Importance of spliceosomal RNP1 motif for intermolecular T-B cell spreading and tolerance restoration in lupus

Abstract

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.

Figure 1

Synthetic peptides used in this study. (a) Peptides of U1-A, U1-70K, hnRNP-A2 and La proteins containing or corresponding to RNP1 motifs. The RNP1 motif (complete or partial) is highlighted by an empty box. In peptides corresponding exactly to the motif (gray boxes) amino acid similarities are in bold face. The serine¹⁴⁰ residue, which is phosphorylated in the P140 peptide is underlined. (b) Sequence of other peptides used in this study.

Figure 2

Spontaneous T-cell reactivity to peptides containing or corresponding to RNP1 motifs in unprimed MRL/lpr mice. The proliferative response of peripheral blood lymphocytes (PBLs) from 8-, 10-, 12- and 14-week-old mice was measured in the presence of U1-70K, U1-A and hnRNP-A2 peptides (100 μM) encompassing completely or partially the RNP1 motif of each protein (a) or corresponding exactly to the RNP1 motif of each protein (b). The results are expressed as SI corresponding to the ratio cpm in the culture with peptide to cpm in the culture without peptide. A mean SI > 2 was considered to be positive (horizontal line). The average tritiated thymidine incorporation in the absence of peptide and in the presence of Con-A was 50 and 3,000 cpm, respectively. This experiment is one of two individual experiments that showed similar results. Bars show the mean ± SEM.

Figure 3

Reactivity of rabbit antibodies directed to peptide 131–151 of the U1-70K protein with RNP1-peptides and spliceosomal proteins. (a) The antiserum was diluted 1/20,000 and incubated first for 1 h at 37°C and then overnight at 4°C with increasing amounts of different RNP1-peptides used as competitors in the fluid-phase. The homologous peptide 131–151 was used as control. The mixtures were then added to microtiter plates pre-coated with 2 μM of immunizing peptide 131–151. The results are expressed as the percentage of inhibition of the ELISA reaction measured without competitor peptide. (b) U1-70K, U1-A, U1-C, and SmD1 proteins were subjected to electrophoresis, transferred to nitrocellulose, and colored with Ponceau red (lanes 1–4). The blotted strips were then incubated with the serum (diluted 1/500) from rabbits that received either peptide 131–151 (lanes 5–8) or CFA alone (lanes 9–12). IgG antibodies only were tested. ECL reagents were used to reveal positive reactions.

Figure 4

Effect of a brief peptide P140 therapy on the spontaneous T cell spreading in MRL/lpr mice. Mice were either treated with peptide P140 (hatched bars) administrated intravenously in saline at weeks 5, 7 and 9 or received saline alone (solid bars). The proliferative response of PBLs was measured ex vivo at 10 weeks in the presence of selected peptides (100 μM) containing (P140, 35–54 U1-A, RNP1-U1A, 35–55 hnRNP-A2, RNP1-A2) or not (183–202 of the U1-70K protein, 97–119 SmD1) an RNP1 motif. A mean SI > 2 was considered to be positive (horizontal line). The average tritiated thymidine incorporation in the absence of peptide and in the presence of Con-A was 50 and 2,000 cpm, respectively. Bars show the mean ± SEM. Significant differences are indicated. ns: non-significant reduction.

Figure 5

Brief peptide P140 therapy does not affect immune responses to viral challenge in MRL/lpr mice. P140-treated (closed symbols) and untreated MRL/lpr mice (open symbols) (10 mice/group) were challenged intra-nasally with infectious influenza virus. (a) Thirteen days after viral challenge, the proliferative response of PBLs to increasing concentrations of HA peptide 307–319 was measured ex vivo. Results are expressed as SI. Bars show the mean ± SEM. The average tritiated thymidine incorporation in the absence of peptide and in the presence of Con-A was 50 and 4,000 cpm, respectively. (b) Weight loss pattern after intra-nasal viral challenge. The weight of MRL/lpr mice treated or untreated with P140 peptide was compared.

Figure 6

Model illustrating the possible role of RNP1 motif in the initiation of T-B cell spreading pathway (adapted from [13]). Epitopes containing the RNP1 motif (in dark blue) are presented to specific T cells that in turn activate B cells to produce anti-RNP1 antibodies (Abs). These B cells then bind and process the RNP1 epitope present within RNP1+ proteins, such as U1-70K (in blue) and U1-A (in green), but also the whole spliceosomal particle that contains RNP1-proteins, such as U1-C (in yellow) and SmD1 (in pink) proteins. This leads to the activation of Th and B cells and results in the production of diverse sets of auto-antibodies, which then deposit in tissues (IC, immune complexes) and trigger organ damage.