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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2010 August 10.
Published in final edited form as:
PMCID: PMC2919051
NIHMSID: NIHMS217652

CD4+ T Cells Target Epitopes Residing within the RNA-Binding Domain of the U1-70-kDa Small Nuclear Ribonucleoprotein Autoantigen and Have Restricted TCR Diversity in an HLA-DR4-Transgenic Murine Model of Mixed Connective Tissue Disease1

Abstract

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease with significant morbidity and premature mortality of unknown pathogenesis. In the present study, we characterized U1-70-kDa small nuclear ribonucleoprotein (70-kDa) autoantigen-specific T cells in a new murine model of MCTD. These studies defined 70-kDa-reactive T cell Ag fine specificities and TCR gene usage in this model. Similar to patients with MCTD, CD4+ T cells can be readily identified from 70-kDa/U1-RNA-immunized HLA-DR4-transgenic mice. Using both freshly isolated CD4+ T cells from spleen and lung, and T cell lines, we found that the majority of these T cells were directed against antigenic peptides residing within the RNA-binding domain of 70 kDa. We also found that TCR-β (TRB) V usage was highly restricted among 70-kDa-reactive T cells, which selectively used TRBV subgroups 1, 2, 6, 8.1, 8.2, and 8.3, and that the TRB CDR3 had conserved sequence motifs which were shared across different TRBV subgroups. Finally, we found that the TRBV and CDR3 regions used by both murine and human 70-kDa-specific CD4+ T cells were homologous. Thus, T cell recognition of the 70-kDa autoantigen by HLA-DR4-transgenic mice is focused on a limited number of T cell epitopes residing primarily within the RBD of the molecule, using a restricted number of TRBV and CDR3 motifs that are homologous to T cells isolated from MCTD patients.

Mixed connective tissue disease (MCTD)3 is a systemic autoimmune disease characterized immunologically by the presence of autoantibodies reactive with U1 ribonucleoprotein (U1-RNP) polypeptides, including the U1-70-kDa (70-kDa) polypeptide, and their associated U1-RNA (1). Clinically, MCTD is characterized by manifestations that overlap systemic lupus erythematosus (SLE), scleroderma, inflammatory myopathy, and rheumatoid arthritis (2, 3). The primary disease-related cause of death in MCTD is pulmonary disease including pulmonary hypertension, which distinguishes it from SLE where pulmonary disease is uncommon (4).

The putative target of autoimmunity in MCTD is the U1-RNP Ag which is a U1-RNA-small nuclear RNP complex that is normally contained within the nucleus of eukaryotic cells and whose biologic function is to convert pre-mRNA to mature mRNA (57). The 70-kDa polypeptide of the U1-RNP Ag is a dominant autoantigen in MCTD and consists of a 437-residue polypeptide which noncovalently associates with U1-RNA through an RNA-binding domain (RBD) on the polypeptide spanning residues 92–202 (8).

In a recent genome-wide association study, we found that genetic association of MCTD with the MHC which is consistent with previous candidate gene studies where association of MCTD with HLA-DRB1*04 alleles was found (2, 9, 10). Furthermore, HLA-DR4-restricted CD4+ T cells reactive with U1-RNP polypeptides including 70 kDa have been isolated from PBMC of MCTD and characterized in considerable detail (1114). We have shown that these human CD4+ T cells can provide help to anti-70-kDa autoantibody producing B cells, are restricted in Ag presentation by HLA-DR, have TCR fine specificity for peptides encoded within the RBD of 70 kDa, and have limited TCR-β (TRB) V and CDR3 usage (1117).

To further advance our current understanding of the pathogenesis of MCTD, we have developed a model of MCTD in mice that expresses a transgene (Tg) encoding the HLA-DR4 molecule (HLA-DRA*0101/DRB1*0401) by immunizing them with the p205 fusion protein of the 70-kDa polypeptide and its associated U1-RNA (18). These mice develop sustained anti-U1-RNP Abs following a single exposure to the 70-kDa polypeptide/U1-RNA autoantigen (1921). Further distinguishing the model, these mice develop pulmonary inflammatory infiltrates characteristic of MCTD but do not develop anti-Sm-Abs or anti-DNA Abs which distinguishes the model from SLE (24, 21).

In the present study, we characterized the peptide-TCR molecular interactions of 70-kDa-reactive CD4+ T cells in this novel model of MCTD, including characterizing the CD4+ T cell Ag fine specificities and TCR usage in 70-kDa autoantigen recognition. We found that similar to patients with MCTD, CD4+ T cells can be readily identified from HLA-DR4-Tg mice following a single exposure to 70-kDa/U1-RNA and that the majority of these T cells are specific for antigenic peptides encoded within the RBD of 70 kDa. Also similar to MCTD, we found by examining 70-kDa-specific T cell lines that TRBV usage was highly restricted among 70-kDa-reactive murine T cells. TCR from 70-kDa-reactive CD4+ T cells demonstrated selective use of TRBV subgroups as well as common structural CDR3 motifs across different TRBV subgroups. Finally, we found that TRBJ subgroups and TRB CDR3 used by human and murine CD4+ T cells were homologous.

Materials and Methods

Mice

C57BL/6Ntac-(KO)Abb-(Tg)DR-4 mice were purchased (Taconic Farms). The transgenic strain uses a hybrid MHC class II molecule composed of the peptide-binding domains of human HLA-DR4 and the membrane proximal domains of mouse I-E. Native MHC class II is not expressed as these have been genetically inactivated (I-E β and I-A α deficient). As the α2 and β2 domains of mouse MHC class II are preserved in the transgene, interactions with CD4 coreceptors on murine T cells are maintained (18). As previously reported the HLA-DR4-Tg mice in our colony have not developed serologic or clinical manifestations of spontaneous autoimmunity at up to 9 mo of age (19, 20).

70-kDa and U1-RNA Ags

Fusion protein for the 70-kDa Ag, as well as control maltose-binding protein, was produced and characterized as described previously (11, 17, 21). In brief, the p205 peptide of the 70-kDa protein, spanning amino acids 63–205, was expressed as a maltose-binding protein fusion protein in Escherichia coli, affinity purified over amylose columns and gel-purified. For ELISA studies, maltose-binding protein was cleaved from the 70-kDa peptide with Genenase (New England Biolabs), and the resulting 70-kDa product was gel-purified. The identity and purity of all products was confirmed by immunoblot against standard 70-kDa-recognizing sera, and each was confirmed for the absence of endotoxin contamination with a Limulus assay (Cambrex Bio Science). U1-RNA was produced by in vitro transcription of a Sp64 plasmid (Promega) containing a 165-base insert corresponding to the sequence of the human U1-RNA, as previously described (22). U1-RNA was tested for contamination with endotoxin as described above.

Immunization of mice

Mice were immunized s.c. once between 8 and 12 wk of age with 50 μg of a purified 70-kDa fusion protein containing 70-kDa residues 63–205 in 50 μl of PBS. Ags were instilled mixed with 50 μg of U1-RNA in 50 μl of sterile PBS. Blood and urine samples were collected immediately before immunization and monthly thereafter. Urine was tested for blood and protein using Multistix 10 SG Reagent Strips (Bayer). Except for animals that died or required euthanasia for humanitarian reasons before completion of the protocol, all mice were sacrificed at 2 mo after immunization. All mouse studies were approved by the Institutional Animal Care and Use Committee at the University of Miami Miller School of Medicine, and all animals were housed in American Association for Accreditation of Laboratory Animal Care-approved facilities.

ELISA

Anti-70-kDa and -maltose-binding protein assays were performed as previously described (4, 1921). Briefly, 96-well flat-bottom microtiter plates were incubated overnight at 4°C with purified Ag in PBS. After washing in PBS/0.05% Tween 20 buffer, plates were blocked with 3% powdered milk in PBS/Tween 20, incubated with mouse sera at final dilutions of 1/100, and developed with HRP-linked Fc region-specific goat anti-mouse-IgG secondary Ab followed by orthophenylenediamine. Absorbance at 450 nm was measured in a microtiter plate reader (Bio-Tek). Anti-dsDNA assays were performed using a commercial kit for murine dsDNA IgG (Alpha Diagnostic International), according to the manufacturer's instructions. All ELISA were performed in duplicate wells. Standard positive and negative control sera and test antisera were assayed on each plate.

Histology

Mice were euthanized by cervical dislocation after induction of anesthesia using isoflurane inhalation 2 mo after immunization, and organs were immediately harvested and fixed in UMFIX (20). Lungs were inflated with fixative at the time of harvest as previously described (20). Paraffin-embedded sections were stained using H&E or using a series of mAb, and imaged with a Nikon CoolPix 990 camera mounted on an Olympus BH2 microscope. All slides were graded at the same session, blinded to the mouse strain and immunization condition of the animals. Histologic assessments were confirmed by an additional independent blinded trained pathology review. For publication purposes, images were cropped using Microsoft Powerpoint; no additional image manipulation was performed.

Peptide design and synthesis

Peptides were synthesized using N-[9-fluorenyl]methoxycarbonyl solid-phase chemistry on the AAPPTEC Apex 396 or the Applied Biosystems 433A model peptide synthesizer. Peptides were analyzed for purity and sequence fidelity using HPLC and mass spectrometry, as described previously (17).

T cell isolation

Cells were purified from spleen and lymph nodes by mechanical separation and passage through a 100-μm mesh filter (BD Biosciences). Cells were treated with RBC lysis buffer (Sigma-Aldrich), washed in RPMI 1640 with 2 mM l-glutamine, supplemented with 20 μg/ml gentamicin and 10% FCS, and then passed over Histopaque 1083 (Sigma-Aldrich) for further purification of lymphocytes. Cells were taken from the Histopaque interface and washed in RPMI 1640 followed by an additional wash in MACS buffer (Miltenyi Biotec). The cells were then incubated with anti-CD4 mAb (BD Biosciences) coupled to iron beads (Miltenyi Biotec) for 15 min in MACS buffer. Following incubation of the cell-Ab-bead conjugates, cells were positively selected on an AUTOMACS cell separator (Miltenyi Biotec) magnetic column and their purity was confirmed by flow cytometry; AUTOMACS isolation typically yielded CD4+ T cells of >95% purity. Highly purified CD4+ T cells were resuspended at the appropriate concentration and used in proliferation experiments as described below.

Murine lung T cell isolation

T cells were obtained at necropsy from lung tissue by mechanically disrupting the tissue, following which it was filtered through a sterile 100-μm nylon mesh filter and then subjected to density gradient centrifugation using Histopaque (Sigma-Aldrich). Purified CD4 cells were stimulated with plate-bound anti-CD3 and mRNA was isolated as described (16). All T cells studied were from mice with histologically confirmed pulmonary inflammatory infiltrates.

Murine T cell lines

Murine T cell lines used in these studies were generated and characterized using an approach similar to that which has been used extensively for generating human autoantigen-specific T cell lines and clones with slight modifications (1113). In brief, spleen and lymph node cells were obtained at necropsy, mechanically disrupted, filtered through a sterile 100-μm nylon mesh filter and then subjected to density gradient centrifugation using Histopaque (Sigma-Aldrich). Cells obtained from immunized mice as responder cells were used immediately in proliferation or for the generation of T cell lines. Cells obtained from naive mice were irradiated with 30 Gy and used as APC to stimulate T cells and in proliferation assays. Approximately 5 × 106 cells were cultured in DMEM with 2 mM l-glutamine (complete medium), supplemented with 20 μg/ml gentamicin, 15% FCS, and containing fusion protein at a final concentration of 50 μg/ml. As Ag, 70-kDa fusion protein was used, as described (1113, 17). 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 × 106 murine APC that had been irradiated with 30 Gy plus Ag in fresh medium on days 7–10. Following two cycles of stimulation, cells were rested for 10 days and then either tested in a standard proliferation assay or stimulated with plate-bound anti-CD3 with 20 U/ml IL-2 in complete medium and used for TCR analysis. RNA was extracted from the cells which had been stimulated with plate-bound anti-CD3 in the absence of APC at 48 h for TCR analyses using RNeasy (Invitrogen).

Proliferation assay

Approximately 2 × 104 CD4+ T cells from immunized mice were cultured with ~1 × 105 APC in complete medium. APC were obtained from naive DR4-Tg mice which had been irradiated with 30 Gy. T cells and APC were subsequently incubated for 48 h in 96-well flat-bottom tissue-culture plates in the presence of Ag or medium control (MC). Cells were then pulsed for an additional 18 h with 1 μCi/well [3H]TdR. Cells were harvested and [3H]TdR incorporation detected by liquid scintillation counting (17).

Flow cytometry

The TRBV expression on T cells was identified using flow cytometry and a series of group-specific mAbs (mouse VB TCR screening panel, Technical Bulletin 557004, www.bdbiosciences.com and Ref. 23). Briefly, following density gradient separation cells were stained using mAb specific for TCR VB 2, 3, 4, 5.1, 5.2, 6, 7, 8.3, 9, 10, 11, 12, 13, 14, and 17a (BD Biosciences) and counterstained with anti-CD4-PE (BD Biosciences). Isotype controls were conjugate coupled IgG2b (BD Biosciences) and IgG2b (BD Biosciences). Cells were enumerated on a LSRII cytometer and results were analyzed using FACSDiva software (BD Biosciences). Following initial gating on the lymphocyte population, cells were enumerated for those which were CD4+ with each of the group-specific TRBV mAbs. The absolute number of each cell subset was calculated based on the absolute number of CD4+ cells and the total lymphocyte count.

PCR analysis of TCR

mRNA was extracted from cell pellets using an oligo(dT)-cellulose mi-croaffinity column adsorption method (MicroFastTrack 2.0; Invitrogen). First-strand synthesis of cDNA was conducted using reverse transcriptase and RNaseH (SuperScript First-Strand Synthesis System for RT-PCR; In-vitrogen Life Technologies). Aliquots of cDNA were PCR amplified in the presence of primers specific for 1 of 22 TRBV groups of the TCR (Table I) (1517, 23, 24). As a positive control, the amplification of a portion of the TCR-α C region was conducted in parallel. As described previously, extensive precautions were taken to ensure that cross-contamination of samples did not occur (1517). The amplified DNA was subjected to gel electrophoresis in 3% Nusieve, 1% SeaKem agarose and visualized by staining with ethidium bromide. PCR-amplified fragments were purified using the QIAquick PCR purification kit (Qiagen) and cloned into pCR-Blunt II-TOPO vector using the Zero Blunt TOPO PCR cloning kit with One Shot chemically competent E. coli (Invitrogen). DNA sequencing was performed as described previously (10, 1517).

Table I
TRBV primers

GenBank accession numbers

Accession numbers assigned to novel TCR gene sequences identified in this study were EF457520-EF457550 and EU518945-EU519008.

Statistical analysis of data

The Graph Pad Prism software package was used to analyze data.

Results

70-kDa/U1-RNA-immunized HLA-DR4-Tg mice develop high-titer IgG anti-70-kDa autoantibodies following immunization

Following immunization with 70-kDa/U1-RNA, mice developed high-titer IgG reactivity with 70-kDa as detected by ELISA and immunoblotting. Fig. 1 illustrates the presence of IgG anti-70-kDa specific Abs as detected by immunoblotting of sera from 70-kDa/U1-RNA-immunized HLA-DR4-Tg mice. Shown are typical immunoblotting results using sera diluted 1/5000 from 70-kDa/U1-RNA-immunized HLA-DR4-Tg mice to probe Jurkat cell extract. Sera from immunized mice but not naive mice had high levels of specific IgG reactivity with the 70-kDa protein. The IgG reactivity against 70 kDa, as detected by immunoblotting and ELISA, was typically detectible within 1 mo following immunization and continued to rise over time with the highest levels present at the terminal bleeding of immunized mice.

FIGURE 1
Immunoblot of sera from immunized mice and controls. IgG anti-70-kDa Abs detected by immunoblotting of Jurkat extract using sera from 70-kDa/U1-RNA-immunized HLA-DR4-Tg mice which were diluted 1/5000. Lane 1, A positive control serum; lane 2, A serum ...

70-kDa/U1-RNA-immunized HLA-DR4-Tg mice develop T cell infiltration of the lungs similar to MCTD

Fig. 2 illustrates the characteristic histologic findings in HLA-DR4-Tg mice immunized with 70-kDa/U1-RNA including the presence of substantial numbers of T cells (20). Fig. 2A illustrates H&E staining of representative lung tissue demonstrating the characteristic perivascular and interstitial mononuclear cell infiltration of the lung; Fig. 2B is a section corresponding to A which was stained with enzyme-coupled anti-CD3 illustrating immunohistochemical findings of CD3+ T cell staining of lung tissue at sites of inflammation as seen on H&E staining. Fig. 2C illustrates a representative section of lung from a naive unim-munized HLA-DR4-Tg mouse showing the absence of significant tissue staining. This demonstrates that immunized HLA-DR4-Tg mice developed T cell-enriched perivascular and interstitial mononuclear cell infiltration of lung tissue similar to that found in involved human lung from MCTD patients (R. W. Hoffman, unpublished observations; and Refs. 2 and 3).

FIGURE 2
Lung histologic findings in the 70-kDa/U1-RNA-immunized HLA-DR4-Tg murine model. A, H&E staining of representative lung tissue demonstrating mononuclear cell infiltration of the lung. B, Section of lung corresponding to A which was stained immunohistochemically ...

Immunized HLA-DR4 Tg mice develop 70-kDa-reactive CD4+ T cells

To determine whether 70-kDa-reactive CD4+ T cells could be detected from 70-kDa/U1-RNA-immunized mice, purified CD4+ T cells were incubated with a series of pooled, overlapping peptides, 15 residues in length (shown in Table II), spanning the 70-kDa protein in the presence of irradiated syngeneic APC. [3H]TdR incorporation was measured and a stimulation index calculated for each sample. CD4+ T cells reactive with 70-kDa peptides could readily be detected in all mice studied. Representative results from three of six mice are shown in Fig. 3. Murine CD4+ T cells proliferated consistently to pools 1, 4, 9, and 10, while individual mice variously responded to pools 5, 7, and 11. Although clear findings of CD4+ T cell-proliferative responses to 70 kDa were obtained, the high background levels of proliferation of T cells from some mice made precise interpretation of T cell epitope mapping results difficult using the peptide pools.

FIGURE 3
Murine CD4+ T cells proliferate to 70-kDa Ag-peptide pools. Shown are results from three mice using purified CD4+ T cells incubated with a series of individual overlapping peptides spanning the 70-kDa protein in the presence of irradiated syngeneic APC. ...
Table II
Peptide pool sequences

70-kDa-reactive T cells target RBD on Ag

To confirm findings from experiments using pooled peptides and to more precisely determine the T cell epitopes recognized by CD4+ T cells from immunized DR4-Tg mice, purified CD4+ T cells were incubated with a series of individual peptides spanning the entire 70-kDa protein and overlapping each other in sequence, in the presence of irradiated syngeneic APC. [3H]TdR incorporation was measured and results shown as stimulation indices. Representative results from three mice are shown in Fig. 4. These three representative results (n = 6) confirmed results from experiments using pooled peptides that there were at least seven regions which could be reliably identified as reactive with CD4+ T cells in immunized mice. The majority of T cell epitopes resided within the RBD (shown in brackets) of the 70-kDa protein. There were a limited number of T cell epitopes which resided in regions within either the N or C terminus of the 70-kDa protein and which were outside the RBD that were identified in some mice.

FIGURE 4
Murine CD4+ T cell response to individual 70-kDa peptides. Purified CD4+ T cells were incubated with a series of individual peptides, overlapping in sequence, spanning the entire 70-kDa protein in the presence of irradiated APC. The [3H]thymidine incorporation ...

70-kDa epitope 2 is the most frequently recognized T cell epitope in immunized HLA-DR4-Tg mice

To further define the frequency of T cell epitope reactivity against different regions of 70 kDa in immunized HLA-DR4-Tg mice, we examined the T cell proliferative responses in a panel of 10 mice to the five dominant T cell epitopes previously identified in MCTD patients which were termed epitopes 1–5. Freshly isolated CD4+ T cells from all mice demonstrated a proliferative response to 70-kDa Ag. As summarized in Table III, we found that similar to MCTD CD4+ T cells from immunized mice recognized all five T cell epitopes within the RBD although with slightly different frequency than that found in the small group of MCTD patients previously studied (17).

Table III
Epitopes on UI-70-kDa autoantigen recognized by CD4+ T cells

TRBV subgroups use in CD4+ T cells from 70-kDa-immunized mice is restricted to select TRBV subgroups and CDR3 usage appears nonrandom

We next examined TCR usage by T cells reactive with 70-kDa autoantigen in the context of HLA-DR4 in the Tg mice to determine whether TRBV usage by 70-kDa-reactive CD4+ T cells was randomly distributed or showed discernable bias of either TRBV subgroup or CDR3 usage. Previously, we had found in MCTD patients that the TRBV gene usage in 70-kDa-specific CD4+ T cell clones was not randomly distributed and that certain TRBV structural motifs were more common among patients compared with controls (16). To identify TRBV used by CD4+ T cells in the MCTD murine model, we first performed two-color flow cytometry on T cells stained for CD4 that were also stained with individual mAbs specific for TRBV used by the C57BL/6 strain of mice: BV 2, 3, 4, 5.1, 5.2, 6, 7, 8.3, 9, 10, 11, 12, 13, 14, or 17. Table IV summarizes the TRBV gene usage for BV 2, 3, 4, 5.1, 5.2, 6, 7, 8.3, 9, 10, 11, 12, 13, 14, and 17 found using splenic T cells. Using flow cytometry to analyze TRBV genes from splenic T cells, it was observed that there were slight variations between immunized and naive mice with increases in TRBV 4 and 8.3 and decreases in TRBV6, 7, and 10, but these differences were not statistically significant for either the percentage or absolute number of CD4+ T cells (Table IV).

Table IV
TRVB expression in CD4+ T cells from immunized and naive mice

We also examined TRBV used by T cells isolated from lymph nodes of immunized and control unimmunized mice and found no statistically significant differences (data not shown). Next, we performed RT-PCR using TRBV group-specific primers to examine TRBV usage compared with the flow cytometry results and to interrogate those TRBV subgroups where TCR group-specific mAbs were not available. Findings by RT-PCR were similar to those obtained by flow cytometry with no discernable expansion of any TRBV subgroup (data not shown). Although the difference between immunized and unimmunized mice was not statistically significant, such an approach at the bulk cell level might not be adequate to identify clonal expansion by a highly restricted set of T cells.

We next examined TRBV distribution from short-term CD4+ T cell lines which were generated in vitro using 70-kDa Ag stimulation. Using this approach, we found that the short-term CD4+ T cell lines selectively used TRBV1, VB2, VB6, and VB 8.1/8.2/8.3. We then cloned and sequenced TCR from these lines and found that the CDR3 were highly restricted (Table V), characteristic of an Ag-driven T cell response. As controls, randomly cloned and sequenced CDR3 from unselected CD4+ T cells from spleen were examined. Unlike the short-term T cell lines, T cells from unfrac-tionated whole spleen did not demonstrate any identifiable skewing of structural motifs or CDR3 length (data not shown).

Table V
TCR deduced amino acid sequence from CD4+ T cells isolated from U1-70-kDa-immunized HLA-DR4-transgenic mouse spleen

TRBV use by CD4+ T cells from 70-kDa-immunized HLA-DR4-Tg mice demonstrates homology to TCR use by human T cell clones from MCTD patients

To determine whether there was a relationship between TCR usage by 70-kDa-reactive human and murine T cells, we compared TRBV use by human CD4+ T cell clones reactive with 70-kDa protein with those of TRBV derived from short-term CD4+ T cell lines derived from HLA-DR4-Tg mice. As we have in part reported previously, sequences from 18 individual human T cell clones derived from patients with and without HLA-DR4 revealed three unique TRBV sequences (17). When these were compared with the unique murine CD4+ TRBV sequences significant homology was identified (see Table VII). The murine sequences with the greatest homology are shown aligned to the human sequences and the regions of shared sequence identity are underlined (see Table VII). Also, it was notable that TRBJ2S7 was highly represented in both the human (7 of 18; 56%) and murine spleen (24 of 115; 21%) TCR identified.

Table VII
Alignment of deduced amino acid sequences of human and murine TCR from 70-kDa T cells

TRBV use by CD4+ T cells from 70-kDa-immunized HLA-DR4-Tg mouse lungs demonstrates homology to TCR use by murine splenic CD4+ T cells

Although murine 70-kDa-specific T cell lines demonstrated TRBV and CDR3 homology with TCR from human 70-kDa-reactive T cell clones, it remained possible that this could be attributable to antigenic selection and expansion in vitro. To more directly address this possibility, CD4+ T cells were isolated directly from the lungs of mice found to have pulmonary inflammation histologically (17). Lung tissue mRNA was extracted and TRBV usage was characterized by RT-PCR and DNA sequencing using group-specific primers. When the human sequences were compared with murine CD4+ TRBV sequences from lung tissue, significant homology was identified (Tables VI and VII) (16). The murine sequences with the greatest homology are shown aligned to the human sequences and the regions of shared sequence identity are underlined (Table VII). As was seen in splenic tissue (TRBJ2S7 present in 24 of 115 (21%) of isolates), it was again notable that TRBJ2S7 was highly represented in both the human (7 of 18; 56%) and lung (19 of 81; 24%) TCR identified (Table VII).

Table VI
TCR deduced amino acid sequence from CD4+ T cells isolated from U1-70-kDa-immunized HLA-DR4-transgenic mouse lung

Discussion

HLA-Tg mice immunized with a specific autoantigen have been reported as potentially powerful models for dissections of molecular interactions between defined autoantigen-MHC complexes and the TCR (18, 2528). Such an approach holds substantial promise for the detailed analysis of TCR-antigenic peptide-human MHC interactions in those unique instances where the autoantigen is known and structurally well-characterized such as MCTD.

We have developed a new murine model of systemic autoimmunity, using HLA-DR4-Tg mice, which is induced following a single immunization with the 70-kDa polypeptide of U1-RNP and its associated U1-RNA. Immunization induced high levels of IgG anti-U1-RNP Abs characteristic of MCTD which increased over time without additional boosting; however, these immunized animals did not develop anti-dsDNA or anti-Sm autoantibodies which are characteristic of SLE and are found in murine models of SLE (19, 20).

In addition to the development of high titers of autoantibodies, 70-kDa/U1-RNA-immunized HLA-DR4-Tg mice develop additional features characteristic of MCTD. A distinguishing feature of the model is that mice develop pulmonary inflammation, including mononuclear cell perivascular and interstitial infiltration of the lungs which typifies MCTD but is uncommon in SLE (2, 3). Pulmonary infiltrates in mice contained a substantial proportion of lymphoid cells including T cells (Fig. 2). These finding are similar to those in pulmonary tissue in MCTD patients where T lymphocytes are prevalent (R. W. Hoffman, unpublished observations; and Refs. 2 and 3). Furthermore, in preliminary studies, we have found that adoptively transferred CD4+ T cells alone or in conjunction with B cells can transfer disease to naive mice (29). While beyond the scope of the present report, this is currently an area of active investigation in our laboratory.

To more fully define the nature of the autoantigenic peptides recognized by CD4+ T cells in the model, we have tested the proliferative responses of CD4+ T cells isolated from immunized mice against a large panel of synthetic peptides spanning the 437-residue 70-kDa polypeptide (8, 17). As shown in Figs. 3 and and44 and summarized in Table III, we found that the T cell proliferation could readily be detected using CD4+ T cells from whole spleno-cytes and lymph nodes. T cell epitopes recognized by CD4+ T cells from mice were similar to those previously identified in MCTD patients in that most resided within the RBD of 70 kDa (8). There was also limited immune spreading to T cell epitopes outside of those contained within the immunizing fragment of 70 kDa to residues contained within the N and C terminus of the full-length molecule. Thus, while immune spreading can occur, the T cell response in the murine model is similar to human MCTD and remains focused primarily on the RBD.

The importance of the RBD as a shared target of autoimmune B cell responses to complex nuclear Ags has been emphasized by Monneaux and Muller, and Mamula et al. (30, 31). The RBD has also been identified as a dominant T cell epitope in nonautoim-mune-prone CBA normal mice immunized with 70 kDa, as well as in the MRL/n and MRL/lpr spontaneous murine models of SLE, and among T cells reactive with 70 kDa isolated from patients with MCTD (17, 32, 33). The mechanistic basis of T cell responses focusing on the RBD could reflect indirect influences of B cell epitope selection and Ag presentation to T cells, preferential Ag protection/processing of the RNA-RBD complex by dendritic cells or other APC, or other mechanisms (17, 31). Interestingly, Muller and colleagues (33) have demonstrated that immune tolerance to 70 kDa can be restored in lupus prone mice through immunization using peptides derived from the RBD; therefore, understanding the molecular basis of these interactions may have important implications for future T cell directed therapy.

Toward the goal of further defining the molecular details of the interactions between the TCR and the 70-kDa autoantigen, TCR use by 70-kDa-reactive T cells was examined using several approaches. We discovered that TRBV usage by CD4+ T cells was restricted to a select subgroup of TRBV genes (Tables V and andVI)VI) and that common structural motifs could be identified within the CDR3 across the different TRB subgroups identified. The use of selective TRB or TRA genes has been described in a number of autoimmune diseases including SLE, autoimmune thyroiditis, multiple sclerosis, ulcerative colitis, and polymyositis (1416, 3438). These studies suggested that even for a structurally complex autoantigen, which has the potential to generate a large number of peptide-TCR interactions, the T cell response in these autoimmune diseases may be focused on a surprisingly limited number of epitopes and/or use a limited number of TCR motifs. When we compared the TRB and CDR3 from 70-kDa-reactive CD4+ T cells in HLA-DR4-Tg mice with those we have previously identified from MCTD patients, we found striking similarities between TCR use in MCTD and the murine model (Table VII).

Few previous studies have been published examining detailed molecular interactions between an autoantigen and the TCR and even fewer have directly compared findings in the murine model with human disease. One of the most striking findings of the present study was the fact that highly restricted TRBV genes used in the model were highly similar to those found in MCTD patients (Table VII) (16). Common TCR use of TRBV and select CDR3 in the model was homologous to TCR use in MCTD (16). The similarity of this and other aspects of the model to human disease validates its relevance to human disease pathogenesis and helps establish its potential future utility as a preclinical model for manipulation of T cell responses (39).

Acknowledgments

We gratefully acknowledge the excellent technical assistance of Judith Pignac-Kobinger and Irina Fernandez.

Footnotes

1This work was supported by grants from the National Institutes of Health, Lupus Foundation of America, Lupus Research Institute, and Department of Veterans Affairs.

3Abbreviations used in this paper: MCTD, mixed connective tissue disease; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; RBD, RNA-binding domain; Tg, transgenic; TRB, TCR-β; MC, medium control.

Disclosures: The authors have no financial conflict of interest.

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