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

Abstract

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.

Regulation of autoimmune arthritis by the pro-inflammatory cytokine interferon-γ

“…Our results show that high levels of the pro-inflammatory cytokine IFN-γ are associated with recovery/resistance in AA, and that the IFN-γ induced by the diseaseregulating peptide 465-479 of self (rat) hsp65 (R465) [34] downmodulates AA in part by suppressing IL-17. These results warrant a revisitation to the mechanisms underlying the pathogenesis of AA, which is considered to be a typical Th1-mediated disease.…”

“…To avoid potential toxicity, we explored a more practical approach for immunomodulation than the use of IFN-γ per se, namely, using a peptide of Rhsp65, R465-479 (R465), which we have previously shown to downmodulate the course of AA [34]. However, in that earlier study, neither the ability of R465-primed lymphoid cells to transfer protection against AA nor the cytokine profile of R465-reactive cells was determined.…”

“…Prior to performing the adoptive transfer experiment, we independently validated the previously reported [34] AA-protective effect of the R465 peptide preparation used in this study (Fig. 4A).…”

“…Specific peptides containing the amino acid (a.a.) sequences of Bhsp65 and Rhsp65 were obtained from Macromolecular Resources and Global Peptide Services (both at Fort Collins, CO) [34,35]. These include Bhsp65 peptides 180-188 (B180), 177-191 (B177), 333-379 (B333); Rhsp65 peptide 465-479 (R465); and HEL peptide 65-78 (HEL65).…”

“…The recombinant Bhsp65 was expressed and purified in our laboratory as described in detail elsewhere [34,35]. Specific peptides containing the amino acid (a.a.) sequences of Bhsp65 and Rhsp65 were obtained from Macromolecular Resources and Global Peptide Services (both at Fort Collins, CO) [34,35].…”