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* Division of Immunology and Rheumatology and
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212; and
Harry S. Truman Memorial Veterans Hospital, Columbia, MO 65201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Autoantibodies to spliceosomal small nuclear ribonucleoprotein (snRNP)3 Ags frequently develop in patients with connective tissue disease (CTD) including systemic lupus erythematosus (SLE), mixed CTD, and systemic sclerosis (7, 8). Abs to the 70-kDa subunit of U1 snRNP (U1-70kDa) are often the first snRNP Abs to appear in patients referred for autoantibody testing, consistent with a role for U1-70kDa as an initial immunogen in CTD-associated anti-snRNP responses (9). U1-70kDa has been shown to undergo Ag modifications including cleavage by caspases at residue 341 in apoptosis, by granzyme B at residue 409 in cell-mediated cytotoxicity, and by metal-catalyzed oxidation at undetermined sites (4, 10, 11). Apoptosis-specific Abs to U1-70kDa have been shown to exist that are distinct from Abs to the intact molecule (7), and Abs to the oxidatively cleaved form have been shown to be distinct from the apoptotic form (12).
Previously, we have shown that T cell immunity against the U1-70kDa
snRNP polypeptide can be detected in PBMC from CTD patients, and that T
and B cell immunity against U1-70kDa are linked in vivo
(13). We also reported that U1-70kDa-reactive T cells have
a typical Th phenotype and produce cytokines that are important in B
cell help and differentiation, including IFN-
, IL-4, and IL-2
(13). Additionally, we have shown that TCR usage by
U1-70kDa reactive T cells is highly restricted and by analysis of
complimentarity-determining region 3 deduced amino acid sequences found
a pattern characteristic of an Ag-driven immune response
(14). Finally, in the MRL/Mp-lpr murine model
of lupus, we have found that tolerization with a U1-70kDa fusion
protein delayed the development of anti-snRNP Abs
(15).
The present study was designed to characterize the range of T cell immune responses against the U1-70kDa Ag encountered in CTD patients with anti-U1-70kDa Abs, including determining the precise Ag peptides recognized and the common features of antigenic structure recognized. To do this, we derived T cell clones using PBMC from CTD patients and pools of peptides encompassing the entire U1-70kDa protein, presented by autologous APCs.
Despite the diversity of Ag forms available, the only clones that we generated from CTD patients reacted exclusively against determinants within the RNA binding domain (RBD) of the U1-70kDa polypeptide, spanning residues 92–202 (16). Five distinct T cell epitopes were identified within the RBD, including two that were recognized by T cells from multiple patients. This focused T cell reactivity directed against a limited portion of the molecule was in contrast to the diversity of patterns of Ab reactivity that we observed. Binding studies confirmed that U1-RNA bound with high avidity to the intact form of U1-70kDa, as well as to the modified forms of the polypeptide that have been implicated in the pathogenesis of CTDs. Thus, the high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All samples and data for this study were obtained through protocols approved by the University of Missouri Institutional Review Board (Columbia, MO). Sera from patients seen in the Division of Immunology and Rheumatology at the University of Missouri (Columbia, MO) were characterized for the presence of autoantibodies reactive with the U1-70kDa polypeptide using immunodiffusion, immunoblotting, and ELISA as previously described (17, 18). Sera were also characterized for reactivity with U1-RNA using an assay for binding to radioactively labeled U1-RNA, as described previously (19). Patients (P) (P1 to P6) selected for study all had IgG Abs against U1-70kDa. P4 had IgG Abs to U1-RNA. All patients were female. The P1, P2, and P4 were classified as mixed CTD, P3 and P5 were classified as SLE, and P6 was classified as undifferentiated CTD (20, 21). The patients exhibited mild to moderate disease activity with disease activity indexes ranging from 0–6 and prednisone doses ranging from 0–20 mg per day (22). All of the patients were receiving 400 mg hydroxychloroquine per day. None of the patients were receiving additional immunosuppressive drugs during the study. HLA typing was done using serologic and DNA-based typing methods as described previously (23). In brief, this was done using a combination of subfamily-specific PCR and direct DNA sequencing of PCR-amplified products (24, 25). The HLA-DRB1 genotypes of the patients were: P1, DRB1*0101; P2, DRB1*0401, 1502; P3, DRB1*0101, 1302; P4, DRB1*0401, 1301; P5, DRB1*0301, 0401; and P6, DRB1*0301, 1301. The HLA genotypes of the controls were HLA-DRB1*1501, 0401; HLA-DRB1*0401, 0401; and HLA-DRB1*0701, 1501.
Recombinant Ag
Control unconjugated maltose-binding protein (MBP), U1-70kDa-MBP fusion protein, and C-terminal truncated forms of U1-70kDa-MBP fusion protein were expressed from the pMAL plasmid in Escherichia coli and affinity purified over amylose columns as previously described (14). For some experiments, forms of U1-70kDa-MBP were further purified by elution of the relevant band cut from 10% SDS-PAGE preparative gels as previously described (26). Caspase-cleaved U1-70kDa was produced by incubating intact U1-70kDa fusion protein with caspase-3 (R&D Systems, Minneapolis, MN) in buffer containing 10 mM HEPES (pH 7.4), 2 mM EDTA, 5 mM DTT, and 1% Nonidet P-40 as previously described (6). To produce the oxidatively cleaved form of U1-70kDa, we incubated purified recombinant full-length U1-70kDa fusion protein with 100 µM iron (II) sulfate and 2 mM sodium ascorbate in buffer containing 20 mM HEPES (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, 1.45 µM pepstatin, 10 µM leupeptin, 20 µM chymostatin, 3.2 µM antipain, and 1 mM PMSF for 30 min at room temperature. The reaction was terminated by the addition of EDTA to 10 mM plus Laemmli sample buffer, and boiled for 3 min.
C-terminal truncation mutants were generated from the pMALc2g-61-70kDa
expression plasmid using Quik-Change mutagenesis methodology
(Stratagene, La Jolla, CA). Briefly, complimentary primers were
designed with the stop codon at the desired location within 12–15
flanking bases of plasmid-complementary sequence. A mixture of these
primers, expression plasmid, dNTP, and a proofreading DNA polymerase
(Pfu Turbo; Stratagene) was cycled 18–20 times to generate
full-length copies of the plasmid containing the desired mutation. The
parental template was digested away with DpnI and the
remaining DNA was transformed into TOP10 F' competent bacteria
(Invitrogen, Carlsbad, CA) and plated on Luria-Bertani agar plates with
ampicillin selection. Colonies were grown in liquid selective medium,
and plasmid DNA was obtained by alkaline lysis miniprep. The plasmids
were sequenced, and those containing the proper sequence were used to
transform competent BL21 Codon-Plus arginine proline E.
coli for expression. After overnight selection with
chloramphenicol and ampicillin, a colony was picked to inoculate Luria
Bertani without antibiotics. After allowing growth to
OD600 = 0.5, expression was induced by addition
of isopropyl 
-D-thiogalactopyranside to 0.5 mM
and allowed to proceed for 5 h. Bacterial lysates from the
expression were purified over amylose resin (NEB, Worcester,
MA).
Immunoblotting protocol
Immunoblots were performed as previously described (12). Briefly, control, apoptotic, oxidatively cleaved, and granzyme B cleaved cell lysates or purified Ag forms were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 3% BSA, membranes were exposed to 1/10,000 dilutions of test sera, incubated with HRP-linked goat anti-human secondary Ab (Southern Biotechnology Associates, Birmingham, AL), and visualized with chemiluminescence (Pierce, Rockford, IL). Cell lysates were prepared from Jurkat cells using previously described protocols (4, 12). To generate apoptotic samples, cells were exposed to 1,650 J/m2 of UV-B light and incubated for 16 h before harvesting. To make oxidatively cleaved and granzyme B-cleaved samples, cell lysates were incubated with 100 µM iron (II) sulfate and 2 mM sodium ascorbate for 30 min, or with 42 nM granzyme B in the presence of 2 mM iodoacetamide for 1 h, respectively.
Peptide design and synthesis
Peptides were synthesized using N-(9-fluorenly) methoxycarbonyl solid-phase chemistry on an Applied Biosystems Model 432A Peptide synthesizer (Applied Biosystems, Foster City, CA) or were purchased (Research Genetics, Huntsville, AL) (27). Peptides were analyzed for purity and sequence fidelity using HPLC and mass spectrometry. The peptides that were used were required to have at least 90% purity and unambiguous sequence fidelity. For experiments using pooled peptides, each peptide was synthesized and validated individually, and equal molar quantities of every peptide in the pool were used.
Generation of human T cell clones
T cell clones used in these studies were generated as described previously (28). In brief, cells were obtained by aphaeresis and then subject to density gradient centrifugation using Histopaque (Sigma-Aldrich, St. Louis, MO). Patient PBMC were used immediately or cryopreserved for use as APCs to restimulate clones. A total of 5 x 106 cells were cultured in RPMI (Life Technologies, Rockville, MD) with gentamicin 20 µg/ml, 2 mM L-glutamine, 15% AB-positive human serum, and pooled peptides. As Ags, a series of synthetic peptides 25 residues in length were used. Each peptide spanned the previous peptide in the series beginning at residue 13. The entire series of peptides was designed to span the U1-70kDa polypeptide. Cells in a final volume of 5 ml were placed in a 25-cm2 flask and incubated in 5% carbon dioxide at 37°C. Cells were restimulated with 5 x 106 autologous APCs irradiated with 30 Gray and Ag in fresh medium on day 7. On day 14, T cell blasts were separated by centrifugation on a density gradient (Percoll; Amersham Pharmacia Biotech, Piscataway, NJ) and cloned by limiting dilution in the presence of pooled peptides, fresh irradiated autologous APCs, and 10 U/ml IL-2 (R&D Systems) as described previously (13, 27, 28). Cloning was attempted twice with every donor. Cells were cloned by limiting dilution and those that were positive for growth, seeded at the lowest number of cells per well, were selected for expansion. Typically, 10–20 wells were expanded and tested for reactivity with U1-70kDa peptides. Those clones that exhibited strong reactivity with U1-70kDa and no reactivity with APCs alone were studied further. Although clones reactive with autologous APCs alone were identified, they were not characterized further. Cell surface phenotypes of clones were determined by flow microfluorometry as described previously (13).
Proliferation assay
A total of 2 x 104 T cells in complete medium were cultured for 48 h in 96-well flat-bottom tissue culture plates and then pulsed for 18 h with 1 µCi/well of TdR. Cells were harvested and TdR incorporation detected by liquid scintillation counting (13).
MHC blocking of Ag-induced proliferation and MHC restriction element mapping
A series of HLA-homozygous lymphoblastoid cell lines (LCLs) of known HLA genotypes and HLA-DR, DQ, and DP-specific mAb were used in Ag stimulation and mAb blocking experiments to define the restriction elements used by individual T cell clones, as described previously (28).
Searches for similarity with known DNA and protein sequences
Searches for similarities between T cell epitopes identified in the present study and all DNA and protein sequences in GenBank Database were done using basic local alignment search tool (National Center for Biotechnology Information, Bethesda, MD). Searches for similarity were done using the core T cell epitopes identified and then repeated using the information derived from alanine substitution analysis to distinguish residues that tolerate amino acid substitution (e.g., searches of sequences HMVYSKRSGKPR and XXVYXXRXGKPR against GenBank Database for homology).
Determination of U1-RNA binding avidity to U1-70kDa polypeptide
U1-RNA was produced and purified as previously described (19), except 35S-labeled UTP (NEN, Albany, MA) was used in the preparation of the labeled product. Briefly, the RNA was produced by in vitro transcription from the corresponding DNA contained in linearized SP64 (a kind gift from Dr. S. Deutscher, University of Missouri), followed by template digestion with DNase I and purification by Sephadex spin column chromatography.
Saturation binding experiments were modified from Query et al. (16) and Kessler (29). In each experiment, a constant amount of unlabeled U1-RNA was used to dilute different amounts of 35S-labeled U1-RNA of known specific activity in NET-2 buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl (pH 7.4) with 0.05% Nonidet P-40). This was allowed to interact with 1 pmol of test protein for 20 min at room temperature, after which the mixture was immunoprecipitated by a high-titer anti-U1-70kDa positive antiserum previously documented to have no reactivity to U1-RNA. This was precipitated with washed Pansorbin (Calbiochem, San Diego, CA), and the complex washed four times with ice-cold NET-2 after removal of supernatants for liquid scintillation counting. The washed pellets were also counted, and total bound and free fractions were then calculated. This was used to determine the Kd using a one-site binding nonlinear regression model with Prism 3.0 (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U1-70kDa-specific human T cell clones were successfully generated
from six patients (P1 to P6) against a pool of peptides, each 25
residues in length, spanning the entire U1-70kDa molecule. Each peptide
overlapped the previous peptide in the series by 13 residues.
Representative U1-70kDa reactive T cell clones that were generated and
studied from three patients are shown in Fig. 1
. As shown in Fig. 1, all clones
demonstrated a strong proliferative response to a pool of U1-70kDa
peptides or PHA, but did not respond to APCs alone or APCs with an
irrelevant peptide. All clones examined had a CD4-positive cell surface
phenotype. These clones and others from P1 to P6 were subsequently
characterized to determine more precisely which region of U1-70kDa they
were responding against. Sera from the patients studied were noted to
be diverse with regard to immunoblot recognition of the apoptotic,
oxidatively cleaved, and granzyme B-cleaved forms of U1-70kDa (Table I).
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Three separate attempts were made to generate T cell clones from three normal control subjects using U1-70kDa pooled peptides. These normal control subjects were matched for the presence of the HLA-DRB1*0401 or HLA-DRB1*1501 responder genotype. Although we could generate T cell clones from these donors specific for a control Ag (tetanus toxoid or heterogeneous nuclear ribonucleoprotein), no U1-70kDa-specific clones could be generated from normal controls.
Five T cell epitopes were identified on the U1-70kDa polypeptide by T cell clones derived from patients
An iterative process was used to define the regions of the
U1-70kDa polypeptide recognized by human T cell clones. Clones were
initially analyzed for proliferation against a pool of peptides 25
residues in length, overlapping by 13 residues with the previous
peptide in the protein, which taken as a set span the entire U1-70kDa
polypeptide. Clones were subsequently tested against smaller pools of
peptides and eventually against individual peptides 15 residues in
length. Finally, select clones were tested against additional peptides
truncated from either the amino or C terminus to define the minimal
epitopes recognized (Fig. 2). Five T cell
epitopes were identified by U1-70kDa-specific T cell clones on the
U1-70kDa protein using the 25-residue and 15-residue synthetic
peptides. Peptides encoding these five epitopes (E) 1–5 reside,
respectively, between residues 97–111, 112–136, 133–147, 151–165,
and 173–187 of the full-length U1-70kDa protein. As shown in Table II, clones that recognized E1 were
generated from P1, clones that recognized E2 were generated from P2,
clones that recognized E3 were generated from P2 and P3, clones that
recognized E4 were generated from P4, and clones that recognized E5
were generated from P1, P2, P3, P5, and P6.
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The three T cell epitopes against which T cell clones were most
frequently generated (E1, E3, and E5) were analyzed further to
determine the minimal T cell epitope recognized by the clones. These
minimal epitopes are summarized in Fig. 2
. To determine the minimal
region within the U1-70kDa-E1 required for T cell recognition,
truncated variants of the 15 residue peptide AQGDAFKTLFVARVN were
synthesized and tested. As shown in Fig. 3A, stimulation of clones was
observed only when the truncated peptides contained the core sequence
DAFKTLFVARVN (contained in the full-length U1-70kDa polypeptide at
residues 100–111). To determine the minimal region within the
U1-70kDa-E3 required for T cell recognition, truncated variants of the
15 residue peptide HMVYSKRSGKPRGYA were synthesized and tested. As
shown in Fig. 3B, stimulation of clones was observed only
when the truncated peptides contained the core sequence
HMVYSKRSGKPR (contained in the full-length U1-70kDa polypeptide at
residues 133–144). To determine the minimal region within the
U1-70kDa-E5 required for T cell recognition, truncated variants of the
15-residue peptide GRRVLVDVERGRTVK were synthesized and tested;
stimulation of clones was observed only when the truncated peptides
contained the core sequence VLVDVERGRTV (contained in the full-length
U1-70kDa protein at residues 174–184; data not shown). These minimal
epitopes are shown in Fig. 2; all reside within the RBD.
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A, alanine substitutions of
the core peptide DAFKTLFVARVN for minimal E1 were synthesized and
tested for their ability to induce proliferation of T cell clones.
Substitution of the residues at positions 1, 3–6, 8, and 10–12 with
alanine abolished T cell reactivity with DAFKTLFVARVN (E1). Positions 2
and 9 (underlined in Fig. 4A) that already contained alanine
could not be evaluated. Alanine substitutions of HMVYSKRSGKPR (E3)
revealed that alanine substitution at positions 3, 4, 7, and 9–12
abolished the ability of the peptide to stimulate the clone (Fig. 4B).
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C). Alanine
substitution at position 4 was partially inhibitory, while substitution
of valine to alanine at position 1 was well tolerated. HLA-DR is the MHC restriction element used in T cell recognition of U1-70kDa peptides
The MHC restriction element used by T cell clones was determined using Ag-induced proliferation in the presence of blocking concentrations of mAb specific for HLA-DR, HLA-DQ, or HLA-DP, or using a series of well-characterized homozygous LCL of known HLA genotypes as APCs. The HLA class II blocking mAb used were specific for either HLA-DR, DQ, or DP framework determinants. These were used to determine which HLA class II molecule was the restriction element for T cell recognition. It was found that all clones tested used HLA-DR as a restriction element for Ag recognition. A panel of LCL was then used to determine the specific HLA-DR allele used by the T cell clone. It was found that HLA-DRB1*0401 was used as the restriction element by clones from the two patients analyzed in detail. The complete HLA genotypes of all six of the donors were determined, and these are given in Materials and Methods. Interestingly, five of the six patients shared the closely related genotypes HLA-DRB1*0401 (P2, P4, and P5) and HLA-DRB1*0101 (P1 and P3).
U1-70kDa polypeptides show homology with other spliceosomal proteins but not with other sequences in GenBank Database
As a possible explanation for the limited targeting of anti-U1-70kDa T cell clones, we considered whether anti-U1-70kDa T cells evolved due to molecular mimicry with a microbial Ag. However, comparisons of the five T cell epitopes and their minimal peptides from truncation experiments with GenBank Database revealed homology only with other functionally related snRNPs from humans and other vertebrate species. Similar results were obtained when searches were performed with only the essential contact residues retained in the backbone of the peptide derived from alanine scanning experiments (e.g., searches of sequences HMVYSKRSGKPR and XXVYXXRXGKPR).
U1-RNA binds with similarly high avidity to the intact U1-70kDa polypeptide and to multiple modified forms of U1-70kDa implicated in the development of autoimmune diseases
As shown in Table I, the patients exhibited a broad spectrum of
profiles of Abs reactive with different modified forms of U1-70kDa
(caspase-cleaved, granzyme B, or oxidatively cleaved), but had a
limited T cell response directed solely at the RBD (Fig. 2). As a
possible explanation for the predominance of T cells reactive against
the RBD of U1-70kDa among anti-U1-70kDa positive CTD patients, we
considered whether similar T cell clones could be generated against the
endogenous forms of the U1-70kDa Ag implicated in autoimmune disease
pathogenesis. To assess for conserved structure of the RBD in modified
forms of the molecule, we investigated whether the RBD demonstrated
conserved U1-RNA binding affinity between the intact, caspase-cleaved,
and oxidatively cleaved forms of the molecule. The caspase-cleaved and
oxidatively cleaved forms of the molecule were generated by exposing
the purified full-length form of U1-70kDa to caspase-3 and
iron/ascorbate, respectively, following standard protocols (4, 6). Using C-terminal truncation mutants, we identified the
site(s) of oxidative cleavage of U1-70kDa to be distal to residue 205,
preserving the RBD sequence (Fig. 5).
Thus, all modified forms retained the RBD.
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). The
Kd of U1-RNA with each of the forms of
U1-70kDa tested (Table III) falls in the
10 pmol range. These values are similar to the dissociation constant
reported for the homologous and highly avid interaction of the U1-A
protein with U1-RNA (30). Thus, all forms of modified
U1-70kDa bound to U1-RNA with similar high affinities.
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Because double-stranded and stem-loop RNAs have been shown to have direct immunostimulatory effects (unrelated to TCR recognition of Ag), and since the U1-RNA possesses a stem-loop structure, we examined whether U1-RNA acted as an immune adjuvant (31). To test for this possibility in vitro, we tested the proliferative response of U1-70kDa-specific human T cell clones to U1-70kDa peptides presented by autologous irradiated APCs in the presence or absence of equimolar quantities of U1-RNA. No increase in the proliferative responses to the U1-70kDa peptides could be observed with the addition of U1-RNA compared with peptides alone (data not shown). The kinetics of survival of U1-RNA was not examined and these experiments could have failed due to RNA degradation. Studies are now in progress to examine the mechanism(s) by which U1-RNA may influence T cell epitope selection and/or T cell activation. Possible mechanisms could include influences on Ag processing or costimulation (31, 32).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The finding that there are a limited number of T cell epitopes on U1-70kDa is similar to results of T cell epitope mapping of another antigenic spliceosomal polypeptide, the Sm Ag. We have recently reported that there were three T cell epitopes on the Sm-B polypeptide and two on the Sm-D polypeptide (33). We have also observed RBD-specific T cell clones directed against the heterogeneous nuclear ribonucleoprotein A2 autoantigen (34). The findings of the present study are also consistent with T cell epitope mapping of other autoantigens, including myelin basic protein in multiple sclerosis and topoisomerase I in systemic sclerosis, where only limited numbers of T cell epitopes were found to be present on the autoantigen (35, 36).
To further define the minimal epitope recognized by T cells and to identify the essential contact residues on the Ag, truncated peptides and alanine substitutions of the core peptides were synthesized and tested for their ability to stimulate T cell clones. These studies revealed substitutions of most residues were not well-tolerated, although there were specific exceptions. The findings were consistent with the presumed MHC binding motifs for the peptides, and were similar to those recently reported for the Sm-B and Sm-D polypeptides, where alanine substitutions were well-tolerated in select positions of the peptide, but not tolerated in other positions (33). If the results of the current study accurately reflect the diversity of T cells involved in the pathogenesis of anti-U1-70kDa autoimmunity, epitope-specific T cell immunomodulatory therapies may be plausibly envisioned. This is in contrast to B cell immunity to U1-70kDa, where multiple, widely scattered epitopes have been identified (37).
Experiments using blocking mAb against HLA framework determinants and LCL homozygous for HLA demonstrated that anti-U1-70kDa clones studied were restricted in Ag presentation by HLA-DRB1*0401. Based upon the patients’ genotypes (see Materials and Methods), other restriction elements presumably are used in patients lacking HLA-DRB1*0401; although notably five of six patients share the HLA-DRB1*0401 or the closely related genotype, *0101. Studies directly testing HLA binding and TCR interactions, using the combination of HLA-transfected L cells with site-directed mutagenesis of cloned TCR genes expressed in TCR-deficient Jurkat cell lines are underway to more precisely define the molecular interactions between U1-70kDa self-reactive peptides, HLA, and the TCR (38, 39). However, the fact that T cell clones exclusively targeting the RBD of U1-70kDa were isolated from a patient (P6) without HLA-DRB1*0401 or HLA-DRB1*0101 suggests that a single restrictive HLA genotype is unlikely to fully explain the preferential targeting of CTD patient T cells to the RBD.
The properties of the RBD of the U1-70kDa polypeptide that preferentially target it for immune recognition have not been identified. Because the pooled peptides used to select the clones did not have RNA bound to them, it is unlikely that bound RNA plays a direct role in T cell recognition of Ag. However, RNA binding could influence the shaping of the initial anti-U1-70kDa T cell repertoire. For example, U1-70kDa bound to U1-RNA may be processed differently than other parts of the polypeptide. For example, such an effect could potentially occur in peripheral APCs relevant to the initiation of an autoimmune response if RNA binding protects this region of the protein from proteolysis. Alternatively, preferential T cell targeting of the RBD could be due to relative inefficient thymic deletion of autoreactive T cells against this region, potentially because the thymic Ag-processing pathway is inefficient in proteolyses of the RBD epitope or if it remains intact in a subset of peripheral APC. The uptake of RNA-complexed modified Ag as dead cell fragments in the periphery (such as in the case of apoptotically or granzyme B-exposed cells) may also contribute to differential central vs peripheral tolerance induction. Finally, the proposed target of the RBD could be due to effects entirely unrelated to the binding of the RNA itself; for example, the RBD could have promiscuous binding to MHC class II molecules, similar to that described for superantigen or the HLA class II-associated invariant chain-derived peptide (40, 41).
Because the peptide library against which T cells were screened did not include posttranslationally modified peptides, we cannot exclude the existence of additional T cell epitopes on the U1-70kDa molecule. Potentially relevant posttranslational modifications of this and related snRNP polypeptides have been reported including glycosylation, phosphorylation, and arginine methylation (42, 43, 44). However, many autoantibodies targeting unmodified U1-70kDa peptides have been identified from diverse sections of the molecule both within and outside of the RBD (45).
In summary, we found that there was a highly restricted set of U1-70kDa self peptides recognized by T cells from CTD patients, with T cell recognition limited to five epitopes. All T cell epitopes were found to be located within the RBD of the U1-70kDa polypeptide. These findings, in conjunction with our recently reported findings that T cells against the Sm proteins preferentially target the areas of highly avid protein-protein interactions between these molecules (33) raise the question whether protein-nucleic acid or protein-protein interactions may be a general feature of T cell targets in autoimmunity. The T cell autoimmune response to U1-70kDa in CTDs appears to have less diversity than is seen in the humoral response and therefore may be a favorable target for therapeutic intervention.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert W. Hoffman, Division of Immunology and Rheumatology, MA427 Health Sciences Center, University of Missouri, 1 Hospital Drive, Columbia, MO 65212. E-mail address: hoffmanr{at}health.missouri.edu
3 Abbreviations used in this paper: snRNP, small nuclear ribonucleoprotein; CTD, connective tissue disease; E, epitope; LCL, lymphoblastoid cell line; MBP, maltose binding protein; P, patient; RBD, RNA binding domain; SLE, systemic lupus erythematosus; U1-70kDa, 70-kDa subunit of U1 snRNP.
Received for publication March 21, 2002. Accepted for publication July 12, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and chains from human T-cell clones specific for small nuclear ribonucleoprotein polypeptides Sm-D, Sm-B and U1-70kDa: TCR complementarity determining region 3 usage appears highly conserved. Scand. J. Immunol. 54:204.[Medline]
-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease. Arthritis Rheum. 42:703.[Medline]
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