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The Regulatory C-Terminal Determinants
within Mycobacterial Heat Shock Protein 65
Are Cryptic and Cross-Reactive with the
Dominant Self Homologs: Implications for
the Pathogenesis of Autoimmune Arthritis
Malarvizhi Durai, Hong Ro Kim and Kamal D. Moudgil
J Immunol 2004; 173:181-188; ;
doi: 10.4049/jimmunol.173.1.181
http://www.jimmunol.org/content/173/1/181
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
The Regulatory C-Terminal Determinants within
Mycobacterial Heat Shock Protein 65 Are Cryptic and
Cross-Reactive with the Dominant Self Homologs: Implications
for the Pathogenesis of Autoimmune Arthritis1
Malarvizhi Durai, Hong Ro Kim, and Kamal D. Moudgil2
A
djuvant-induced arthritis (AA)3 is a well-studied experimental model of human rheumatoid arthritis (RA), and
it can be induced in the Lewis rat (RT.1l) by immunization with heat-killed M. tuberculosis (Mtb) in oil (1, 2). The
mycobacterial 65-kDa heat shock protein (Bhsp65) has been implicated in the pathogenesis of AA as well as RA (3– 8). AA is a
self-limiting disease, which makes it an attractive model for studies on immune regulation. Pretreatment of naive Lewis rats with
certain peptides of Bhsp65 can provide protection against subsequent induction of AA (4, 9 –11). However, the physiologic significance of these disease-regulating T cell epitopes of Bhsp65 in
AA is not fully defined. We have previously reported that there is
diversification (epitope spreading) of the T cell response to Bhsp65
C-terminal determinants (BCTD) during the course of AA in the
Lewis rat, and that immunization of Lewis rats with a mixture of
Department of Microbiology and Immunology, University of Maryland School of
Medicine, Baltimore, MD 21201
Received for publication January 20, 2004. Accepted for publication April 19, 2004.
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 work was supported by grants (to K.D.M.) from the National Institutes of
Health (AI-47790), Arthritis Foundation (Atlanta, GA), and the Maryland Chapter of
Arthritis Foundation and the Maryland Arthritis Research Center (Baltimore, MD).
2
Address correspondence and reprint requests to Dr. Kamal D. Moudgil, Department
of Microbiology and Immunology, University of Maryland School of Medicine, BRB
13-019, 655 W. Baltimore Street, Baltimore, MD 21201. E-mail address:
[email protected]
3
Abbreviations used in this paper: AA, adjuvant-induced arthritis; BCTD, Bhsp65
C-terminal determinant; Bhsp, mycobacterial heat shock protein; DDA, dimethyldistearoylhydroxyethyl ammonium chloride; HEL, hen egg lysozyme; LNC, lymph
node cell; Mtb, Mycobacterium tuberculosis; RA, rheumatoid arthritis; RCTD,
Rhsp65 C-terminal determinant; Rhsp, rat heat shock protein; S.I., stimulation index.
Copyright © 2004 by The American Association of Immunologists, Inc.
peptides comprising BCTD affords protection against AA (4). Furthermore, arthritic Lewis rats in late phase of the disease spontaneously raised T cell response to the corresponding C-terminal
epitopes of the homologous self (rat) hsp65 (Rhsp65) (4). (Rat
hsp60 (Rhsp60) (12) has been referred to as Rhsp65 to match
Bhsp65.) Unlike in experimental models and patients with other
autoimmune diseases (e.g., multiple sclerosis (13–15) and type I
diabetes mellitus (16, 17)), in which the spreading of T cell response to neoepitopes has been invoked in the progression of the
disease, the diversification of response to BCTD in arthritic Lewis
rats was associated with regression of acute AA (4).
It is not clear as to why Lewis rats fail to raise T cell response
to BCTD in the early phase of AA following injection of Mtb, and
what might be the likely mechanism underlying emergence of T
cell response to the same epitopes in the late phase of the disease.
Furthermore, the role of self hsp65 (Rhsp65), if any, in diversification of response to BCTD is not yet known. In this context,
defining the fine specificity and functional attribute of the T cell
repertoire directed against C-terminal epitopes of Bhsp65, and examining the interrelationship between the C-terminal epitopes of
mycobacterial and self hsp65 are critical for fully understanding
the immunological basis of epitope spreading during the course of
AA and regulation of this disease.
In this study, we describe that the disease-regulating T cells
potentially reactive against BCTD are available in the mature T
cell repertoire of the Lewis rat, and that these T cells can be primed
by peptides comprising BCTD, but not by native Bhsp65. Therefore, BCTD represent cryptic epitopes of Bhsp65. Importantly, the
T cells directed against cryptic BCTD can be stimulated by naturally processed (dominant) homologous C-terminal determinants
of self hsp65 (Rhsp65), or by the corresponding peptides of
0022-1767/04/$02.00
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The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in
the Lewis rat (RT.1l) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T
cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides
representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the
BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response
to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer
protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD,
but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally
generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain
individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a
model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic
BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The
identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis. The Journal of Immunology, 2004, 173: 181–188.
182
REGULATION OF AUTOIMMUNE ARTHRITIS BY CRYPTIC EPITOPES
Rhsp65 and vice versa. Furthermore, like BCTD mixture (4), certain individual peptides representing the C-terminal epitopes of
foreign/self hsp65 can also suppress the course of subsequent AA.
Our results suggest that the unmasking of previously cryptic
BCTD within Bhsp65, and enhanced expression of endogenous
self hsp65 leading to efficient display of its dominant Rhsp65 Cterminal determinants (RCTD) (that are cross-reactive with
BCTD) during the acute phase of AA contribute to the observed
epitope spreading to BCTD during the late phase of this disease.
Our study demonstrates that cryptic epitopes of a disease-related
Ag could be immunoregulatory in nature. The regulatory aspect of
cryptic determinants and the involvement of self hsp65 in diversification of the T cell response to Bhsp65 in AA provide novel
insights into the pathogenesis of autoimmune arthritis, and can be
exploited for immunotherapy of RA.
were tested at concentrations that were found to be optimal in pilot experiments.) The results were expressed either as cpm or as a stimulation index
(S.I. ⫽ ratio of cpm in the presence of Ag and cpm of cells in medium
only).
Adoptive transfer of Ag-primed LNC into Lewis rats
Rats were immunized s.c. at the base of the tail with either BCTD in IFA
(experimental group) or PBS/IFA (control group), as described above. On
day 9, the draining LNC were cultured (3 ⫻ 106 cells/ml) for 48 h in
complete RPMI 1640 (RPMI 1640 medium supplemented with heat-inactivated FCS (10%) (Life Technologies, Rockville, MD), L-glutamine (1%),
penicillin-streptomycin (1%), and 5 ⫻ 10⫺5 M 2-ME) in the presence of
Con A (2.5 ␮g/ml). Thereafter, cells were collected, washed thoroughly,
and transferred i.v. into naive Lewis rats. On the day of cell transfer, the
recipient rats were immunized with Mtb (2 mg/rat) s.c. for induction of
AA, and all rats were then observed regularly for signs of arthritis. The
severity of the disease was graded, as described above.
Preparation and use of heat-stressed APCs
Materials and Methods
Rats
Ags/mitogen/adjuvant
Bhsp65 (18) was prepared from BL21 (DE3) pLysS cells (Novagen, Madison, WI) transformed by the vector pET23b-GroEL2 (Colorado State University, Fort Collins, CO), whereas Rhsp65 (12) (rat hsp60 (Rhsp60) (12)
has been referred to as Rhsp65 to match Bhsp65) was expressed from
pTrcHisA-transformed BL21 cells. The transformed Escherichia coli BL21
cells were cultured with isopropyl-D-thiogalactopyranoside (0.4 mM) (Sigma-Aldrich, St. Louis, MO) for expression of recombinant proteins. Thereafter, the histidine-tagged recombinant proteins were purified using ProBond nickel resin (Invitrogen, Carlsbad, CA). Any contaminating
endotoxin was removed using Acticlean Etox Kit (Sterogene Bioseparations, Carlsbad, CA). The identity of the purified proteins was confirmed on
Western blot by using the appropriate anti-hsp60 mAb (clone LK-1; StressGen Biotechnologies, Victoria, Canada) (for Rhsp65) or an anti-his tag
mAb (Novagen) (for Bhsp65). Heat-killed Mtb H37Ra was obtained from
Difco Laboratories (Detroit, MI), whereas hen egg lysozyme (HEL), Con
A, and IFA were obtained from Sigma-Aldrich. Synthetic peptides of
Bhsp65 (18) and Rhsp65 (12) were obtained from Macromolecular Resources and Global Peptide Services (both at Fort Collins, CO). BCTD
(equal to a mixture of five peptides of Bhsp65: 417– 431, 441– 455, 465–
479, 513–527, and 521–535) and RCTD (equal to a mixture of five Cterminal peptides of Rhsp65, namely 418 – 432, 441– 455, 465– 479, 512–
526, and 521–535) (Fig. 1) were prepared by mixing equal volume of each
of the five respective peptides (stock; 5 mg/ml of each peptide) to a final
concentration of 1 mg/ml each peptide. Therefore, rats (adults/pups) immunized with 200 ␮l of this mixture emulsified in adjuvant (1:1; v/v)
received 100 ␮g of each peptide in the mixture.
Induction and evaluation of AA
Lewis rats were injected s.c. with 200 ␮l (2 mg/rat) of Mtb in oil at the base
of the tail, and thereafter, these rats were observed regularly for signs of
arthritis, such as erythema, swelling, and induration (4). The severity of the
disease in each paw was graded on a scale from 0 to 4. The maximum
arthritic score for each rat was 16.
Lymph node cell (LNC) proliferation assay
Lewis rats were injected with Ag (100 –200 ␮g/rat) in IFA (the reason for
using IFA instead of CFA in this and other experiments was to avoid any
concurrent stimulation of T cells by Bhsp65 of Mtb within CFA). The rats
were sacrificed after 8 days, and the draining LNC (inguinal, para-aortic,
and popliteal) of these rats were harvested and tested in a proliferation
assay at 5 ⫻ 105 cells/well in HL-1 serum-free medium (BioWhittaker,
Walkersville, MD) in the presence or absence of Ag (4). Con A was used
as a positive control, whereas HEL served as a negative control. (The Ags
used for both immunization and in vitro recall in this and other experiments
Neonatal tolerization of Lewis rats against BCTD/RCTD
Newborn pups of Lewis rats were given two injections i.p., one within 24 h
and the other within 72 h after birth, of either BCTD/IFA or RCTD/IFA
(100 ␮g of each peptide in the mixture, as described above) following the
procedure described elsewhere (20). After 4 wk, a subgroup each of these
tolerized rats was immunized s.c. with either BCTD/IFA or RCTD/IFA.
After 8 days, the draining LNC of these rats were tested in a proliferation
assay using BCTD and RCTD as recall Ags, as described above.
Pretreatment of Lewis rats with individual peptides comprising
BCTD/RCTD for determining their protective effect against AA
Lewis rats were immunized intradermally twice at 1-wk interval with either
a peptide or PBS, each suspended in GERBU adjuvant 100 (GERBU Biochemicals, Gaiberg, Germany) containing N-acetylglucosaminyl-(b,1– 4)N-acetylmuramyl-L-alanyl-D-isoglutamine (2 ␮g/rat) and dimethyldistearoylhydroxyethyl ammonium chloride (DDA) (10 ␮g/rat). (In previous
studies in the AA model by others (10) and us (4), PBS/DDA-treated rats
were used as controls, and the course of AA in these rats was comparable
to that of rats injected with an irrelevant Ag in DDA. Therefore, we have
used PBS/DDA as a control for antigenic challenge.) After 4 wk of the
second injection, these rats were challenged s.c. at the base of the tail with
Mtb in oil for induction of AA, and then observed regularly for signs of
arthritis. The severity of the disease was graded, as described above.
Results
BCTD represent cryptic determinants within Bhsp65
As described above, Mtb-injected arthritic Lewis rats in the late
phase of AA, but not those in early phase of the disease, raised T
cell response to BCTD (4). We reasoned that this pattern of T cell
response to BCTD in AA might be owing to the inability of
Bhsp65 within Mtb to reveal these epitopes. To test this proposition, Lewis rats were immunized with native Bhsp65/IFA, and the
draining LNC of these rats were tested in a proliferation assay
using native Bhsp65 and BCTD (Fig. 1) as recall Ags. Another
group of Lewis rats was challenged with BCTD (positive control).
The results (Fig. 2A) show that Lewis rats challenged with Bhsp65
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Inbred Lewis (LEW/SsNHsd) (RT.1l) rats (4 – 6 wk old, male, 150 –200 g)
were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed
in a conventional environment in the vivarium of the University of Maryland School of Medicine (Baltimore, MD). All experimental procedures
performed on these animals were in compliance with the guidelines of the
Institutional Animal Care and Use Committee. Newborn pups were obtained by breeding male and female Lewis rats (8 –10 wk old) in the vivarium of the University of Maryland School of Medicine.
Splenic APCs of a naive Lewis rat were prepared following the procedure
described elsewhere (19), but with minor modifications, and the final preparation consisted mostly of macrophages and dendritic cells. These APCs
were stained with mouse anti-rat CD11b/c Ab (BD PharMingen, San Diego, CA) and then analyzed by flow cytometry to confirm their identity.
Thereafter, one aliquot of these cells was heat shocked by incubating at
43 ⫾ 1°C for 20 min in a water bath (heat-stressed APCs; experimental
group), whereas the other aliquot of cells was kept at room temperature
(unstressed APCs; control). After incubation, fresh complete RPMI 1640
medium was added and cells were then incubated again for 1 h at 37°C
(recovery period) in a CO2 incubator. The viability of these APCs was
checked by trypan blue staining before their use in a proliferation assay,
which was performed essentially as described above, but with the difference that LNC were cultured with APCs (2.5 ⫻ 105 cells/well) without
addition of an exogenous Ag (unless indicated). The results were expressed
as an S.I. (S.I. ⫽ cpm with heat-stressed APCs ⫼ cpm with unstressed
APCs).
The Journal of Immunology
183
raised a vigorous T cell response to the intact immunogen, but not
to BCTD. Moreover, there was no recall response to any of the five
C-terminal peptides representing BCTD when tested individually
(data not shown). Immunization of rats with different doses (50 or
200 ␮g/rat) of Bhsp65 also failed to induce a T cell response to
BCTD (data not shown). On the contrary, BCTD per se were immunogenic (Fig. 2B), demonstrating that the T cell repertoire potentially reactive with BCTD could be primed only by the preprocessed (peptide) form of BCTD, but not by native Bhsp65. In this
regard, the positive in vitro T cell recall response to the immunogen in Bhsp65-immunized rats could be accounted for by response
to epitopes of Bhsp65 other than BCTD (4). Taken together, these
results show that potentially immunogenic BCTD are cryptic
epitopes within native Bhsp65, and therefore, Lewis rats fail to
raise a T cell response to BCTD early after Mtb injection during
the acute phase of AA.
The T cells primed by peptides comprising BCTD can adoptively
transfer protection against AA: BCTD-reactive T cells are
disease regulating in nature
To determine the functional attribute of the BCTD-reactive T cells,
a group each of Lewis rats was challenged with either BCTD/IFA
or PBS/IFA. The draining LNC of these rats were harvested and
cultured (activated) in vitro with Con A for 48 h. Thereafter, these
two pools of LNC were adoptively transferred i.v. separately into
naive Lewis rats, and on the day of cell transfer, the recipient rats
were immunized with Mtb s.c. for induction of AA. All rats were
examined regularly for clinical signs of AA. The results (Fig. 3)
show that Lewis rats that had received BCTD-primed LNC were
significantly protected from subsequently induced AA compared
with the control recipient rats. These results demonstrate that the T
cells specific for cryptic BCTD were capable of conferring protection against AA, and therefore, activation of this repertoire in
vivo might be involved in regulation of AA.
The T cells against BCTD are cross-reactive with peptides
representing the homologous C-terminal epitopes of self Rhsp65
and vice versa
We have previously shown that arthritic Lewis rats in the recovery
phase of AA develop T cell response not only to BCTD, but also
to the corresponding C-terminal epitopes of self hsp65, Rhsp65
(4). Considering that hsp are highly conserved proteins, we tested
whether the T cells primed by BCTD can also be restimulated by
their self homologs, Rhsp65 C-terminal determinants (RCTD), and
vice versa. Lewis rats were immunized with BCTD/IFA, and after
8 days, the LNC of these rats were tested in a proliferation assay
using BCTD and RCTD (separately) as recall Ags. A similar set of
experiments was performed using RCTD/IFA as the immunogen.
Interestingly, BCTD-primed T cells could be restimulated in vitro
by RCTD (Fig. 4A) and vice versa (Fig. 4B), demonstrating the
cross-reactivity between BCTD and RCTD despite a limited sequence homology between them (Fig. 1). However, in each case,
the T cell recall response to the immunogen was relatively higher
compared with that to the homologous peptides. Overall, RCTD
were more immunogenic compared with BCTD.
The T cell repertoire against BCTD can be primed by the
naturally generated (dominant) RCTD from native self Rhsp65
We next tested whether the T cells potentially reactive against
BCTD could also be stimulated following challenge with native
Rhsp65. Lewis rats were immunized with native Rhsp65/IFA, and
9 days later, the draining LNC of these rats were tested in a proliferation assay using BCTD (test Ag) and RCTD (positive control) as recall Ags. In another experiment, native Bhsp65/IFA was
used as the immunogen instead of Rhsp65 for comparison. The
results (Fig. 5) show that Lewis rats immunized with Rhsp65, but
not Bhsp65, raised T cell response to both RCTD and BCTD. The
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. Amino acid sequence of the C-terminal determinants of Bhsp65 and Rhsp65. The corresponding C-terminal regions (aa 400 –535) of Bhsp65
(GenBank accession no. P06806) and Rhsp65 (ACCN CAA37654) were aligned using the LALIGN program, and the homology of amino acids is depicted
as follows: (:), identical; (.), conservative; and ( ), entirely different. BCTD is a mixture of five peptides of Bhsp65, namely 417– 431 (a), 441– 455 (b),
465– 479 (c), 513–527 (d), and 521–535 (e), and RCTD mixture contains the corresponding five peptides of Rhsp65: 418 – 432, 441– 455, 465– 479,
512–526, and 521–535. The aa sequence of these epitopes is shown in boldface. The overall amino acid sequence homology between native Bhsp65 and
Rhsp65 is 48%, whereas that between the region 417–535 containing BCTD/RCTD is 40%. The homology between the corresponding individual peptides
of BCTD and RCTD (derived separately using the LALIGN program) ranged between 16.7 and 58.3%, with relatively higher level of homology in the pairs
represented by a (58.3%), d (50%), and e (50%) compared with b (16.7%) and c (28.6%).
184
REGULATION OF AUTOIMMUNE ARTHRITIS BY CRYPTIC EPITOPES
failure of Bhsp65 to induce a T cell response directed against either BCTD or RCTD can be explained on the basis of crypticity of
BCTD described above.
BCTD-primed T cells can be restimulated in vitro by naive
syngeneic heat-stressed APCs
In another set of experiments, we tested whether endogenous self
hsp65 could also restimulate T cells primed by BCTD. For this
purpose, naive heat-stressed syngeneic APCs (heat-stressed APCs
express higher level of cellular hsp65 compared with unstressed
APCs) (21) were cultured with BCTD-primed LNC of Lewis rat
without addition of any exogenous Ag. HEL/IFA-primed or naive
LNC served as controls. The results (Fig. 6) reveal that stressed
APCs induced a significant restimulation of BCTD-primed, but not
HEL-primed or naive LNC compared with unstressed APCs. Thus,
following heat shock, naive APCs could present to BCTD-primed
T cells the naturally processed self epitopes (presumably RCTD)
that mimic the synthetic BCTD peptides.
The BCTD-reactive T cell repertoire consists of shared crossreactive and specific noncross-reactive subsets of T cells
We have described above that T cells against BCTD are crossreactive with RCTD and vice versa. To further define the fine
specificity of the T cell repertoire directed against BCTD, we performed neonatal tolerization experiments. A cohort of newborn
Lewis pups was tolerized with BCTD/IFA, and 4 wk later, a group
each of these rats was challenged s.c. with either BCTD/IFA or
RCTD/IFA. After 8 days, the draining LNC of these rats were
tested in a proliferation assay using both BCTD and RCTD separately as recall Ags. In a parallel experiment, RCTD were used as
the tolerogen instead of BCTD. The results show that BCTD-tolerized rats (Fig. 7A) failed to raise a T cell response upon immunization with BCTD, but responded vigorously upon challenge with
RCTD. However, these RCTD-reactive T cells were not crossreactive with BCTD. Similarly, RCTD-tolerized rats (Fig. 7B) responded to challenge with BCTD, but not RCTD, and these
BCTD-reactive T cells failed to recognize RCTD in vitro. These
results demonstrate that the T cell repertoire against BCTD consists of BCTD-specific as well as BCTD-RCTD cross-reactive T
FIGURE 3. Modulation of AA in the Lewis rat by adoptive transfer of
BCTD-primed LNC before induction of disease. A group of Lewis rats was
immunized with BCTD/IFA (experimental donors). Another group of control donor rats was injected with PBS/IFA. The draining LNC of these rats
were harvested on day 9 postinjection and then cultured separately for 48 h
with Con A. Thereafter, a total of 2 ⫻ 107 BCTD-primed LNC (f, n ⫽ 3)
(experimental recipient group) or PBS/IFA-primed LNC (䡺, n ⫽ 3) (control recipient group) was injected i.v. separately into naive Lewis rats. On
the day of cell transfer, all recipient rats were injected s.c. with Mtb for
induction of AA and then observed regularly for clinical signs of AA. The
difference in the mean arthritic score of experimental and control recipient
rats was statistically significant from days 12 to 31 (value of p, ⬍0.0009 to
⬍0.05). The difference in the sum of the arthritic scores over the entire
course of AA between the two groups was also significant (p ⬍ 0.05) by
Wilcoxon rank sum test. Similar results were obtained on repeat testing.
cells, and that both these subsets of T cells are efficiently tolerized
upon neonatal exposure to BCTD, leaving only RCTD-specific T
cells unaffected. The same explanation applies to RCTD-reactive T
cell repertoire in RCTD-tolerized rats: in this case, the BCTDspecific T cells escaped tolerance induction. The presence in the
repertoire of T cells that are specific for BCTD only or RCTD only
demonstrates the unique identity of the C-terminal epitopes of foreign vs self hsp65 despite cross-reactivity between these epitopes.
However, the fine specificity of the T cell repertoire directed
against individual component peptides of BCTD or RCTD remains
to be determined.
The protective efficacy against AA of individual components of
cryptic BCTD and dominant RCTD
To further examine the functional significance in AA of the T cell
repertoire directed against individual peptides representing C-terminal determinants of Bhsp65 and Rhsp65, subgroups of Lewis
rats were pretreated with individual peptides of BCTD and RCTD
separately. The control rats were challenged with PBS/DDA. (In
previous studies in the AA model by others (10) and us (4), Lewis
rats immunized with PBS/DDA before Mtb injection were found
to serve as a reliable control group, comparable in their disease
course to rats challenged with an irrelevant Ag before disease induction.) Four weeks later, these rats were challenged with Mtb for
induction of AA, and then observed regularly for clinical signs of
disease. The results show that certain individual C-terminal peptides of both Bhsp65 (Fig. 8A) and Rhsp65 (Fig. 8B) could induce
protection against AA. On the basis of their protective efficacy
against AA, each set of these C-terminal peptides of Bhsp65 and
Rhsp65 could be separated into two groups: three of five peptides
each of BCTD (417– 431, 465– 479, and 521–535) and RCTD
(441– 455, 465– 479, and 521–535) induced a significant ( p ⬍
0.05) protection against AA compared with the control rats. Interestingly, two of these peptides (namely, 465– 479 and 521–535)
constituting BCTD and RCTD each are in the same region of the
respective native protein. These results demonstrate that the T cell
repertoire against both cryptic BCTD and dominant RCTD is of
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FIGURE 2. BCTD are immunogenic as peptides and represent cryptic
determinants within native Bhsp65. Lewis rats were injected s.c. at the base
of the tail either with native Bhsp65/IFA (n ⫽ 3) (A) or BCTD/IFA (n ⫽
4) (B). On day 9, the draining LNC of these rats were tested in a proliferation assay using native Bhsp65 and/or BCTD as recall Ags. HEL served
as a negative control. (The Ags used for both immunization and in vitro
recall in this and the following experiments were tested at concentrations
that were found to be optimal in pilot experiments.) In A, the cpm value for
each of the five individual BCTD peptides tested was comparable to that of
the medium background, and therefore, none of these peptides gave an S.I.
value above 1.0 (data not shown). The cpm values for medium background
were 12,479 (A) and 1,799 (B). S.I. for Con A were 31 (A) and 372 (B),
respectively. Similar results were obtained in repeat experiments.
The Journal of Immunology
185
FIGURE 5. Native Rhsp65 can prime T cells that are cross-reactive
with BCTD. Lewis rats were immunized s.c. with either native Rhsp65/IFA
(n ⫽ 3) or Bhsp65/IFA (n ⫽ 3). On day 9, the draining LNC of these rats
were tested in a proliferation assay using the indicated Ags. The results are
shown as an S.I. (mean ⫾ SEM). The cpm value for medium background
was 1,560 for Rhsp65-immunized and 12,479 for Bhsp65-immunized
groups. The cpm values were 104,918 for native Rhsp65 and 176,687 for
Bhsp65.
functional significance in AA, and further highlight the physiologic relevance of the T cell repertoire shared between BCTD and
RCTD in diversification of T cell response to Bhsp65 as well as in
regulation of AA.
Discussion
We observed that Lewis rats immunized with native Bhsp65 failed
to raise a T cell response to BCTD; however, the peptides comprising BCTD were immunogenic and induced a potent T cell
response. Furthermore, adoptive transfer into naive Lewis rats of T
cells primed by BCTD could afford significant protection to the
recipients from subsequent induction of AA. Therefore, the T cell
repertoire potentially directed against BCTD is available in Lewis
rats, but it can be stimulated by preprocessed (peptide) form of
BCTD, but not by native Bhsp65. In this regard, BCTD are cryptic
in nature. At this time, we do not know the precise reasons for
crypticity of BCTD, which may include one or more of the following: relatively poor binding to MHC molecules of these
epitopes (22), hindrance in the interaction of the epitopes with the
MHC or the TCR by amino acid residues flanking the core determinant (23), destruction of the epitope owing to presence of a
proteolytic cleavage site within the core epitope (24, 25), the influence of the HLA-DM (H-2M) molecule on determinant selection and presentation (26, 27), or a low frequency of T cells reactive against these epitopes (28), etc. Contrary to the crypticity of
BCTD within Bhsp65, the corresponding determinants within the
C-terminal region of Rhsp65 (RCTD) are dominant. Thus, identical epitope regions within a pair of self and foreign homologs
display differential determinant hierarchy (crypticity/dominance)
(22, 29, 30).
Importantly, the T cells directed against BCTD can be restimulated by the naturally generated self epitopes (presumably RCTD)
within both exogenously administered and endogenous (within
heat-stressed APCs) self hsp65. Moreover, the T cells primed by
peptides comprising RCTD can be restimulated by peptides constituting BCTD and vice versa (two-way cross-reactivity). These
results demonstrate that the T cell repertoire potentially reactive
against cryptic determinants within a foreign Ag (Bhsp65) could
be engaged and primed by the corresponding dominant determinants of the self homolog (Rhsp65). Molecular mimicry between
microbial and host Ags has been proposed as a mechanism for the
initiation of autoimmunity in several animal models as well as in
humans (31–35). In contrast, our results suggest that cross-reactivity between homologous foreign and self hsp65 plays a role in
diversification of T cell response to disease-regulating epitopes of
foreign hsp65 during the course of AA, and in regulation of autoimmune arthritis. Thus, defining the T cell repertoire against a
disease-related Ag is critical for better understanding of both the
induction as well as regulation of autoimmunity (36 –38).
The results of our neonatal tolerance experiments showed that
the T cell repertoire directed against BCTD and RCTD each is
comprised of a shared cross-reactive component and a specific
noncross-reactive subset of T cells. In this context, we suggest that
the observed response to BCTD in the late phase of AA (epitope
FIGURE 6. Heat-stressed naive APCs can restimulate BCTD-primed T
cells of the Lewis rat. LNC (5 ⫻ 105 cells/well) derived from Lewis rats 8
days after challenge s.c. with BCTD/IFA were cocultured with either naive
heat-stressed (experimental) or unstressed (control) APCs (macrophages
and dendritic cells) (2.5 ⫻ 105 cells/well each) in a proliferation assay
without addition of any exogenous Ag. LNC of naive Lewis rats and those
from HEL-immunized Lewis rats cultured separately with heat-stressed or
unstressed APCs served as controls. The results are expressed as an S.I.
(cpm with heat-stressed APCs ⫼ cpm with unstressed APCs) (mean ⫾
SEM). The results of a representative experiment are shown. Similar results were obtained in repeat experiments. The difference between BCTD
vs HEL (or naive) groups was statistically significant (ⴱ, p ⬍ 0.005). However, HEL-primed LNC gave a vigorous proliferative response when cultured in presence of HEL, indicating optimal in vivo priming of Ag-specific
T cells (data not shown).
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FIGURE 4. The T cells directed against BCTD are cross-reactive with
RCTD and vice versa. Lewis rats were challenged s.c. with either BCTD/
IFA (n ⫽ 5) (A) or RCTD/IFA (n ⫽ 4) (B). After 8 days, the draining LNC
of these rats were tested in a proliferation assay using a panel of recall Ags.
The results are shown as an S.I. (mean ⫾ SEM). The cpm values for
medium background were 1799 (A) and 1639 (B). The S.I. for Con A were
372 (A) and 505 (B).
186
REGULATION OF AUTOIMMUNE ARTHRITIS BY CRYPTIC EPITOPES
spreading) (4) could arise by at least three pathways: 1) up-regulation of the previously cryptic BCTD under the inflammatory environment of acute AA and activation of BCTD-specific T cells as
well as T cells cross-reactive with self hsp65; 2) enhanced cellular
expression of endogenous Rhsp65 under the inflammatory conditions during the acute phase of the disease leading to priming
(around the peak or late phase of AA) of T cells specific for RCTD
as well as T cells that are cross-reactive with BCTD; the latter
subset of T cells can be restimulated in vivo by the up-regulated
BCTD; and 3) restimulation of the cross-reactive subset of BCTDprimed T cells by endogenous self hsp65. In addition, Abs to
Bhsp65 (5), and one or more of the mechanisms underlying
epitope spreading in other models of autoimmunity (13, 14, 39 –
41) might also contribute to induction of T cell response to BCTD
during AA. The spontaneous or deliberate priming of T cell response to previously cryptic/subdominant antigenic determinants
(22, 30) has generally been associated with induction and/or propagation of autoimmunity (pathogenic cryptic/subdominant
epitopes) (13, 14, 42– 45). However, our results described above
show that T cell responses to cryptic BCTD are AA protective in
nature (regulatory cryptic epitopes) (4, 46). Nevertheless, using the
appropriate antigenic challenge regimen (dose and form of the Ag,
the type of adjuvant, and the route of immunization, etc.), downmodulation of an autoimmune disease has been achieved by using
either pathogenic (14, 44, 47) or regulatory (4) cryptic/subdominant epitopes of the appropriate Ag.
We observed that T cells primed by BCTD could adoptively
transfer protection against subsequent AA. The control Mtb-immunized Lewis rats (5– 6 wk) displayed disease characteristics
(such as the mean peak severity and the duration of the disease)
that were somewhat similar to that observed in another laboratory
(10), but were less pronounced than that described by other investigators (5, 48); moreover, the severity of AA was more in younger
(5– 6 wk) Lewis rats compared with slightly older (10 –11 wk) rats
FIGURE 8. The protective efficacy against AA of individual peptides of
BCTD/RCTD. Lewis rats (n ⫽ 8 –17) were immunized intradermally twice
at 1-wk interval with an individual peptide of either BCTD (A) or RCTD
(B), each emulsified in GERBU adjuvant 100, followed 4 wk later by
challenge s.c. with Mtb. Control rats received PBS/DDA before Mtb challenge. Thereafter, all rats were observed and scored for clinical signs of
AA. The arthritic score (mean ⫾ SEM) of the AA-protected Lewis rats was
significantly (p ⬍ 0.05) reduced for rats preimmunized with peptide 417–
431, 465– 479, or 521–535 of Bhsp65 (with a prefix B) (A), and peptide
441– 455, 465– 479, or 521–535 of Rhsp65 (having a prefix R) (B) compared with controls, as analyzed using the nonparametric Wilcoxon rank
sum test and Student’s t test.
(data not shown). The differences in disease severity mentioned
above could be attributed to one or more of the following factors:
the age and sex of Lewis rats, the dose (0.5–2 mg/rat) of Mtb used,
the route (s.c. or intradermal) of injection of Mtb, the housing
environment (conventional vs pathogen free), and presumably inherent differences in susceptibility to AA of Lewis rats from different vendors. A variation in disease susceptibility in different
animal colonies has already been reported for experimental autoimmune encephalomyelitis in the Lewis rat (49) and type I diabetes in the nonobese diabetic mouse (50).
Our results demonstrate that certain individual peptide components of both the cryptic C-terminal epitopes of Bhsp65 and the
corresponding dominant epitopes of Rhsp65 are disease regulating
in nature. We suggest that T cell responses to BCTD/RCTD are
also involved in natural remission from acute AA. However, it is
likely that T cells specific for other regulatory epitopes of Bhsp65
(10) and mammalian self hsp65 (48, 51–54), including those
against not yet identified epitopes within these proteins, that might
be primed or restimulated following immunization with Mtb (5,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. Neonatal tolerization reveals the fine specificity of the T
cell repertoire directed against BCTD/RCTD. Newborn Lewis pups were
injected twice (i.p.) with either BCTD/IFA (A) or RCTD/IFA (B) within 24
and 72 h after birth. After 4 wk, these rats were injected s.c. with either
BCTD/IFA or RCTD/IFA. On day 9, the draining LNC were harvested and
tested in a proliferation assay using the indicated Ags. HEL was used as an
irrelevant recall Ag control, whereas Con A served as a positive control.
The results are expressed as an S.I. (mean ⫾ SEM). The S.I. values for Con
A stimulation of BCTD- and RCTD-primed cells were 47.2 and 52.6 (A),
and 69.9 and 61.2 (B), respectively.
The Journal of Immunology
Acknowledgments
We thank Jan Cerny, Martin Flajnik, Donna Farber, Gregg Hadley, and
Noel Rose for critical reading of this manuscript.
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