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The Journal of Immunology, 2004, 173: 181-188.
Copyright © 2004 by The American Association of Immunologists

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

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adjuvant-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, 4, 5, 6, 7, 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, 10, 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 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, 14, 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 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 C-terminal 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rats

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.

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 C-terminal 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 µl of this mixture emulsified in adjuvant (1:1; v/v) received 100 µg of each peptide in the mixture.



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  		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%).

 
Induction and evaluation of AA

Lewis rats were injected s.c. with 200 µl (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 µg/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 x 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 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 x 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 x 10–5 M 2-ME) in the presence of Con A (2.5 µg/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

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 x 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).

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 µg 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 µg/rat) and dimethyldistearoylhydroxyethyl ammonium chloride (DDA) (10 µg/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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 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 µg/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.



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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 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.



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  		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 x 107 BCTD-primed LNC ({blacksquare}, n = 3) (experimental recipient group) or PBS/IFA-primed LNC ({square}, 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.

 
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.



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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).

 
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 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.



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  		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.

 
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.



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  		FIGURE 6. Heat-stressed naive APCs can restimulate BCTD-primed T cells of the Lewis rat. LNC (5 x 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 x 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 (*, 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).

 
The BCTD-reactive T cell repertoire consists of shared cross-reactive and specific noncross-reactive subsets of T cells

We have described above that T cells against BCTD are cross-reactive 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 cross-reactive 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 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 BCTD-specific 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.



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  		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 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 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.



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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 32, 33, 34, 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, 37, 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 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 BCTD-primed 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, 40, 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, 43, 44, 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.), down-modulation 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 (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, 52, 53, 54), including those against not yet identified epitopes within these proteins, that might be primed or restimulated following immunization with Mtb (5, 48), might also contribute to natural regression of acute AA. Besides T cells against hsp65, Abs against Bhsp65 may also contribute to protection against AA (5, 55). Moreover, other mycobacterial and self hsp (e.g., hsp90, hsp70, hsp10) besides hsp65 may also contribute to regulation of acute AA (56, 57, 58, 59). Apparently, the regulation of AA is quite plastic and harnesses redundant antigenic systems (46), with contribution of one Ag/epitope reinforcing or compensating for the other missing component under different experimental conditions. This situation is reminiscent of regulation of diabetes in the nonobese diabetic mouse by different Ags/epitopes (47, 60, 61, 62, 63, 64, 65, 66).

In summary, the results of this study showing that the C-terminal epitopes of Bhsp65 are cryptic in nature, provide one explanation for the inability of the Lewis rats to raise T cell response to these regulatory epitopes early following disease induction (by Mtb injection). The acute inflammatory phase of AA provides the milieu for both up-regulation of previously cryptic BCTD and increased cellular expression of self hsp65, revealing its dominant epitopes (54) that are cross-reactive with BCTD, leading to the observed regulatory epitope spreading in the late phase of AA. Our results suggest that cryptic epitopes of arthritis-related Ags should also be considered and exploited for immunotherapy of autoimmune arthritis.


   Acknowledgments
 
We thank Jan Cerny, Martin Flajnik, Donna Farber, Gregg Hadley, and Noel Rose for critical reading of this manuscript.


   Footnotes
 
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). Back

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: kmoud001{at}umaryland.edu Back

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. Back

Received for publication January 20, 2004. Accepted for publication April 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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