By
From the Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095-1489
Recent evidence indicates that chronic autoimmune disease can result from breakdown of regulation and subsequent activation of self-reactive T cells. In many murine autoimmune disease
systems and in the Lewis rat, antigen-specific T cells utilizing the T cell receptor (TCR) V8.2
gene segment play a major role. In the myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE) model in H-2u mice, we had shown that T cells recognizing a
peptide determinant within the framework 3 region of the V
8.2 chain have a critical role in
influencing the course of the disease. Here, we report experiments in another disease system,
collagen II (CII)-induced arthritis (CIA) in DBA/1LacJ (H-2q) mice, indicating a remarkably
parallel control circuit to that found for EAE. A critical role is played by CII-specific V
8.2bearing T cells in the CIA system, which we have confirmed. Animals treated with the superantigen SEB before CII administration are significantly protected from CIA. Next, we tested
the ability of peptides encompassing the entire V
8.2 chain to induce proliferative responses.
Only TCR peptide B5 (amino acids 76-101), a regulatory peptide in EAE, induced proliferation. B5 was then used to vaccinate DBA/1LacJ mice and was shown to reduce greatly the severity and incidence of CIA as measured by joint inflammation or histology. Furthermore, similar protection was found when B5 was administered after CII immunization. It was shown that
there is physiological induction of a proliferative response to B5 during CIA and that the determinant within B5 is produced from a single chain TCR construct containing the entire V
8.2
chain. Finally, the regulation of CIA is discussed in the context of other experimental autoimmune diseases, especially EAE, with emphasis on what appear to be strikingly common mechanisms.
In recent years, a number of antigen-induced and spontaneous autoimmune diseases in susceptible strains of rodents have been extensively studied. The accumulated evidence, as well as clinical studies have led to the inference that
each of these autoimmune diseases is a representative model
for the human condition it most closely resembles. For example, experimental autoimmune encephalomyelitis (EAE)1
for multiple sclerosis, collagen II (CII)-induced arthritis (CIA) for rheumatoid arthritis and diabetes in nonobese diabetic
(NOD) mice for insulin-dependent juvenile diabetes. A common theme found in studies with mice of the TCRV Self-reactive T cells appear to be pivotal in the development of several human autoimmune disorders, including
multiple sclerosis, rheumatoid arthritis, and type 1 diabetes.
The plethora of observations linking a large number of
experimental autoimmune diseases in mice and rats with
the use of TCR encoded by the V CIA in rodents is an acute and severe experimental
model of autoimmune polyarthritis that develops following
immunization with heterologous type II collagen in adjuvant (8); homologous CII appear to induce chronic and
progressive arthritis (7). Both MHC class II and non-MHC
genes play important roles in the susceptibility to disease
induction, with H-2q and H-2r being the most susceptible
MHC haplotypes (12). Although the relative contribution
of antibody and cellular mechanisms in disease is not yet
clear, there is evidence that T cells are critically involved in
the pathogenesis of CIA (13).
It has clearly been shown that the depletion of CD4+ T
cells in mice attenuates the incidence and delays the onset of
CIA (13). Similarly, CIA has been induced in rats by adoptive transfer of CD4+ T cells specific for CII (14, 15). It has
been suggested that in susceptible mouse strains expressing
either the complete TCR V Mice.
DBA/1LacJ mice (H-2q) were obtained from The
Jackson Laboratory (Bar Harbor, ME), and were bred at UCLA
under specific pathogen-free conditions. 8 to 12 wk-old male
mice were used for induction of arthritis.
Induction of CIA in DBA/1LacJ Mice.
Arthritis was induced in
DBA/1LacJ male mice after injection of 100 µg of heterologous
bovine CII. The bovine CII (Institut Jacques Bois, Reims, France)
was dissolved in 0.01 M acetic acid at 4°C overnight and emulsified in an equal volume of CFA (DIFCO, Detroit, MI). 3 wk after the primary injection, mice were again immunized with 100 µg
CII emulsified in IFA, intraperitoneally. A second injection with
collagen was necessary to induce severe and reproducible disease.
Peptide Synthesis.
Peptides were synthesized as described earlier (5). The following are amino acid sequences of the TCR peptides and are shown in the single letter amino acid code. B1 (1-30L),
EAAVTQSPRNKVAVTGGKVTLSCNQTNNHNL; B2 (21-
50), LSCNQTNNHNNMYWYRQDTGHGLRLIHYSY; B3
(41-70), HGLRLIHYSYGAGSTEKGDIPDGYKASRPS; B4 (61-
90), PDGYKASRPSQENFSLILELATPSQTSVYF; B5 (76-101),
LILELATPSQTSVYFCASGDAGGGYE.
T Cell Proliferation Assay.
Mice were immunized as above
with CII, or subcutaneously with 3-7 nmol of TCR peptide emulsified in CFA (DIFCO, Detroit, MI). Draining (para-aortic and
inguinal) lymph node cells were obtained 9-10 d later, and used
in antigen-induced proliferation assays. 5 × 105 lymph node cells
were cultured in 0.2 ml of serum-free medium (HL-1; Ventrex,
Portland, ME or X-Vivo 10; BioWhittaker, Walkersville, MD)
medium alone, containing 2 mM glutamine, or with varying concentrations of collagen or specific TCR peptide in 96-well culture dishes for 4 d. Antigen-induced proliferation was assessed by
the incorporation of 1 µCi of [3H]thymidine during the last 18 h
of culture. For splenic proliferation assays, erythrocytes were lysed
and the residual cells were plated as above in HL-1 or X-Vivo 10 medium at a density of 1 × 106 cells in a total volume of 200 µl per
well in a 96-well plate with varying concentrations of antigens.
Flow Cytometry Analysis.
Antibodies were purified from hybridoma supernatants by protein A chromatography. Anti-CD4-
PE (GK1.5) was acquired from Becton-Dickinson (Mountain View,
CA). Anti-V Anti-CII Antibody Response.
Mice were bled retro-orbitally
and serum samples were collected from individual mice and
stored at As mentioned earlier, treatment of mice with antibodies
against the TCR V Table 1.
Administration of SEB 10 d Before CII Challenge
Protects Mice from CIA
To identify TCR determinants, we have
chemically synthesized five overlapping peptides (26-30
mers) encompassing the entire variable region of the TCR
V
In an attempt to influence the course of
CIA, DBA/1LacJ mice were vaccinated with 7 nmol of
B5/IFA twice, before challenge with bCII. Mice in the
control group were challenged with PBS/IFA or B4/IFA.
The effect of vaccination on the severity as well as on the
incidence of arthritis was examined in double-blind experiments. We have performed three different experiments
with 5-8 mice in each group. A representative experiment
with B5 vaccination is shown in Fig. 2. In Table 2, data
pooled from three independent experiments are shown: 34 of 36 mice in the control groups (PBS/IFA and B4/IFA)
developed severe arthritis by 8-9 wk after CII immunization, whereas only 6 of 18 mice in the B5-vaccinated group
displayed CIA. Importantly, B5-vaccinated mice contracted
less severe arthritis than mice in the control group (Table 2).
A majority of animals in the control group developed severe arthritic symptoms in both front and hind limbs. In
contrast, arthritic symptoms were rarely detected in the
front limbs of mice vaccinated with B5. These data demonstrate the efficacy of B5-specific T cells in modulating CIA
in DBA/1LacJ mice.
Table 2.
Vaccination with TCR Peptide B5 Prevents CIA in
DBA/1LacJ Mice
We have tested several variations of the B5 vaccination
protocol described in the legend for Fig. 2. Mice injected
only once with 14 nmol of B5 in IFA are not protected
from CIA. Furthermore, mice given only a single injection
of B5 even at a much higher concentration (50 nmol) are
not protected, but rather appear to have increased severity
of the disease (data not shown). Finally, we have asked
whether treatment with B5 after CII injection can protect
mice from CIA. In two independent experiments, DBA/
1LacJ mice challenged with B5 post-CII priming (days
+10, +20, +30, Fig. 3 A; or days +30, +40, +50, Fig.
3 B) show significant amelioration of disease. These data
clearly establish that B5-specific T cells are able to down
modulate CIA, even after CII-induced disease has been initiated.
Histologically, joints from untreated or control peptide
(B4)-treated animals were severely damaged by the rapidly
expanding synovial pannus. Mononuclear cell infiltration,
thickening of the synovial membrane, and bone marrow
erosion by osteoclasts, as well as cell exudate and polymorphonuclear cell accumulation in the synovial space were
present in the controls. In contrast, there were no signs of
an inflammatory process in 4 of 5 joints examined from
mice vaccinated with B5. Thus, lack of clinical disease in
B5-treated mice could be attributed to the absence of inflammation in the synovium (Fig. 4). Interestingly, the
anti-CII IgG response in groups treated with B5/IFA or
B4/IFA or PBS/IFA did not differ significantly, as shown
in Fig. 5. No significant difference in anti-CII titers was
seen between arthritic and non arthritic (B5-vaccinated)
mice, even though levels in individual mice varied considerably.
Although after peptide immunization (see Fig. 1), a proliferative response was detected only to a single TCR-peptide,
B5, in DBA/1LacJ mice, it was important to determine
whether B5-specific T cells are activated physiologically
during the course of disease, in the absence of any challenge with the peptide itself. Mice were challenged with
CII for the induction of CIA. 35 d after primary immunization with CII, at which time mice had already developed
arthritic symptoms, peripheral T cells proliferated in vitro
in response to B5 (Fig. 6). Proliferative responses to any of
the other TCR peptides from the V
The dynamics of spontaneous activation of B5-specific T
cells was studied further by following spontaneous proliferation to B5 in lymph node and spleen cells isolated from
DBA/1LacJ mice (three mice in each group) 2, 10, 20, 30, 40, and 50 days after CII injection. In contrast with studies
in the EAE model, the proliferative response to B5 was relatively lower (stimulation index [S.I.] of 2.5-3.0) until day
30. Interestingly, responsiveness to B5 continues to increase even beyond day 50 (S.I. of 4-5). An indolent expansion of B5-reactive T cells could perhaps explain the
chronic nature of CIA, in that Treg are not able to expand quickly enough to control the damage induced by CII-
reactive T cells.
The existence of primed B5-specific T cells
in animals with CIA suggests that T cells specific for TCR
determinant(s) are physiologically primed. To examine further immunodominance as well as the naturally processed
form of TCR determinants, soluble TCR-recombinant
single-chain TCRs containing appropriate V
The studies reported in this paper demonstrate a remarkably parallel strategy in the regulation controlling bovine
CIA in H-2q mice and the previously examined MBP-
induced EAE in H-2u mice. In EAE, the major disease-
inducing, MBP-specific T cells are highly restricted in their
TCR V gene usage, predominantly using the V Earlier studies from some laboratories (21), but not
from others (27) showed that CII-reactive T cell hybridomas preferentially use the TCR V It is a striking characteristic of experimentally induced
autoimmune diseases in mice (and the Lewis rat) bearing
the V For this reason, it is of interest to consider EAE induction in SJL (H-2s) and CIA in BUB (H-2q) mice, because
both these strains are of the TCRV It is remarkable that the B10.PL (H-2u) mouse and the
DBA/1LacJ (H-2q) mouse appear to use apparently similar
regulatory mechanisms in the physiological control of EAE
and CIA. In each case, a regulatory circuit is spontaneously
activated during the course of the disease after antigenic
challenge. T cells specific for the framework 3 region determinant derived from the V The mechanism by which B5-specific Treg mediate protection from CIA is not yet clear. Preliminary data indicate
that B5 vaccination does not result in deletion of most
V With regard to regulation of EAE in B10.PL mice, regulatory T cells are specific for the peptide sequence derived
from the framework 3 region of the Vb
haplotype but of many different H-2 haplotypes and in
Lewis rats is the key role of T cells using the V
8.2 gene
segment in primary recognition of autoantigens (1; see Discussion). These results suggest a common regulatory system
that controls potentially pathogenic self-reactive T cells. Experimental support for this idea comes from studies reported here with CIA in H-2q mice that appear to employ
similar regulation to that found for myelin basic protein
(MBP)-induced EAE in H-2u mice.
8.2 gene segment has
prompted us to make a detailed comparison of initiation of disease and regulation of potentially pathogenic self reactive T cells in two well-defined murine experimental models,
namely CIA and EAE. It has previously been shown that T
cells using the TCR V
8.2 gene segment are required for
induction of EAE in H-2u mice (2, 3). Antigen-induced
EAE, using MBP or peptide Ac1-9 of MBP as antigen, was
strongly inhibited with pretreatment by anti-V
8.2. We
have recently demonstrated in mice that the response to
self-MBP is controlled by potent regulatory T cell circuitry,
which is based on recognition of different determinants derived from the TCR V
8.2 chain, in the context of class I
and class II MHC molecules (4). This TCR-based regulation appears to be involved in maintaining peripheral tolerance to MBP, thus protecting mice from autoimmune
demyelination. Furthermore, TCR peptide-specific regulatory T cells appear to be responsible for natural recovery
from antigen-induced EAE in B10.PL mice (4).
gene repertoire (TCRV
b)
or lacking part of the V
gene segments (TCRV
a), CIA is
mediated by CII-specific T cells using a limited set of TCR
V gene segments (17). These findings are in agreement with the hypothesis that pathogenic T cells use a restricted
repertoire of V gene segments, as has previously been demonstrated in the EAE system. It has been shown that T cells
specific for CII in DBA/1 mice as well as T cells infiltrating
the joints in B10.Q mice use a limited number of TCR V
genes (20, 21). The significant reduction in the incidence
of CIA in both DBA/1 as well as in B10.RIII mice treated
with anti-V
8 antibodies indicates that T cells expressing
V
8 TCR are important in the development of CIA (20-
23). Selective depletion of V
8-expressing T cells has been
shown to be as effective as the depletion of all
T cells in
B10.R III mice (23). Here, using superantigenic stimulation of T cells in vivo, we show that CII-specific T cells
expressing V
8.2 genes are crucial for the induction of CIA
in DBA/1LacJ mice. Our studies indicate that TCR V
8.2
chain-derived determinants are physiological targets for the
regulation of CIA and TCR-peptide-based regulation may
be critical for the maintenance of peripheral tolerance to
CII. These data also show that the same framework 3 region of the V
8.2 chain appears to be crucially involved in
regulation of both CIA and EAE.
8.2 (F23.2) (24) was used for TCR staining after
SEB treatment; The F23.2-producing hybridoma was provided
by Dr. M. Bevan (University of Washington, Seattle, WA). Antibodies were used in PBS containing 1% fetal bovine serum. 1 × 106 cells were stained with 0.5 µg of antibody or biotinylated Ab in a total volume of 50 µl at 4°C for 30 min. Cells were washed twice with PBS and then resuspended in 50 µl of a 1:50 dilution of either FITC-conjugated streptavidin or goat anti-mouse Ig- FITC (Southern Biotechnology, Birmingham, AL). After 20 min
at 4°C, cells were washed, fixed with 1% paraformaldehyde in
PBS, and analyzed using a Becton-Dickinson cytofluorograph.
70°C until assay. A standard ELISA assay was used to
quantify the antibody response to CII. In brief, microtiter plates
were coated overnight with CII at a concentration of 10 µg/ml
in a total volume of 50 µl/well in PBS. After blocking remaining
protein-binding sites with BSA, diluted serum samples were incubated and the amount of bound antibody determined by incubation with goat anti-mouse IgG covalently coupled to alkaline
phosphatase (Southern Biotechnology, Birmingham, AL). A serum pool collected from hyperimmune mice was used as a standard.
Superantigen-induced Tolerance and Susceptibility to CIA.
8.2 gene segment has been shown to
reduce significantly the incidence of arthritis (21). Also,
the majority of T cells recognizing bovine CII (bCII) utilize V
8.2 and V
11 gene segments in the DBA/1LacJ mouse
(21). In several experimental models, it has been demonstrated that injection of mice with the superantigen, SEB,
results in an initial expansion of V
8.2 T cells followed by
clonal exhaustion and nonresponsiveness (25). We have
asked whether the disease-mediating response to bCII predominantly uses T cells expressing the V
8.2 gene segment; if so, clonal exhaustion or tolerance induction among
V
8.2 T cells after SEB challenge might result in protection from CIA. As shown in Table 1, DBA/1LacJ mice
given a single injection of SEB are significantly protected from disease. In the SEB-treated group, 3 of 10 mice displayed mild arthritic symptoms (arthritic index, 1.1). In
contrast, within control groups, including the SEA-injected
group, 8 of 10 mice developed severe arthritis (arthritic index, 6.0). These data implicate V
8 T cells in mediating
CIA. Interestingly, mice injected with SEB 10 d after CII/
CFA injection were not protected (8 of 8) but rather developed more severe arthritis (arthritic index, 9.9) than those
in the control group (arthritic index, 5.3).
Treatment group
Incidence of disease
Arthritis severity
PBS
8/10
6.0
SEB
3/10
1.1
SEA
8/9
5.7
Mice were injected with 50-100 µg of superantigen per animal 10 d
before the injection of CII/CFA for induction of arthritis. The percentage of CD4+, V 8.2+ T cells was analyzed by flow cytometry and
found to decrease significantly 48 h after SEB injection (7.9 ± 1.5% in
controls versus 1.8 ± 0.3% in SEB-treated group). Arthritis severity was determined as in Fig. 2.
Fig. 2.
Vaccination with TCR-peptide B5 reduces severity and incidence of CIA. Groups of DBA/1LacJ mice (eight in each group) were immunized subcutaneously with 100 µg of CII in one hind footpad. Disease was monitored in the three remaining limbs in a double-blind manner by two individuals. Each limb was graded with a score of 1 through 4, the maximum score being 12 for each mouse. The mean arthritis index
was determined by summation of the total score of each joint in each
group of mice and dividing by the total number of animals in each group.
For vaccination experiments, mice were injected with 7 nmol B5/IFA at
days 10 and +10 with respect to the primary CII injection (
). Control
mice were immunized with PBS/IFA (
).
[View Larger Version of this Image (12K GIF file)]
8.2 Chain in
DBA/1LacJ Mice.
8.2 chain. To characterize the immunogenicity of these
TCR peptides, mice were immunized subcutaneously with 7 nmol of individual peptides in CFA. 10 d later, proliferative responses in the draining lymph nodes were assayed
in response to an in vitro challenge. We found that only a
single peptide (B5) was able to elicit a lymph node proliferative response in DBA/1LacJ mice (Fig. 1). Interestingly,
T cells specific for the same TCR peptide, B5, have been
shown to regulate autoimmunity to MBP and are primed
naturally in B10.PL mice recovering from antigen-induced
EAE (4). Polyclonal T cell lines specific for B5 did not
proliferate in response to an adjacent overlapping peptide
B4 (data not shown).
Fig. 1.
Immunogenicity of TCR-peptides from the V8.2 chain in
DBA/1LacJ mice. DBA/1LacJ mice were immunized with 7 nmol each
of the five overlapping TCR-peptides. 10 d later, draining lymph node cells were assayed for proliferation in response to the immunizing peptide
at a concentration of 3.5 µM. [3H]thymidine incorporation was measured
by standard techniques. Individual responses in two mice are shown. The
amino acid sequences of the TCR peptides are given in Materials and
Methods.
[View Larger Version of this Image (14K GIF file)]
Treatment
CIA
Incidence
Arthritis severity*
Control (PBS/IFA or B4/IFA)
34/36
9.8
B5/IFA
6/18
2.1
*
Mean arthritic index was determined at 9 wk after CII immunization,
as described in the legend for Fig. 2. Mice were injected with B5/IFA
or B4/IFA or PBS/IFA at days 10 and +10 with respect to the primary CII injection, as described in the legend for Fig. 3.
Fig. 3.
DBA/1LacJ mice challenged with TCR peptide B5, after
CII immunization, are significantly protected from CIA. After immunization with bovine CII, mice in each group (n = 9) were challenged with B5/IFA () or B4/IFA (
) on days +10, +20, +30 (A), or days +30, +40, +50 (B) relative to primary immunization with CII/CFA. These immunizations were done as described in the legend for Fig. 2.
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
The infiltration of inflammatory cells into the synovium and
cartilage or bone erosion does not occur in B5-treated mice. Four mice
from each group treated with PBS/IFA, B4/IFA, or B5/IFA, were killed
12-14 wk after collagen II immunization. Hind limbs were formalinfixed, decalcified, embedded in paraffin, and stained in hematoxylin and
eosin. Tissue sections were examined and results are illustrated with a representative example from each group.
[View Larger Version of this Image (94K GIF file)]
Fig. 5.
In B5-treated mice, the IgG anti-collagen II response is not
inhibited. DBA/1LacJ mice were vaccinated with B5/IFA, B4/IFA, or
PBS/IFA as described in the legend for Fig. 2. Individual serum samples,
collected 8 wk after CII injection, were analyzed for the presence of antiCII IgG, using a standard ELISA assay. For the quantitation of IgG responses, a standard curve was generated using a hyperimmune antiserum
to CII. The values are represented in arbitrary units reflecting titers of the
anti-CII antibody in different groups of mice.
[View Larger Version of this Image (8K GIF file)]
8.2 chain were not
detected. Also, nonimmunized mice as well as mice immunized with HEL showed no response to B5.
Fig. 6.
Physiological induction of a proliferative response to B5 during CIA. DBA/1LacJ mice were immunized with CII for the induction of CIA. 35 d later, splenic cells were assayed for proliferation in response
to a concentration (7 µM) of different TCR peptides, B1 and B5.
[3H]thymidine incorporation was measured by liquid scintillation counting. There was no significant response to B5 in nonimmunized mice, nor
in mice challenged with HEL, 35 d after immunization. Individual responses of eight individual mice are shown.
[View Larger Version of this Image (9K GIF file)]
and V
chains (26), produced in Escherichia coli and provided by Dr.
E. Sally Ward (Dallas, TX), were used to challenge DBA/
1LacJ mice. An in vitro recall response was tested with various V
chain peptides. In parallel, TCR molecules containing a V
chain other than V
8.2 were tested for specificity, to establish whether B5-reactive T cells could be
stimulated by TCR molecules containing the appropriate V
domain. As shown in Fig. 7, lymph node cells from
B5-primed mice gave good proliferative recall to the immunizing peptide B5, as well as to soluble recombinant
scTCR molecules containing the V
8.2 chain (V
8.2-V
4).
There was no proliferative recall to a different TCR peptide (B3) or to scTCR molecules lacking a V
8.2 chain,
such as V
4. These data clearly established that TCR determinants within B5 are processed and presented following CII injection.
Fig. 7.
TCR determinant(s) within B5 are naturally processed and
presented. DBA/1LacJ mice were immunized with 7 nmol of B5 in CFA. 10 d later, draining lymph node cells were assayed for proliferation in
response to varying concentration of different antigens (3.5-14 µM).
[3H]thymidine incorporation, measured by standard techniques, at an optimum concentration, 7 µM, is shown. Responses in lymph node cells
pooled from two mice are shown. B5 and B3 are synthetic TCR peptides, whereas V8.2-V
4 and V
4 are scTCR molecules containing both V domains or only a single V domain, respectively.
[View Larger Version of this Image (11K GIF file)]
8.2 gene
segment. A variety of experiments point to potent regulation by naturally primed CD4 T cells that recognize a
framework 3 region determinant of the V
8.2 chain. Here,
we show that TCR peptide-specific T cells are implicated
in the physiological control of CII-reactive T cells, which
mediate autoimmune arthritis in DBA1/LacJ mice. Our
data support earlier studies (21) suggesting the in vivo
involvement of V
8.2 T cells in CIA induced by CII, which
is accompanied by activation of T cells specific for a determinant derived from the framework 3 region (peptide B5,
amino acids 76-101) of the V
8.2 chain. Vaccination with
TCR peptide B5 before (Fig. 2) or after CII immunization
(Fig. 3) protects mice significantly from arthritis and prevents inflammation in the synovia. With the use of recombinant scTCR protein containing the V
8.2 chain, we were
able to determine that the framework 3 region determinant is immunodominant and can be processed and presented
from the whole V
8.2 chain.
8.2 gene segment and
also that anti-V
8.2 treatment resulted in a significant reduction in the incidence of arthritis in DBA/1LacJ mice.
Consistent with the role of V
8.2 T cells, our data demonstrate that mice treated with the superantigen, SEB, but not
with SEA, 10 d before antigenic challenge, are significantly protected from CIA (Table 1), presumably by exhaustive
activation (25) of potential disease-causing T cell precursors. SEB appears to exacerbate disease when given after
CII injection, perhaps by expanding/activating a previously
primed CII-reactive V
8.2 T cell population. These studies further confirm the involvement of V
8.2 T cells in
CII-induced arthritis in DBA/1LacJ mice.
8.2 gene that the autoimmune response appears to
focus on T cells utilizing the TCR V
8.2 gene segment. In
the Lewis rat, MBP induced a remarkably homogeneous
response in which the induced T cells were specific for
peptides 68-88 and most used the TCR V
8.2 gene segment (28). In H-2u mice, the response to MBP focuses on
the NH2-terminal peptide, Ac1-9, and again, the TCR
V
8.2 gene segment is predominantly used (3, 4). In the
Lewis rat, experimental autoimmune neuritis (EAN), induced by the myelin P-2 protein or peptides 53-78 of this
protein, and experimental autoimmune uveoretinitis (EAU) induced by S antigen or interphotoreceptor retinoid binding protein (IRBP), are both characterized by the high frequency of antigen-specific T cells using the V
8.2 gene
segment in their TCRs (29, 30). EAU in B10.A mice is not
as well characterized with regard to T cell usage, but there
is evidence that T cells using TCR V
8.2 are also a major
component in this autoimmune disease (31). In BALB/c
mice, which are resistant to active EAE induction by MBP,
T cell clones specific for peptides 59-76 of mouse MBP
have been isolated that induce EAE upon transfer to naive
recipients (32). These clones are I-Ad-restricted and mainly
use the TCR V
8.2 gene segment. Models of Sjogren's
syndrome have been developed in several autoimmuneprone strains (NZB × NZW)F1, MRL/lpr, and MRL(+/+)
and one strain (NFS/sld) not prone to autoimmunity (33,
34). The disease is characterized by lymphocytic infiltration
of the lacrimal glands and infiltrating T cells have been
identified bearing the TCR V
8.2 gene segment. In NOD
mice, early induction of diabetes by a single injection of cyclophosphamide is prevented by treatment with anti-V
8.2 (35). In the autoimmunity-prone mouse strain, MRL/lpr,
there is amelioration of clinical autoimmunity by treatment
with anti-V
8.2 or with SEB. Finally, with regard to CIA,
although some groups have failed to demonstrate an effect
of anti-V
8.2 (27), in other studies a clear-cut inhibition of
CIA was shown. Clearly, our studies indicate a major involvement of T cells utilizing V
8.2 in CII-induced arthritis in DBA/1LacJ mice. It is not yet known why autoantigenspecific V
8-expressing T cells play a major role in mediating
different experimental autoimmune diseases in rodents carrying V
8 genes and also why V
8-expressing T cells constitute such a major proportion (10-30%) of the peripheral T cell repertoire.
a haplotype and lack
V
8 genes. In SJL/J mice, MBP-induced EAE is characterized by T cells specific for peptides 89-101/As. There is
oligoclonality with predominant usage of the V
4 or V
17a
gene segment (36). In BUB mice, CII injection results in
inflammatory polyarthritis. TCR usage of T cells isolated
from arthritic joints appears to be limited to V
3 and V
10
gene segments (19). These results suggest that oligoclonality need not be restricted to the TCR V
8 gene family and
may be characteristic of many autoimmune T cell responses.
It is significant that BUB mice, which lack the V
8.2 gene,
contract severe CIA in contrast with the minimal response
in DBA/1LacJ mice in which V
8.2-bearing T cells have
been greatly reduced by SEB treatment (Table 1) or by
anti-V
8 treatment. Because genes coding for TCR V
chains used in the BUB mice are present in DBA/1LacJ
mice, it would appear that in V
8.2+ mice, a functional hierarchy is established, perhaps during early development, in
which V
3 or V
10 TCR specific for CII are excluded.
8.2 chain are also spontaneously primed and involved in regulating EAE in the Lewis
rat model (37). It is interesting that despite the differences
in species, MHC haplotypes, background genes, and disease systems, T cells specific for the TCR determinants
within the same framework 3 region (B5 TCR-peptide,
amino acids 76-101), but not necessarily the same determinant, become activated spontaneously. There could be at least three relevant aspects to the broad use of B5, in particular, that could explain its predominant use in regulatory
circuitry. First, the likelihood is that B5 is efficiently processed in a variety of mouse strains and becomes readily
available, leading to its immunodominance within the V
8.2
TCR. Consistent with this, lymph node cells from both
B10.PL and DBA/1LacJ mice challenged with B5 respond
to recombinant single-chain TCR molecules containing the
entire V
8.2 chain in recall proliferation assays. This indicates that this TCR determinant can be processed and presented from the whole V
8.2 chain. Second, the possibility
exists that B5 possesses one or more promiscuous determinants, able to bind to various class II MHC molecules.
Third is the unusual feature of an apparent inability to induce tolerance. Notably, we have not been able to induce
proliferative tolerance to this TCR peptide in neonatal
DBA/1LacJ mice or in the B10.PL mouse model (7; our unpublished observations). In contrast, proliferative tolerance to all other immunogenic TCR peptides or peptides from
any other antigen tested (from MBP, HEL, cyt C) could be
successfully induced after neonatal exposure.
8.2-expressing T cells in DBA/1LacJ mice. This is in
contrast with the mechanism suggested in BUB mice,
where immunization of mice with peptide 25-46 derived from the TCR V
10 chain resulted in deletion of most T
cells bearing TCR V
10, presumably by induction of an
anti-V
10 antibody response (38). Whether T cells specific
for the TCR V
10-peptide are spontaneously primed and
are physiologically involved in regulation of CIA in BUB
mice remains unknown. Similarly, it is not known whether
TCR determinants on other V
(and V
) chains are naturally involved in such regulation (38, 39) or whether the
immune system focuses on T cells expressing the V
8.2
chain to reestablish effective regulation of autoimmunity.
Currently, experiments are in progress to investigate further whether CII-specific and V
8.2-bearing T cells are deleted, or deviated to produce noninflammatory cytokines, e.g., IL-4, after B5-induced regulation. Our preliminary
data indicate that regulatory mechanisms in EAE, as well as
in the CIA system, result in antigen-specific immune deviation (Kumar, V., unpublished observations). CIA appears
to be mediated by CII-specific Th1-like cells, with antigen-specific Th2 cells being protective.
8.2 chain, and does
not include the D-J region sequence, i.e., a sequence on all
V
8.2 TCRs is recognized. Nevertheless, the vast majority
of V
8.2 cells are unaffected by vaccination with B5 or the
scTCR containing the V
8.2 chain (4-7, Kumar, V., E. Coulsell, B. Ober, G. Hubbard, E. Sercarz, and E. Sally
Ward, manuscript submitted for publication). In our preliminary studies in the CIA model, only activated antigenspecific T cells appear to be downregulated after vaccination of mice with B5, suggesting a similar mechanism to
that involved in the EAE model. These findings are consistent with earlier indications that it is only activated T cells
which are subject to interaction with Treg (40). With relation to the treatment of chronic autoimmune diseases, it is
noteworthy that vaccination of mice with TCR peptide B5
as late as 30 d after CII immunization leads to a significant inhibition in the incidence and severity of arthritis (Fig. 3). It is likely that the common regulatory principles we have
described in the control of two distinct experimental autoimmune conditions define a generalized TCR centered
regulatory mechanism employed by the immune system.
This mechanism may be exploitable through specific therapeutic strategies in several human autoimmune diseases.
Address correspondence to Dr. Vipin Kumar, Division of Immune Regulation, La Jolla Institute for Allergy and Immunology 10355 Science Center Drive, San Diego, CA 92121.
Received for publication 21 February 1997.
1Abbreviations used in this paper: bCII, bovine collagen II; CIA, collagen IIinduced arthritis; CII, collagen II; EAE, experimental autoimmune encephalomyelitis; EAN, experimental autoimmune neuritis; EAU, experimental autoimmune uveoretinitis; IRBP, interphotoreceptor retinoid binding protein; MBP, myelin basic protein; NOD, nonobese diabetic; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; S.I., stimulation index.We thank Dr. E. Sally Ward for providing recombinant single chain TCR proteins and Dr. Thomas Goldschmidt for help in establishing a reproducible scoring system for CIA.
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