Intramolecular T cell spreading in unprimed MRLlpr miceImportance of the U1-70k protein sequence 131151.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 10, October 2004, pp 3232–3238 DOI 10.1002/art.20510 © 2004, American College of Rheumatology Intramolecular T Cell Spreading in Unprimed MRL/lpr Mice Importance of the U1-70K Protein Sequence 131–151 Fanny Monneaux, Véronique Parietti, Jean-Paul Briand, and Sylviane Muller Conclusion. This is the first study to demonstrate that intramolecular T cell spreading effectively occurs in MRL/lpr mice with lupus, and that region 131–151 is important in the cascade of events observed in the murine lupus response. This sequence might originate a mechanism of tolerance spreading that leads to the beneficial effect observed in MRL/lpr mice after treatment with the phosphorylated peptide 131–151. Objective. To analyze spontaneous T cell spreading against determinants of the U1-70K protein in young autoimmune MRL/lpr lupus mice, in comparison with the T cell spreading occurring in normal BALB/c mice immunized with peptide 131–151 of this protein. Methods. Peripheral blood lymphocytes (PBLs) from both unprimed MRL/lpr mice and immunized BALB/c mice were tested for their ability to proliferate ex vivo in response to 18 overlapping peptides of the U1-70K spliceosomal protein, using assays for lymphocyte proliferation and secretion of interleukin-2. Results. The proliferative response to peptides of the U1-70K protein evolved rapidly in MRL/lpr mice tested at different ages. At least 5 peptides were recognized by PBLs from 8-week-old autoimmune mice, whereas a different peptide was recognized by PBLs from MRL/lpr mice at 12 weeks of age. At 15 weeks, the proliferative response was weak or negative when assessed with any of the test peptides. At least 2 major peptides recognized by MRL/lpr PBLs were also recognized by PBLs generated in the BALB/c mice primed with peptide 131–151. We further demonstrated that, in preautoimmune MRL/lpr mice, repeated administration of phosphorylated peptide 131–151 (called P140), which was shown previously to be protective, transiently abolished T cell intramolecular spreading to other regions of the 70K protein. Autoimmunity toward the different components of small nuclear RNP (snRNP) particles is typically observed in certain autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE), as well as in experimental models of lupus. Whereas the so-called anti–U1 RNP antibodies target the 70K, A, and C proteins, which are present specifically within the U1 snRNP complex, anti-Sm antibodies predominantly react with the Sm B/B⬘ and D common proteins, which are also present in the other spliceosomal snRNP particles (1). The autoimmune response to snRNP observed in human and murine lupus is complex in terms of its specificity, and to date, the precise mechanism leading to the broad diversity of antibody specificities is largely unknown. It is now widely believed that this diversification of the antibody response results from a typical phenomenon of B and T cell epitope spreading, which is preceded by the initiation of immunity to a single or a few self components (2–6). A model of intra- and intermolecular diversification of the antibody response has been proposed and is supported by several experimental studies in naive animals immunized with self antigens (7–16). Longitudinal studies of sera from patients with lupus and from lupus mice have also demonstrated that, with time, the immune response matures in an apparently ordered manner and gives rise to a diversified, non–cross-reacting antibody population Supported in part by a grant from the Association de Recherche sur la Polyarthrite. Dr. Monneaux’s work was supported by a postdoctoral grant from the Centre National de la Recherche Scientifique and BioDelivery Systems (Wittelsheim, France). Fanny Monneaux, PhD, Véronique Parietti, BSc, Jean-Paul Briand, PhD, Sylviane Muller, PhD: Centre National de la Recherche Scientifique, Strasbourg, France. Address correspondence and reprint requests to Sylviane Muller, PhD, Institut de Biologie Moléculaire et Cellulaire, UPR9021, Centre National de la Recherche Scientifique, 15 rue René Descartes, 67084 Strasbourg, France. E-mail: S.Muller@ibmc.u-strasbg.fr. Submitted for publication November 27, 2003; accepted in revised form June 4, 2004. 3232 INTRAMOLECULAR T CELL SPREADING IN MRL/lpr MICE (5,17–19), thus reinforcing the concept of intra- and intermolecular B cell epitope spreading. In the prevalent view of the mechanisms involved in epitope spreading, T helper cells play a central role. Although spreading of the antibody response has been extensively studied in immunized and lupus-prone animals, little is known concerning the spreading of the T cell response in lupus. The only data suggesting the existence of T cell diversification in the case of lupusassociated antigens were obtained in normal animals immunized with peptide containing a single T helper cell epitope from lupus autoantigens (20,21). To our knowledge, there is no study published, to date, in which the natural T cell spreading that occurs in unprimed lupus mice is discussed. Our group has identified an epitope within the 131–151 sequence of the U1-70K spliceosomal protein, which is recognized very early in the course of the disease by CD4⫹ T cells from MRL/lpr and (NZB ⫻ NZW)F1 mice with lupus (22,23). Peptide 131–151 binds to diverse murine and human class II major histocompatibility complex molecules and is recognized by IgG antibodies from lupus mice and from patients with lupus. Furthermore, we recently demonstrated that administration of peptide 131–151 phosphorylated on Ser140 (called peptide P140) in preautoimmune MRL/lpr mice reduced the production of anti–double-stranded DNA (anti-dsDNA) antibody, delayed the development of proteinuria, and significantly prolonged survival (24). The mechanism by which the phosphorylated sequence 131–151 may restore tolerance in autoimmune mice is not yet known. This P140 peptide encompasses a conserved RNP1 motif in residues 139–151 that interacts with RNA within the U1 RNP particle. This motif is also found in other RNP and non-RNP proteins, and we previously postulated that autoreactive T cells to this candidate sequence might drive the diversification of the autoimmune response at an early, asymptomatic stage of the disease (14,25). To investigate this possibility experimentally, we tested the T cell immune response in MRL/lpr mice longitudinally with a set of 18 overlapping peptides of the U1-70K protein, and followed up the hierarchy of the appearance and disappearance of T cell reactivity over time. We compared the results with the T cell reactivity in normal mice immunized with peptide 131– 151 administrated in adjuvant. The findings of the present study support our model and emphasize the importance of the region 131–151 in the cascade of events observed in the murine lupus response. 3233 MATERIALS AND METHODS Peptides. The eighteen 16- to 25-mer peptides corresponding to the murine sequence of the snRNP 70K protein have been described previously (22,24). The homogeneity of each purified peptide was checked by analytical highperformance liquid chromatography, and the identity of each peptide was assessed by matrix-assisted laser desorption and ionization time-of-flight (TOF) mass spectrometry using a Protein TOF apparatus (Bruker Spectrospin, Bremen, Germany). Mice. Female BALB/c (H-2d) and MRL/lpr (H-2k) mice, 5–6 weeks old at the beginning of the experiments, were purchased from Harlan (Gannat, France). Lymphocyte proliferation assays and measurement of interleukin-2 (IL-2) secretion. To test the immunogenicity of peptide 131–151 in normal mice, BALB/c mice (7 per group) were immunized subcutaneously at the base of the tail and hindfoot pads with 100 g of peptide 131–151 or peptide 21–41 (used as a control), which was dissolved in water and mixed (volume/volume) with Freund’s complete adjuvant. After 13 days, blood was withdrawn from the BALB/c mice and lymphocytes were purified by density separation (Lympholyte-M, density ⫽ 1.0875; Cedarlane, Hornby, Ontario, Canada). Peripheral blood lymphocytes (PBLs) were collected and washed 3 times in L-alanyl-L-glutamine–enriched RPMI 1640 medium (Biomedia, Boussens, France) containing 10% fetal calf serum (FCS; Biomedia), 10 g/ml gentamycin, 10 mM HEPES, and 5 ⫻ 10⫺5M ␤-mercaptoethanol, and resuspended at 3 ⫻ 106 cells/ml in the above-described medium. The proliferative response to the 18 overlapping peptides of the 70K protein was measured in duplicate using 3 ⫻ 105 cells/well and a single peptide concentration (120 M). After 72 hours, the cultures were pulsed for 18 hours with tritiated thymidine (specific activity 6.7 Ci/mmole; 1 Ci/well) and DNA-incorporated radioactivity was measured using a Matrix 9600 direct beta counter (Packard, Meriden, CT). The SD value in duplicate cultures was always less than 20% of the mean value. Control tests were performed by adding concanavalin A (100 l/well; 5 g/ml) to cells during the time course (90 hours) of the culture. In some experiments, proliferation and IL-2 secretion from lymph node cells were measured 10 days after immunization, as previously described (22). To study the natural spreading occurring in autoimmune mice, blood was regularly withdrawn from the MRL/ lpr mice (at weeks 5, 8, 12, and 15), and lymphocyte proliferation was measured as described above for the BALB/c mice, except that peptides of the 70K protein were tested at various concentrations, rather than at a single concentration. In some experiments, anti-CD4 monoclonal antibody GK1.5 (10 g/ml; PharMingen, San Diego, CA) was added to the culture. To study the effect of P140 peptide administration on spontaneous T cell spreading occurring in lupus-prone mice, preautoimmune MRL/lpr mice were injected intravenously (IV) with 100 g of peptide P140 in saline (9 mice), or received phosphate buffered saline (PBS) alone (10 mice), at weeks 5, 7, 9, and 13, as described previously (24). Blood was then regularly obtained from the treated mice at 10, 12, or 14 weeks, and each peptide (at a concentration of 80 M) was tested in the cultures for its ability to induce proliferation of PBLs. 3234 MONNEAUX ET AL RESULTS Intramolecular T cell spreading in unprimed MRL/lpr mice. Blood samples were obtained from 20 young, nonimmunized MRL/lpr mice at weeks 5, 8, 12, and 15, and pooled PBLs were systematically tested for their ability to proliferate in response to 2 concentrations (80 M and 120 M) of each of the 18 overlapping 16–25-mer peptides spanning the mouse U1-70K protein. Responses were considered to be positive only when stimulation indices were higher than 3 in the proliferation assays using either 80 M or 120 M of the recall peptides, and a dose-dependent response was measured. As shown in Figure 1A, no proliferative response of PBLs purified from 5-week-old MRL/lpr mice was observed. In contrast, a major shift occurred at week 8 (Figure 1B). Eight peptides of the 70K protein, namely peptides 57–77, 96–116, 131–151, P140, 219–238, 231– 250, 407–428, and 426–448, were found to be able to stimulate proliferation of PBLs from 8-week-old MRL/ lpr mice. At 12 weeks, most of the peptides that had shown a positive response with the T cells from 8-week-old mice failed to stimulate proliferation of MRL/lpr PBLs, whereas one peptide, encompassing residues 183–202, which had been unable to recall a proliferative response with T cells from the 5- and 8-week-old mice, produced a strongly positive response in the 12-week-old mice (Figure 1C). Thus, at 12 weeks, T cells proliferated in the presence of peptides P140 and 183–202 in the cultures. Peptides 201–220 and 303–320 were considered to yield negative results, since they did not produce a positive dose-dependent response (this was confirmed in another independent experiment in which both also showed a negative response). Finally, at 15 weeks, 4 peptides, 96–116, P140, 183–202, and 303–320, were able to stimulate proliferation with indices higher than 5, but only at the high peptide concentration of 120 M (Figure 1D); therefore, in accordance with our criteria, the results in the 15-week-old mice have to be regarded as negative. In these assays, T cell proliferation was dose dependent (as illustrated with a few examples in Figure 2A) and was found to be inhibited by an anti-CD4 monoclonal antibody (Figure 2B). These results provide strong evidence that the main proliferative cell population corresponds to CD4-positive T cells. T cell spreading in BALB/c mice immunized with peptide 131–151. We recently proposed a model in which the RNP1 motif present in sequence 131–151 of Figure 1. Spontaneous T cell spreading in unprimed MRL/lpr mice. The proliferative response of peripheral blood lymphocytes from 5(A), 8- (B), 12- (C), and 15-week-old mice (D) was measured in the presence of 80 M and 120 M of each of 18 overlapping peptides of the U1-70K protein. The results are expressed as the mean and SEM stimulation index, calculated as the cpm in cultures with peptide divided by the cpm in cultures without peptide. A mean stimulation index ⬎3 was considered to be positive (horizontal line). The average tritiated thymidine incorporation in the absence of peptide was 100 cpm. This experiment is 1 of 2 individual experiments that showed similar results. the U1-70K protein could be responsible for the spreading of the autoimmune response directed against spliceosomal proteins (14,25). To evaluate this hypothesis and perform a comparison with the findings obtained in the unprimed lupus mice, we immunized BALB/c mice subcutaneously with peptide 131–151 or the control peptide 21–41 in Freund’s complete adjuvant, and 13 INTRAMOLECULAR T CELL SPREADING IN MRL/lpr MICE 3235 114–132 and 317–334 (at the limit of positivity) showed a positive response, as defined by our stringent criteria, in the proliferation assay (Figure 3A). These results suggest a mechanism of T cell spreading, rather than simply representing a crossreaction between the 3 peptides. First, sequence analysis, as shown in Figure 3A, revealed no overlapping stretches of residues between peptides 57–77, 131–151, and 219–238. The 3 peptides are hydrophilic, but Figure 2. Dose-dependent proliferative response to U1-70K peptides in unprimed MRL/lpr mice, with mediation by CD4 T cells. A, The proliferative response of peripheral blood lymphocytes from 8-weekold MRL/lpr mice was measured in the presence of various concentrations of peptides 96–116 (solid diamond), 131–151 (open triangle), P140 (open circle), and 219–250 (solid square) of the U1-70K protein. The results are expressed as the mean ⫾ SEM stimulation index, calculated as the cpm in cultures with peptide divided by the cpm in cultures without peptide. A mean stimulation index ⬎3 was considered to be positive (horizontal line). The average tritiated thymidine incorporation in the absence of peptide was 100 cpm. This experiment is 1 of 2 individual experiments that showed similar results. B, Proliferative response to peptide P140 (120 M) in 8-week-old MRL/ lpr mice measured in the presence (⫹) or absence (⫺) of neutralizing anti-CD4 monoclonal antibodies (10 g/ml). days after administration, we studied the response of PBLs to the 18 overlapping peptides of the U1-70K protein. When peptide 21–41 was used as the immunogen, no or very weak proliferation was measurable with 17 of the peptides, with the one exception being in response to the homologous peptide (results not shown). In contrast, when peptide 131–151 was used as the immunogen, we found that not only did the P140 phosphorylated analog yield positive results, but also 2 of the other unrelated peptides of the U1-70K protein, namely peptides 57–77 and 219–238, as well as peptides Figure 3. T cell spreading in BALB/c mice immunized with peptide 131–151 of the 70K protein. A, The proliferative response of peripheral blood lymphocytes from BALB/c mice immunized with peptide 131– 151 was measured 13 days after immunization, in the presence of 120 M of each of the 18 overlapping peptides of the U1-70K protein. The results are expressed as the mean ⫾ SEM stimulation index. A mean stimulation index ⬎3 was considered to be positive (horizontal line). The average tritiated thymidine incorporation in the absence of peptide was 120 cpm. The sequence of 3 strongly positive peptides is shown (note that, in this experiment performed 13 days after immunization, the response to the homologous peptide is weak or negative). B, Lymph node cells from BALB/c mice immunized with peptide 131–151 were recalled ex vivo after 10 days in the presence of increasing concentrations of peptides 131–151 (solid diamond), 57–77 (open square), and 219–238 (open triangle). The average tritiated thymidine incorporation in the absence of peptide and in the presence of concanavalin A was 2,500 cpm and 14,000 cpm, respectively. A mean stimulation index ⱖ2.0 in the proliferation test was considered to be positive (horizontal line). Secretion of interleukin-2 (IL-2) was measured using CTL-L cells. Bars show the mean ⫾ SEM. 3236 whereas peptide 131–151 is largely basic (ratio of positive charge to negative charges of 5), peptides 57–77 and 219–238 are rather acidic or neutral (ratio of 0.67 and 1.2, respectively). Second, as shown in Figure 3B, we demonstrated unambiguously that lymph node T cells generated against peptide 131–151 in BALB/c mice cannot be activated ex vivo by either of the other 2 peptides, 57–77 and 219–238. Finally, we also showed that whereas peptide P140 was strongly recognized by PBLs from unprimed 15-week-old MRL/lpr mice, peptides 57–77 and 219–238 were weakly or not at all recognized by the same cells (Figure 1D). Effect of P140 therapy on spontaneous T cell spreading. We recently demonstrated that IV administration of the phosphorylated peptide P140 to healthy (preautoimmune) MRL/lpr mice reduced the production of high titers of anti-dsDNA antibodies, delayed the appearance of severe proteinuria, and significantly enhanced the survival of treated mice (24). The cellular and molecular mechanisms involved in this apparent restoration of tolerance are still unknown. It is particularly striking to observe that the IV administration of a single 21-mer peptide of the U1 snRNP particle seems to affect the autoimmune response to native DNA, as well as important clinical features, leading to a substantial improvement in the treated animals. We have proposed several potential mechanisms, which are presently under investigation in our laboratory, to explain these observations. We have used the term “tolerance spreading,” as introduced by Kaliyaperumal et al (26), to describe the possible tolerogenic effect of peptide P140 on autoimmune B and T cell responses (24). With the intention of experimentally investigating the effect of peptide P140 on spontaneous T cell spreading, we studied the proliferative response of PBLs collected serially from lupus mice that were first subjected to P140 treatment. Preautoimmune MRL/lpr mice received 4 IV injections of peptide P140 at weeks 5, 7, 9, and 11 and blood was withdrawn at weeks 10, 12, and 14 (for review, see ref. 24). The capacity of a set of selected peptides to recall the T cell response of PBLs ex vivo was then tested (Figure 4). As expected, at week 10, in the mice that received 3 administrations of PBS only, PBLs proliferated in response to the 5 selected peptides 96–116, P140, 219– 238, 407–428, and 426–448 (Figure 4A). However, in the mice that received 3 administrations of P140, we observed that, with the exception of peptide 96–116, the proliferative response was significantly lower, and mean stimulation indices were even under the level of positiv- MONNEAUX ET AL Figure 4. Effect of brief phosphorylated peptide P140 therapy on the spontaneous T cell spreading in MRL/lpr mice. Mice either were treated with peptide P140 (hatched bar), administrated intravenously in saline at weeks 5, 7, 9, and 13 (as indicated by the arrows), or received phosphate buffered saline (PBS) (solid bar) alone at the same time points. The proliferative response of peripheral blood lymphocytes from 10- (A), 12- (B), and 14-week-old mice (C) was measured in the presence of 6 selected peptides (80 M). A mean stimulation index ⬎3 was considered to be positive (vertical line). Bars show the mean ⫾ SEM. Values adjacent to bars are the percentage reduction in treated mice versus nontreated mice. NS ⫽ nonsignificant reduction; na ⫽ not applicable. ity, with peptides P140, 219–238, 407–428, and 426–448 (31–51% decrease) (Figure 4A). The same drop in proliferative response was observed in 12-week-old mice with peptide 183–202 (85% decrease) (Figure 4B). At 14 weeks (1 week after the fourth and last injection), PBLs from MRL/lpr mice that received PBS or P140 proliferated equally well in response to recall peptides P140 and 183–202 (Figure 4C). DISCUSSION In recent years, several laboratories have been interested in the molecular and cellular mechanisms of autoantibody diversification in response to SLE autoantigens. As methods are developed to rapidly screen epitopes of self proteins with miniaturized test formats, such studies will emerge that have the potential to generate results with implications for the analysis of patients’ sera. These previous studies led to the proposal of a model of intra- and intermolecular diversification based on the particle hypothesis introduced by Hardin INTRAMOLECULAR T CELL SPREADING IN MRL/lpr MICE 3237 (27), which associates antigen-reactive B cells and T cells providing help to these B cells. Although the diversification of serum circulating autoantibodies has been studied, particularly in murine lupus, that of autoreactive T cells is largely unknown in lupus. Nevertheless, in nonobese diabetic mice that develop a type 1 diabetes mellitus–like disease, for example, this phenomenon has been studied in detail, and the results revealed the progression from a Th1 type of response directed to a single determinant (peptide 524–543 of glutamic acid decarboxylase [GAD]) in 4-week-old mice to a diversified response to additional GAD determinants as well as to other beta cell antigens such as insulin B chain and the 65-kd heat shock protein in 12-week-old mice (28). Such sequential spreading of T cell autoreactivity has also been observed in patients with multiple sclerosis during disease progression, as well as in the experimental autoimmune encephalomyelitis (EAE) murine model. The present study shows that in MRL/lpr mice, the T cell repertoire evolves rapidly in a few weeks, between weeks 7–8 and 12–13. The behavior of peripheral T cells then changes, and at week 15, when the lupus disease is established in most MRL/lpr mice, T cells become ignorant ex vivo in the presence of peptides of the U1-70K protein. We have observed that certain peptides are recognized very early by peripheral MRL/ lpr T cells, and then with the emergence of new reactivity, a regression of some, but not all, of the primary reactivity occurs. Thus, as shown previously in EAE (29), there is a cascade of primary and secondary determinants as well as more stable determinants that remain during the progression toward lupus. Regression of T cell autoreactivity with age, visualized ex vivo, may result from different mechanisms, such as T cell exhaustion due to a chronic stimulation in vivo, a modified balance of the peripheral apoptotic cells associated with disease onset, and in the MRL/lpr mouse model, the well-known accumulation of CD4⫺/CD8⫺ double-negative T cells. The maturation of MRL/lpr T cell reactivity toward determinants of the U1-70K protein occurs concomitantly with the autoantibody reactivity described previously (25). It is notable that sequences 96–116 and 131–151 contain both a B cell epitope and an epitope recognized by peripheral T cells. However, the peptide 21–41, which was recognized very early by IgG antibodies from 8-week-old mice and by antibodies from as many as 9 of twenty 22-week-old mice tested serially (25), was unable to activate peripheral MRL/lpr T cells ex vivo. Conversely, with the exception of peptides 96–116 and 131–151, several peptides able to recall peripheral MRL/lpr T cells ex vivo were not recognized by antibodies. Furthermore, in contrast to the emergence/regression type of T cell spreading observed in the case of the 70K protein, B cell spreading arising in 8–22-week-old MRL/lpr mice in response to this antigen was shown to progress without disappearance of existing autoantibody subsets (25). In the present study, we have shown that normal mice that were immunized with peptide 131–151 in adjuvant developed a peripheral T cell response revealed with the P140 peptide, but also with 2 peptides that have no structural similarities with, and are located at a distance from, the sequence 131–151. Most importantly, these 2 peptides were also strongly recognized by peripheral T cells from MRL/lpr mice. Such a result strongly supports our model of spreading based on the RNA binding domain (RBD) motif called RNP1, present in the sequence 131–151 of the U1-70K protein as well as in other lupus common antigens such as the U1A and hnRNPA2/B1 proteins (14,25). Recent observations made by Greidinger et al also support our hypothesis (30). Using T cell clones derived from patients with SLE and mixed connective tissue disease, those authors identified 5 T cell epitopes on the U1-70K protein, and all 5 of these epitopes reside in the RBD motif of the protein. The most important finding in this report is the effect of phosphorylated peptide analog P140 on the diversification of the T cell response. We clearly demonstrated that administration of peptide P140 (in saline) at least transiently abolishes the intramolecular spreading to other regions of the 70K protein. Although there is no available information suggesting any putative role of CD4⫹ T cell clones to RNP proteins in the pathogenic events shown in lupus, this result supports the view that a single peptide can effectively modulate T cell reactivity to other regions of the protein and potentially to other RNP1⫹ and RNP1⫺ proteins of the same particle (14), and thus can regulate the possible functions of autoreactive T cell clones on the B cell response (26,31). This result, shown experimentally for the first time, is crucial, since it demonstrates that epitope spreading is not a severe limitation to the treatment of autoimmune individuals with a single peptide, or with a small cocktail of well-selected peptides. 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