14-J281 TCR chains paired with a
relatively restricted pool of TCR ß-chains (23). NK T
cells are diminished in nonobese diabetic mice, a defect that has been
linked to the high susceptibility of spontaneous diabetes in this mouse
strain (24, 25, 26). NK T cell-mediated protection from
diabetes has been reported in two studies (27, 28), but a
CD4+ non-NK T cell population was found to be
responsible for diabetes protection in another study (29).
However, we and others have shown that spontaneous EAE does not occur
in MBP-specific TCR transgenic mice lacking
ß2-microglobulin, which have a severe
deficiency in both CD8+ T cells and the
CD1-restricted NK T cells (16). Thus, if the regulatory T
cells that protect from spontaneous EAE bear a canonical TCR, it is
different from the one expressed by NK T cells.
We have previously shown that while MBP-specific TCR transgenic mice
(referred to as T/R+) do not develop EAE
spontaneously, 100% of MBP-specific TCR transgenic/recombinase
activating gene-1 (RAG1)-/- mice (referred to
as T/R-) as well as 100% of MBP-specific TCR
transgenic
mice/TCR-/-/TCRß-/-
mice (referred to as
T/-ß-) develop EAE
spontaneously by the age of 3 mo in the C57BL genetic background
(7, 16, 17). Thus, crossing T/R+
mice with RAG1-/- mice or with
TCR-/- and TCRß-/-
mice blocks the development of regulatory CD4+ T
lymphocytes, which express TCRs encoded by the endogenous TCR and
TCRß loci. Furthermore, a single administration of as few as 2
x 105 splenic or thymic
CD4+ T cells from normal mice into
T/R- or
T/-ß- is sufficient
to confer life-long protection from EAE.
Taking advantage of this spontaneous EAE experimental system, we
demonstrate in this report that while a strictly monoclonal
OVA-specific T cell population was unable to confer protection,
regulatory cells were effectively generated in mice expressing a
restricted T cell repertoire. CD4+ T cells
lacking N nucleotides as well as T cells exclusively expressing the
V4 TCR chain protected from EAE. In addition, MBP-specific
transgenic T cells expressing a second TCR did not acquire protective
capacity. We conclude that T cell specificity, and not space filling,
is required for effective protection and that regulatory T cells do not
express canonical TCR chains.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MBP-specific trangenic mice with a disrupted RAG1 gene
(T/R-) and TCR-/-
mice have been described previously (16).
T/R- mice in the B10.PL background were crossed
with B10.D2 RAG1-deficient mice to yield
B10.H-2d/u mice. T/R- mice
used in all experiments were younger than 31 days old and EAE free.
DO11.10 anti-OVA TCR transgenic mice (30) backcrossed
into B10.D2 were obtained from Dr. Dan Littman (New York University),
with permission from Drs. Kenneth Murphy and Dennis Loh, and crossed to
RAG1-/- mice (31) in the B10.PL
background to obtain B10 H-2d/u mice.
TdT-/- mice were generated in Dr. Diane
Mathis’ laboratory (32) and were kindly supplied by Dr.
Mark M. Davis (Stanford University) and backcrossed with B10.PL mice.
The TCR V4 transgenic mouse line was generated with the same TCR
-chain construct used to generate the MBP-specific TCR transgenic
mice, as described (7). MHC H-2u and
TCR-/- was introduced by crossing with
B10.PL TCR-/- mice (33). All
mice were backcrossed into the C57BL genetic background
(N6 to N10 generation).
Mice were kept under specific pathogen-free conditions in individually
ventilated cages (Thoren) at the Skirball Institute Central Animal
Facility, New York University Medical Center.
Disease evaluation
EAE was scored as previously described (34): level 1, limp tail; level 2, weak or partial leg paralysis; level 3, total hind leg paralysis; level 4, hind leg paralysis and weak or partial front leg paralysis. All protocols involving mice handling were approved by New York University’s Institutional and Animal Care Use Committee.
Generation of bone marrow chimeras
Bone marrow precursors cells were obtained from the tibia and femur bones by conventional procedures. Bone marrow precursors were incubated for 45 min at 4°C in the presence of anti-Thy 1.2 99TIB hybridoma supernatant (American Type Culture Collection, Manassas, VA), followed by 30 min incubation at 37°C with Low-Tox guinea pig complement (Cederlane, Hornby, Ontario, Canada). As indicated, 1 x 106 or 1 x 107 cells were transferred i.v. into sublethally irradiated recipient mice (a total of 500 rad; 100 rad/min).
Adoptive transfer experiments and immunizations
Donor splenocytes from TdT-/- and V4
TCR transgenic/TCR-/- mice were obtained by
conventional procedures. Total splenocytes
(107/mouse) were transferred i.v. into
<31-day-old, disease-free T/R-
H-2u/u. Donor splenocytes and mesenteric lymph
node cells from anti-OVA TCR
transgenic/RAG1-/- mice were collected, and the
proportion of CD4+ KJ1-26+
cells was determined by FACS analysis. Total splenocytes and lymph node
preparations containing 3–5 x 106
CD4+ KJ1-26+ cells were
transferred i.v. into <31-day-old, disease-free
T/R- H-2d/u. Two and 12
days after adoptive transfer, mice were immunized i.p. with 100 mg of
OVA (Sigma, St. Louis, MO) adsorbed in aluminum hydroxide (EM Science,
Gibbstown, NJ). Aluminum hydroxide resuspended in PBS was used as
control for the immunizations.
Abs and FACS analysis
Anti-MBP TCR clonotypic Ab (3H12) was generated in our laboratory as described (16). Anti-OVA TCR Ab (KJ1-26) was purchased from Caltag (Burlingame, CA). Anti-CD3 and anti-CD4 Abs were obtained from PharMingen (San Diego, CA). To determine the proportion of CD4+ KJ1-26+ cells in anti-OVA transgenic/RAG1-/- mice, splenocytes were stained with anti-CD4 and KJ1-26 Abs. For reconstitution analysis, peripheral blood was stained 45 min with the Ab mixture, lysed and fixed with FACS lysis solution (Becton Dickinson, San Jose, CA), and subsequently washed twice with PBS. Samples were analyzed in a FACScalibur instrument (Becton Dickinson). Up to 5000 lymphocytes were acquired. Splenocytes were stained similarly with incubation at 4°C, not lysed, and resuspended in propidium iodide to gate out dead cells. Up to 20,000 cells were acquired.
Infiltrating cells from the CNS were prepared as described (35). Briefly, anesthetized mice were perfused through the left ventricle of the heart with PBS/EDTA. Discoloration of the liver was a good indication of a successful perfusion. After dissection of the brain and spinal cord, single-cell suspensions were prepared mechanically, resuspended in 38% in Percoll (Pharmacia, Piscataway, NJ), and spun for 20 min at 1750 rpm in a benchtop centrifuge (Sorvall RT7). The pellet was washed twice and stained as described above for live splenocytes.
Cell sorting and proliferation assay
Single-cell suspensions of splenocytes and mesenteric lymph
nodes from MBP-specific/OVA-specific TCR
H-2d/u/RAG1-/- mice were
obtained following conventional procedures. Stained cells were sorted
in a Coulter EPICS Elite cell sorter (Palo Alto, CA). Sorted
populations from the double TCR transgenic mice were washed with PBS,
analyzed by FACScalibur for purity (>99%), and counted.
CD4+ cells (1.25 x
104 per well) were incubated in 96-well plates in
the absence or presence of the peptides
OVA323–339 (1 µM) or
MBPAc1–17 (5 µM). Proliferation to
anti-CD3 Ab was used as control. Irradiated nontransgenic
H-2d/u/TCR-/-
splenocytes (2 x 105 cells per well) were
added as APCs. After 48 h, the plates were pulsed for 6 h
with [3H]thymidine (1 µCi per well; NEN,
Boston, MA), cells were harvested, and the radioactivity was counted in
a Betaplate (Wallac, Gathersburg, MD). Each sample was done in
triplicate.
Generation of OVA-specific Tr1-like and Th2 cells
Anti-OVA CD4+ cells were obtained from
spleen cells from anti-OVA TCR
transgenic/RAG1+ mice. Spleen cells (2 x
106/ml) were cultured in the presence of
OVA323–339 peptide and either 200 U/ml of IL-4
(PharMingen) and 10 µg/ml anti-IFN-
(PharMingen) to
generate Th2 cells (36, 37) or 100 U/ml IL-10 (PharMingen)
and 10 µg/ml anti-IL-4 (PharMingen) to generate Tr1-like cells.
Cultures set in the absence of anti-IL-4 yielded cells with a Th2
cytokine profile. Cultures were restimulated weekly with irradiated
syngeneic APCs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have previously reported that ß
CD4+ T cells are capable of protecting monoclonal
anti-MBP TCR trangenic mice with a disrupted RAG1 gene
(T/R- mice) from developing spontaneous EAE
(16, 17), whereas TCR transgenic mice carrying at least
one functional copy of the RAG1 gene (T/R+ mice)
are resistant to spontaneous EAE. It is not yet known whether the
protective CD4+ TCR ß+
cells present in T/R+ mice require a particular
TCR specificity for their function or if they protect from spontaneous
EAE nonspecifically, for instance by competing with the effector cells
for growth factors and/or space.
To address this issue, we generated mice in which MBP-specific T cells developed alone (T/R- mice) or together with three different sources of CD4+ T cells: CD4+ T cells bearing a fully diverse T cell repertoire (from normal mice), CD4+ T cells bearing a repertoire dominated by OVA-specific T cells (from anti-OVA TCR transgenic/RAG1+ mice), and CD4+ T cells bearing exclusively OVA-specific T cells (from anti-OVA TCR transgenic/RAG1-/- mice).
RAG1-/- mice were reconstituted with T cell-depleted bone marrow cells from T/R- mice in the absence or presence of bone marrow precursors from mice carrying the three different CD4+ T cell repertoires. Bone marrow from DO11.10 TCR transgenic mice (30) was used as a source of OVA-specific T cells. As the MBP-specific T cells are restricted by I.Au, and the OVA-specific T cells are restricted by I.Ad, all recipient and donor mice used throughout this set of experiments bore MHC H-2d/u. In addition, to avoid potential minor histocompatibility effects, all mouse strains were repeatedly backcrossed onto a C57BL background (N6 to N10 generations).
Recipient mice reconstituted with bone marrow cells from
T/R- mice started to develop signs of EAE around
35 days after transplantation, reaching an incidence of 100% 
20
days later (Fig. 1
A and data
not shown). Mice reconstituted with T/R- and
anti-OVA TCR transgenic/RAG1-/- bone marrow
cells developed EAE with a similar incidence and severity as control
mice that received only T/R- cells (Fig. 1A). Disease was also observed when recipient mice received
T/R- cell precursors together with bone marrow
cells from TCR-/- mice, which generate cells
capable of occupying and competing for space in the bone marrow and
thymic cortex, but cannot generate mature T cells (Fig. 1A).
In contrast, disease development was controlled when recipient mice
were reconstituted with T/R- and normal bone
marrow or T/R- and anti-OVA TCR
transgenic/RAG1+ bone marrow (Fig. 1C).
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D, as few as 13% of
clonotype-negative CD4+ T cells were sufficient
to confer protection from EAE in mice reconstituted with bone marrow
from T/R- and anti-OVA TCR
transgenic/RAG1+ mice. In these recipient mice,
anti-MBP T cells constituted about 40% of the total
CD4+ T cell population. In contrast, as many as
43% of anti-OVA CD4+ T cells from
OVA-specific TCR transgenic/RAG1-/- animals
were not capable of preventing disease in mixed chimeras with
T/R- bone marrow (Fig. 1B). The lack
of protective capacity of monoclonal OVA-specific T cells is not due to
their inability to reach the CNS. Fig. 1E shows that >5%
of the T cells obtained from a perfused CNS preparation are OVA
specific, whereas the blood of the same mouse contains about 25%
OVA-specific T cells. The fact that OVA-specific T cells are able to
migrate into the CNS is in accordance with published observations
(34, 38, 39). According to those studies, persistence in
the CNS but not immigration from the blood require Ag specificity.
However, it is possible that the capacity to control EAE may be
acquired early during ontogeny. For instance, it has been shown that
certain T cell populations such as the first two waves of 
T
cells only develop from fetal liver precursors and not from bone marrow
precursors (40), and other lymphocytes such as B-1A cells
are better generated from fetal liver precursors than from bone marrow
precursors (41). In addition, the fetal thymus provides a
unique environment that may be required for the development of
regulatory T cells, as is the case for the development of early
T cells (40). To assess whether OVA-specific T cells could
acquire regulatory properties if they could be generated from the
earliest possible developmental stages, we used anti-OVA TCR
transgenic RAG1-/- and
RAG1+ as recipients for MBP-specific T cell
precursors. This experimental system also enabled us to obtain mice
that displayed very high ratios of OVA-specific T cells to MBP-specific
T cells.
As shown in Fig. 2A,
reconstitution of sublethally irradiated anti-OVA TCR
transgenic/RAG1-/- mice with bone marrow from
T/R- mice resulted in EAE development, whereas
reconstitution of sublethally irradiated normal or anti-OVA TCR
transgenic/RAG1+ mice did not result in EAE (Fig. 2A). Disease developed in anti-OVA TCR
transgenic/RAG1-/- harboring as few as 20% of
the cells expressing the anti-MBP TCR, but was prevented in normal
or anti-OVA transgenic/RAG1+ recipient mice
even when 32% of the total CD4+ T cells were
specific for MBP (Fig. 2B).
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In vivo-activated OVA-specific CD4+ T cells do not confer protection from spontaneous EAE
In the previous section we demonstrated that a monoclonal OVA-specific CD4+ T cell population cannot confer protection from spontaneous EAE. However, the regulatory potential of this monoclonal T cell population may not be realized in the absence of its cognate Ag. Because OVA is not present in the environment of our mouse colony, most OVA-specific T cells remain naive throughout life. To assess whether activation of OVA-specific T cells renders them regulatory, we activated them through immunization. Toward this end, we chose to transfer mature OVA-specific CD4+ T cells into T/R- mice and immunize the transferred animals. This system enables us to clearly visualize the expansion of the OVA-specific T cells brought about by immunization.
Mice were immunized i.p. with OVA adsorbed to alum 2 and 12 days after
transfer of OVA-specific T cells. The effects of immunization are
evident by the marked expansion of circulating OVA-specific T cells 5
days after immunization (Fig. 3B). Control animals were left
untreated or received cells but were not immunized, or were given alum
without OVA. As shown in Fig. 3A, spontaneous EAE developed
in all immunized and control animals. A modest delay in disease onset
was observed in all alum-treated animals, including animals receiving
no cells and was more pronounced in animals receiving OVA-specific
cells and immunized with OVA adsorbed to alum. The observed delay in
disease onset can be attributed to Th2 priming of MBP-specific T cells
in the presence of alum, as this adjuvant is known to induce Th2
responses (42, 43, 44). Because all alum-treated animals
eventually develop severe EAE, this effect is clearly different from
the protection conferred by regulatory T cells, which is long lasting
even after a single administration. Furthermore, MBP-specific Th2 cells
have been shown to cause a delayed form of EAE in immunodeficient
recipient mice (35) (see below).
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In vitro-generated OVA-specific Tr1-like or Th2 cells do not confer protection from spontaneous EAE
It has recently been shown that anti-OVA CD4+ T cells with a Tr1 phenotype (i.e., slow growing cells with high IL-10/low IL-4 production) prevent the development of inflammatory bowel disease in mice (10). In contrast, the role of Th2 cells in the protection from Th1-mediated autoimmune diseases such as EAE is controversial (reviewed in Refs. 45, 46, 47, 48, 49). Therefore, we decided to assess the protective capacity of Tr1 and Th2 cells by transferring in vitro-generated OVA-specific Tr1 and Th2 cells into T/R- mice. Using the published procedure with the addition of neutralizing anti-IL-4 Abs (see Materials and Methods), we generated OVA-specific Tr1-like cells that share with bona fide Tr1 cells their growth properties and bear similarities in the cytokine secretion pattern. However, the most conspicuous difference between the reported cytokine secretion profile of Tr1 cells and the cells we obtained is a lower level of IL-10 secretion in our Tr1-like T cells (data not shown).
Following established procedures, we generated OVA-specific Th2 cells.
H-2d/u Tr1-like and Th2 cells were transferred
into H-2d/u T/R- mice, and
the progression of spontaneous EAE was monitored in the injected mice.
OVA was added to the drinking water to keep the antigenic stimulation
of the injected cells. As shown in Fig. 4, neither the Tr1-like nor the Th2
population was capable of controlling disease development.
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One of the differences between the disease-free
T/R+ strain of mice and the disease-prone
T/R- mice is the presence in the former of B
cells, T cells, and ß T cells bearing TCRs not encoded by
the TCR transgenes. We have previously shown that among those cells,
only CD4+ ß T cells can confer protection
from EAE (16, 17). Another difference between the
T/R+ and T/R- strains is
the presence of cells bearing two TCRs in the
T/R+ mouse strain. In the nonselecting
H-2b/b MHC background, all MBP-specific cells on
the same RAG1+ TCR transgenic mouse line express
two TCRs (50). In the selecting
H-2u/u MHC background, we have estimated that
about 10% of the MBP-specific T cells in T/R+
mice express two TCRs (16). Because a fraction of T cells
from normal mice also expresses two TCRs, it is possible that this cell
type could be important in the control of autoimmune disease.
To investigate the role of cells expressing two TCRs in the regulation
of EAE, we generated double TCR transgenic
RAG1-/- mice expressing both the anti-OVA
and the anti-MBP TCRs. Peripheral blood staining with the
anti-clonotypic Abs KJ1-26 and 3H12 (anti-OVA and anti-MBP
TCR, respectively) revealed that double transgenic
H-2d/u/RAG1-/- mice
harbor a large CD4+ T cell population (40%) with
high expression of anti-OVA TCR and intermediate levels of
anti-MBP TCR
(OVAhighMBPint) (Fig. 5A). However, this population
decreased to 2% in H-2u/u mice, which lack
I.Ad, the MHC molecule restricting the transgenic
OVA-specific T cells. Another population expressing preferentially the
anti-MBP TCR with low to intermediate levels of anti-OVA TCR
(OVAintMBPhigh) was present
in both the H-2d/u and the
H-2u/u mice (32% and 47%, respectively). A
third population, which only expresses the anti-MBP TCR, was
present in both types of mice. The percentage of this population was,
expectedly, higher in H-2u/u than in
H-2d/u mice (40% and 16%, respectively).
Finally, a small population expressing only the anti-OVA TCR
(0.12%) was exclusively observed in H-2d/u
mice.
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B, both
OVAintMBPhigh and
OVAhighMBPint proliferated
in response to OVA peptide 323–339 and to MBP peptide Ac1–17.
However, when compared with equal numbers of
OVAintMBPhigh T cells, the
OVAhighMBPint cells
responded better to OVA than to MBP, a predictable result based upon
the expression levels of both TCRs as well as the diminished frequency
of OVAhighMBPint T cells in
H-2u/u mice. Our results indicate that
OVA-specific, MBP-specific double TCR
transgenic/RAG1-/- mice bear
CD4+ T cells coexpressing two functional
TCRs. However, the presence of numerous double TCR-expressing cells did not affect the outcome of disease, as 100% of OVA-specific, MBP-specific double TCR transgenic/RAG1-/- H-2d/u mice developed spontaneous EAE (average score of 2.0 ± 0.3 at 60 days of age; n = 9). Moreover, in vivo activation of the doubleTCR-expressing cells by immunization with OVA adsorbed in alum before disease onset did not prevent spontaneous EAE (data not shown). These results demonstrate that, in this spontaneous EAE system, the presence of lymphocytes expressing two TCRs does not confer protection from disease.
TCR on regulatory T cells do not require N nucleotides or a
canonical TCR -chain
T/R+ mice are resistant to spontaneous EAE
due to expression of TCR - and ß-chains encoded by the endogenous
loci. Expression of these TCR chains confers some diversity to an
otherwise strongly biased MBP-specific T cell repertoire. In contrast,
as shown above, OVA-specific CD4+ T cells do not
prevent disease, suggesting that TCR specificity is important for
regulatory cell function. To determine what kind of repertoire
restrictions can be tolerated by regulatory T cells without loss of
activity, we adoptively transferred T cells displaying two different
repertoire limitations, one caused by the disruption of the TdT gene
(from TdT-/- mice), and the other caused by the
usage of a single TCR -chain (from TCR-/-
V4 Tg mice, see below).
Splenocytes from adult TdT-/- mice lack N
nucleotides in the joining regions of the TCR and Ig genes, thus
displaying an embryonic-type T and B cell repertoire (32).
Splenocytes from TCR-/- V4 Tg mice
contain CD4+ T cells expressing TCRs consisting
of ß-chains that pair exclusively with the transgene-encoded V4
chain. The V4 chain is the same used by the MBP-specific T cells,
and the exclusive usage of the transgene-encoded TCR -chain on all
ß T cells was ensured by the cross with
TCR-/- mice (33).
Adoptively transferred splenocytes from TdT-/-
or TCR-/- V4 Tg mice prevented the
development of spontaneous EAE in T/R- mice as
effectively as splenocytes from normal donors (Fig. 6, A and B). These
results demonstrate that the generation of regulatory cells is possible
in mice expressing a single TCR V4 chain or in mice lacking N
nucleotides.
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4 chain does not confer protection to mice
that also express the Vß8.2 TCR chain used by the MBP-specific T
cells, as spontaneous EAE occurs in MBP-specific TCR transgenic mice
crossed with TCR-/- mice (16, 17). Regarding the TCR ß-chain, we have previously shown that
approximately one-third of the mice bearing T cells that exclusively
express the Vß8.2 TCR chain from MBP-specific T cells remain EAE
free. This result indicated that, at least in those disease-free
animals, the Vß8.2 TCR chain from MBP-specific T cells was able to
pair with TCR -chains encoded by the endogenous loci, thus
generating regulatory T cells (16). Taken together, our
experiments show that the regulatory CD4+ T cells
that are able to prevent EAE in T/R+ mice do not
express canonical or ß TCR chains. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have addressed this issue by studying the incidence and course of EAE in a biclonal T cell experimental system. Using TCR transgenic RAG1-/- mice, we combined different proportions of disease-causing MBP-specific T cells with OVA-specific T cells. Both MBP-specific and OVA-specific T cells are positively selected in H-2d/u mice, therefore the endogenous peptide(s) required for thymic selection are present in these mice. However, OVA-specific T cells did not prevent EAE caused by MBP-specific T cells, even when they largely outnumbered the effector T cells. OVA-specific T cells were found in the CNS, lymph nodes, thymus, and spleen of animals that developed EAE, indicating that the absence of protection is not due to lack of access of OVA-specific T cells to the target tissue. Therefore, we conclude that a monoclonal population of OVA-specific CD4+ T cells is unable to regulate self-reactive T cells despite its ability to compete for space. The same conclusion was previously reached by Suri-Payer et al. (12) using an experimental system in which autoimmune gastritis is induced after neonatal thymectomy and can be prevented by the transfer of CD4+CD25+ T cells. It is noteworthy that CD4+CD25+ T cells do not appear to play a role in the protection of T/R+ mice, as depletion of CD25+ T cells does not diminish the protective capacity of spleen cells injected into T/R- mice (D. Olivares-Villagómez and J. J. Lafaille, unpublished observations).
Further demonstration that regulatory T lymphocytes do not act simply by expanding and occupying available niches comes from experiments in which T/R- mice were protected from EAE using 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester-labeled CD4+ T cells from normal donors. Among donor-derived T cells, those which expanded the most in the recipient mice (thus becoming 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester low-negative) exhibited lower disease-protective capacity in secondary T/R- recipients than the original unsorted populations (D. Olivares-Villagómez and J. J. Lafaille, unpublished observations). Therefore, the capacity to expand in T/R- recipients is not a requirement for the protective function of regulatory T cells.
While monoclonal OVA-specific T cells were unable to protect from EAE,
a small number of T cells bearing TCR - and ß-chains encoded by
the endogenous TCR loci, which are present in OVA-specific TCR
transgenic RAG1+ mice, was sufficient to confer
protection. However, the lack of protection by space-filling
OVA-specific T cells and the need for specific recognition by
regulatory T cells does not rule out that regulatory T cells act by
preventing expansion of MBP-specific T cells. For example, either
TCR-mediated recognition of expanding T cells (for instance,
recognition of activation markers) or unique properties of a putative
regulatory cell lineage (for instance, overexpression of growth factor
receptors) could lead to an impaired peripheral T cell expansion of
potentially effector T cells and, consequently, lack of disease. In the
latter instance, TCR specificity is required at least at the time of
thymic selection, when the T cells become committed to a regulatory
lineage.
The regulatory mechanisms that operate in T/R+ mice do not permanently inactivate MBP-specific T cells. First, disease-free T/R+ mice harbor at least as many MBP-specific T cells as disease-prone T/R- mice, indicating that the regulatory mechanism is nondeletional (7). Second, T cells from either mouse readily proliferate to MBP in vitro, and a virulent disease can be induced in T/R+ mice by administration of MBP, adjuvants, and pertussis toxin (6, 7). Finally, adoptive transfer experiments have also shown that MBP-specific T cells from T/R+ mice retain the potential to cause EAE in RAG1-/- recipients (D. Olivares-Villagómez and J. J. Lafaille, unpublished observations).
Activation of OVA-specific T cells in vivo or in vitro failed to render monoclonal OVA-specific T cells protective. While the inability to protect was observed using several immunization protocols (s.c., i.p., i.v., and gastric), we cannot formally eliminate the possibility that OVA-specific T cells could protect from spontaneous EAE if primed in different ways than the ones we attempted here.
T/R- mice injected with alum (with or without OVA-specific T cells) showed a delay in EAE onset; however, all mice eventually succumbed to EAE. Alum has been shown to be an effective adjuvant for the induction of IgE (42) as well as other Th2-mediated responses (43, 44). We have previously shown that MBP-specific Th2 cells do not protect from EAE, but instead cause a delayed disease in T cell-deficient animals (35). A further contributing factor to the generation of a Th2 environment could be the presence of Ag-stimulated OVA-specific Th2 cells, themselves generated in the presence of alum. Such bystander effects have been described in a number of experimental systems, including Ag-induced EAE (51). Thus, OVA-specific T cells do not behave like regulatory T cells even after they encounter their cognate Ag.
Postthymic OVA-specific T cells primed in vitro in the presence of IL-10 were able to prevent colitis (10). In contrast, in autoimmune disease caused by neonatal thymectomy, OVA-specific T cells failed to confer protection (12). These discrepancies indicate that CD4+ regulatory T cells are heterogeneous, and different experimental systems reveal the predominant effect of regulatory T cells displaying different properties. In spontaneous EAE, our data supports a model in which regulatory T cells display regulatory properties upon leaving the thymus, as has been previously proposed for transplantation tolerance models (52).
A major difference between spontaneous EAE-susceptible
T/R- or
T/-ß- mice and
spontaneous EAE-resistant T/R+ mice is the
presence, in the latter, of a proportion of T cells expressing two TCRs
(16). One of the hallmarks of double TCR-expressing cells
is that each TCR is expressed on the cell surface at lower levels than
the level of the same TCR in single TCR-expressing cells, presumably
due to competition for limiting CD3 components. Cells expressing two
TCRs may be important in regulation, because the presence of a second
TCR may alter the amount of signaling transduced inside the cells,
particularly under suboptimal stimulatory conditions. This quantitative
change, in turn, may lead to qualitative changes in effector functions,
for instance, alterations of cytokine production. Such signaling
effects in cells with two TCRs could be analogous to those demonstrated
when T cell responses to nominal Ags or altered Ags were studied
(53, 54), or when T cell responses in mice lacking CD4
(55, 56) or Itk (57) were analyzed.
Earlier descriptions of cells with two TCRs highlighted the potential hazard of activation of a self-reactive cell through a second TCR recognizing, for instance, a foreign pathogen (58, 59). More recent work has demonstrated that cells with two TCRs can escape tolerance through low-level expression of the self-reactive receptor (60, 61). Although cells with two TCRs have been shown to be more pathogenic than their single TCR-autospecific T cells in the absence of immunization (61, 62, 63), we have previously shown that spontaneous autoimmune disease arises at high frequency in the absence of double TCR-expressing T cells (7). Therefore, expression of two TCRs is not required for autoimmune disease development. In contrast, it has been shown that cells with two TCRs can become protective upon immunization in a double TCR transgenic system (63).
By crossing OVA-specific and MBP-specific TCR transgenic mice in the RAG1-/- background, we have generated mice in which a large proportion of the T cells expresses two TCRs. No protection from EAE was observed in this system even when the mice were immunized with OVA. These results indicate that the presence of a second TCR is not sufficient to generate regulatory T cells.
The reasons for the discrepancy regarding the protective role of double
TCR-expressing lymphocytes are presently not clear. One of the main
differences between the two experimental systems is that in the system
of Fossatti et al. (63), all T cells exhibit a uniform
double TCR staining, whereas in our double transgenic system we can
identify four T cells populations that express different levels of each
TCR (Fig. 5A). It is also possible that mixed TCRs are
formed in one experimental system more than the other, but no data is
available for either system. Given the fact that only about 10% of the
T cells in EAE-free T/R+ mice express two TCRs,
it is unlikely that the lack of protection that we observed in the
double TCR transgenic system is due to a lower proportion of double
TCR-expressing cells.
Spontaneous EAE occurs in all T/R- and
T/-ß- mice, and in
the vast majority of
T/-ß+ mice, but not
in T/R+ mice (16). Having shown that
a monoclonal OVA-specific T cell population lacked protective capacity,
we wished to test the protective capacity of T cells bearing a limited
but not monoclonal T cell repertoire. In one case, we used cells
lacking N nucleotides obtained from TdT-/-
mice, because N nucleotides account for a very significant portion of
TCR diversity in adult animals (64). One of the possible
explanations for the occurrence of organ-specific autoimmune disease in
neonatally thymectomized animals is that regulatory
CD4+CD25+ T cells are
generated in the thymus after birth (65), and,
consequently, their V(D)J junctions contain N nucleotides. According to
this scenario, the removal of the thymus soon after birth would lead to
a repertoire hole that includes the specificity of regulatory cells,
thereby leaving the animals unprotected from disease. However, T cells
from TdT-/- mice effectively protected
T/R- mice from EAE, indicating that a fetal-type
repertoire has the potential to generate regulatory T cells.
In the other case, we used T cells that could only express a single TCR
-chain, obtained from MBP-specific TCR -only transgenic mice
crossed with TCR-/- mice. These T cells also
exhibited a protective capacity that was not significantly different
from that of cells of normal mice.
The elimination of TCR diversity from T/R+
mice leads to EAE in the vast majority of the mice, whereas the
elimination of TCR ß diversity leads to both protected and diseased
animals (16). However, the data presented in this
manuscript argues against the existence of regulatory T cells
expressing canonical TCR -chains. Instead, our results showed that
protection from EAE can be mediated by T cells expressing the same TCR
-chain as the effector MBP-specific cells, as long as the diversity
of the TCR ß-chain is constrained only by pairing with the single TCR
-chain. In contrast, the same single MBP-specific TCR -chain is
ineffective in the generation of regulatory cells when the diversity on
the TCR ß-chain is further constrained by the presence of a
rearranged transgene-encoded MBP-specific TCR ß-chain.
In this manuscript, we have shown that control of spontaneous EAE by CD4+ T cells does not rely on niche occupancy, but instead depends on regulatory T cells that require ligand specificity to exert their protective function. However, the nature of ligand(s) that trigger regulatory cell activity remains a major unresolved issue. While in two reports it was described that regulatory T cells recognize the same target organ as the effector T cells (66, 67), another publication refuted this idea (68). Interestingly, the EAE susceptibility of T/R- mice and resistance of T/R+ mice were observed even when animals are kept under germ-free conditions (J. J. Lafaille, F. Van de Keere, and S. Tonegawa, unpublished observations), suggesting that the ligands recognized by T regulatory cells are self-Ags. A better knowledge of regulatory T cell repertoire and specificity will greatly enhance our capacity to prevent or treat chronic inflammatory autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Juan J. Lafaille, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016.
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; RAG1, recombinase activating gene-1; T/R-, MBP-specific TCR transgenic mice with mutated RAG1 genes; T/R+, MBP-specific TCR transgenic mice with at least a normal RAG1 gene; int, intermediate.
Received for publication December 15, 1999. Accepted for publication March 3, 2000.
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; n = 6) or in the presence of
107 bone marrow cells from OVA-specific
transgenic/RAG1-/- mice (
; n = 6)
or 107 from TCR
;
n = 4). Mice were observed for a period of 80 days
and EAE was scored as described in Materials and
Methods. Data represent the average of each group. All mice are
B10.H-2d/u. B, Donor cell reconstitution
from the three groups in A was analyzed 42 days after
bone marrow transplantation. Peripheral blood was stained for CD3, CD4,
anti-OVA TCR (KJ1-26), and anti-MBP TCR (3H12). Plots show a
representative mouse from each group and indicate
CD3+CD4+ gated cells. Five thousand lymphocytes
were acquired. C, Recipient mice, as in
A, were reconstituted with 106 T
cell-depleted bone marrow cells from T/R- donors alone
(
; n = 4), or left unimmunized (
;
n = 6). Mice were observed for a period of 12 wk,
and EAE was scored as described in Materials and
Methods. Data represent the average of each group. All mice are
B10.H-2d/u. B, Donor cell expansion from
nonimmunized, alum-immunized, or OVA/alum-immunized mice was analyzed 5
days after the first immunization. Peripheral blood was stained for
CD3, CD4, anti-OVA TCR (KJ1-26), and anti-MBP TCR (3H12). Plots
show a representative mouse from each group and indicate
CD3+CD4+ gated cells. Seven thousand
lymphocytes were acquired.
