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The Journal of Immunology, 2002, 168: 5876-5884.
Copyright © 2002 by The American Association of Immunologists

HLA Class II Influences the Immune Response and Antibody Diversification to Ro60/Sjögren’s Syndrome-A: Heightened Antibody Responses and Epitope Spreading in Mice Expressing HLA-DR molecules1

Tawatchai Paisansinsup2,*,{ddagger}, Umesh S. Deshmukh2,{dagger},{ddagger}, Vaidehi R. Chowdhary*, Harvinder S. Luthra*, Shu Man Fu{dagger},{ddagger} and Chella S. David3,*,{ddagger}

* Division of Rheumatology, Department of Immunology, Mayo Clinic, Rochester, MN 55905; {dagger} Division of Rheumatology and Immunology, University of Virginia, and {ddagger} University of Virginia Specialized Center in Systemic Lupus Erythematosus, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sjögren’s syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61–185) and the carboxy portion (aa 381–525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281–315 and 401–538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)4 is a multisystem autoimmune disease characterized by the presence of autoantibodies and tissue damage from immune complex deposition (1). One of the hallmark features of SLE is the development of autoantibodies directed to multiple cellular components, including DNA, histones, small nuclear ribonucleoproteins (snRNPs), ribosomes, and the Ro/SSA and La/SSB RNP complex (2). The development of these autoantibodies is not a randomized process. They often arise in grouped, or linked, sets that target proteins which are associated physically in vivo as part of functional particles or colocalize within the same subcellular components (2, 3). The examples of these include anti-Smith Ags (Sm) and anti-RNP Abs directed to proteins which comprise spliceosome, anti-Ro, and anti-La Abs which bind a group of RNPs. Autoantibodies to the Ro/SSA and La/SSB RNPs are commonly found in sera of patients with SLE and Sjögren’s syndrome (SS) (4, 5, 6). Anti-Ro (SS-A) Ab immunoprecipitates a single antigenic protein of m.w. 60,000 (Ro60) and several small discrete RNAs from human cell extracts (7). Their presence is strongly correlated with distinguishable clinical subsets of SLE and SS including sicca, neonatal lupus, and subacute cutaneous lupus (8). Whether the Ro (SS-A) Ag is directly involved in initiating an autoimmune response is still not clear. Indeed, Ab to Ro is demonstrable in healthy individuals more than any other rheumatic autoantibodies (9).

The availability of recombinant proteins/peptides from snRNPs and the Ro/La-RNP complex has provided the opportunity to explore the underlying mechanisms by which these autoantibodies arise (10, 11, 12, 13, 14, 15, 16, 17, 18). The data from these studies suggest that diversification of these autoantibodies can be initiated by challenging the animals with a single subcomponent through a process called intramolecular and intermolecular epitope spreading within the physically associated multimeric complex (10, 11, 12, 13, 14, 17, 18). In addition, we have also demonstrated that the diversification of the autoantibodies beyond the nonphysically associated molecules can occur through Abs, which can recognize the conformational epitopes shared among these molecules (10, 11).

The question of what factor(s) governs the extent of epitope spreading has remained. The extent of intermolecular spreading of autoimmunity differs between various strains of mice, suggesting that genetic factors are important in modulating the patterns of autoimmune responses (12, 18). Corroborating with these data, there is a marked variation of autoantibody levels and specificities in SLE and SS patients and those patients with certain levels of autoantibody diversification remain stable in their autoantibody patterns when followed over time (19). The most likely genetic candidate controlling this serological phenomenon is HLA class II. Indeed, the association of HLA class II molecules with SLE is stronger for subsets of autoantibodies than with the disease itself (20).

Population-based studies have demonstrated the association of HLA-DR2, DR3, and DQ1/DQ2 with anti-Ro in the Caucasian population (6, 8, 20). The results from these studies can potentially be confounded by the coexpression of other class II molecules and the strong linkage disequilibrium among the HLA region genes (21). Moreover, the in vivo experiment to prove this finding is still lacking. To circumvent this difficulty, we used HLA class II transgenic mice in the absence of the endogenous mouse class II molecules. These animals provided an opportunity for a differential evaluation of different HLA class II molecules, particularly HLA-DR2, DR3, DQ6, and DQ8, to the production and diversification of Ab to human Ro60 (hRo60) protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The production and characterization of A{beta}0DR2 (DRB1*1502), A{beta}0DR3 (DRB1*0301), A{beta}0DQ6 (DQA1*0103/DQB1*0601), and A{beta}0DQ8 (DQA-1*0301/DQB1*0302) mice lacking endogenous class II molecules have been described previously (22, 23, 24, 25, 26, 27, 28). The HLA class II transgenic mice used in the present work were backcrossed to B10 mice for 10 generations, thus carry B10 genetic backgrounds. For convenience, these HLA class II transgenic mice will be called DR2, DR3, DQ6, and DQ8, respectively. All mice used in this study were bred and maintained in the pathogen-free Immunogenetics Mouse Colony at Mayo Clinic (Rochester, MN). The Institutional Animal Care and Use Committee approved the protocol of this study.

Monoclonal Abs

All of the mAb cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA). These included anti-DR mAb L227 (29) and anti-DQ mAb IVD12 (30).

Immunization

For in vitro lymph node cell (LNC) proliferative response, 8- to 12-wk-old HLA class II transgenic mice were immunized with 100 µg of rhRo60 emulsified in CFA (Difco, Detroit, MI) in one hind footpad and at the base of the tail. For analysis of Ab production, mice were immunized initially as described above. They were subsequently boosted i.p. with 50 µg of rhRo60 emulsified in IFA (Difco) on day 14, 30, 60, and 90. Controls were immunized with only adjuvant in a similar way. Tail bleeds were done at different time points postimmunization, and sera were assayed for specific Abs.

rAgs and synthetic peptides

The production and purification of r6XHis-tagged hRo60, 6XHis-tagged mouse Ro60 (mRo60), 6XHis-tagged mouse La (mla), and 6XHis-tagged group 2 allergen of Dermatophagoides pteronyssinus (Der p 2) were previously described (10). The sequence of synthetic overlapping peptides spanning the entire sequence of hRo60 were previously reported (10) and synthesized at the Peptide Core Facility at Mayo Clinic (Rochester, MN). The peptides are 17–25 aa in length and contain a 10 aa residue overlapping sequence with the previous peptide in the panel.

LNC proliferation assays

Two weeks after immunization, draining lymph nodes were removed and single-cell suspensions were prepared for in vitro cultures according to a previously described protocol (10).

ELISA

The purification of the hRo60 protein and its use in a solid phase assay for anti-hRo60 Abs was performed as previously described (10). Briefly, microtiter plates were coated with either rhRo60 (10 µg/ml) or overlapping peptides (20 µM) in carbonate buffer, pH 8.4, overnight at 4°C. The wells were subsequently blocked with 3% BSA in PBS for 2 h at room temperature (RT). Diluted sera (2-fold dilution started at 1/100 for anti-hRo60 Ab titration, and 1/100 for anti-peptide Abs) in 3% BSA were added to Ag-coated wells, incubated at 37°C for 1 h, and washed 5 times with PBS containing 0.05% Tween 20. Subsequently, wells were incubated with peroxidase-conjugated goat anti-mouse Ig (BD PharMingen, San Diego, CA) for 1 h, and washed. The 3,3',5,5' tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO) was added and the absorbance spectrum was determined with an automated spectrophotometer (Bio-Rad, Hercules, CA). Our preliminary studies suggested that, according to our immunization protocol, maximal Ab level was obtained at day 60. Sera at this day were used in most of the subsequent studies or stated otherwise. Controls were sera from mice immunized with only adjuvant. For anti-hRo60 IgG subclass analyses, bound mouse Abs were detected with alkaline phosphatase-conjugated goat anti-mouse IgG1, IgG2a, and IgG2b (Southern Biotechnology Associates, Birmingham, AL). Mouse mAbs of IgG1, IgG2a, and IgG2b subclasses reactive with hRo60 (kind gifts of Dr. J. Lewis, University of Virginia, Charlottesville, VA) were used as standards.

Immunoprecipitation (IP)

The cDNAs encoding mRo60 and mouse Ro52 (mRo52) were previously described (10, 11). They were subcloned into the pGEM7 vector and used for in vitro transcription and translation. The [35S]methionine (Perkin-Elmer, Wellesley, MA) -labeled hRo60, mRo60, and mRo52 proteins used for IP were generated by using the Quick TNT lysate kit (Promega, Madison, WI), following manufacturer’s instructions. Different amounts of diluted sera (in PBS) were incubated with 20 µl of protein A-Sepharose beads for 1 h at RT. The beads were washed once in 0.5 M NaCl/NET buffer (0.3% Nonidet P-40, 2 mM EDTA, 50 mM Tris-Cl, pH 7.4) followed by the addition of [35S]methionine-labeled proteins. After a 2-h incubation in the cold room, beads were washed three times with 0.5 M NaCl/NET buffer, followed by a wash in NET buffer (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl, pH 7.4). The precipitated proteins were separated on a 10% SDS-PAGE and visualized by autoradiography. The control sera included the Centers for Disease Control reference human anti-Ro sera (Atlanta, GA) which had been shown to immunoprecipitate hRo60, hRo52 (data not shown), and mRo60 (see Fig. 4Go below) in our preliminary studies, and sera from mice immunized with only adjuvant alone.



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  		FIGURE 4. IP of mRo60 by sera from HLA class II transgenic mice immunized with hRo60. Semiquantitation of immunoprecipitating anti-mRo60 Abs. Pooled sera obtained from different HLA class II transgenic mice at day 60 after the initial immunization with hRo60 were tested for immunoprecipitating reactivities to endogenous mRo60 (the first two rows). The numbers indicate the amount of sera used in the assays. The exposure time is similar for all the gels. The data represent the same results of two different experiments performed in different days. The control sera (the third row) included the Centers for Disease Control reference human anti-Ro sera which had been shown to immunoprecipitate mRo60 (shown), hRo60, hRo52 (data not shown), and sera from mice immunized with only adjuvant alone (DR2, DR3, DQ6, and DQ8).

 
Competitive inhibition and slot blots

Pooled sera from transgenic mice at appropriate dilutions were preincubated with either different concentrations of rhRo60 (0.2–5 µM) or rDer p 2 with a 6XHis-tag (control protein, 5 µM) and reactivities to mla were tested in slot blot as described previously (10, 11).

Immunofluorescence

Hep-2 cell line slides (Bio-Rad) were used as substrates for antinuclear Ab (ANA) detection. Sera diluted in PBS containing 1% BSA were added, incubated for 1 h at RT, and washed 3 times in PBS for 15 min. Bound Abs were detected with FITC-coupled goat anti-mouse IgG (Accurate Chemical and Scientific, Westbury, NY). Stained slides were washed three times in PBS for 15 min and examined under fluorescent microscope.

Statistical analysis

The titer of anti-hR060 was analyzed initially for difference by Kruskal-Wallis one-way ANOVA, followed by Dunn’s multiple comparison procedure (Sigma Stat, Chicago, IL) (31). Values of pcorr < 0.05 are considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The immune responses of HLA class II transgenic mice to hRo60 were directed to multiple T and B epitopes

To localize the T cell epitopes recognized by HLA-DR2, DR3, DQ6, and DQ8 molecules, transgenic mice were immunized with hRo60 protein and the draining LNCs were challenged in vitro with a panel of synthetic peptides spanning the entire sequence of hRo60 molecule (538 aa). In preliminary experiments, the peptide concentration of 20 µM provided the optimal recall of T cell responses in vitro (data not shown). This concentration was used in subsequent experiments.

As shown in Figs. 1Go and 2Go, dominant T cell epitopes, which are dependent on the HLA-D alleles, are located mainly in the amino portion (N), corresponding to aa 61–185, and the carboxy portion (C), corresponding to aa 381–525, of the hRo60 molecule. Although T cell epitopes recognized by DR2 and DQ6 molecules are present in both the N and C portions, T cell epitopes recognized by DR3 and DQ8 molecules are predominantly located in the C portion of the hRo60 molecule. Many of the peptides can recall a T cell proliferative response in two or more of the transgenic mice. Because of the lengths of the peptides, further studies with truncated peptides are needed to determine whether some of the epitopes are recognized by multiple DR and/or DQ alleles. Apart from these, there are peptides containing T cell epitopes specific for several DR and DQ molecules: peptide 241–265 contains a T cell epitope for DR3 molecule while peptides 61–105 and peptide 161–185 contain T cell epitopes for DQ6 and DR2, respectively. Fig. 2Go summarizes all Ro60 peptides containing T cell epitopes for one or more DR and/or DQ molecules.



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  		FIGURE 1. T cell epitope mapping on hRo60 protein in different HLA class II transgenic mice. In vitro LNC proliferative responses were recalled at day 14 after hRo60 immunization. Single-cell suspension from regional lymph nodes (3 x 105 cells/well) was incubated in triplicate with synthetic overlapping peptides at 20 µM concentrations for 96 h. [3H]Thymidine (0.5 µCi/well) was added during the last 16 h of culture. Cultured cells were harvested onto glass fiber filters and radioactivity was measured by liquid scintillation counting. Results are expressed as SI, a ratio of the mean triplicate cpm obtained with peptide and the mean triplicate cpm without peptides. Results are shown as mean triplicate SI ± SEM from four to five independent experiments conducted on different days. Peptides giving a SI >= 2 (line) are considered stimulatory.

 


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  		FIGURE 2. A summary of T cell epitopes on hR060 in the context of different HLA class II molecules. A, Schematic representation of multiple T cell epitopes of hRo60 (shown in Fig. 1Go) in relationship to the structural features of hRo60 (RNP-80, the RNA-binding motifs, and zinc finger). Peptides giving a SI of 2–4 ({square}) and a SI > 4 ({blacksquare}) are shown. B, A summary of T cell epitopes on hRo60 and their amino acid sequences. Peptides giving a SI >= 2 are shown.

 
The B cell epitopes on hRo60 protein were mapped by ELISA using the same panel of peptides (10). In contrast to the location of T cell epitopes, B cell epitopes are localized throughout the whole hR060 protein. Interestingly, the earliest dominant B cell epitopes (reactivities detected in sera on day 14 after immunization, Fig. 3Go, {blacksquare}) of all HLA class II molecules tested are localized in the mid portion of hRo60 corresponding to aa 281–315 and the C portion of hRo60 corresponding to aa 401–538 and remain dominant up to 3 mo after immunization (Fig. 3Go, {square}). In DQ8 mice, an additional dominant early B cell epitope is also present on peptide 1–25. The peptide reactivities diversified over time to involve the whole molecules of hRo60 protein in all HLA class II transgenic groups except for DQ6 whose peptide reactivities remain confined predominantly in the middle and C portion of the hRo60 protein, even up to 5 mo after immunization (data not shown).



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  		FIGURE 3. B cell epitope mapping on hRo60 in different HLA class II transgenic mice immunized with hRo60. Sera (1/100 dilution) from different HLA class II transgenic mice (8–10/group), at day 14 after the initial immunization with rhRo60 were pooled and reactivity to the overlapping synthetic peptides of hRo60 was determined by ELISA. Results are expressed as mean duplicated OD450 and are shown for sera obtained at day 14 ({blacksquare}) and at day 90 ({square}). Control sera from mice immunized with only adjuvant gave an OD450 of less than 0.17 (line).

 
HLA class II molecules determine the magnitudes of Ab responses to hRo60 and mRo60

ELISA was used with the rRo60 as the substrate to determine the magnitudes of the immune responses in the four strains of transgenic mice. Immune sera, obtained 60 days after the initial immunization, were used after a pilot study showed that the titers of the immune sera peaked at this time point. Individual sera were titered with the end points at which the OD readings were about three times greater than those for the sera from mice treated with adjuvant alone. The median titers were 25,600, 25,600, 4,800, and 102,400 for DR2, DR3, DQ6, and DQ8 transgenic mice, respectively. The difference between the median titers for DR2, DR3, and DQ8 transgenics and those for the DQ6 transgenic mice was statistically significant at the pcorr < 0.05 level. The difference between the titers for the DR2 and DR3 transgenics and those for the DQ6 transgenic mice was also statistically significant at the same level. IgG subclass analyses of the anti-hRo60 Abs did not show significant differences between different strains of mice except for an enhanced IgG2b response in DR3 mice (data not shown). The anti-Ro60 titers did not correlate with the titers against the 6XHis peptides. However, they did, in general, correlate with the pooled serum titers for individual Ro60 peptides (data not shown). These data indicate that the anti-Ro60 titers represent Ab titers against the core Ro60 sequence and not against the 6XHis peptides, Abs which may arise as a result of immunization against the rRo60 with a 6XHis tag.

All mice produced Abs which could immunoprecipitate both in vitro-translated [35S]-labeled hRo60 and mRo60; Fig. 4Go shows IP of mRo60 from a representative experiment. The pooled immune sera from all groups of mice precipitated various amounts of in vitro-translated [35S]-labeled mRo60, suggesting the presence of Abs that could also bind the native mRo60 molecules after immunization with rhRo60. Interestingly, despite the finding of the highest production of anti-hRo60 Abs in the DQ8 group, as detected by ELISA, the DR2 and DR3 groups produced the highest levels of immunoprecipitating Abs to mRo60 (Fig. 4Go) and hRo60 (data not shown). The DQ6 group produced the lowest amount of Ab responses (anti-hRo60 and anti-mRo60 Abs). No immunoprecipitating Abs were detected in sera from mice immunized with only adjuvant. Limited studies with individual sera showed similar results.

HLA class II molecules influence intermolecular Ab diversification induced by immunization with hRo60

To further evaluate the role of HLA class II molecules in Ab diversification after hRo60 immunization, sera from mice immunized with hRo60 were tested for reactivities to other RNPs by IP. All DR2 sera (8/8) and only one of DQ8 sera (1/10) strongly immunoprecipitated [35S]-labeled mRo52. There was no immunoprecipitating reactivity to mRo52 detected in DR3 (0/10) and DQ6 (0/10) groups. The results of sera from five representative mice from each immunized group of transgenics are shown in Fig. 5GoA. No immunoprecipitating reactivities to [35S]-labeled mouse A-RNP, SmB, and SmD were detected in the sera of any mice (data not shown).



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  		FIGURE 5. Intermolecular epitope spreading to Ro52 is readily detected in DR2 mice. A, IP of mRo52. Sera from individual HLA class II transgenic mice at day 90 after the initial hRo60 immunization were tested for immunoprecipitating Abs to in vitro-translated and [35S]-labeled mRo52. Each lane represents 2.5 µl of serum from an individual mouse. The data is representative of two different experiments performed on different days. B, The kinetics of immunoprecipitating anti-mRo60 and anti-mRo52 Abs in DR2 mice. Pooled sera at different time points after the initial immunization with hRo60 were tested for IP of [35S]-labeled mRo60 (upper panel) and [35S]-labeled mRo52 (lower panel). Each lane represents 2.5 µl of serum from an individual mouse. The exposure time is similar for all the gels. Lane 1 is only protein A beads incubated with labeled proteins; lanes 2–6 are sera at days 0, 14, 30, 60, and 90, respectively; lane 7 is blank; lane 8 is day 90 pooled sera from mice immunized with only adjuvants. The results are representative of two different experiments performed on different days.

 
To determine the kinetics of anti-Ro52 Ab generation in the DR2 mice, pooled sera of each mouse at different time points were tested in IP. As shown in Fig. 5GoB, anti-mRo60 immunoprecipitating reactivity was first detected at day 30 postimmunization and increased at later time points. However, anti-mRo52 immunoprecipitating reactivity was detected at day 60 after the initial immunization. The different kinetics between anti-mRo60 and anti-mRo52 immunoprecipitating reactivities and the lack of inhibition of anti-mRo52 by mRo60 proteins (data not shown) strongly indicate the true intermolecular epitope spreading induced specifically in HLA-DR2 transgenic mice.

Despite our exhaustive experiments, we were unable to detect autoantibodies reactive with La-RNP either by IP using [35S]-labeled mla or by Western blotting using WEHI 7.1 cell extracts, in the sera of any mice (data not shown). We thus used slot-blot analysis using recombinant mla with 6XHis-tag as a substrate. Abs reactive to mla were detected in all strains of mice by day 30 postimmunization (data not shown). To determine, whether this reactivity represented epitope spreading, the ability of rhRo60 to inhibit the binding of immune sera to mla was determined and data from a representative experiment using pooled sera obtained 90 days postimmunization are shown in Fig. 6Go. In DR2 mice, preincubation of sera with rhRo60 did not entirely inhibit Ab reactivity to mla, despite complete inhibition of Ab reactivity to mRo60. In DR3 and DQ8 mice, rhRo60 in a dose-dependent manner, completely inhibited the binding of Abs to both mRo60 and mla. In DQ6 mice, reactivity to mla was not detected at the dilution of sera used for competitive inhibition. The rDer p 2, with a 6XHis-tagged protein used as a control, inhibited minimal Ab reactivity to mla. These data suggest that in DR2, DR3, and DQ8 mice, cross-reactive Abs between the immunogen and mla were generated. Although some of these Abs recognized the common 6XHis-tag, others recognized cross-reactive epitopes between mla and mRo60 as we described previously (11). Only in DR2 mice were specific Abs to mla detected.



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  		FIGURE 6. Analysis of anti-mla Ab reactivity in HLA class II transgenic mice. Pooled sera from HLA class II transgenic mice immunized with hRo60 were diluted appropriately and preincubated with different concentrations of either rhRo60 (0.2 to 5 µM) or rDer p 2 (5 µM). Reactivity of untreated and treated sera with mRo60 and mla was tested in slot blots. Note in DR2 mice that although reactivity to mRo60 is completely inhibited, reactivity to mla persists. Control sera at similar dilutions from HLA class II mice immunized with only adjuvant did not show any reactivity to mRo60 or mla.

 
Immunization with hRo60 induces ANAs in HLA class II transgenic mice

Although none of the animals developed clinical illness during several months of observation (up to 12 mo after the initial immunization; data not shown), ANAs could be detected in mice immunized with hRo60 when determined by immunofluorescence (Fig. 7Go and Table IGo). Three patterns of ANAs, namely speckled (Fig. 7GoB), cytoplasmic (Fig. 7GoC), and homogenous (Fig. 7GoD), were found in various numbers of mice immunized with hRo60 but were undetectable in mice immunized with only adjuvants (Fig. 7GoA) or in preimmune sera (data not shown). The pattern of ANA is not uniform. Even among the same group of HLA class II transgenic mice, different ANA patterns were found. The different rates of appearance and the incidences of ANAs (Table IGo) concur with the data showing different immune responses to hRo60 in HLA class II transgenic mice as described above. Although all DR2, DR3, and DQ8 mice developed ANAs by 4 mo after the initial immunization, only 20% of DQ6 mice developed ANA. Indeed, the incidence of ANA in the DQ6 group was not increased when followed up to 6 mo after the initial immunization while all of the mice in other groups still maintained their presence of ANA. There was no detectable anti-dsDNA Abs as detected by the anti-Crithidia luciliae in any of the immunized mice. None of the naive or immunized mice produced anti-ssDNA Abs (data not shown).



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  		FIGURE 7. The development of ANAs following immunization with hRo60. Sera obtained at different time points after immunization with hRo60 were used at 1/40 dilution for ANA detection using Hep-2 cell lines as substrates. The representative results of sera from DR3 mice at day 60 after immunization with hRo60 are shown. A, Sera from mice immunized with only adjuvant. B, C, and D, Sera from different mice immunized with hRo60.

 

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  		Table I. The incidence and pattern of ANAs in HLA class II transgenic mice immunized with hRo60

 
There is lack of correlation between class II Ag expression and the anti-Ro60 responses in the transgenics

We evaluated the level of surface expression of HLA class II molecules in the four transgenics by flow cytometry using mAb L227 (anti-DR) and IVD12 (anti-DQ). In the DR transgenic groups, there is a comparable level of expression of HLA-DR in PBMCs (DR2: 23.78 ± 4.9%, DR3: 20.9 ± 7.7%, p = NS). The level of HLA-DQ surface expression in PBMCs is approximately two and one-half times the level of HLA-DR surface expression (DQ6: 53 ± 7.1%, DQ8: 56 ± 9.2%, p < 0.001 compared with the DR groups). Despite the higher levels of DQ expression, the DR transgenics produced much more precipitating Abs and the DQ6 transgenic mice produced the least amount of anti-Ro60 Abs measured by both ELISA and IP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we used the MHC class II knockout mice expressing HLA-DR2, DR3, DQ6, and DQ8 molecules to map T cell and B cell epitopes and to study the role of these particular HLA class II molecules in controlling the immune response to hRo60 protein. The data from this study has validated the use of this animal model to prove that HLA class II is the important molecule, which controls not only the magnitude but also the diversification of the immune response to hRo60. This is also the first report of T cell epitopes of hRo60 in the context of specific HLA class II molecules.

We previously reported the T cell epitopes of hRo60 in several mouse strains (10). In this study, using synthetic overlapping peptides spanning the whole hRo60 molecule, we are able to detect T cell epitopes in multiple regions of the hRo60 molecule. Consistent with the T cell epitope data in mice, the C portion of hRo60 (aa 381–525) contains multiple common T cell epitopes recognized by all HLA class II molecules examined (Figs. 1Go and 2Go) suggesting that this region is highly immunogenic and naturally processed. Additionally, few dominant T cell epitopes which DR2 and DQ6 molecules can recognize are present in the regions within or near the RNA-recognition motif (RNP-80) in the N portion of the hRo60 molecule (aa 61–185). In contrast, peptide 241–265, the only peptide in the middle portion of hRo60, gave a weak proliferative response (stimulation index (SI) = 2.5) and contains a unique T cell epitope for the DR3 molecule. Interestingly, most of these peptides containing T cell epitopes could readily elicit variably weak responses (SI = 2–3) in the naive HLA class II transgenic mice (data not shown) indicating incomplete negative selection of these potential autoreactive T cells in these animals.

Previous studies have variably identified B cell epitopes of hRo60 from sera of patients with SLE and SS (32, 33, 34, 35, 36, 37, 38, 39). The discrepancies probably reflect the different populations studied and the types of assays and Ags selected. Although the Ag(s), which initiates the anti-Ro remains to be determined, our B cell epitope mapping data suggested that HLA class II molecules could be another important factor responsible for the discrepancies. Peptide reactivities were found in multiple regions throughout the whole hRo60 molecules in the DR2, DR3, and DQ8 groups while peptide reactivities in the DQ6 group is limited only to the middle and C portions (Fig. 3Go). In addition, peptide 1–25 gave a very strong reactivity early during the immune response in the DQ8 group but only weak reactivities in the DR2 and DR3 groups, and was nonreactive in the DQ6 group. Despite these different reactivities, the positions of major early hRo60 B cell epitopes are consistent and in agreement with the data derived from patients. A major region in the middle portion of hRo60 (aa 155–326) has been identified in six of seven studies using anti-Ro positive sera from patients with SLE and SS (32, 33, 34, 35, 36, 37). Our identified dominant B cell epitopes corresponding to aa 281–315 lie within this region. This region is the most hydrophilic and presumably the exposed part of the protein (38). In contrast, this region contains no T cell epitopes recognized by any HLA class II molecules examined in this study indicating that this region may not be naturally processed. It is thus conceivable that the reactive Ab-producing B cells of peptides 281–315 most likely received help from T cells recognizing other dominant T cell epitopes.

A previous study has shown that the significantly low level of class II surface expression (7–15% of control) can impair Ag presentation, thus influencing cellular immune response (40). However, our data strongly suggest that the type of HLA class II molecule, rather than the quantity of its expression, influences the immune responses to hRo60. Indeed, the levels of HLA class II expressions in these mice are enough to induce the selection of CD4+V{beta}TCR+ cells and to restore the CD4+ T cell population in the periphery, which are sufficient to elicit in vitro Ag proliferation and to induce clinical diseases in several animal models reported previously from our laboratory (27, 41). There is a hierarchy of immune responses to hRo60 within the groups, which have a comparable level of HLA class II expression. The DQ8 group elicited much higher responses than the DQ6 group despite the comparable levels of HLA class II expression. In addition, the stronger immune responses in the DR2 and DR3 groups compared with DQ6, despite lower levels of HLA class II expression, highlights the importance of the type of HLA class II molecule in mediating the immune response to hRo60. The lowest immune response of the DQ6 group is not universal. The mice carrying this HLA-DQ6, but not HLA-DQ8, have been demonstrated as the allergen-susceptible mice models reported previously (41). The difference in immune response to hRo60 is also not attributed to transgene-related influence in hemopoiesis because T and B cell maturation and monocyte numbers are comparable among all backcrosses (data not shown).

Several population-based studies have demonstrated the significant association of anti-Ro Abs, with or without anti-La, with HLA-DR3 and/or HLA-DR2 in Caucasians (6, 8, 20). Moreover, the highest level of anti-Ro Abs and the presence of anti-Ro and anti-La are likely to be found in patients who are heterozygous for the serologically defined HLA-DQ1 and HLA-DQ2 (6). Subsequent molecular analysis has revealed that the presence of glutamine residue at position 34 of the outermost domain of the DQA1 chain and/or a leucine at position 26 of the outermost domain of DQB1 chain is associated with anti-Ro (42). Despite the lack of information regarding which Ag(s) triggers the development of anti-Ro, our data using hRo60 protein as the immunogen in HLA class II transgenic mice have provided the experimental evidence to support the association of HLA-DR2 and HLA-DR3 with the development of anti-Ro Abs. The finding of strong immune response in HLA-DQ8, but not HLA-DQ6, transgenic mice is not unexpected. HLA-DQ8 (DQB1*0302) is found in both Caucasians and African-Americans with anti-Ro lacking alleles comprising HLA-DQ1 and HLA-DQ2 (42). In addition, HLA-DQ8 (DQB1*0302), but not HLA-DQ6 (DQB1*0601), transgenic mice in this study contained leucine at position 26 of the outermost domain of DQB1 chain (43), which has been postulated to be associated with anti-Ro Ab production.

The evidence from the previous study has suggested that the anti-Ro response to the 60 kDa molecule is driven by the native 60 kDa (44). Thus, we evaluated whether mice immunized with hRo60 could generate an anti-Ro response recognizing the native endogenous mRo60 by IP. Our data clearly showed that all mice immunized with hRo60 not only produced anti-Ro responses to the immunogen (recombinant and native molecules), but also anti-Ro Abs which could recognize the native endogenous mRo60, suggesting possible pathological relevance and providing us the chance to use these animal models to study the role of HLA class II molecules in controlling the autoantibody diversification. The data showed a strong correlation between high levels of anti-mRo60 (Fig. 4Go) and anti-hRo60 (data not shown) immunoprecipitating Abs and HLA-DR2 and HLA-DR3, while HLA-DQ6 consistently produced the lowest level of anti-Ro response. In addition, we have also found an exclusive association of the appearance of anti-mRo52 immunoprecipitating Abs with HLA-DR2 and much less of an appearance with HLA-DQ8, but not with HLA-DR3 and HLA-DQ6 (Fig. 5GoA). Our kinetic studies of the development of anti-mRo52 (Fig. 5GoB) and the inhibition studies (data not shown) strongly suggested that there was a true intermolecular epitope spreading at day 60, 30 days after the appearance of anti-mRo60. Interestingly, only in DR2 mice were specific Abs to mla detected, whereas in other strains, the Abs reactive with mla were cross-reactive with the immunogen (Fig. 6Go).

Previous studies have demonstrated that the presence of the HLA-DR3-DQ2 haplotype is highly associated with a more diversified La/Ro RNP response, whereas the haplotype HLA-DR2-DQ1 is associated with a less diversified La/Ro RNP response (5, 45, 46). Our previous data (47) also confirmed the association of HLA-DR2 or HLA-DR3 with more diversification of hRo60-induced autoantibodies to involve other RNPs including mRo52, mla, and mouse Sm as detected by immunoblotting. It is important to note that those association studies have been based entirely on either solid-phase immunosorbent assays or Western immunoblotting, which detect reactivities to the denatured forms of proteins. Assays to measure the reactivities of native isoforms to confirm this finding have not been systematically evaluated. However, we could not confirm this association by using the IP method which detects Abs recognizing the native proteins. The prominent role of HLA-DR regions in determining the magnitude of an anti-Ro60 precipitating Ab response and B cell epitope spreading to Ro52 and La is in agreement with the recent finding that HLA-DR2 and HLA-DR3 play a more significant role than HLA-DQ regions in SLE disease susceptibility in multiplex SLE family studies (48). Although the exact initiating Ag(s) leading to the development of anti-Ro remains to be determined, it most likely involves the sum of interactions among susceptible/protective gene products and triggering events (6, 15, 49). In SLE and SS patients, native Ro protein, molecular mimics or the combination can be the potential triggers/perpetuators of the development of anti-Ro Abs (15, 44). Although immunization of mice with recombinant proteins cannot mimic the way autoimmunity to Ro-RNP initiated in SLE patients, our model system has provided the first experimental evidences to show that, once initiated, HLA class II molecules dictate the magnitude and the extent of Ab diversification in the Ro-RNP system. Moreover, we have for the first time reported HLA-restricted T cell epitopes on hRo60. This allows us the opportunity to study the role of these T cell determinants in the initiation of anti-Ro60 immune responses in future.

Our ANA patterns are consistent with the findings in previous studies using hRo60 (whole protein or hRo60-derived peptides) as immunogen in other animal models (13, 50). The intracellular localization of Ro (SS-A) Ag by immunofluorescence techniques is controversial and has been shown to be present in both nuclear (9, 50) and cytoplasmic (51) locations. mAbs recognized hRo60, but producing different patterns of ANAs could be detected in mice immunized with hRo60 (50). It is evident that Ro (SS-A), La (SS-B), and other snRNP Ags can be translocated to the cell surface during apoptosis (52). Thus, it is conceivable that these RNPs can be shuttled between different subcellular components depending on the state of activation of the cell. In contrast, these Abs might represent different populations of Abs with the same or different specificities. Whether any of these Abs detected by immunofluorescence represent specific binding to Ro (SS-A), its variance or other molecules associated with Ro molecule remain to be demonstrated.

In summary, we have provided an in vivo experiment in HLA class II transgenic mice lacking endogenous class II molecules for studying the immune response to hRo60 protein. Our data clearly indicate that HLA class II molecules are important in controlling the magnitude and the extent of immune response to hRo60 and have validated the use of these animal models to study the development of autoantibodies in autoimmune diseases.


   Acknowledgments
 
We thank Dan McCormick, Jane Liebenow, and Denise Walker (Peptide Core Facility, Mayo Clinic) for support in synthesizing and purifying the peptides used in this study, Michele Smart for tissue typing of transgenic mice, Julie Hanson and her staffs for mouse husbandry, and Mary Brandt for preparation of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R01 AR-42465, AI-14764, and P50 AR-45222. T.P. was the recipient of the Ziegler Family Research Award from the North Central Minnesota Chapter of the Arthritis Foundation and was supported by a Clinical Investigator Fellowship from the Mayo and General Mills Foundations. U.S.D. was supported by a Scientist Development Grant from the American Heart Association. Back

2 T.P. and U.S.D. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Chella S. David, Mayo Clinic, 311C Guggenheim, 200 First Street, SW, Rochester, MN 55905. E-mail address: david.chella{at}mayo.edu Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; RNP, ribonucleoprotein; snRNP, small nuclear RNP; SS, Sjögren’s syndrome; Sm, Smith Ag; LNC, lymph node cell; mla, mouse La; IP, immunoprecipitation; RT, room temperature; ANA, antinuclear Ab; hRo60, human Ro60; mRo60, mouse Ro60; mRo52, mouse Ro52; N, SI, stimulation index. Back

Received for publication January 23, 2002. Accepted for publication April 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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