By testing a series of overlapping peptides, we identified an epitope present in residues 131-151 of the spliceosomal U1-70K small nuclear ribonucleoprotein (snRNP), recognized very early by IgG antibodies and CD4+ LN T cells from both H-2k MRL/lpr and H-2d/z BW lupus-prone mice (Monneaux et al. 2000, 2001). Fibroblasts transfected with MHC class II molecules were used to demonstrate that peptides 131-151 readily binds I-Ak, I-Ek, I-Ad and I-Ed murine MHC molecules (Monneaux et al. 2000, 2001). We further showed that an analogue of this sequence phosphory-lated on Ser140 (named peptide P140) was strongly recognized by LN and peripheral CD4+ T cells and by IgG antibodies from MRL/lpr mice (Monneaux, 2003, 2004). This analogue and the cognate peptides 131-151 were used in therapeutic trials in lupus-prone mice to investigate their ability to restore tolerance. Young MRL/lpr mice were given the peptides i.v. in saline (4 X 100 |xg), and we found that P140 peptide, but not the non-phosphorylated peptide 131-151, reduced proteinuria and dsDNA IgG antibody levels and significantly enhanced the survival of treated mice (Monneaux et al. 2003). When administrated s.c. in Freund's adjuvant, P140 peptide accelerated lupus nephritis.
Our studies revealed that P140 peptide and the non-phosphorylated corresponding sequence behave as promiscuous epitopes (Monneaux et al. 2005). As in the mouse model, we found that peptides 131-151 of the U1-70K protein induces ex vivo proliferation of CD4+ T cells from lupus patients. Interestingly, however, we observed that phosphorylation of Ser140 prevented proliferation while favouring secretion of high levels of regulatory cytokines, which are produced specifically when lupus patients' peripheral CD4 T cells are incubated in the presence of P140 analogue (Monneaux et al. 2005).
The identification of a tolerogenic CD4+ T cell epitope within P140 peptide is remarkable, because this sequence, which is completely conserved in the mouse and human U1-70K protein, contains an RNA-binding motif called RNP1, also present in other sn/hnRNPs and often targeted by antibodies from lupus patients and mice. The 131-151 sequence of the spliceosomal U1-70K protein is located within an 80-90 amino acid-long RNA-binding domain. It encompasses a conserved sequence, called RNP1 motif, which is also present in other RNA-binding proteins, such as snRNP (e.g. U1-A) and heterogeneous nuclear (hn)RNP (e.g. hnRNP-A2/B1) proteins.
Starting from the observation that sequences containing this RNP1 motif are often targeted by antibodies from lupus patients and mice, we hypothesized that the RNP1 motif could be involved in the earliest stages of the T-B intramolecular diversification process to other regions of one of the spliceosomal proteins containing this unique motif and might promote intermolecular spreading to epitopes of other proteins present within the same spliceosomal particle and containing or not an RNP1 motif (Monneaux and Muller 2001, 2002). We experimentally demonstrated that an intramolecular T and B cell spreading effectively occurs in MRL/lpr mice tested at different ages (Monneaux et al. 2004). Moreover, we showed that repeated administration of phosphorylated analogue P140 in saline into pre-autoimmune MRL/lpr mice transiently abolishes both T cell intramolecular spreading to other regions of the U1-70K protein (Monneaux et al. 2004) and T cell intermolecular spreading to regions of other spliceosomal proteins, suggesting that the P140 analogue might originate a mechanism of so-called "tolerance spreading."
These results are extremely promising. From a conceptual point of view, however, we have to determine how in such a polymorphic and multifactorial pathology, administration of a single peptide (P140 peptide, histone peptides or peptides from other self-antigens) can be sufficient to suppress a complex autoimmune response to multiple cell components. Several possible pathways by which peptide P140 might exert a modulating effect are currently envisaged. P140 peptide could act as a partial agonist of the autoreactive T cell receptor, as suggested by our results obtained with lupus patients' T cells (Monneaux et al. 2005). It may also expand the regulatory T cell pool and/or restore defective regulatory T cell function as seen in the case of therapeutic histone peptides (see above). These and other possible mechanisms are presently under evaluation.
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