Importance of spliceosomal RNP1 motif for intermolecular T-B cell spreading and tolerance restoration in lupus
© Monneaux et al.; licensee BioMed Central Ltd. 2007
Received: 05 July 2007
Accepted: 26 October 2007
Published: 26 October 2007
We previously demonstrated the importance of the RNP1 motif-bearing region 131–151 of the U1-70K spliceosomal protein in the intramolecular T-B spreading that occurs in MRL/lpr lupus mice. Here, we analyze the involvement of RNP1 motif in the development and prevention of naturally-occurring intermolecular T-B cell diversification. We found that MRL/lpr peripheral blood lymphocytes proliferated in response to peptides containing or corresponding exactly to the RNP1 motif of spliceosomal U1-70K, U1-A and hnRNP-A2 proteins. We also demonstrated that rabbit antibodies to peptide 131–151 cross-reacted with U1-70K, U1-A and hnRNP-A2 RNP1-peptides. These antibodies recognized the U1-70K and U1-A proteins, and also U1-C and SmD1 proteins, which are devoid of RNP1 motif. Repeated administration of phosphorylated peptide P140 into MRL/lpr mice abolished T-cell response to several peptides from the U1-70K, U1-A and SmD1 proteins without affecting antibody and T-cell responses to foreign (viral) antigen in treated mice challenged with infectious virus. These results emphasized the importance of the dominant RNP1 region, which seems to be central in the activation cascade of B and T cells reacting with spliceosomal RNP1+ and RNP1- spliceosomal proteins. The tolerogenic peptide P140, which is recognized by lupus patients' CD4+ T cells and known to protect MRL/lpr mice, is able to thwart emergence of intermolecular T-cell spreading in treated animals.
Longitudinal studies of spontaneously lupus-prone inbred mouse strains and patients with systemic lupus erythematosus (SLE) consistently show an ordered appearance of typical auto-antibodies in the serum of individuals [1–4]. With time the fine specificity of the antibody response initially focused against one or few autoepitopes diversifies to other epitopes of the same protein (intramolecular spreading) and to other components that are physically associated within the same antigenic macromolecular particles, such as nucleosome, spliceosome, and Ro particle (intermolecular spreading). Epitope spreading is thus a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become major targets of an ongoing immune response. This phenomenon is not limited to autoimmunity; it has also been described in experimental and natural situations as a consequence of acute or persistent infection. Although the concept of epitope spreading was introduced more than 15 years ago , the cellular components that catalyze the spreading hierarchy have not been well defined, and certain aspects of this process remain unexplained. Recent studies suggest that autoreactive B cells are important cellular mediators contributing to autoreactive T-cell response diversification via their functions that mediate antigen processing and presentation [6, 7].
Previous work from our laboratory demonstrated that peptide 131–151 of the spliceosomal U1-70K protein as well as a peptide analogue containing a phosphoserine residue at position 140 (peptide P140) are recognized by CD4+ T cells from lupus mice. Both peptides were shown to behave as promiscuous epitopes and bind a large panel of murine and human MHC class II molecules [8–11]. Administration into young MRL/lpr lupus-prone mice of P140 peptide in saline, but not of the non-phosphorylated peptide 131–151, led to a dramatic amelioration of the clinical and biological manifestations of treated animals and significantly prolonged their survival . The peptide P140 administrated in Freund's adjuvant (FA) accelerated the renal disease in MRL/lpr mice . Our studies revealed further that peripheral CD4+ T cells from lupus patients, but not from patients with other autoimmune diseases (such as rheumatoid arthritis, primary Sjögren's syndrome, autoimmune deafness, polymyositis, primary billiary cirrhosis and autoimmune hepatitis) or infectious diseases, very specifically recognized the 131–151 and P140 peptides, and that phosphorylation of Ser140 prevented ex vivo proliferation of lupus patients' CD4+ T cells but not secretion of high levels of regulatory cytokines .
The 131–151 sequence of the spliceosomal U1-70K protein is located within a 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 small nuclear (sn)RNP (such as U1-A) and heterogeneous nuclear (hn)RNP (such as 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 that contain 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 [12, 13]. We demonstrated that an intramolecular T and B cell spreading effectively occurs in MRL/lpr mice tested at different ages and emphasized the importance of the RNP1 region in the cascade of events observed in the murine lupus response . In the present study, we investigated the potential capacity of peptide 131–151 to generate antibodies that react with spliceosomal peptides and proteins encompassing, or not, the RNP1 motif. We tested the reactivity of peripheral blood lymphocytes (PBLs) from unprimed lupus mice to react with RNP1-containing peptides from several spliceosomal proteins. We also examined whether administration of protecting P140 peptide into MRL/lpr mice could modulate intermolecular epitope spreading in vivo by preventing T-cell response to peptides from heterologous spliceosomal proteins that do or do not contain an RNP1 motif. We provide data showing that P140 peptide treatment efficiently suppresses T-cell responses to other, unrelated spliceosomal epitopes, in addition to the tolerizing peptide. Remarkably, however, the B and T cell immune response to foreign antigens (such as viral particles) remains unaffected in treated animals.
Materials and methods
Peptides and proteins
On days 1, 21 and 42, outbred Fauves de Bourgogne female rabbits (Dombes Romans, Chatillon, France) were injected subcutaneously (sc) with peptide 131–151 of the U1-70K protein (100 μg/rabbit/injection) emulsified in complete FA (CFA) for the first injection and incomplete FA for the subsequent injections. Rabbits were bled before immunization and then regularly throughout the protocol.
ELISA and Western immunoblotting
Standard ELISA procedures with peptide 131–151 (2 μM in 0.05 M carbonate buffer, pH 9.6) or dsDNA (100 ng/ml in 25 mM citrate buffer, pH 5.4) directly coated onto polyvinyl microtiter plates (Falcon, Oxnard, California, USA), and goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch Laboratories, West Groves, Pennsylvania) diluted 1:50,000 in phosphate-buffered saline containing 0.05% Tween (PBS-T) were used to measure antibody reactivity in rabbit antisera . The final reaction was visualized with H2O2 and 3,3',5,5'-tetramethyl benzidine used as chromogen, and absorbance was measured at 450 nm. For competitive ELISA, increasing amounts of inhibitor peptides were first incubated for 1 h at 37°C and then overnight at 4°C with constant dilutions of rabbit antisera in PBS-T. The mixtures were subsequently transferred to polyvinyl Falcon plates pre-coated with peptide 131–151, and post-coated with bovine serum albumin in PBS-T (0.4% w/v). After a 1 h incubation at 37°C and washing, bound antibodies were detected as indicated above. Preliminary experiments were conducted in a direct format to define the working dilution of rabbit antiserum. For Western immunoblotting, U1-A, U1-C, U1-70K, SmBB' and SmD1 proteins were first subjected to electrophoresis in 12.5% SDS-polyacrylamide gels and then transferred to nitrocellulose. The blotted strips were saturated in Tris-buffered saline, pH 7.5, containing 0.5% Tween (TBS-T) and 5% milk for 1 h at room temperature (RT), and then incubated with rabbit antisera diluted 1:500 in TBS-T-milk for 1 h at RT. After washing, strips were incubated with HRP-conjugated second antibodies to rabbit IgG (1:5,000 in TBS-T). Enhanced chemiluminescent (ECL™) reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK) were used to reveal positive reactions.
To study the natural T-cell reactivity occurring in unprimed lupus-prone mice, MRL/lpr mice were bled at weeks 8, 10, 12 and 14, and lymphocytes were purified by density separation (Lympholyte-M, d = 1.0875; Cedarlane, Hornby, Canada). PBLs were collected, washed three times, and resuspended at 3 × 106 cells/ml in L-alanyl-L-glutamine-enriched RPMI 1640 medium (Cambrex, Verviers, Belgium) containing 10% fetal calf serum (FCS; Dutscher, Brumath, France), HEPES, gentamycine, and β-mercaptoethanol. The proliferative response to peptides was measured in duplicate using 3 × 105 cells/well and a single peptide concentration (100 μM). After 72 h, the cultures were pulsed for 18 h with [3H]-thymidine (6.7 Ci/mmol; 1μCi/well) and DNA-incorporated radioactivity was measured using a Matrix 9600 direct beta counter (Packard, Meriden, Connecticut, USA; cpm range from 100 to 30,000). The SD of duplicate cultures was always below 20% of the mean. Control tests were performed by adding Con-A (100 μl/well; 5 μg/ml) to cells during the time of the culture (90 h).
To analyze the effect of P140 peptide administration on spontaneous T cell spreading occurring in lupus-prone mice, MRL/lpr mice received peptide P140 (100 μg in saline/mouse/injection; 10 mice) or PBS alone (10 mice) intravenously (iv) at weeks 5, 7 and 9 . Treated mice were bled at week 10, and peptides of interest (100 μM in the cultures) were tested for their ability to induce proliferation of PBLs, as described above.
To examine the possible effect of P140 treatment on the ability of mice to mount a normal immune reaction after viral infection, P140-treated MRL/lpr mice (18-week-old at the time of the experiment) were challenged intranasally (in) with A/NT/60/68 influenza virus (H3N2 strain) in allantoic fluid using a dose predetermined to provoke ~30% of weight loss . The required dose was of the order of 40 μl of undiluted allantoic fluid (HA titer 1260) per mouse. As a control, a group of untreated MRL/lpr mice was challenged in parallel. Mice were followed daily for their body weight. The capacity of PBLs from challenged mice to proliferate and secrete INF-γ ex vivo in the presence of increasing concentrations of the HA peptide 307–319 (PKYVKQNTLKLAT ) representing a promiscuous T-helper epitope from influenza virus HA, was evaluated 13 days after challenge, as described above. The anti-virus antibody production in the serum from challenged mice was measured in ELISA  28 days after challenge.
All animal experiments were performed with the approval of the local Institutional Animal Care and Use Committee (CREMEAS).
Analysis for statistically significant differences was performed with Student's t-test. P values < 0.05 were considered significant.
Recognition by T cells from unprimed MRL/lpr mice of peptides containing or corresponding exactly to the RNP1 motif
In order to determine whether MRL/lpr T cells precisely recognized the RNP1 motif present in the respective spliceosomal proteins, we synthesized three additional peptides corresponding exactly to the RNP1 motifs of the U1-70K, U1-A and hnRNP-A2 proteins, called RNP1-70K, RNP1-U1A and RNP1-A2, respectively (Figure 1a), and tested, as above, the ability of these peptides to stimulate the proliferation of PBLs from 8-14-wk-old MRL/lpr mice (Figure 2b). No or very weak proliferation was observed in response to the RNP1-70K peptide 139–151 at any age. A modest but significant proliferative response with RNP1-A2 peptide 45–57 was measurable with PBLs from 10, 12 and 14-wk-old MRL/lpr mice. In contrast, as early as 8 weeks of age, and at least until 14 weeks, a strong proliferative response was observed when MRL/lpr PBLs were cultured in the presence of the 13-residue-long RNP1-U1A peptide 47–59 (Figure 2b; maximum at week 12). This response was inhibited in the presence of neutralizing anti-CD4 monoclonal antibody GK1.5 (10 μg/ml; not shown) indicating that the main cell population reacting with the RNP1-U1A peptide 47–59 does correspond to CD4+ T cells.
Reactivity of rabbit antibodies to peptide 131–151 of the U1-70K protein with RNP1-peptide
Antibodies generated in outbred rabbits to peptide 131–151 of the U1-70K protein react with the cognate protein and with other spliceosomal proteins
We then questioned whether antibodies raised to peptide 131–151 are able to recognize the whole cognate protein and other proteins present in the same spliceosomal particle (Figure 3b). Ponceau red staining of membranes blotted with the three recombinant proteins U1-70K, U1-A, and U1-C and with the long synthetic fragment 21–119 of SmD1 revealed a single band at 65 kD, 35 kD, 20 kD and 12 kD, respectively, as determined with MW markers (Figure 3b, lanes 1–4). IgG antibodies from the rabbit immunized against peptide 131–151 in CFA (see above) strongly reacted with the U1-70K (lane 5) and the U1-A (lane 6) proteins, as well as with the U1-C (lane 7) and SmD1 (lane 8) proteins, which do not contain any RNP1 motif, suggesting the establishment of a heterologous B-cell epitope spreading phenomenon, and not solely immune cross-reactivity. No reactivity was found with SmBB' protein (not shown) or with any of the five proteins when tested with the serum from an outbred rabbit that received several injections of CFA alone (lanes 9–12). This rabbit did not develop any IgG reactivity to dsDNA (as measured by ELISA), and no clinical sign of autoimmunity was measurable 8 weeks after the last of three peptide administrations (result confirmed in several outbred rabbits). New Zealand White rabbits were not used in this set of experiments because they have a natural tendency to produce auto-antibodies . A similar result was obtained with two outbred mice (ICR, Harlan) immunized with peptide 131–151, for which antisera react in Western immunoblot with the U1-70K protein and also with U1-A and U1-C proteins (data not shown).
Effect of P140 therapy on spontaneous T cell spreading
As U1-A and hnRNP-A2 peptides do contain an RNP1 motif, we could not rule out the possibility that the observed drop of responsiveness was simply due to the extinction of P140-specific T cells by a homologous-like, cross-reactive effect. To ensure that P140 treatment was leading to a true (non-cross-reactive) mechanism of 'intermolecular tolerance spreading', the goal was thus to demonstrate that T cell response to spliceosomal proteins that do not contain an RNP1 motif was also abolished in P140-treated MRL/lpr mice. U1-C and SmD1 proteins, which are associated to the U1-snRNP particle and are devoid of RNP1 motif, were appropriate candidates in this context. Epitopes recognized by CD4+ T cells are not known in U1-C protein. However, an epitope recognized by T cells from SLE patients and (NZBxNZW)F1 mice has been identified in the C-terminal end of SmD1 [22, 23]. This epitope is present in peptide 83–119, which encompasses the sequence 97–119 shown earlier in our laboratory to contain a B-cell epitope recognized by IgG antibodies from MRL/lpr and (NZBxNZW)F1 lupus mice and from patients with lupus [3, 10, 15]. In preliminary experiments with peptide dose-response measurements, we found that SmD1 peptide 97–119, but not SmD1 peptide 77–96, was efficiently recognized by MRL/lpr PBLs (not shown). With this new target, as above, we analyzed the reactivity of MRL/lpr PBLs (the peptide 77–96 was used as control) after P140 treatment. As shown in Figure 4, we observed that PBLs from P140-treated MRL/lpr mice did no longer proliferate ex vivo in response to SmD1 peptide 97–119 used as recall antigen in the culture (81% decrease; P = 0.0005).
A crucial question in our understanding of SLE is to identify the early cellular and molecular events that predispose to the loss of tolerance in individuals and those that trigger the transition from a preclinical status to overt disease and perpetuate the immune-mediated, inflammatory response. The concept of the T-B cell spreading phenomenon based on an initial acute autoimmune response restricted to a few epitopes using a limited number of T-cell clones for expansion is now widely accepted. However, the possible initiator antigens and the mechanism that render this(these) antigen(s) immunogenic in a predisposed context is not clear [1, 24–26]. It is also not clear what the link is that drives T-B cell diversification from these putative initial epitopes to a particular set of antigens. Our hypothesis is that the RNP1 motif, which is present in a number of RNA-binding proteins and represents a frequent target for B- and T-cell response, might be central in this process and partly explain autoimmunity to RNP proteins in lupus [12, 13].
In this model, the RNP1 motif initiates the spreading of the immune response to the whole protein (intramolecular spreading), then proceeds in an ordered manner to other proteins containing the RNP1 motif, and finally, to proteins that do not contain any RNP1 motif but are colocalized in the same particle (intermolecular spreading). In our previous work , we clearly demonstrated that the first step of this process effectively occurs in MRL/lpr lupus mice, in which the immune response diversifies from the RNP1 motif to the whole U1-70K protein (intramolecular spreading). In the present study, we validated the second step of our proposed model  by demonstrating that an immune response induced against the RNP1 motif can drive the diversification to other spliceosomal proteins that may or may not contain an RNP1 motif, such as U1A, hnRNP-A2, U1C and SmD1 proteins (intermolecular spreading).
Moreover, we not only confirmed that the sequence 131–151 of the U1-70K protein contains a recurrent epitope recognized by CD4+ T cells from MRL/lpr mice, but we also showed that the RNP1 motif present in other spliceosomal proteins can be recognized by MRL/lpr PBLs. Clearly, the RNP1 sequence 47RSLKMRGQAFVIF59 present within the U1-A protein induced a stronger proliferation of MRL/lpr PBLs compared to other RNP1 sequences, which were much less efficient and for some, very poor inducers. It has to be noted that the T cell epitopes of U1-A characterized previously by others [27–29] in MRL/lpr and (NZBxNZW)F1 mice and patients with SLE or mixed-connective tissue disease did not or only partially encompass the RNP1 motif. Epitopes recognized by autoreactive CD4+ T cells either in patients or in murine models of lupus or rheumatoid arthritis are not known in the case of the hnRNP-A2 protein.
The RNP1 motif is present in B-cell epitopes recognized in different proteins by antibodies from autoimmune mice and patients [; and references therein]. In the present study, we show that antibodies elicited after immunizing a rabbit with the peptide 131–151 of the U1-70K protein cross-react with the short RNP1 motif contained within the immunizing sequence but significantly less well with the RNP1 motifs of U1-A and hnRNP-A2 proteins, indicating that in solution these short peptides do adopt different conformations. In Western immunoblotting, antibodies from this immunized outbred rabbit reacted strongly with the cognate protein U1-70K and with U1-A, but also with U1-C and SmD1, which are both devoid of RNP1 motif. These results fit well with the previous findings from several groups, which showed similar intermolecular antibody diversification in longitudinal studies of lupus patients and mice [1, 30, 31]. However, in our rabbits immunized with the 131–151 peptide, we found no antibodies to dsDNA or chromatin, no antibodies to recombinant SmBB', and no clinical sign of autoimmunity. Immunization of experimental rabbits and mouse models with the proline-rich peptide PPPGMRPP, another key sequence, which is present in several spliceosomal proteins and was reported to trigger spreading in immunized animals [32, 33], gave contradictory results in independent studies regarding the presence or not of anti-DNA antibodies and signs of lupus-like autoimmunity [34–36]. To our knowledge, PPPGMRPP peptide administrated in lupus mice was never shown to possess protective or tolerogenic properties.
The reasons that underlie the successful treatment of MRL/lpr mice with peptide P140, which significantly retards the emergence of dsDNA IgG antibodies and the progression of nephritis and prolongs the survival of treated mice, are not yet fully elucidated. We showed here that P140 administration not only abolishes T cell intramolecular spreading to other regions of the cognate U1-70K protein, but also leads to an impressive unresponsiveness of PBLs reacting with RNP1-peptides or longer peptides containing the motif, and with a peptide from the C terminus of SmD1 protein, newly characterized in this study and originated from a spliceosomal protein that does not contain any RNP1 motif. Thus, in treated animals, P140 peptide elicits clearly a state of bystander suppression leading to the downregulation of autoreactive T- and B-cell response to other self-antigens (so-called tolerance spreading ). Important questions remain at this stage as to whether P140 peptide induces initial tolerance by playing the role of antagonist or partial agonist of the receptor of autoreactive T cells, or by using another mechanism, as previously discussed . The fact that P140-treated animals could mount a normal immune response to foreign antigens is not an unexpected result if we consider that P140 peptide primarily target the TCR of specific autoreactive T cells.
= enzyme-linked immunosorbent assay
= Freund's adjuvant
= peripheral blood lymphocyte
= systemic lupus erythematosus
This work was supported by CNRS and a grant from the Fondation pour la Recherche Médicale. VP was supported by a fellowship from the Association de Recherche sur la Polyarthrite (ARP). We thank M. Valette (Faculté de Médecine RTH Laennec, Lyon, France) for providing influenza virus (strain A/NT/60/68).
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