In our previous studies, we demonstrated that both monomeric DR2-derived and I-As
-derived RTLs could protect and treat the clinical and histological signs of EAE in DR2 Tg mice (39
) and SJL mice (40
), respectively. In the current study, we used a similar approach, constructing monomeric I-Aq
-derived RTLs covalently linked with bCII257–270 peptide (RTL2001) (US patent no. 6,270,772). Biochemical and biophysical characterization indicated the recombinant molecule retained structural integrity. In our initial animal study, RTL2001 did not appear to protect the animals from CIA (data not shown). Previous studies (49
) suggested that the side chains of isoleucine at position 260 and phenylalanine at position 263 of the bCII257–270 peptide are the important P1 and P4 anchor residues for binding to the I-Aq
molecule. Lysine 264 (K264) appears to be the major T cell recognition site of CII257–273 and CIA is in particular associated with recognition of K264 after posttranslational hydroxylation and subsequent attachment of a β
-D-galactopyranosyl moiety (31
). A number of publications indicated that the posttranslational modification of arthritogenic peptide bCII257–270 may be required to induce a protective or suppressive effect. Glycosylation of the key TCR contacting residue K264 within the bCII257–270 peptide resulted in a reduction of Ab response to arthritogenic peptide and a decrease in T cell response to the glycopeptides (45
). We have not yet overcome the technical hurdles required for engineering a recombinant molecule with a glycosylation at only this single amino acid residue. A previous study showed that substituting a few amino acid residues on both the amino terminus (position 257) and carboxy terminus (positions 268 and 269) of the determinant core peptide (bCII260–267) could significantly alter Ag-specific T cell responses (49
). Moreover, a recent study involved in the TCR specificity suggested that certain amino acid side chains on the peptide/MHC surface can actually ‘disrupt’ the TCR-peptide-MHC interaction, even though the side chains are not involved in binding to the TCR (52
). Thus, we modified RTL2001 by substituting four amino acid residues (E257A, G265A, G268A, and P269A) within the arthritogenic bCII257–270 peptide (termed RTL2001MII). Our results showed that the modified murine I-Aq
-derived RTL, RTL2001MII, could protect the animals from CIA. The results strongly support our hypothesis that regulating the context in which the complex of peptide/MHC interacts with the TCR could control the fate of the pathogenic T cells.
In the current study, the pretreatment with murine versions of RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term immunoregulation against arthritogenic Ags without need for further boosting. The ability of RTL2001MII to suppress arthritis appears to be correlated with a change in the cytokine response both systemically ( and ) and locally () and a reduction in infiltrating pathogenic cells in synovial joint tissue (). The dramatic reduction of infiltrating pathogenic cells in the local joint tissue at day 70 post immunization is the most significant change resulting from RTL treatment, and may explain the corresponding reduction in expression of proinflammatory cytokines. Our previous EAE study in SJL mice showed that treatment with I-As
-derived RTL covalently linked to PLP139–151 peptide reduced the expression of adhesion molecules including the very late activation Ag-4 (VLA-4; CD49d) and lymphocyte function-associated Ag-1 (LFA-1; CD11a) in the CNS (40
). These results suggest that the RTL treatment affected the disease systemically and also at the local level. Long-term protection against arthritogenic Ags supports the clinical application of this novel class of peptide/MHC class II construct in patients with RA.
In the CIA model, CD4+ T cells are initially activated by presentation of disease-associated CII Ags by I-Aq class II molecules, with concomitant activation of B cells that later will play an important role in the course of the disease by producing CII-specific autoantibodies. We thus examined the effect of the RTLs on the bCII-specific Ab response. Our results demonstrated that the RTL2001MII treatment significantly increased the bCII-specific IgG1 isotype and reduced the IgG2a isotype that is usually associated with a pathogenic response. However, the reduction of IgG2a was minimal, which may in part explain the residual disease score and 22% incidence of CIA in the RTL2001MII treated group. An approach that could specifically target Ag-specific B cells or plasma cells may be needed in combination with the RTLs for successful treatment of RA.
Using peptide/MHC complex as a direct approach toward Ag-driven immunosuppression has been studied previously. It has been demonstrated that partial activation of pathogenic T cells through the Ag-specific TCR without costimulators could induce anergy (34
) or apoptosis (33
). In the current study, we produced monomeric murine I-Aq
-derived RTLs and our results suggested that treatment of CIA with I-Aq
-derived RTL linked to the CII peptide not only down-regulated two key proinflammatory cytokines, IL-17 and IFN-γ
, but also up-regulated anti-inflammatory cytokines, IL-10 and IL-13, in the spleens of DBA/1LacJ mice. Interestingly, we previously reported that the treatment of EAE with monomeric I-As
-derived RTL covalently linked to PLP139–151 peptide also resulted in a switch from proinflammatory to anti-inflammatory cytokines and reduced expression of the adhesion molecules, VLA-4 and LFA-1, in the CNS (40
). In both studies, these two different murine monomeric RTLs did not significantly reduce T cell proliferation responses to bCII257–273 or PLP139–151, respectively (data not shown). These data suggest that systemic shifting the cytokine profile may be an important regulatory mechanism of these monomeric RTLs. It may be that reduction in IL-17 and IFN-γ
and induction of anti-inflammatory cytokines, IL-10 and IL-13, are of particular importance because these changes were observed only after pre-treatment with the protective RTL2001MII. In contrast, both RTL2001MII and the “empty” RTL2000 reduced expression of the proinflammatory cytokines IL-1β
and IL-6, thus suggesting that these changes were not directly related to protection against CIA.
In the joint tissue, treatment with RTL2001MII but not RTL2000 resulted in a significant reduction in the expression of three key proinflammatory cytokines, IL-1β, IL-6, and IL-23. These selective changes thus may have contributed to reduced disease scores and pathology in the joints. In contrast, both RTL2000 and RTL2001MII induced lower expression of TNF-α, although the inhibition of TNF-α by RTL2001MII was much more pronounced than for RTL2000. It is noteworthy that the pattern of inhibition mediated by treatment with RTL2001MII differed systemically vs locally in the joint. This discrepancy could be due to regional differences or perhaps might reflect a disparity between protein secretion (measured in spleen cell cultures stimulated with CII peptide) vs mRNA expression in whole joint tissue.
Recently, numerous publications suggested that the IL-23/IL-17 axis is “evil” in organ-specific autoimmune diseases (17
). It has been demonstrated that IL-12 primarily acts on naive T cells and plays a critical role in determining the differentiation and generation of Th1 cell populations, whereas IL-23 preferentially acts on memory T cells and is an essential cytokine for the development of organ specific inflammatory autoimmune disease such as EAE (54
) and CIA (55
). Production of IL-23 is an important pathway that drives a subset of the highly pathogenic CD4+
T cell population (termed the Th17 subset) to produce IL-17A, IL-17F, and TNF-α
, but not IFN-γ
or IL-4. Both IL-17A and IL-17F are members of the IL-17 family, which are homodimeric cytokines and play an important role in the regulation of autoimmunity (53
). A recent study showed that IL-17 enhanced osteoclastogenesis by inducing the receptor activator of NF-κ
B (RANKL) on mesenchymal cells (17
). Moreover, both IL-1β
and IL-6 have been suggested to promote the development of the Th17 subset (61
). Clearly, the reduction in IL-1β
, IL-6, and IL-23 could have profound effects on joint inflammation and bone destruction in CIA. These results, in combination with RTL-induced systemic inhibition of IL-17 and IFN-γ
and up-regulation of IL-10 and IL-13 suggest that long-term tolerance against arthritogenic Ags may be mediated through a “cytokine switch” mechanism.
Expression of the FoxP3
gene in splenocytes was significantly increased in the RTL2001MII treated group vs the controls or “empty” RTL2000 treated mice. These data suggest that RTL treatment may up-regulate the Treg population, although we were unable to detect an increase in the number of FOXP3+
Treg cells in spleen. Tregs are critical regulators of immune tolerance (63
), and their suppressive control of effector T cells was observed in both experimental systems (63
) and humans (64
). Tregs are defined by their function, and express the transcription factor FoxP3
and/or suppressive cytokines (IL-10 and TGF-β
). The process for induction of FoxP3
gene expression is still unclear. In vitro studies suggested that the expression of FoxP3
was inducible, and a suboptimal stimulation by anti-CD3/CD28 Abs could activate the expression of FoxP3
). Moreover, a recent study demonstrated that de novo generation of Ag-specific CD4+
regulatory T cells from human CD4+
cells could be achieved through an Ag specific TCR with a cognate peptide/MHC complex (70
). We believe that the RTLs may be capable of providing such a suboptimal signal as suggested by our previous study in which the RTLs led to a partial calcium signal and transient T cell signaling (47
). We postulate that when a peptide/MHC class II complex interacts with cognate TCR in the absence of costimulators, such engagement could result in a suboptimal signal in the T cell that could trigger FoxP3
In conclusion, the current study demonstrates that the murine I-Aq-derived RTLs could reduce the incidence of the disease, suppress the clinical and histological signs of CIA and induce long-term clinical benefits. These effects on CIA appeared to be mediated by a cytokine switch mechanism involving a systemic increase of anti-inflammatory factors, IL-10, IL-13, and FoxP3, coupled with both systemic and local reduction of proinflammatory factors. Additionally, RTL treatment resulted in increased IgG1 and decreased IgG2a specific for bCII. This is the first report demonstrating effective treatment of joint inflammation and clinical signs of CIA with I-Aq-derived RTLs, thus supporting its possible clinical use for treating rheumatoid arthritis in humans. We believe that the general strategy developed in this study may have application to other organ specific autoimmune diseases with risk associated MHC class II alleles for which potential T cell target Ags are known or suspected.