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The Journal of Immunology, 2003, 171: 636-644.
Copyright © 2003 by The American Association of Immunologists

CD4+ T Cells from (New Zealand Black x New Zealand White)F1 Lupus Mice and Normal Mice Immunized Against Apoptotic Nucleosomes Recognize Similar Th Cell Epitopes in the C Terminus of Histone H31

Sylvie Fournel*, Sarah Neichel*, Hayet Dali*, Sandrine Farci{dagger}, Bernard Maillère{dagger}, Jean-Paul Briand* and Sylviane Muller2,*

* Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9021, Centre National de la Recherche Scientifique, and Université Louis Pasteur, Strasbourg, France; and {dagger} CEA Saclay, Département d’Ingénierie et d’Etudes des Protéines, Gif-sur-Yvette, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2d/d) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4+ T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F1 lupus mice (H-2d/z). None of the 11 H4 peptides was recognized by CD4+ T cells from (NZB x NZW)F1 mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-{gamma} upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-{gamma} and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF1 (H-2d/q) and MRL/lpr (H-2k/k) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F1 mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Evidence accumulated in recent years suggests that the nucleosome, the fundamental unit of chromatin, plays a pivotal role in the induction phase and pathogenesis of systemic lupus erythematosus (SLE)3. High levels of nucleosome-specific Abs are often detectable in the serum of patients and young mice with lupus before the appearance of Abs to its individual components, dsDNA and histones (1, 2). Nucleosomes are also important for mediating tissue lesions, especially glomerulonephritis (3, 4, 5, 6). Histones and nucleosomes are present in glomerular deposits of patients with lupus nephritis, and Abs to DNA, histones, and nucleosomes have been eluted from glomeruli in lupus nephritis. Moreover, elevated plasma levels of nucleosomes have been observed in lupus patients (7, 8), and a positive correlation (9, 10) or absence of correlation (11) has been found between the level of these circulating nucleosomes and active disease. Compelling evidence is also accumulating to suggest that some (but not all) anti-nucleosome Abs are nephritogenic and that their level can be increased in active lupus (3, 4, 11, 12). Amoura et al. (11) further demonstrated an inverse correlation between nucleosome plasma levels and serum anti-nucleosome Ab activity.

Apoptotic cells, and more particularly the apoptotic blebs, are probably an important source of autoantigens in SLE (13). It has been speculated that apoptosis defects such as highly accelerated rates of apoptosis, or defect in the clearance of apoptotic bodies by macrophages, as well as abnormal sites or abnormal processing of apoptotic cells might lead to an autoantibody response in patients with SLE and in lupus mice (14, 15, 16, 17, 18). It has also been demonstrated that the i.v. injection of syngeneic apoptotic cells in normal mice generated the production of anti-nuclear Abs and IgG deposition in the glomeruli (19).

In SLE, T lymphocytes do not appear to play a direct role in tissue damage. Recently, it has been reported that in autoimmune mice, immune complexes containing IgG2a associated to chromatin material accumulate and stimulate a T cell-independent immune response probably because they synergistically engage both the B cell receptor (first signal) and a Toll-like receptor 9 (20). In SLE, however, cognate interaction between autoreactive Th cells and autoreactive B cells is probably initially involved in the production of autoantibodies (21). Autoreactive Th cells directed against nucleosomes have been described in lupus patients (22). The presence of pathogenic autoantibody-inducing Th cells specific for chromatin subparticles or histones has also been described in patients with lupus as well as in lupus mice (23, 24, 25). The origin of signals giving rise to their stimulation is still speculative (26). Using overlapping histone peptides covering the four core histones, H2A, H2B, H3, and H4, autoreactive Th cells were characterized at the level of epitopes in normal and (SWR x New Zealand Black (NZB))F1 (SNF1; haplotype H-2d/q) lupus mice as well as in patients with lupus. Kaliyaperumal et al. (27) identified three epitopes, localized in residues 10-33 of H2B, and 16-39 and 71-94 of H4, recognized by nephritogenic autoantibody-inducing Th cell clones derived from SNF1 mice. These peptides triggered the pathogenic Th cells of SNF1 lupus mice in vivo to induce the development of severe lupus nephritis. It was further shown that repetitive i.v. injections of the three peptides (and particularly peptide 16-39 of H4) to 3-mo-old prenephritic SNF1 mice that already produced pathogenic Abs delayed the onset of severe lupus nephritis (28). Our group has recently described a minimal T cell epitope recognized by nucleosome-induced Th cells from normal BALB/c mice of H-2d/d haplotype in residues 88-99 of H4 (29). This epitope overlaps with the region 71-94 identified in SNF1 mice (25, 27). Finally, Datta and coworkers (25) identified several epitopes recognized by nucleosome T cells from lupus patients. They encompassed residues 10-33 of H2B, 34-48 of H2A, 85-99 and 94-108 of H3, as well as 16-39 (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), 49-63, and 71-94 of H4. Interestingly, most of these T cell epitopes are located in regions of histones that have been shown previously to be readily accessible at the surface of nucleosome (30). In the present study, we tested 11 peptides from H4 and 15 peptides from H3 for recognition by CD4+ T cells from unprimed (NZB x New Zealand White (NZW))F1 mice (BW; H-2d/z), a classical lupus-prone mouse model that was not previously investigated for reactivity of anti-histone T cells. These mice share the H-2d allele with SNF1 and BALB/c mice. None of H4 peptides was recognized by autoreactive Th cells from BW mice, whereas CD4+ T cells from these mice recognized several peptides within the region 53-85 of H3. H3 peptides were also tested with the sera from BW mice bled longitudinally for 30 wk, and several peptides, including the peptide 53-70 within the region 53-85, were found to contain B cell epitopes. Moreover, immunization of BALB/c mice with syngeneic nucleosomes or apoptotic cells was found to induce Th cell response against peptides of the same region in H3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides, histones, and nucleosomes

Eleven overlapping peptides covering the whole sequence of histone H4 and fifteen overlapping peptides covering that of H3 (Fig. 1; calf thymus sequence) were synthesized using classical F-moc (N-[9-fluorenyl] metoxycarbonyl) solid-phase chemistry (31). The purity of each purified peptide was assessed by analytical HPLC and was found to be superior to 95%. Their identity was checked by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry using a protein time-of-flight apparatus (Brucker Spectrospin, Bremen, Germany). Individual histones were prepared from calf thymus and purified, as described previously (32). There is no change in the primary structure of mouse and calf core histones. Nucleosomes were prepared, as described previously (30), from mouse liver, and purified on a 5-29% (w/v) sucrose gradient. The nucleosome preparations were characterized by 1.5% agarose gel electrophoresis, and the content of histones was checked by 18% SDS-PAGE (29).



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  		FIGURE 1. Sequence of H4 and H3 peptides. Schematic representation of the 11 overlapping peptides covering the whole sequence of H4 and of the 15 overlapping peptides covering the whole sequence of H3.

 
Induction and measurement of apoptosis

Apoptotic cells were obtained by culturing BALB/c spleen cells (2 x 106 cells/ml) during 48 h in the presence of 5 µg/ml etoposide (ICN, Orsay, France). After 48 h, dead cells were removed from nonapoptotic cell culture by centrifugation on a layer of Ficoll (Lymphoprep, d = 1.077; ICN). Cell death was then evaluated by the detection of phosphatidylserine expression by flow cytometry after addition of FITC annexin V (33, 34).

Mice and immunization protocols

Female BALB/c, NZB, NZW, BW, MRL/lpr, and male SWR mice were purchased from Harlan (Gannat, France). SNF1 mice were bred and maintained in our animal facilities in the Molecular and Cellular Biology Institute (Strasbourg, France). For T cell experiments using BALB/c and NZW mice, two different immunization protocols were used. First, to test the T cell response against nucleosomes and apoptotic cells, three 8- to 10-wk-old mice/Ag/experiment were injected i.p. with nucleosomes (20 µg/mouse, expressed as DNA concentration), nonapoptotic spleen cells (107 cells/mouse), or etoposide-induced apoptotic spleen cells (107 cells/mouse) suspended in PBS and mixed (v/v) with CFA. Second, to test responses against H3 and H4 peptides and whole histone H3, two 8- to 10-wk-old mice/Ag/experiment were immunized s.c. at the base of the tail and hind footpads with 100 µg of unconjugated peptide or histone diluted in H2O and mixed (v/v) with CFA. To test the production of Abs, BW mice were bled each week from wk 10 until they died from their disease.

Lymphocyte proliferation assay and IL-2 secretion

The study of T cell activation in BW and other autoimmune mice was performed, as described previously (35). For each experiment, inguinal, popliteal, periaortic, and axial lymph nodes and spleens were removed from 10 nonimmunized mice aged from 5 to 12 wk. CD4+ T lymph node cells (LNCs) were purified on magnetic beads coated with anti-CD4 mAbs (Dynabeads; Dynal, Olso, Norway). Mitomycin C-treated spleen cells from the respective mice were used as autologous APCs. In each well, 5 x 105 CD4+ T cells were cultured with 105 APCs in the presence of peptide, Con A (2.5 µg/ml; Sigma-Aldrich, St. Louis, MO), or medium alone without Ag. As an additional control, APCs were cultured alone.

Some experiments were performed using purified B cells as APCs. B cells were prepared by positive selection using magnetic beads coated with anti-B220 mAbs (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). This fraction contained >95% of B220+ cells. The remaining fraction obtained after removal of B220+ cells (<3% of B220+ cells) was referred as B- cells and was used in the same conditions. In each well, 5 x 105 CD4+ T cells were cultured either with 105 B cells or 105 non-B cells or with 0.5 x 104 B cells plus 0.5 x 104 non-B cells. These cells were used as APCs and treated with mitomycin C. Cultures were maintained in humid atmosphere containing 5% CO2. After 24, 48, or 72 h, cell culture supernatants were collected to detect IFN-{gamma}, IL-4, and IL-10 secretion by ELISA (see below) or to test the production of IL-2 using CTL-L cells (36). Proliferative responses were measured after culture for 96 h. Cells were pulsed with 1 µCi/well of tritiated thymidine (ICN) during the last 20 h of culture, and [3H]thymidine uptake was measured using a Matrix 9600 direct beta counter (Packard, Meriden, CT). The results are expressed as the arithmetic mean of thymidine uptake expressed as cpm. Proliferative responses were considered to be significantly positive when the tritiated thymidine uptake was equal to or above twice the uptake by LNC cultured in medium alone without Ag (stimulation index (SI) ≥ 2). The SD of triplicate cultures was always below 20% of the mean.

In the case of BALB/c and NZW normal mice immunized with the different peptides, the culture conditions and the proliferation assay were performed, as described previously (29, 36). To 100 µl of LNCs suspended at a concentration of 5 x 106 cells/ml, different concentrations of peptide or protein were added. Each Ag concentration was tested in triplicate, and tests were repeated at least three times in independent experiments. After 48 h, 50 µl supernatant was taken off to test the production of IL-2 using CTL-L cells. To study the T cell reactivity from BALB/c immunized with nucleosomes and nonapoptotic and apoptotic cells, spleen CD4+ T cells were isolated after 10 days, as described above. CD4- T cells obtained after removal of CD4+ T cells were treated with mitomycin C and used as APCs. In each well, 2 x 105 CD4+ T cells were mixed with 105 APCs in the presence of various H3 peptides. After 48 h, 50 µl supernatant was taken off to test the production of IL-2 using CTL-L cells. After 72 h, the proliferative response was measured, as described above, and the results are expressed, as described.

ELISPOT assay for the detection of Ag-dependent IFN-{gamma}-secreting Th cells

Nitrocellulose-bottomed plates (Multiscreen-HA; Millipore, Bedford, MA) were coated with purified anti-mouse IFN-{gamma} Ab (BD PharMingen, San Diego, CA) by incubating the plate overnight at 4°C with 100 µl/well of Ab at 6 µg/ml in PBS, pH 7.4. After six washes in PBS, medium containing 10% FCS was added to each well during 2 h at 37°C. LNCs from BALB/c or NZW mice immunized with the different peptides were then added to the wells at densities ranging from 0.5 to 2 x 106/well in the presence of the homologous peptide at 50 µM in a final volume of 200 µl. A negative control containing 2 x 106 cells/well without any peptide and a positive control containing 0.25 x 106 cells/well incubated in the presence of 2.5 µg/ml Con A were tested in the same conditions. After 24 h at 37°C in humid atmosphere containing 5% CO2, cells were removed by extensive washes in PBS containing 0.05% Tween 20, and biotinylated anti-IFN-{gamma} Abs (100 µl/well, 2 µg/ml) were added to each well. After an overnight incubation at 4°C, 100 µl of alkaline phosphatase-conjugated extravidin (0.3 µg/ml; Sigma-Aldrich) was added to each well and incubated for 1 h at 37°C. After extensive wash in PBS, spots were developed by the addition of 100 µl/well of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (liquid substrate system; Sigma-Aldrich). After spot development, the plates were rinsed thoroughly with H2O. The results are expressed as the number of spots, corresponding to the number of IFN-{gamma}-secreting cells for 106 total cells.

ELISA for cytokine measurements

IFN-{gamma}, IL-4, and IL-10 secretion was evaluated by sandwich ELISA using commercial Abs from BD PharMingen and polyvinyl plates (Falcon, Oxnard, CA; reference 3912). Cytokines were tested in supernatants collected at 24, 48, and 72 h. Standard curves performed with known concentrations of recombinant cytokines (BD PharMingen) were used for the test calibration. All steps were performed according to manufacturer’s recommendations. In these conditions, the minimal levels of detectable cytokine were 180 pg/ml IFN-{gamma}, 100 U/ml IL-4, and 150 pg/ml IL-10.

ELISA for Ab measurements

For the test of mouse sera, polyvinyl microtiter plates (Falcon) were coated overnight at 37°C with 2 µM histone peptide diluted in 0.05 M carbonate buffer, pH 9.6. The conditions used for the coating of native DNA and individual histones were described previously (30). In each assay, sera were also tested in a noncoated well incubated with coating buffer alone as a control. The subsequent steps of the test were performed, as described previously (37), using mouse sera diluted at 1/1,000 in PBS containing 0.05% Tween and 1% BSA and HRP-conjugated goat anti-mouse IgG diluted 1/5,000. Mouse sera were considered positive when the OD values were higher than the mean OD values + 2 SD of sera from 12 normal BALB/c mice, i.e., OD values ≥0.3 in the case of all histone peptides tested in this study. The same procedure was applied with patients’ sera (serum dilution 1/1,000) in using goat anti-human IgG conjugated to HRP (diluted 1/35,000) to reveal Ab binding. To exclude a possible interference of DNA/nucleosome components in serum samples leading to false positives, some sera were also treated with DNase I (5 U/ml for 1 h at 37°C; Sigma-Aldrich) as control. The cutoff points of each assay were determined with the sera from 20 normal donors tested in the same conditions.

HLA-DR peptide-binding assays

Purification of HLA-DR molecules and peptide-binding assays was performed, as previously described (38, 39). Briefly, HLA-DR molecules purified from EBV homozygous cell lines by affinity chromatography were incubated with different concentrations of competitor peptide and an appropriate biotinylated peptide. The biotinylated peptides were the following: HA 306-318 (PKYVKQNTLKLAT) for DRB1*0101 (1 nM, pH 6), DRB1*0401 (30 nM, pH 6), DRB1*1101 (20 nM, pH 5), and DRB5*0101 (10 nM, pH 5.5); YKL (AAYAAAKAAALAA) for DRB1*0701 (10 nM, pH 5); A3 152-166 (EAEQLRAYLDGTGVE) for DRB1*1501 (10 nM, pH 4.5); MT 2-16 (AKTIAYDEEARRGLE) for DRB1*0301 (200 nM, pH 4.5); B1 21-36 (TERVRLVTRHIYNREE) for DRB1*1301 (200 nM, pH 4.5); LOL 191-210 (ESWGAVWRIDTPDKLTGPFT) for DRB3*0101 (10 nM, pH 5.5); and E2/E168 (AGDLLAIETDKATI) for DRB4*0101 (10 nM, pH 5). Quantity of bound peptide was evaluated in an ELISA fluorescence assay. Data were expressed as the peptide concentration that prevented binding of 50% of the labeled peptide (IC50). Maximal binding was determined by incubating the biotinylated peptide with the purified MHC class II molecule in the absence of competitor. Averages were deduced from at least two independent experiments. Unlabeled forms of the biotinylated peptides were used as reference peptides to assess the validity of each experiment. Their IC50 variation did not exceed a factor of 3.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of H4 and H3 epitopes recognized by CD4+ T cell from unprimed BW mice

Based upon previous studies showing that lupus-prone SNF1 mice and immunized normal BALB/c mice generate a T cell response to histone peptides, we anticipated that BW mice that share the H-2d allele with these mice could also mount a CD4+ T cell response to histones. In a first set of experiments, purified CD4+ LNCs from unprimed BW mice were cultured in the presence of each H4 peptide and mitomycin-treated spleen cells from BW mice used as APCs. Although a weak response was occasionally observed with peptides 18-34, 28-42, and 71-94, none of the 11 overlapping H4 peptides tested in this study did induce a significant proliferative response of autoreactive CD4+ T cells from BW mice (data not shown). This absence of proliferative response was observed with 5-, 7-, 10-, and 12-wk-old BW mice and with a peptide concentration range varying from 1 to 200 µM. Secretion of IL-2, IL-4, IL-10, and IFN-{gamma} was undetectable. Unmanipulated BALB/c mice tested in parallel were also negative (proliferation and cytokine secretion). However, as shown previously by Datta and coworkers (27), we confirmed that CD4+ T cells from 12-wk-old SNF1 mice proliferate ex vivo in the presence of 50 µM of H4 peptides 71-94, 72-89, or 88-99 with SI varying from 3 to 5 (data not shown). We thus concluded from this first set of experiments that in contrast to the results found in SNF1 mice, none of the H4 peptides was able to recall any significant positive response of purified CD4+ T cells from BW mice.

We then examined the capacity of 15 overlapping H3 peptides to activate the response of CD4+ T cells from BW mice of different ages in the presence of BW total APCs. Three peptides, namely H3 peptides 53-70, 64-78, and 68-85, were found to induce a peptide dose-dependent proliferation of unprimed BW CD4+ T cells (Fig. 2, A–C). In the case of peptides 64-78 and 68-85, the optimal response was measured with CD4+ T cells from 7-wk-old BW mice, while CD4+ T cells from 7- to 12-wk-old mice responded to peptide 53-70 (Fig. 2B). As reported in Table I, the three H3 peptides, 53-70, 64-78, and 68-85, as well as peptide 58-75 were unable to stimulate CD4+ T cells from unmanipulated, MHC haplotype-matched BALB/c mice (H-2d/d) or SNF1 (H-2d/q) lupus-prone mice, or from non-MHC-related MRL/lpr mice (H-2k/k).



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  		FIGURE 2. T cell response from BW mice against peptides of histone H3. A, The proliferative response of CD4+ T cells from 7-wk-old BW mice was measured ex vivo in the presence of 50 µM of each H3 peptide. T cell proliferation is expressed as SI corresponding to cpm in cultures with peptide/cpm in cultures without peptide. Error bars denote the SD of triplicate cultures. This experiment is representative of three independent experiments performed with a pool of 20 mice in each experiment. B, The proliferative response of purified CD4+ T cells from 5-, 7-, 10-, and 12-wk-old BW mice was cultured in the presence of six peptides covering the region 53-85 of H3 (50 µM). The results are expressed as the mean SI + SD of four to five separate experiments with a pool of 10 mice per experiment. C, CD4+ T cells from 7-wk-old BW mice were cultured in the presence of six H3 peptides tested at various concentrations. The results are expressed as in B. The SD values were comprised between 0.02 and 0.80 (not shown for clarity). The mean tritiated thymidine incorporation in the absence of peptide was 50 cpm. The maximal response of T cells measured in the presence of Con A corresponded to an mean SI of 15. Index values in the gray area (SI <2) are considered as negative.

 

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  		Table I. H3 epitopes recognized by CD4+ T cells from unprimed lupus-prone mice and normal mice immunized against H3 peptides, H3, nucleosomes, and apoptotic cells

 
Culture supernatants of purified BW CD4+ T cells incubated in the presence of six H3 peptides of the region 53-85 or Con A were collected after 24, 48, or 72 h, and tested for their cytokine content (Table II). Depending on each individual H3 peptide or Con A, maximal cytokine secretion was observed at different incubation times, and distinct patterns of cytokine secretion were observed: 1) when CD4+ T cells from BW mice were activated by Con A, although it is well known that autoimmune T cells respond relatively weakly in these conditions (40), we detected a significant production of IL-2, IFN-{gamma}, IL-4, and IL-10 with a peak of secretion at 48 or 72 h of culture; 2) peptide 53-70 induced a secretion of IL-2 and IFN-{gamma}, but no IL-4 and IL-10; 3) peptides 64-78 and 68-85 induced the production of IL-2, IFN-{gamma}, and IL-10 (but no IL-4); 4) peptide 58-75, which induced no proliferative response of BW CD4+ T cells, did not induce any production of IL-2, IFN-{gamma}, IL-4, or IL-10; however, 5) peptides 56-73 and 61-78, which were also negative in the proliferation test, generated the production of IFN-{gamma} and IL-10, respectively, suggesting that they are recognized by CD4+ T cells.


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  		Table II. Cytokine production of CD4+ T cells from BW mice in response to peptides spanning the 53-85 region of histone H3a

 
Presentation of H3 peptides by APCs from H-2d and H-2z normal mice

To address the question of the presentation of H3 peptides by APCs, we first tested whether the six overlapping H3 peptides covering the region 53-85 could recall a T cell response generated in normal mice with the homologous peptides (Fig. 3 and Table I). Groups of two 8- to 10-wk-old BALB/c (H-2d/d) and NZW (H-2z/z) mice were immunized s.c. with each of the overlapping H3 peptides (100 µg/mouse) in the presence of CFA. Mice injected with CFA alone were used as control. After 10 days, draining lymph nodes were removed and each suspension was tested ex vivo for its ability to proliferate (Fig. 3, A and B) and produce IL-2 (Fig. 3, C and D) and IFN-{gamma} (Fig. 3, E and F) in the presence of the respective priming peptides. The results are summarized in Table I. First, in normal NZW mice, peptide 68-85 was immunogenic because it induced in draining lymph nodes CD4+ T lymphocytes that proliferated and produced IL-2 and IFN-{gamma} ex vivo in the presence of the homologous peptide in a dose-dependent manner. Although peptide 53-70 induced no proliferation and IFN-{gamma} secretion, it was also considered to be immunogenic because it generated a significant secretion of IL-2. Peptide 64-78 generated very low levels of IL-2, but no proliferation and IFN-{gamma} production. Second, in BALB/c mice, peptide 53-70 induced CD4+ T lymphocytes that proliferated weakly, and produced positive IL-2 and IFN-{gamma} levels. Peptide 56-73 induced CD4+ T lymphocytes that proliferated weakly, and produced positive IL-2 levels, but no IFN-{gamma}, and peptide 61-78 generated secretion of IL-2 without significant proliferation. The other peptides, namely peptides 58-75, 64-78, and 68-85, failed to induce a Th cell response in BALB/c mice. The major conclusions raised from this set of experiments are that: 1) peptide 53-70 can be presented by APCs from both H-2d and H-2z mice; 2) peptides 64-78 and 68-85 can be presented by APCs from H-2z mice; 3) peptides 56-73 and 61-78 can be presented by APCs from H-2d mice (H-2z was not tested); 4) peptide 58-75 is not presented by H-2d (H-2z was not tested).



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  		FIGURE 3. T cell response to peptides of the 53-85 region of H3 in nonlupus-prone BALB/c and NZW mice immunized with H3 peptides. The proliferative response (A and B), the production of IL-2 (C and D) of Th cells, as well as the number of IFN-{gamma}-producing Th cells (E and F) in the LNC population from NZW mice (A, C, and E) and BALB/c mice (B, D, and F) immunized with 100 µg/mouse of each H3 peptide were measured ex vivo in the presence of homologous peptides. IL-2 production in 48-h culture supernatants was assessed with the CTL-L cell line. The number of IFN-{gamma}-producing cells was measured by ELISPOT after 24 h in the presence or in the absence of homologous peptide at 50 µM. Results are respectively expressed as the mean SI of proliferation, the mean secretion of IL-2 (in U/ml), and the mean number of IFN-{gamma} spots per 106 cells. The results of two to three independent experiments (pool of two mice in each experiment) are shown. The SD values were generally comprised between 0.10 and 0.70 for SI (A, B) and 0.02–0.30 for IL-2 production (C, D). Injection of CFA alone did not induce T cell proliferation, IL-2, or IFN-{gamma} secretion in the presence of the various Ags. The maximal proliferative response of T cells measured in the presence of Con A corresponded to an average SI of 16.3 ± 3.4 for BALB/c mice and 17.8 ± 5.9 for NZW mice. The average maximal production of IL-2 measured in the presence of Con A was 6.3 ± 1.1 U/ml for BALB/c mice and 5.8 ± 1.2 for NZW mice. The maximal number of IFN-{gamma} spots measured in the presence of Con A was 431 ± 46 for BALB/c mice and 192 ± 24 for NZW mice. Index values and IL-2 concentration in the gray area (SI <2 and IL-2 concentration <0.1 U/ml) are considered as negative.

 
To further determine the nature of APCs engaged in H3 peptide presentation to BW Th cells, we next purified B cells from the spleen of BW mice and used the purified B cell fraction or the B cell-depleted fraction for their ability to present peptides 64-78 and 68-85 to BW CD4+ T cells. As shown in Fig. 4, the response to both peptides was completely abolished when either the B cell fraction or the non-B cell fraction was used as APCs. The reactivity of BW CD4+ T cells to both peptides was restored after remixing the B cell and B cell-depleted fractions. We concluded from these observations that B cells alone cannot present the H3 peptides 64-78 and 68-85 to autologous BW CD4+ T cells and that other APCs and/or costimulation molecules are required for activating T cell response.



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  		FIGURE 4. Response to H3 peptides in the presence of B or non-B cells used as APCs. Proliferative response of CD4+ T cells from 7-wk-old BW mice to peptides 64-78 (25 µM) and 68-85 (12.5 µM) was measured ex vivo in the presence of B cells (B+), B cell-depleted fraction (B-), or the reconstituted cell mixture (B+ + B-). Each experiment was performed with the cells from a pool of 10 mice. Index values in the gray area (SI <2) are considered as negative.

 
Priming of normal mice with syngeneic nucleosomes and apoptotic cells induces activation of CD4+ T cells reacting with peptides of the region 53-85

Next, we sought to establish whether systemic exposure to H3, nucleosomes, or apoptotic cells could evoke in normal mice a T cell response similar to the one observed in unprimed BW mice. We immunized 8- to 10-wk-old BALB/c mice with these different Ags (three mice/Ag), and tested the proliferative response as well as the IL-2 secretion of purified CD4+ T cells in the presence of six peptides spanning the region 53-85 of H3.

We first immunized BALB/c mice with H3 protein in CFA, and found that while CD4+ cells responded in a specific and Ag dose-dependent manner to H3, no proliferation and IL-2 secretion were observed in the presence of any of the H3 peptides used as recall Ags (data not shown). However, in the case of BALB/c mice injected i.p. with syngeneic liver nucleosomes in CFA, a population of CD4+ T lymphocytes was generated, which proliferated and secreted IL-2 in a peptide dose-dependent manner mainly in the presence of peptides 61-78 and 64-78 (Fig. 5A).



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  		FIGURE 5. T cell response to peptides of the 53-85 region of H3 in BALB/c mice immunized with syngeneic nucleosomes or apoptotic cells. A, Eight- to 10-wk-old BALB/c mice received either 20 µg nucleosome/mouse (filled bars) or CFA alone (open bars). IL-2 production of purified CD4+ T cells was measured in the presence of 50 µM of each of the six peptides covering the 53-85 region of H3. B–D, Eight- to 10-wk-old BALB/c mice received either 107 nonapoptotic spleen cells (gray bars) or 107 apoptotic spleen cells (shaded bars). The percentage of apoptotic cells in the two spleen cell preparations was determined by annexin V staining (B and C; control, dotted line; annexin staining, dark line). Proliferative response and IL-2 production of purified CD4+ T cells were measured ex vivo in the presence of 50 µM of each peptide. These experiments correspond to the mean response of three independent experiments (pool of three mice in each experiment). For IL-2 secretion, results are expressed as the mean secretion of IL-2 (in U/ml) in cultures with peptide-IL-2 secretion measured in cultures without peptide. IL-2 concentration in the gray area (<0.1 U/ml) is considered as negative. The background IL-2 secretion in the absence of peptide was near 0 for mice injected with CFA alone or with nucleosomes, 3.5 ± 0.3 U/ml for mice injected with nonapoptotic cells, and 0.3 ± 0.1 U/ml for mice injected with apoptotic cells. The mean maximal IL-2 production by T cells measured in the presence of Con A was 8.3 ± 0.8 U/ml for mice injected with CFA alone, 6.7 ± 0.5 for mice injected with nucleosomes, 13 ± 6 U/ml for mice injected with nonapoptotic cells, and 12 ± 5 U/ml for mice injected with apoptotic cells. The proliferative response is expressed as the mean SI. SI <2 is considered as negative. The mean tritiated thymidine incorporation in the absence of peptide was 681 ± 146 cpm for mice injected with nonapoptotic cells, and 936 ± 500 cpm for mice injected with apoptotic cells.

 
We then immunized BALB/c mice i.p. with nonapoptotic or with etoposide-induced apoptotic syngeneic spleen cells and tested the reactivity of T cells against the complete H3 peptide set. Apoptotic cells (corresponding to 81% of total cells in the suspension, as determined by annexin V staining) and nonapoptotic cells (containing 30% of apoptotic cells) were injected in CFA (Fig. 5, B and C). In these conditions, while nonapoptotic cells failed to induce a proliferative T cell response against any of the H3 peptides (a weak response was shown inconstantly with peptide 83-100), syngeneic apoptotic cells did generate CD4+ T lymphocytes that proliferated and secreted IL-2 ex vivo in a peptide dose-dependent manner in the presence of peptide 1-21 (not shown) and the six H3 peptides of the region 53-85 (Fig. 5D). No significant response (proliferation and IL-2 secretion) was detectable in the presence of the other H3 peptides.

Peptide 53-70 does contain a B cell epitope recognized by IgG Abs from BW mice

The Ab reactivity of sera collected from 28 BW mice was tested longitudinally in ELISA with native DNA, the four core histones, H3, H4, H2A, and H2B, as well as with only 10 of 15 H3 peptides described above due to a limited volume of serum available from each mouse. As expected, at wk 10, 50% of the mice developed a strong anti-DNA IgG Ab response (74% at 16 wk). Although in this series of mice 10% only of animals at 16 wk produced IgG Abs reacting in ELISA with any of the four individual histones, H2A, H2B, H3, and H4, in 60% of 16-wk-old mice was a positive IgG Ab reactivity found against three H3 peptides, namely peptides 1-21, 53-70, and 111-130 (Fig. 6). The results obtained with sera preincubated with DNase I did not differ from those obtained with nontreated sera (not shown). As typically observed also in patients with SLE, the kinetic of appearance and fluctuations of levels of these Ab subsets differed from one mouse to another, as illustrated with the three distinct Ab profiles shown in Fig. 6, A–C. Despite these individual differences, Fig. 6D shows that from wk 10 to 30, IgG Abs reacting with these three peptides were revealed in a high proportion of BW mice. We found no correlation between the presence of IgG Abs reacting with H3 or any H3 peptides, or between the presence of these Abs and any pathological signs, such as the proteinuria level or appearance.



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  		FIGURE 6. Longitudinal analysis in ELISA of Abs to H3 peptides produced in 28 BW mice. Serum samples collected fortnightly from weeks 10 to 30 were diluted 1:1000 and allowed to react with 10 overlapping H3 peptides, as indicated within the figure. The IgG response only was measured. A–C, A total of 3 of 28 mice is shown as an example. D, Number of positive mice among the 28 mice followed over the time for mortality and Ab reactivity with peptides 1-21, 53-70, and 111-130. OD values in the gray area (OD450 < 0.3) are considered as negative.

 
Recognition of H3 peptides by Abs from lupus patients and HLA class II DR molecules

All these findings suggest that the H3 region 53-85 may be critical in the autoimmune response in BW mice. We thus investigated more closely whether H3 peptides spanning residues 53-85, which have been highly conserved between mouse and human, are also recognized by HLA-DR molecules. As shown in Table III, peptides 53-70, 56-73, and 58-75 efficiently bind to DR11, DR13, and DRB5 and with intermediate affinity to DR1. The 53-70 peptide is also a good binder for DR7, while the 56-75 and 58-75 peptides are good binders for DR15. The 61-78 and 64-78 peptides are very efficient to bind to DR11 and DR13, while the 68-85 peptide is very active for DR3 and DRB5. Altogether, these results show that the H3 region 53-85 contains multiple potential T cell epitopes in humans.


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  		Table III. Binding capacities of H3 peptides to bind to HLA-DR moleculesa

 
H3 peptides were also tested in ELISA for their ability to be recognized by IgG Abs from 155 nonselected patients with SLE and 30 normal individuals used as control (not shown). No reactivity was found with the sera from normal donors. In contrast, as much as 56.8% of patients’ sera reacted with peptide 47-64 with OD values ≥OD obtained for the 20 normal human sera tested in parallel + 2 SD (i.e., a cutoff value set at 0.30 OD unit with sera diluted 1/1000). Peptides 1-21, 53-70, 64-78, and 118-135 were recognized by 8-15% of sera, and less than 5% of sera reacted with the other H3 overlapping peptides. The presence of anti-dsDNA Abs (in 58.8% of 155 SLE sera, as measured by ELISA) and Abs reacting with H3 peptide 47-64 in the same sample was not correlated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because Th cells from mice and subjects with SLE proliferate poorly and produce low levels of IL-2 and IFN-{gamma} to antigenic challenge ex vivo, very few T cell epitopes have been precisely characterized in autoantigens typically associated to lupus (41). In the case of nucleosome, however, important results have been obtained both in lupus patients and SNF1 mice (25, 27). In H4, two peptides spanning residues 16-39 and 71-94 were found to be recognized by pathogenic SNF1 Th cells (27). Interestingly, peptide 71-94 overlaps with peptide 88-99 recognized by CD4+ T cells from normal BALB/c mice immunized with nucleosomes (29). In the present study, we have demonstrated that polyclonal purified CD4+ T cells from another well-characterized lupus-prone mouse model, the BW mouse of H-2d/z haplotype, fail to recognize any of the 11 overlapping peptides of H4 tested. Although we confirmed the previous results described by Datta and coworkers (27), showing that peptide 71-94 is well recognized by SNF1 CD4+ T cells, this peptide was not recognized by BW CD4+ T cells. Thus, our data clearly show that autoreactive CD4+ T cells from SNF1, BW, and nucleosome-immunized BALB/c mice do not recognize the same epitopes in H4. This suggests strain-specific differences in Ag processing and presentation or in T cell repertoires.

In contrast to the lack of reactivity of BW Th cells with the set of H4 peptides tested, in the present study we have demonstrated that CD4+ T cells from BW mice recognize several epitopes within the region 53-85 of H3 (see Table I for a summary). Tested with T cells from immunized BALB/c and NZW mice, five overlapping peptides of the region 53-85, but not peptide 58-75, generated a response suggesting strongly that they are presented by H-2d and/or H-2z MHC molecules. Three of these five peptides encompassing residues 53-70, 64-78, and 68-85 did induce proliferative response of CD4+ T cells from unmanipulated BW mice, and this proliferation was associated with different patterns of IL-2, IFN-{gamma}, and IL-10 secretion. Interestingly, peptides 56-73 and 61-78 located in the immediate vicinity of peptides 64-78 and 68-85 do not induce CD4+ T cell proliferative response and IL-2 secretion, but generate the secretion of IFN-{gamma} and IL-10, respectively. These peptides may thus act as partial agonists for the TCR of CD4+ T cells. It should be noticed also that the lack of proliferation to some epitopes may be due to their ability to elicit IL-10 production in culture, which in turn inhibits T cell proliferation (42).

Th cells recognizing peptides 53-70, 64-78, and 68-85 or any peptides of the region 53-85 were not detected in unprimed BALB/c mice and other lupus-prone mice studied (Table I). In the SNF1 model, Th cells from 12-wk-old mice were found previously to react with several 15-mer peptides spanning the C-terminal region 85-135 of H3 (27). However, none of these peptides did stimulate any of the pathogenic Th clones tested, and therefore they were considered as negative by the authors. Purified CD4+ T cells from BW mice described in the present work did not recognize any peptides spanning the H3 region 85-135. Lu et al. (25) have described that H3 peptide 85-99 and overlapping peptide 94-108 were recurrently recognized by CD4+ T cells from patients with lupus. Interestingly, the same authors characterized short-term T cell lines from lupus patients that, in addition to reacting with several peptides of the C-terminal region of H3, recognized the H3 peptides 58-72 and 73-87. Altogether, the region 53-85 of H3 seems to contain important Th cell epitopes in BW mice (this work) and lupus patients. In the present study, we further confirm that this region as well as the N-terminal region 1-21 and the C terminus of H3 also contain B cell epitopes recognized by IgG Abs from BW mice and lupus patients (43, 44). Moreover, the domain 53-70 of H3 has been shown to be accessible at the surface of free mononucleosomes and nucleosomes in long chains of chromatin (30). This suggests that nucleosomes could have initiated the B and T cell response against this H3 region.

In previously reported studies (19, 45), it was shown that injection of apoptotic cells in normal mice can induce the production of Abs against known autoantigens implicated in lupus, and also lead to the development of symptoms associated with lupus disease, such as IgG deposition in glomeruli (19). The direct effect of apoptotic cells was not yet investigated at the T cell epitope level. In this work, we found that immunization of BALB/c mice with syngeneic nucleosomes, but not with the H3 molecule, activates CD4+ T cells reacting with H3 peptides (Table I). Furthermore, we demonstrated that immunization with syngeneic apoptotic cells, but not with nonapoptotic cells, generated the activation of Th cells reacting ex vivo with the H3 peptide 1-21 and the six overlapping peptides spanning the 53-85 region of histone H3. This important result reinforces the hypothesis that apoptotic cells may effectively act as initiator of autoreactive Th cell development in lupus mice.

Injected to BALB/c mice, certain H3 peptides, namely peptides 58-75, 64-78, and 68-85, failed to generate T cells reacting with the homologous peptides used as recall Ag ex vivo. These peptides, however, were able to recall nucleosome and/or apoptotic cell-primed CD4+ T cells generated in BALB/c mice. We can thus hypothesize that nucleosomes and apoptotic cells not only present histone peptides to Th cells, but also give a positive signal, which triggers off breakdown of self-tolerance to histones.

It should be noticed that peptides recognized by CD4+ T cells activated in normal mice by nucleosomes and apoptotic cells, on one hand, and peptides recognized by CD4+ T cells from unmanipulated BW mice, on the other hand, are not exactly the same (Table I). These subtle differences may be the consequence of several causes: 1) they might result from the type of cells involved in the presentation of peptides to the immune system. In particular, it has been shown that the presentation of apoptotic cells by dendritic cells, instead of monocytes, induces a potent immune response against their components (46, 47, 48). Future studies should identify the nature of APCs involved in the presentation of nucleosomes and apoptotic cells and determine their importance in this process; 2) nucleosomes and apoptotic cells have been injected in the presence of CFA, a potent activator of immune response; 3) the Th cell response to nucleosomes and apoptotic cells injected i.p. for technical reasons has been measured in the spleen, while the response in BW mice has been measured using LNCs; 4) BW and BALB/c mice shared only one of the two MHC alleles; 5) BW mice are predisposed to lupus disease, and it is well known that a variety of factors, and not only the immunogen, play a role in the development of the disease. To this regard, recent data have shown that serum from lupus patients induces normal monocytes to differentiate into dendritic cells, which can capture Ags from dying cells and present them to CD4+ T cells (49).

In conclusion, this work describes in the BW lupus model an important region of histone H3 containing several CD4+ T cell epitopes, which is also recognized in normal mice by Th cells generated after immunization with syngeneic nucleosomes and apoptotic cells. Furthermore, the region 53-85 contains epitopes recognized by IgG Abs from lupus patients and potential T cell epitopes in humans recognized by several HLA DR molecules. Identification in BW mice of peptides encompassing these histone Th cell epitopes, as well as the recent delineation of Th cell epitopes in the 70K spliceosomal protein in the same mouse model (50) should allow development of promising therapeutic strategies to restore tolerance conditions in this lupus mouse model. Peptides 56-73 and 61-78, which induce a cytokine-restricted response in BW mice, could also act as partial agonists of the receptor of autoreactive T cells and represent good candidates for peptide therapy. Furthermore, our findings underline for the first time the importance of nucleosomes and apoptotic material not only at the B cell level, but also at the T cell level and potential help. This should allow better understanding of the mechanisms leading to breakdown of self-tolerance in lupus, and the events that initiate and perpetuate the autoimmune response.


   Acknowledgments
 
We thank Chrystelle Mézière and Christine Stemmer, who were involved in the ELISA tests of mouse and patients’ sera; Yann Velten, who was involved in the preliminary steps of this work; David Isenberg (London, U.K.), Jean-Louis Pasquali (Strasbourg, France), and Gilbert Fournié (Toulouse, France) for supplying human sera; and Harry Partidos and Fanny Monneaux for comments and suggestions.


   Footnotes
 
1 This work was supported by institutional funding from Centre National de la Recherche Scientifique. Back

2 Address correspondence and reprint requests to Dr. Sylviane Muller, Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Propre de Recherche 9021, 15 rue René Descartes, 67000 Strasbourg, France. E-mail address: S.Muller{at}ibmc.u-strasbg.fr Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; LNC, lymph node cell; SI, stimulation index. Back

Received for publication December 23, 2002. Accepted for publication May 2, 2003.


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 Results
 Discussion
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