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The Journal of Immunology, 2002, 168: 2530-2537.
Copyright © 2002 by The American Association of Immunologists

Naturally Processed Chromatin Peptides Reveal a Major Autoepitope That Primes Pathogenic T and B Cells of Lupus1

Arunan Kaliyaperumal, Marissa A. Michaels and Syamal K. Datta2

Division of Rheumatology, Department of Medicine, Northwestern University Medical School, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Major autoepitopes for pathogenic Th cells of lupus were previously found in core histones of nucleosomes by testing overlapping synthetic peptides. To detect other dominant epitopes, we eluted peptides from MHC class II molecules of a murine lupus APC line that was fed with crude chromatin. The eluted peptides were purified by reverse-phase HPLC and tested for their ability to stimulate autoimmune Th clones, and then analyzed by mass spectrometry. Amino acid sequences of stimulatory fractions revealed three new autoepitopes. Two of the epitopes were homologous to brain transcription factor BRN-3, whereas the third sequence was homologous to histone H1'22–42. H1'22–42 stimulated autoimmune Th cells to augment the production of pathogenic antinuclear Abs, and was much more potent than other nucleosomal epitopes in accelerating glomerulonephritis in lupus-prone (SWR x NZB)F1 (SNF1) mice. Remarkably, a marked expansion of Th1 cells recognizing the H1'22–42 epitope occurred spontaneously in SNF1 mice very early in life. A significant proportion of H1'22–42-specific T cell clones cross-reacted with one or more core histone epitopes, but not with epitopes in other lupus autoantigens. The H1'22–42 epitope was also recognized by autoimmune B cells, and with the onset of lupus nephritis, serum autoantibodies to the H1'22–42 epitope become increasingly cross-reactive with nuclear autoantigens. Convergence of T and B cell epitopes in H1'22–42 and its ability to elicit a cross-reactive response make it a highly dominant epitope that could be targeted for therapy and for tracking autoimmune T and B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nucleosomes are one of the major immunogens that initiate cognate interactions between autoimmune Th and B cells for the production of pathogenic antinuclear autoantibodies in lupus (1, 2, 3). Nucleosomes are displayed from apoptotic cells in normal subjects without eliciting an immune response (4, 5, 6, 7). However, the spontaneous expansion of nucleosome-reactive T cells is a lupus-specific event (1, 8, 9, 10). A sustained hyperexpression of CD40 ligand by lupus T cells (11) could instigate tolerogenic dendritic cells (12) or resting B cells to present nucleosomes in an immunogenic fashion (13). Nevertheless, only certain epitopes in nucleosomes are dominantly recognized by the autoimmune T and B cells of lupus. Previously, by testing overlapping synthetic peptides spanning the core histones in nucleosomes, we localized autoepitopes for lupus nephritis-inducing Th cells at amino acid positions 10–33 of histone H2B, 16–39 and 71–94 of H4, and 85–102 of histone H3 (8, 9). Autoimmune T cells of lupus-prone (SWR x NZB)F1 (SNF1) mice, as well as human systemic lupus erythematosus, are spontaneously primed to these disease-relevant epitopes (8, 9). Furthermore, in tolerogenic regimens, these peptides could delay the development of lupus nephritis in SNF1 mice, and even check the progression of established disease (14). However, other dominant epitopes for driving the pathogenic autoimmune response in lupus could exist in the chromatin besides those in core histones. Therefore, we searched for peptide epitopes that would be loaded onto MHC class II molecules and preferentially presented after being naturally processed by APCs from lupus mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, Abs, and cloned Th hybridomas

NZB, SWR, and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Lupus-prone SNF1 hybrids were bred at our animal facility. Female mice were used for the experiments. Hybridomas producing the following mAbs: anti-I-Ad (HB3), anti-I-Ab,d,q (TIB120), anti-HSA (TIB183), anti-Thy-1.2 (TIB99), anti-CD8 (TIB211), and anti-CD3 (145-2C11), were obtained from American Type Culture Collection (Manassas, VA). Cloned Th cell lines and hybridomas used herein were derived from SNF1 mice with lupus nephritis, and maintained as described (15, 16).

Preparation of nucleosomes and derivation of nucleosome-specific APC line (1F2.28)

Chromatin and nucleosomes used in the experiments were prepared from chicken erythrocytes, as described (1, 17). B-B cell hybridomas were derived by fusing the hypoxanthine/aminopterin/thymidine-sensitive, A20 B lymphoma (I-Ad) cell line (Ref. 18 ; a gift of Dr. P. Marrack, National Jewish Center for Immunology, Denver, CO) with the splenic B cells from SNF1 mice (I-Ad/q), and then subcloned. The clones expressing high levels of I-A (>600 mean fluorescence intensity (MFI)3) upon staining with anti-IAd-FITC (BD PharMingen, San Diego, CA) were tested for binding to nucleosomes. Mononucleosomes were biotinylated using N-hydroxysuccinimidyl long-chain biotin kit (Pierce Chemicals, Rockford, IL) per manufacturer. Cells were washed once in PBS containing 10% horse serum and 0.1% NaN3 (flow cytometry buffer). The FcR were blocked using 2.4G2 Ab for 15 min on ice, then washed once and incubated with biotinylated mononucleosomes for 30 min followed by streptavidin-PE, and analyzed by flow cytometry. To check the specificity of the nucleosome binding, the staining was also conducted in the presence of anti-Ig, which blocked the binding. The fusion partner, A20 cells, was used as "control" for staining.

Large scale cell culture and isolation of MHC class II molecules

The high I-Ad-expressing and nucleosome-binding B cell hybridoma (1F2.28) was used as the APC line for purification of MHC class II either after chromatin feeding, or as control without chromatin feeding. Large numbers (1010) of cells were grown in DMEM with 10% horse serum in spinner flasks, and 18 h before harvesting, 50 µg/ml of chromatin was added to the cultures. Cells were harvested by centrifugation at 1000 x g and lysed in presence of detergent (1% Nonidet P-40 in PBS) containing 50 mM iodoacetamide, 10 mM sodium orthovanadate, and 1 mM PMSF. The solution was cleared of cell debris by centrifugation (10,000 x g), and frozen at -70°C until used.

MHC class II (I-Ad) molecules from the cell lysates were affinity purified by using a (I-Ad) specific Ab (HB-3) bound Sepharose 4B column as described (19). Briefly, the affinity matrices were charged with lysates in the presence of protease inhibitor mixture (Sigma Aldrich, St. Louis, MO), washed extensively with PBS containing 0.5% Nonidet P-40 and 0.1% SDS, and equilibrated in PBS containing 1% n-octyl glucoside (pH 8.0). The bound peptide-MHC complexes were eluted with 0.15 M sodium chloride containing 50 mM diethylamine and 1% n-octyl glucoside (pH 10.8). The eluate was immediately neutralized with 1/20 volume 2 M Tris (pH 6.8) and concentrated by vacuum dialysis. The protein content was determined using a micro bicinoic acid assay (Pierce Chemicals).

Elution and purification of naturally processed peptides from MHC class II molecules

One milligram of affinity-purified MHC class II molecules (700–900 µg/ml) was concentrated to ~100 µl in a Centricon 3 (Amicon, Beverly, MA). Two milliters of PBS was added and the above procedure was repeated five times. The MHC-bound peptide was eluted by addition of 0.1% trifluoroacetic acid (TFA) in water and incubation at 37°C for 1 h. The sample was concentrated again by ultrafiltration and the flowthrough was collected and concentrated by Speedvac (Thermo-Savant Instruments, Holbrook, NY) to 1/10 of the original volume, and then stored under an atmosphere of nitrogen at -70°C.

The initial separation of eluted peptides (EP) (<3000 m.w.) were conducted on a reverse-phase (C-18) column HPLC (RP-HPLC) (1 x 30 cm) using a gradient of acetonitrile and water containing 0.1% TFA over a time period of 100 min. The flow rate was 1 ml/min. Fractions were collected at 1-min intervals and assayed for stimulating pathogenic autoantibody-inducing Th clones from SNF1 mice. The active (stimulatory) fractions from the initial screening were further separated using a gradient of 0.1% heptafluroacetone and hexafluroacetic acid. The final separation was conducted on a nanobore HPLC (75 µM x 10 mm) and fractions were assayed again for stimulation of pathogenic Th cell clones. The active fractions were sequenced by electrospray ionization mass spectrometry at the Harvard Microchemistry Facility (Cambridge, MA) for a fee. Peptides required in larger quantities for additional experiments were synthesized by F-moc chemistry (Mimotopes, San Diego, CA), purified by HPLC using a gradient of water and acetonitrile, and analyzed by mass spectrometry for purity.

Splenic CD4+ T cells and APCs

The splenic CD4+ T cells from 3- to 4-mo-old SNF1 mice were isolated as described (1, 14). Splenic B cell plus macrophage (B + M{phi}) APC were prepared from 1-mo-old SNF1 mice by treating splenocytes with anti-Thy1.2 (TIB99) and rabbit complement and irradiated (3000 rad). A20 B lymphoma cells were treated with 50 µg/ml of mitomycin-C for 30 min, washed five times, then incubated for 1 h at 37°C and washed twice in complete medium before use as APC. For peptide presentation, either the A20 B cell lymphoma cells or splenic B + M{phi} were used.

Cytokine assays

Fresh splenic CD4+ T cells (5 x 105/well) were cocultured in triplicate with irradiated B + M{phi} or mitomycin-C-treated A20 APC (106 cells) and different concentrations of control or "test" peptide in 200 µl final volume in HL-1 serum-free medium (Hycor Biomedical, Irvine, CA) for 96 h in flat-bottom 96-well plates (Costar, Corning, NY). The culture supernatants were removed from duplicate coculture wells after 24–36 h for cytokine ELISA. Anti-IL-2, anti-IFN-{gamma}, and anti-IL-4, capture and revealing Ab pairs, and the respective standards were from BD PharMingen. Streptavidin-HRP and the substrate tetramethylbenzidine were from Sigma Aldrich. Cytokines were quantitated according to the manufacturer.

Intracellular cytokine assay

Staining for intracellular cytokines and surface markers was done as described (9). T cells were stimulated as described above for cytokine assays with slight modifications. A total of 5 x 106 T cells and 5 x 106 APC were cocultured in a 24-well plate for 6 h and brefeldin A (Sigma Aldrich) was added at a final concentration of 1 µg/ml for the final 5 h of incubation. Four parameter analyses were performed on FACSort (BD Immunocytometry Systems, Mountain View, CA) using FITC and PE as fluorescent parameters. For each analysis, data on 106 CD4+ cells were acquired. Isotype-matched control reagents were used for setting markers to delineate "positive" and "negative" populations.

TCR down-regulation assay

Staining to detect down-regulation of TCR, along with staining for other surface markers were done as described (20). T cells were stimulated for 18 h, as described above for intracellular cytokine assays (ICA), but in the absence of brefeldin A. Cells were stained with anti-CD4-PE and anti-{alpha}{beta}TCR-FITC (H57–597), and analyzed by flow cytometry.

Th cell helper assays and autoantibody quantitation

Ab helper cell assays were conducted as described (21). Briefly, 5 x 106 B cells derived from 4- to 5-mo-old SNF1 mice were mixed with 5 x 106 CD4+ T cell or cloned T cell hybridoma in the presence of 10 µg/ml of the stimulatory or control peptide in DMEM containing 10% FCS and incubated for 7 days. The culture supernatants were then frozen at -20°C and later thawed to measure antinuclear autoantibodies. IgG class autoantibodies to ssDNA, dsDNA, histones, and nucleosomes (histone/DNA complex) in culture supernatants or in sera were quantitated by ELISA (1, 8, 16, 21). Sera were diluted 1/100 and heat-inactivated before use. Serum from normal BALB/c mice was used as negative control.

Antipeptide Ab ELISA

Peptides were coated onto 96-well Maxisorb ELISA plates (Nunc, Roskilde, Denmark) at 10 µg/ml concentration overnight at 4°C in PBS (pH 7.4), and then washed once and blocked with 10% horse serum in PBS. The sera were diluted 1/10 or 1/100 in PBS 10% horse serum and added to the wells and incubated at 4°C overnight and washed five times in PBS. Anti-IgG conjugated to alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) diluted to 1/5000 was added and incubated at room temperature for 1 h. The wells were washed eight times in PBS and the substrate p-nitrophenylphosphate (Sigma Aldrich) was added and developed and read in an ELISA reader (Molecular Devices, Sunnyvale, CA).

Cross-reactivity of antipeptide and anti-DNA autoantibodies

Serum from unmanipulated mice at various ages was collected and assayed for cross-reactivity. Histone H1'22–42 peptide was linked to N-hydroxysuccinimide-activated Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) as described by the manufacturer. DNA-linked cellulose was obtained from Sigma Aldrich and resuspended at 100 mg/ml. A total of 1 ml of the diluted (1/10 in PBS) serum was stirred with 100 µl of the suspended peptide-linked beads or DNA cellulose for 18 h. The beads were washed three times with PBS. The bound Abs were eluted with 800 µl of 0.1 M glycine-HCl (pH 2.8), spun down to remove the beads or cellulose and the supernatant neutralized with 200 µl of 2 M Tris-HCl (pH 9.5). These supernatants were tested in ELISA for the presence of the cross-reactive Abs in the antipeptide or anti-DNA Ab assays described above. Briefly, 100 µl of purified Ab eluted from the peptide-linked beads was dispensed into wells coated with one of the nuclear Ag, namely, dsDNA, ssDNA, histone or nucleosomes, or into wells coated with one of the histone peptide epitopes. After incubation at 4°C for 18 h, the plates were washed and the bound Abs of IgG class were revealed as described above for autoantibody quantitation. Converse assays were conducted with serum Abs eluted from DNA-cellulose for binding to histone peptide-coated plates, as described in antipeptide ELISA.

Pathogenicity of naturally processed chromatin peptides in vivo

Twelve-week-old prenephritic SNF1 females (nine mice per group) were injected with either EP-1, EP-2, or EP-3, or control peptides (100 µg/mouse) emulsified in CFA. The animals received three more booster injections at 2-wk intervals with peptides (50 µg/mouse) adsorbed on alum (Pierce Chemical). The mice were monitored weekly for proteinuria using albustix, and killed when they developed persistent proteinuria (two consecutive weekly readings of >=300 mg/dL). Grading of glomerulonephritis was done as described (1, 8, 16, 21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the naturally processed and presented Th cell epitopes

Purification of MHC class II and elution of bound peptides. Nucleosomal peptides are equally well presented by I-A molecules on splenic APC from SNF1 (I-Ad/q), or on A20, a BALB/c-derived B cell lymphoma (I-Ad) cell (1, 22). A high I-Ad-expressing and nucleosome-binding APC clone, 1F2.28, was derived (see Materials and Methods). 1F2.28 did not secrete Ig, but retained the properties of its fusion partner, A20, in expressing high levels of MHC class II, and being highly efficient in capturing nucleosomal Ags by its surface Ig and presenting it. MHC class II molecules were purified from the 1F2.28 APC after feeding them with a chromatin preparation. Chicken chromatin was used for feeding the APC line, as pathogenic Th clones respond equally well to nucleosomes from chicken (1). Therefore, if any naturally processed stimulatory epitope sequences from the fed chromatin differed in some residues, it could be tracked and distinguished from any nucleosomal peptides derived from autologous cells dying in culture. Fractionation of the EPs (<3000 m.w.) were conducted on a C-18 column RP-HPLC, and then assayed for stimulating ability (Fig. 1GoA).



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  		FIGURE 1. Identification of the naturally processed and presented Th cell epitopes. A, HPLC profile of peptides eluted from purified I-Ad molecules obtained from chromatin-pulsed 1F2.28 APC line. Peptides (<3000 Mr) were purifed on a C-18 column using water and acetonitrile: water containing 0.1% TFA. B, IL-2 release by representative pathogenic Th clone L-3A, after stimulation with HPLC fractions using A20 as APC. C, Homogeneity of one of the stimulatory fraction (no. 23) by mass spectral analysis.

 
Determination of the active fractions. Aliquots of 50 µl from the fractions collected after the RP-HPLC separation were concentrated using Speedvac to 5–10 µl and reconstituted to 25 µl with PBS. A total of 16 Th clones (lines and hybridomas) derived from 10 nephritic SNF1 mice have been characterized for their nucleosome-specificity and TCR structure (1, 8, 16, 22). Four of these Th cell lines were used here in cytokine response assays (IL-2 and/or IL-4) to detect stimulatory activity in the peptide fractions, using A20 or splenic B + M{phi} as APCs. Culture supernatants were collected after 24 h of stimulation and assayed by ELISA. None of the eluate fractions from control APC cultures that were not fed with chromatin stimulated the T cell clones. Only 2/100 fractions of the eluate from chromatin-fed APC stimulated (Fig. 1GoB). These fractions were analyzed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry. One fraction (no. 23) was homogeneous (Fig. 1GoC), whereas the other (fraction no. 50) was heterogeneous. The second active fraction (fraction no. 50) had more than fifty different components, and it was repurified using a microbore HPLC using a gradient of 0.1% heptafluroacetone and hexafluroacetic acid and the fractions were tested for their ability to stimulate the pathogenic Th hybridoma (Fig. 2GoA). The active fraction was further analyzed by electrospray mass spectrometry to check its homogeneity. More than 20 different components were identified by this method (Fig. 2GoB). Further fractionation was done on a nanobore HPLC (0.75 µM x 10 mm; Fig. 2GoC) and tested for its activity as mentioned above. Two major components stimulated the pathogenic Th cell clones (Fig. 2GoD). The amino acid sequences of these active fractions were deduced by electrospray ionization mass spectrometry. Both the active fractions (fractions no. 15 and no. 25 in Fig. 2Go, C and D) contained the same peptides. The fraction that eluted at the earlier time point (fraction no. 15 in Fig. 2Go, C and D) is a product of the oxidation of methionine in the active peptide (Fig. 3Go, sequence EP-3). The peptide sequences thus identified were synthesized and tested for their Th cell stimulatory activity to confirm their functional relevance. As shown in Fig. 3Go, these naturally processed and presented epitope sequences were SQKEEEEGAQREKE (EP-1) and DWMEEEEGAQREKE (EP-2) that were identified from the fraction no. 23 (Fig. 1GoB) and the sequence SASHPTYSEMIAAAIRAEKSR (EP-3) was identified from fraction no. 25 (Fig. 2GoC). Homology search (GenBank) showed EP-2 to be identical in sequence to amino acid position 175–185 in transcription factor BRN-3, except for one residue. EP-1 differed from EP-2 by only three residues. Both were homologous to many sequences (198 hits in basic local alignment search tool search) that were mostly from transcription factors. Interestingly, among these was 81% match with a sequence in cytomegalovirus transcriptional coactivator at amino acid position 115–125. Most likely, these peptides came from some transcription factor in the chromatin that was fed to the APC. The third sequence (EP-3) came from histone H5 (amino acid position 22–42) in chicken nucleosome. EP-3 was homologous to histone H1' sequence of mouse or human at amino acid position 22–42 (STDHPKYSDMIVAAIQAEKNR), as indicated by basic local alignment search tool (identity = 66%, positives = 80%). Functionally, the mouse H1'22–42 peptide was equivalent to the chicken H522–42 peptide in stimulating the pathogenic Th clones from SNF1 lupus mice. Therefore, the mouse H1'22–42 being the autoantigenic peptide in mice, was used for subsequent studies, but still referred to as EP-3. Nested sequences and unrelated sequences that were obtained in the active fractions from the eluates were also synthesized and retested, and they did not stimulate the pathogenic Th clones (Fig. 4Go), indicating epitope specificity and authenticity of the EP sequence EP-3.



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  		FIGURE 2. Repurification of the other active fraction (no. 50) shown in Fig. 1GoB. A, Microbore HPLC (360 µM diameter column) elution profile of peptides obtained from active fraction no. 50 of Fig. 1GoB, using a gradient of hexafluroacetone and heptaflurobutyric acid. Fractions of 20 µl were collected and assayed for Th clone stimulation. B, Active fraction from microbore HPLC was still heterogeneous upon re-analysis by liquid-chromatography mass spectrometry. C, Active fraction from microbore HPLC (A) was then subjected to nanobore HPLC (75 µM column), and 10 µl fractions were collected. D, Fractions were tested for their ability to stimulate pathogenic Th clones to produce IL-2.

 


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  		FIGURE 3. Sequences of MHC class II (I-Ad) bound naturally processed and presented stimulatory peptides and a nonstimulatory control peptide.

 


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  		FIGURE 4. Epitope specificity of the naturally processed peptide EP-3 and requirement of flanking sequences for stimulation. Nested sequences and unrelated sequences found in the active fractions were synthesized and retested for their ability to stimulate the nucleosome-specific, pathogenic Th clones of SNF1 mice. Representative data from a Th clone 3F6 is shown. IL-2 response of this clone to H1'22–42, the murine homolog of EP-3, was 69.8 pg/ml and the responses of other Th clones to H1'22–42 and EP-3 peptides were equivalent (data not shown).

 
We also selected a naturally processed peptide that did not stimulate any of the lupus Th clones. This I-Ad-bound peptide was naturally processed from high-density lipoprotein binding protein (HDL-BP), and it was present in one of the eluted fractions. This HDL-BP825–839 peptide, and a nonstimulatory core histone peptide, H2B59–73, (8) with the sequence AMGIMNSFVNDIFER, were synthesized and used as controls in our subsequent experiments (Fig. 3Go).

Frequency of preexisting CD4+ T cells primed to the peptide autoepitopes

Using synthetic overlapping peptides from core histones, we previously found that T cells from older (4-mo-old), unmanipulated SNF1 mice responded to H416–39, H471–94, H2B10–33, and H385–102 peptides by secreting IL-2 in 24–48 h cultures (8), indicating that priming to those epitopes had occurred in vivo. In our previous study, we had also found that the pathogenic T cells were of Th1 type (8). Herein, we assessed intracellular cytokine (IFN-{gamma}) production in response to each of the histone autoepitopes, by a shorter term assay (ICA) that measures only newly synthesized cytokines. Fresh CD4+ T cells from much younger (preclinical), 4-wk-old, unmanipulated SNF1 mice were assessed for their ability to respond (Fig. 5Go). IFN-{gamma}-producing T cells responding to H1'22–42 peptide autoepitope in the SNF1 were markedly increased and were almost half the number of IFN-{gamma}+ cells responding to anti-CD3. The background frequency of IFN-{gamma}+ CD4+ T cells was relatively higher in the SNF1 mice, probably due to continuous priming to the autoepitopes even at this young age. Nevertheless, the frequency of CD4+ T cells responding to H1'22–42 was increased on average nearly five times (128 cells per 106 CD4+ T cells) in the SNF1 mice (n = 6) when compared with medium or control peptide (23 cells per 106 CD4+ T cells; p < 0.001, Student’s t test, Fig. 5Go). The normal SWR parental strain did not respond to this autoepitope. In these very young SNF1 mice, using this short-term intracellular cytokine assay, responses to the other autoepitopes, namely EP-1, EP-2, and the core histone peptides, H416–39, H471–94, H2B10–33, and H385–102, were all at background level, similar to that of the control peptide (17–23 cells per 106 CD4+ T cells; data not shown). In older SNF1 mice, probably due to continuous presentation of autoepitopes, the background level of IFN-{gamma}+ cells was so high that no further increase in signal could be detected by addition of any of the peptides using this ICA technique (data not shown).



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  		FIGURE 5. Frequency of preexisting CD4+ T cells primed to murine histone H1'22–42 peptide. Irradiated B + M{phi} from 1-mo-old mice were pulsed with 1 µM of the respective peptides and used as APCs to culture with CD4+ T cells from 6-wk-old unmanipulated SNF1 mice. After 6 h of incubation, the cultured cells were stained with anti-CD4-FITC and anti-IFN-{gamma}-PE, washed, and analyzed by flow cytometry. Data on 106 "gated" CD4+ cells were collected from each tube.

 
Ability of EP to augment pathogenic autoantibody production

To test whether the naturally processed and presented peptide could help in augmentation of pathogenic autoantibody production, pathogenic autoantibody-inducing Th clones 1D12, 5E9, and 3F6 that were nucleosome-specific and also responsive to the EP-3 peptide, or the Th clone 1G1 that was also nucleosome-specific but did not respond to EP-3 were used. Each Th clone was cocultured with freshly isolated splenic B cells from SNF1 mice in the presence of EP-3 (H1'22–42). After 7 days, the supernatants were assayed for IgG Abs to nuclear autoantigens. The H1'22–42 peptide stimulated helper function of the first three pathogenic Th clones, but not the nonresponder Th clone, in contrast to whole nucleosome (Fig. 6Go). The H1'22–42 peptide augmented autoantibody-inducing help of responder Th clones, 5E9 and 3F6, almost to the same extent as that by whole nucleosome (data not shown). In the case of clone 1D12, augmentation of autoantibody-inducing help by H1'22–42 peptide was 1.5–2.5 times higher than that by nucleosome (Fig. 6Go).



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  		FIGURE 6. Ability of autoepitope to stimulate T cell help for pathogenic autoantibody production in coculture with freshly isolated B cells. B cells were isolated from 4- to 5-mo-old lupus-prone SNF1 mice and cocultured with nucleosome-specific Th clone 1D12 that responds to the naturally processed and presented peptide (EP-3 or H1'22–42), or with the nucleosome-specific T cell clone, 1G1, that does not respond to the peptide. The B-cells (2 x 106) were cocultured with (2 x 106) Th clones for 7 days in the presence of 10 µg/ml of H1'22–42 peptide or nucleosome (Nuc), or without Ag (control). Supernatants were then assayed for autoantibodies.

 
Acceleration of disease

Twelve-week-old prenephritic SNF1 mice that were immunized with EP-2 or EP-3 in adjuvant (CFA) developed severe nephritis earlier than age-matched SNF1 mice injected with CFA alone or control HDL-BP or H2B59–73 peptide in CFA (p < 0.01, Fisher’s exact test). A total of 80% of the mice injected with H1'22–42 (EP-3) succumbed to severe lupus nephritis within 4 wk of the first injection, before the third booster injection could be given. Indeed, some of the H1'22–42 immunized animals developed severe nephritis in 2–3 wk, just after the first booster immunization. By 28 wk of age, all the EP-3 immunized mice developed severe nephritis. EP-2 also accelerated disease in the initial phase; 60% of the mice developed disease by 20 wk of age. But later on, the incidence of severe nephritis in this group of mice was similar to that of the control group. By contrast, EP-1 immunization did not result in any increase in the rate of disease over control groups, although EP-1 differs in sequence from EP-2 only by three residues (Fig. 7Go).



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  		FIGURE 7. Acceleration of the disease. Incidence of severe nephritis in prenephritic SNF1 mice immunized with naturally processed peptides. Nine mice per group were immunized first at 12 wk of age with 100 µg of the specified peptide in CFA, followed by three injections at 2-wk intervals with 50 µg of peptide adsorbed in alum. The mice received the last injection at 18 wk of age, except for those that had already developed severe nephritis. Mice immunized with EP-3 (chicken H522–42) also developed severe nephritis with an incidence and time course that were indistinguishable from the group immunized with H1'22–42 (data not shown). Moreover, the incidence of nephritis in mice immunized with another control peptide, H2B59–73, was almost identical to that with the HDL-BP825–839 control peptide (data not shown).

 
EPs elicit Th1-type response in proportion to their pathogenicity

The EPs induced IFN-{gamma} synthesis in T cells of mice immunized with the respective peptide (Fig. 8Go). As compared with control, H1'22–42 (EP-3) induced the highest amounts of IFN-{gamma} (p < 0.00001), followed by EP-2 (p < 0.0001), whereas EP-1 elicited the least, but a significant response in terms of IFN-{gamma} production (p < 0.001). The CD4+ T cells did not produce IL-4 or IL-10 in response to the peptide stimulation (data not shown).



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  		FIGURE 8. EPs elicit Th1-type response. Cytokine response to EPs by CD4+ T cells from immunized SNF1 mice. Twelve-week-old SNF1 mice were immunized once with the respective peptide in CFA and the T cells were tested after 8 days for their response. Irradiated B + M{phi} from 1-mo-old mice were pulsed with 1 µM of the respective peptide and used as APCs to coculture with CD4+ T cells. After 36 h of incubation, the supernatants were assayed for IFN-{gamma}, IL-4, or IL-10. There was no increase in production of IL-4 or IL-10 with any of the peptides (data not shown).

 
Cross-reactivity of H1'22–42-specific T cells

The rapid acceleration of lupus nephritis by immunization with the newly identified histone H1'22–42 autoepitope might have been due to extensive cross-reactivity with other autoepitopes. To test this hypothesis, we derived histone H1'22–42-specific Th clones from SNF1 mice. Mice were immunized once with histone H1'22–42 peptide (100 µg/mice, adsorbed in alum) to expand preexisting T cells that were already primed spontaneously in vivo. Eight days after the immunization, CD4+ T cells from the animals were purified and fused with TCR-{alpha}-/TCR-{beta}- BW5147 cell line to generate T cell hybridomas. A total of 453 hybridomas were cloned from the fusions, and 77 of the clones were randomly sampled for reactivity to H1'22–42. Sixty percent (46 of 77 clones) of the hybridomas sampled from the fusion were found to respond to H1'22–42 by IL-2 production.

Cross-reactivity by cytokine response. Th hybridomas specific for histone H1'22–42 were tested as described in Table IGo, but cultured with two different concentrations of each of the test peptides (10 and 100 µg/ml) for 24 h. The supernatants were collected and assayed for IL-2. Eleven hybridomas did not survive storage in liquid nitrogen. Twelve of 35 clones tested (34.3%) cross-reacted with one or more of the previously identified epitopes in core histone peptides, H385–102, H416–39, and H471–94 (Table IGo). The cross-reactions were significantly above background, but relatively weak, suggesting that the latter epitopes were partial agonists. By contrast, we could not detect any cross-reactivity with eight immunodominant T cell epitopes identified in other lupus autoantigens. These noncross-reactive lupus T cell epitopes tested were: anti-DNA Ab, A6 VH peptide from amino acid positions 34–45, 58–69, and 84–95 (23), a consensus peptide derived from anti-DNA VH (24), anti-chromatin VH peptide FR117–39 (25), eluted histone peptide, H2A284–103 from I-Ek of MRL-lpr mice (26), SmD183–119 peptide (27), Sm-B48–96 (28), and Ro60441–465 peptide (29).


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  		Table I. Cross-reactivity of H1'22–42 specific T cell hybridomas1

 
Cross-reactivity by TCR down-regulation. We tested the remaining clones that did not demonstrate cross-reactivity in the IL-2 production assay for their TCR down-regulation in response to the peptides. Of the remaining 23 clones, six clones down-regulated their TCR in response to the other histone autoepitopes. The extent of down-regulation varied between the clones (25–50% reduction in MFI). For example, clone no. 429 down-regulated TCR with histone H416–39, and H471–94, but not with H385–102 peptide, whereas clones no. 442 and no. 297 down-regulated with all three peptides, but to a varying degree (Fig. 9GoB). The response to anti-CD3 used as positive control resulted in 65–70% down-regulation of TCR (Fig. 9GoA). Thus, altogether 51.4% (18/35) of the H1'22–42-specific T cell clones cross-reacted with one or more of the core histone autoepitopes we had characterized previously by overlapping peptide synthesis.



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  		FIGURE 9. TCR down-regulation in response to the autoepitope peptides. Histone H1'22–42 peptide-specific Th hybridoma clones were cocultured with irradiated APCs (B + M{phi}) from SNF1 mice along with 100 µg/ml of the indicated peptide, or with anti-CD3, in serum-free HL-1 medium for 12 h. Cultures were stained with anti-CD4-PE, anti-pan TCR{alpha}{beta}-FITC (H57–597) and analyzed by flow cytometry. T cells from control cultures with HDL825–839 peptide-pulsed APC expressed TCR levels (in MFI) similar to that of T + APC (without any peptide). A, Representative histogram of the TCR down-regulation is shown with Th clone no. 301. B, Numbers no. 429, no. 442, and no. 297 indicate various H1'22–42 peptide-specific clones. Three representative clones are shown here.

 
Flow cytometry staining with a panel of available mAbs to TCR V{beta} and V{alpha} families (BD PharMingen) showed diverse TCR usage among the cross-reactive T cell clones (data not shown).

Helper activity. The ability of cross-reactive vs noncross-reactive H1'22–42-specific hybridomas were compared for providing help to B cells from 3- to 4-mo-old SNF1 mice to produce antinuclear Abs in the presence of the histone H1'22–42 peptide in helper assay cultures. All provided help to the same extent (data not shown).

Histone peptide H1'22–42 contains a B cell epitope

Sera were collected from unmanipulated mice at various ages (10 mice per age group) and assayed either directly or after affinity purification from H1'22–42 peptide-linked beads for the presence of Abs to the peptide epitopes by ELISA. Ab to H1'22–42 peptide was detected as early as 12 wk of age (Fig. 10GoA). The Ab levels rose with age and peaked at 32 wk of age, at which point the levels plateau (Fig. 10GoA), indicating that the antipeptide autoantibodies are produced throughout the natural history of disease development in unmanipulated SNF1 mice. Interestingly, purified anti-H1'22–42 autoantibodies that were eluted from the H1'22–42 peptide-linked beads that had been incubated with sera from SNF1 mice failed to bind to the other pathogenic histone autoepitopes (H2B10–33, H385–102, H416–39, and H471–94; data not shown).



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  		FIGURE 10. A, Histone H1'22–42 peptide contains Ab-binding epitopes. Sera were collected from unmanipulated SNF1 and BALB/c mice at various ages (10 mice per age group). The peptide-reactive Abs were purified from the sera using H1'22–42 peptide-linked beads and tested for binding to the H1'22–42 peptide by ELISA. Sera were also tested directly for binding to the peptide after diluting 1/10 in PBS containing 5% horse serum. Solid lines represent binding by purified Abs and broken lines represent binding of sera. B, Anti-H1'22–42 peptide Abs cross-react with nuclear Ags. The same serum Abs from SNF1 that were immunoaffinity purified using H1'22–42 peptide-linked beads shown in A were also tested for cross-reactivity to various nuclear autoantigens by ELISA. The BALB/c serum eluates did not show any reactivity above background levels (data not shown).

 
Antipeptide Abs cross-react with DNA and nucleosome with age

Competition immunoassays could not be used to detect cross-reactivity, because the H1'22–42 peptide itself binds DNA and vice versa. Therefore, sera from unmanipulated SNF1 mice of various ages were incubated with the peptide-linked beads and antipeptide-reactive Abs were purified by elution, and then tested for their cross-reactivity to various nuclear Ags. The levels of cross-reactive Abs increased with age, peaking around 47 wk. The cross-reactivity increased 3- to 4-fold at the age of 40 wk, as compared with levels between 19 and 35 wk (Fig. 10GoB). In converse experiments with serum Abs eluted from DNA-cellulose, the anti-DNA Abs that were cross-reactive to the peptide remained at a constant level ranging from 115 to 270 U throughout, between 12 and 50 wk of age (data not shown). The eluted anti-DNA Abs could have bound to the H1'22–42 peptide via DNA that might have leached off the DNA-cellulose and occupied the combining sites of the eluted Abs. However, in the case of serum Abs eluted from H1'22–42-linked beads, the temporal profile and the time of reaching peak levels for autoantibodies specific for the H1'22–42 peptide did not coincide with, but preceded that for the cross-reactive population of autoantibodies (Fig. 10Go, A vs B). These results indicate that the cross-reactions of antipeptide autoantibodies were not occurring artificially due to immune complexes, even though the Ab preparations were affinity purified using peptide-linked beads to avoid such a possibility.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have identified new epitopes that drive autoimmune response in lupus which were not detected previously by testing overlapping synthetic peptides representing only the core histones. Because the lupus APC line was fed with crude chromatin, we were able to identify a dominant Th cell epitope in H1 histone. This H1'22–42 epitope is extremely potent in stimulating autoimmune Th cells and accelerating lupus nephritis in the SNF1 mice significantly more than any of the other histone peptides we had identified before. This epitope, either spontaneously or upon immunization, markedly stimulates Th1 cells, which appear to play a crucial role in pathogenesis of lupus nephritis (8, 30, 31, 32). Interestingly, in lupus patients presence of Abs to the whole histone H1 strongly correlates with active disease with major organ involvement, particularly nephritis, and this autoantibody specificity might be the basis for the historical lupus erythematosus cell phenomenon (33, 34). It remains to be seen whether the H1'22–42 peptide epitope is also predominantly recognized by pathogenic T and B cells from humans with lupus.

Remarkably, autoimmune T cell clones specific for the naturally-processed H1'22–42 epitope frequently cross-react with the pathogenic epitopes in core histones described by us previously, although their sequences are distinct except for sharing charged residues. This type of cross-reactive recognition is a feature of other nucleosomal peptide epitopes (8, 22). A single peptide from a histone in the nucleosome can be recognized by multiple autoimmune T cells with distinct TCRs (8). Conversely, individual TCRs of the pathogenic autoantibody-inducing Th cell clones of lupus recognize more than one nucleosomal peptide epitope in a promiscuous fashion, and in the context of diverse class II molecules (22). Considerable cross-reactivity has been shown in several other examples of TCR-MHC/peptide interactions (35). However, it is striking that T cells specific for the H1'22–42 epitope are markedly expanded in the SNF1 mice by 1 mo of age, before responsiveness to the other epitopes could be detected. The cross-reactive T cells became evident after cloning from older mice from 3–4 mo of age. This cross-reactivity might have led to the rapid acceleration of the disease in prenephritic mice when immunized with this autoepitope.

Curiously, among the eluate fractions of class II-bound peptides, we could not detect the core histone epitopes that we had previously identified by overlapping synthesis. However, the naturally processed stimulatory peptides from chromatin were detected here at femtomole to attomole concentrations in the eluate fractions from class II molecules, using extremely sensitive lupus T cell clones. The previously identified core histone epitopes were synthesized and tested at much higher concentrations (1–10 µM). Using assays where the synthetic peptides were fed directly to professional APC, unimmunized SNF1 mice at 4 mo of age did appear to have T cells that had been spontaneously primed to the core histone epitopes in vivo (8, 14). However, at those high concentrations, the core histone peptides were probably being recognized also by cross-reactive T cells, which might have included the H1'22–42 epitope-specific T cells shown here. Thus, the core histone epitopes might have been displayed at levels below the detectable concentration of stimulatory peptides eluted from MHC class II, a result further emphasizing the physiological predominance of the H1'22–42 epitope during natural processing and presentation. In contrast, processing of chromatin by this particular lupus APC line (1F2.28) might have influenced the display of determinants. However, the existence of a much higher frequency of T cells that are spontaneously primed to the H1'22–42 epitope in SNF1 mice and the superior ability of the epitope to accelerate disease indicates its natural predominance in vivo. A histone H2A peptide has been recently detected in eluates from I-Ek molecules from MRL-lpr splenocytes, but functional studies to determine the relevance of that peptide to lupus autoimmunity remains to be done (26).

The H1'22–42 peptide is from the region that contacts DNA in the nucleosome (36, 37, 38), and this T cell epitope is also targeted by autoantibodies from lupus B cells, suggesting how the epitope could be protected, preferentially processed, and presented to the autoimmune Th cells, a basis for its immunodominance. Indeed, the overlapping of epitopes for autoimmune Th cells and autoimmune B cells of lupus make the core histone epitope, H416–39, a highly efficient tolerogen for therapy of murine lupus nephritis, because that peptide could inactivate autoimmune T and B cells in concert (14). Remarkably, IgG autoantibodies to H1'22–42 in sera were relatively specific to the peptide epitope earlier on, but became increasingly cross-reactive with nuclear autoantigens (Fig. 10Go), in parallel with the increasing incidence of lupus nephritis in unmanipulated SNF1 mice (the incidence is identical to the control peptide-injected mice in Fig. 7Go), indicating expansion of a cross-reactive population of B cells that are more pathogenic. Cross-reactive anti-nuclear Abs could be elicited in normal mice upon immunization with a DNA-mimicking peptide isolated from a phage display library (32, 39). It is remarkable that the H-1'22–42 peptide that is naturally processed from a ubiquitous autoantigen can also elicit cross-reactive anti-DNA autoantibodies spontaneously in lupus-prone mice with age. Autoantibodies that bind nucleosome particles are considered to be the earliest nephritogenic population, preceding anti-dsDNA autoantibodies during the pathogenesis of lupus nephritis (10, 40, 41, 42, 43). The H1'22–42 peptide-specific autoantibodies might precede and predict the development of lupus nephritis before autoantibodies directed against other determinants in nucleosomes.

Moreover, the H1'22–42-specific Th cells of lupus are also pluripotent or promiscuous in their helper activity. A single lupus Th clone could help B cells producing autoantibodies to dsDNA, ssDNA, histones, or nucleosomes, probably because each of these B cells, by binding to its respective epitope on the chromatin, could take it up and process and then present the relevant H1 peptide epitope from the chromatin to the Th clone, resulting in intermolecular help. Tolerization of such Th cells would obviously deprive multiple autoimmune B cells of T cell help. The cross-reactive recognition and potent immunogenicity of the H1'22–42 epitope indicates that tolerance therapy could be designed in the future with this epitope for inactivating a broad spectrum of pathogenic Th and B cells of lupus.


   Acknowledgments
 
We thank Eli Shamiyeh for statistical analysis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1-AI41985 and AR39157 (to S.K.D.) and a Systemic Lupus Erythematosus Foundation Inc. grant (to A.K.). Statistical analysis was supported by National Institutes of Health Grant AR30692. Back

2 Address correspondence and reprint requests to Dr. Syamal K. Datta, Division of Rheumatology, Ward 3-315, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: skd257{at}northwestern.edu Back

3 Abbreviations used in this paper: MFI, mean fluorescence intensity; EP, eluted peptide; RP-HPLC, reverse-phase HPLC; TFA, trifluoroacetic acid; B + M{phi}, B cell plus macrophage; HDL-BP, high-density lipoprotein binding protein. Back

Received for publication October 11, 2001. Accepted for publication December 19, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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