Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets
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免疫学杂志 2005年第6期
Abstract
We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 μg nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H471–94 was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN- responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90–100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+CD25+ and CD8+ Treg cells produced and required TGF-1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF--producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.
Introduction
Nucleosomes, derived from apoptotic cells (1), are major immunogens for initiating cognate interactions between autoimmune Th and B cells in systemic lupus erythematosus (SLE)4 (2). CD4+ Th cells drive the production of pathogenic anti-DNA autoantibodies in lupus patients and lupus-prone SNF1 mice (3, 4). Only certain peptides in nucleosomal histones are immunodominant, and spontaneous priming to these particular epitopes occurs in preclinical lupus. The five major autoepitopes for lupus nephritis-inducing Th cells in murine and human lupus are H1'22–42, H2B10–33, H385–102, H416–39, and H471–94 (5, 6, 7). These peptide epitopes are cross-reactively recognized by autoimmune Th cells and B cells. The peptides accelerate lupus nephritis upon immunization, but they delay or even reverse disease upon tolerization in high doses (5, 6, 8). These nucleosomal peptides can be promiscuously presented and recognized in the context of diverse MHC class II alleles, behaving like universal epitopes (9, 10). Thus, universally tolerogenic peptides could be developed for therapy of lupus in humans despite their HLA diversity. High-dose tolerance therapy (300 μg i.v.) with the autoepitopes was effective in halting the progression of established lupus nephritis in SNF1 mice (8). However, high-dose peptide given i.v. may not be suitable in humans. Therefore, in this study, we developed therapy with 300-fold lower doses of the epitopes administrated s.c.
Materials and Methods
Mice
NZB and SWR mice were purchased from The Jackson Laboratory. Lupus-prone SNF1 hybrids were bred and females were used, as approved by the Animal Care and Use Committee.
Peptides
All peptides were synthesized by F-moc chemistry and their purity was checked by amino acid analysis by the manufacturer (Chiron Mimotopes).
Tolerance induction with very low doses of peptides
For long-term experiments, serologically autoimmune, but prenephritic, 12-wk-old SNF1 females (nine mice per group) were injected s.c. with either H1'22–42, H2B10–33, H2B59–73, H416–39, or H471–94 peptide (1 μg/mouse) in saline every 2 wk until the animals died. Control group received only saline. The mice were monitored weekly for proteinurea using albustix (VWR Scientific). Sera were collected every 21/2 months for the determination of total IgG and IgG subclasses of antinuclear autoantibodies. A parallel batch of identically treated mice of each group was followed and sacrificed at different time points for evaluation of renal lesions. To test the immunological consequences of the tolerance therapy early on, another batch of 12-wk-old SNF1 mice (five per group) were treated as above, but they received a total of three injections of each peptide at 2-wk intervals. Ten days after the third injection, these short-term batches of mice were sacrificed for analysis of autoimmune T and B cells and regulatory T (Treg) cells.
Autoantibody quantitation
IgG class autoantibodies to ssDNA, dsDNA, histone and nucleosome (histone-DNA complex) were measured by ELISA (2, 6). Subclasses of IgG autoantibodies were detected by ELISA using alkaline phosphatase (AP)-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechology Associates).
Cell isolation
Total, CD4+ and CD8+ T cells from spleens were purified by appropriate MACS isolation kits using magnetic bead-conjugated Abs specific to each Ag. CD4+CD25+ T cells were purified by a mouse regulatory T cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec). CD28+ and CD28– subsets of CD8+ T cells were separated by using anti-CD28-PE conjugate and anti-PE Microbeads (Miltenyi Biotec). Purity of all isolated cell subsets was >90%.
Adoptive transfer
CD4+CD25–, CD4+CD25+, or CD8+ T cells (1 x 106 cells/mouse) from low-dose peptide (H471–94 or H416–39)-tolerized SNF1 mice were purified by MACS and then immediately injected i.v. into 4-mo-old SNF1 mice. One day after transfer, the mice were immunized i.p. with 100 μg of H1'22–42 peptide in CFA, which accelerates lupus nephritis. Recipient mice were monitored for nephritis and IgG autoantibody levels in serum.
ELISPOT assay
ELISPOT assay plates (Cellular Technology) were coated with capture Abs against IL-2, IL-4, IL-10, or IFN- (BD Pharmingen) in PBS at 4°C overnight. Splenic T cells (1 x 106) from treated mice were cultured with irradiated (3000 rad) splenic APC (B cells, macrophages, and dendritic cells) from 1-mo-old SNF1 mice in the presence of peptides or PBS alone as control. Cells were removed after 24 h of incubation for IFN- and IL-2 or 48 h for IL-4 and IL-10, and the reactions were visualized by addition of the individual anti-cytokine Ab-biotin and subsequent AP-conjugated streptavidin. Cytokine-expressing cells were detected by Immunospot scanning and analysis (Cellular Technology).
Cytokine ELISA
CD4+ T cells (1 x 106) or CD8+ T cells (1 x 106) from low-dose peptide-tolerized or -unmanipulated SNF1 mice were stimulated with H471–94 peptide or Ab to CD3 (1 μg/ml) with splenic APC. Culture supernatants were collected after 48 h (for TGF-1, after 72 h). IL-10 was measured by BD OptEIA ELISA set (BD Pharmingen). For TGF-1, samples were acidified by addition of HCl at 20 mM for 15 min and neutralized by NaOH, and then amounts of TGF-1 were measured by a TGF-1 Emax ImmunoAssay System (Promega).
Helper suppression assay
To detect suppression of autoantibody-inducing help, whole T cells (2.5 x 106/well) or purified CD4+CD25–, CD4+CD25+, or CD8+ T cells (0.3, 0.6, 1.25, or 2.5 x 106/well) from peptide-tolerized or saline-treated mice were cocultured with a helper assay mixture (6) consisting of splenic B cells (5 x 106/well) and Th cells (2.5 x 106/well) from 3- to 5-mo-old, unmanipulated SNF1 mice in 24-well plates (or in 96-well plates with 1/10 cell numbers) for 7 days. The cocultures were performed in the presence of 10 μg/ml cognate peptides or 1 μg/ml nucleosomes. Culture supernatants were collected and assayed for IgG Abs against dsDNA, ssDNA, histones, and nucleosomes as described elsewhere (6). Helper suppression assays were also performed in the presence of anti-IL-10 Ab (10–250 μg/ml), anti-TGF- Ab (10–250 μg/ml), or isotype control for each (R&D Systems) (11).
Transwell experiments
Treg cells (7.5 x 105) from peptide-treated SNF1 mice plus APC (7.5 x 105) were placed in Transwell chambers (12) separated by a 0.4-μm permeable membrane (Corning Costar) from a helper assay coculture of splenic B cells (7.5 x 105/well) from 4- to 5-mo old and T cells (7.5 x 105/well) from 3- to 4-mo-old unmanipulated SNF1 mice. After 7 days of culture, supernatants were assayed for IgG autoantibodies.
Flow cytometry
T cells from tolerized or control mice were stained with PE-conjugated anti-CD62L, CTLA-4, CD69, PD-1, or 4-1BB (BD Pharmingen), TGF-1, latency-associated protein, or glucocorticoid-induced TNFR family related gene (GITR; R&D Systems) at 4°C for 30 min, as described previously (13, 14). Matched PE-conjugated IgG isotype controls were used. Cells were then stained with FITC-conjugated anti-CD25 and CyChrome-conjugated anti-CD4 at 4°C for 20 min. For intracellular CTLA-4 staining, cells were first surface-labeled with FITC-conjugated anti-CD25 and Cy-conjugated anti-CD4 for 20 min at 4°C. Cells were then fixed and permeabilized and then stained with PE-anti-CTLA-4 or PE-IgG isotype control. Cells were analyzed using FACSCalibur (BD Pharmingen).
Real-time RT-PCR
RNA from T cell subsets was isolated by RNeasy kit (Qiagen) and then cDNA synthesized to measure expression of Foxp3, as described previously (15).
Examination of kidneys
Kidney sections were stained with H&E and periodic acid-Schiff and graded 0–4+ for pathologic changes in a blinded fashion, as described elsewhere (6, 8, 16, 17). Immunohistochemistry was done as previously described (6, 17).
ELISA for anti-hen egg lysozyme (HEL) Abs
HEL (10 μg/ml) was coated onto 96-well plates (Nunc). After blocking with 1% BSA in PBS, serially diluted sera were added. Anti-HEL IgG Abs were detected by AP-conjugated anti-mouse IgG (Southern Biotechnology Associates).
Statistical analysis
Chi-square test, log rank test, and the Student two-tailed t test were used. Results are expressed as mean ± SEM.
Results
Very low-dose peptide epitope therapy postpones lupus nephritis and prolongs life span
Twelve-weel-old prenephritic SNF1 females (nine mice per group) were injected s.c. every 2 wk with one of the major autoepitopes, H1'22–42, H2B10–33, H416–39, or H471–91, or a nonstimulatory peptide H2B59–73, each at 1 μg (0.42 nM) of peptide per dose per mouse in saline. A control group received only saline. The control group started developing severe nephritis from 20 wk of age, as documented by persistent proteinurea of >100 mg/dl, and a 3–4+ renal pathology (Fig. 1A). At 31 wk of age, 60% of the saline control group, 40% of the H2B59–73 peptide-injected group, and 33.3% of the H2B10–33-injected group of mice developed severe nephritis (p > 0.2), whereas the H1'22–42, H416–39, or H471–94 peptide-injected mice did not develop disease at this time point (p < 0.01). The largest differences were at 35–38 wk of age, when 80% of the control mice had severe nephritis and the H2B10–33, H2B59–73, or H416–39 group had an incidence of 44.4, 60, or 40%, respectively (p > 0.1–>0.2); whereas in the H1'22–42 or H471–94 group the incidence was nil (p < 0.01). By 47 wk of age, all mice in the saline control group, 88.8% of the H2B10–33 group, 80% of the H2B59–73 group, and 85.7% of the H416–39 group developed severe nephritis, whereas only 40% of the H1'22–42 (p < 0.05) and 20% of the H471–94 peptide-injected groups had severe nephritis (p < 0.01).
FIGURE 1. Beneficial effects of low-dose tolerance therapy. Incidence of severe lupus nephritis (A) and percent survival (B) of lupus-prone SNF1 mice injected with respective nucleosomal histone peptide or saline every 2 wk (nine mice/group). C, Representative kidney sections from peptide-tolerized (left, H471–94 peptide-treated in this example) or control (right, saline-treated) SNF1 mice (H&E; original magnification, x100). The saline control shows marked perivascular and interstitial infiltrate of mononuclear cells, dilated tubules with casts, and hyalinized, sclerotic glomeruli. Lower panels (original magnification, x400) show that in contrast to the peptide-treated mice (left), kidney from control-treated mice (right) shows advanced glomerular lesions with sclerosis, crescent formation, and marked thickening of basement membranes, and perivascular, interstitial infiltrates of mononuclear cells. D, Representative immunohistochemistry (original magnification, x200) shows IgG deposits in glomeruli from both control (right upper panel) and peptide-tolerized (left upper) SNF1 mice. However, marked perivascular cellular infiltrates containing IgG+ plasma cells (upper right panel), as well as CD4+ T (lower left panel), CD8+ T (lower middle panel), and CD138+ plasma cells (lower right panel) were detected only in kidneys of control-treated mice as shown. Positive staining is brown. The results in C and D are representative of five mice per group.
The saline-injected mice rapidly died within 12 mo (Fig. 1B). At this time, 22.2% of the H2B10–33 and 20% of the H2B59–73 peptide-treated animals were alive (p > 0.2), whereas 57.1% of the H416–39-treated (p < 0.05) and 100% of the H1'22–42 and H471–94-treated animals were alive (p < 0.01). At 15 mo, 11.1% of H2B10–33 and 20% of H2B59–73-treated mice were alive (p > 0.2), whereas 42.9% of H416–39 (p < 0.05), 60% of H1'22–42 (p < 0.05), and 100% of H471–94 (p < 0.01)-treated mice were alive. At 21 mo of age, 60% of H471–94 peptide-treated animals were alive (p < 0.05), whereas only 20% of H1'22–42 and 33.3% of H416–39-treated mice were alive; but all other groups were dead. Therefore, very low-dose therapy with H471–94 extended the life span longer than 21 mo, in contrast to the control group that all died within 12 mo. Log rank test for survival was consistent: H1'22–42-treated group (p = 0.000315), H2B10–33-treated group (p = 0.153), H2B59–73-treated group (p = 0.217), H416–39-treated (p = 0.0162), and H471–94-treated group (p = 0.0000182).
Three months after the start of therapy, kidney sections from control mice had an overall score of 3.5 ± 0.5 for nephritis, whereas the H471–94-, H1'22–42-, or H416–39-treated groups showed 1.4 ± 0.8 as the overall score (p < 0.001; Fig. 1C). Glomerular IgG deposits were observed in kidneys from both tolerized and control mice (Fig. 1D), but perivascular and interstitial infiltrations of mononuclear cells containing CD4+ and CD8+ T cells and IgG-producing B cells were markedly reduced in the peptide-treated mice (p < 0.001).
Very low-dose peptide therapy reduces antinuclear autoantibody levels in serum
Sera were first assayed 2 wk after the fifth injection, i.e., 3 mo after starting therapy (at early nephritic age). H471–94 treatment, as compared with H2B59–73 (Fig. 2A), was very effective in reducing levels of autoantibodies pathognomonic of lupus nephritis (18, 19). H471–94 therapy reduced IgG class autoantibodies to dsDNA, ssDNA, nucleosomes, and histones by 41.5, 50, 94.2, and 98.6%, respectively (p < 0.01, p < 0.001, p < 0.001, and p < 0.001, respectively); that of IgG2a subclass autoantibodies by 54, 95.3, 94, and 98%, respectively (p < 0.01, p < 0.001, p < 0.001, and p < 0.001, respectively); that of IgG2b autoantibodies by 82.9, 68, 89, and 88%, respectively (p < 0.01, p < 0.01, p < 0.02, and p < 0.001, respectively); and that of IgG3 autoantibodies by 45, 83.2, 80, and 99%, respectively (all p < 0.001). IgG1 autoantibody levels were already very low in the controls.
FIGURE 2. Low-dose peptide therapy markedly reduces the levels of IgG class autoantibodies (A) and their subclasses (B) in serum. In this sampling, SNF1 mice were bled after 3 mo of treatment (at 6 mo of age) and were assayed for levels of IgG autoantibodies to dsDNA, ssDNA, histone, and nucleosomes. Autoantibody levels (mean ± SEM, mg/dl) are from nine mice per treatment group (key within figure). It should be noted that the commercial Ab reagents used to measure IgG class as a whole vs IgG subclasses were different in sensitivity, thus the standard curves were not comparable.
H416–39 therapy also reduced IgG autoantibodies to nuclear autoantigens as much as H471–94. H1'22–42 therapy reduced the levels of IgG autoantibodies against dsDNA, ssDNA, and nucleosome effectively, but not against histone. H2B10–33 reduced the levels of IgG2a autoantibodies against dsDNA and ssDNA by 25% (p < 0.02) and 85% (p < 0.001), respectively, and that of IgG2b autoantibodies against dsDNA, ssDNA, and nucleosomes by 93, 61, and 82%, respectively (p < 0.001, p < 0.001, and p < 0.05, respectively), but the levels of IgG2a against nucleosome and histone actually increased by 29 and 34%, respectively. The low-dose peptide therapy did not cause IgG1 isotype shift (Th2 deviation), and total polyclonal IgG levels were not significantly different among the groups.
T cell response to autoepitopes was markedly reduced in peptide-treated mice
To test the immunologic consequences of the low-dose peptide therapy early on, a separate set of 12- to 14-wk-old SNF1 mice was injected with the most effective peptide epitope (H416–39 or H471–94), or saline, or H2B59–73 every 2 wk, three times, and then sacrificed. Animals were 18–20 wk old at this time. T cells in unmanipulated SNF1 mice are spontaneously primed to the major nucleosomal peptide epitopes early in life and respond to them in vitro (5, 6). T cells from peptide-treated or control mice in this study were challenged with the epitopes by coculturing with APC in the presence of the peptides or nucleosomes, and their cytokine responses were measured (IL-2, IL-4, IL-10, and IFN-). Only IFN- was detected. T cells from H1'22–42, H416–39, and H471–94-treated mice showed markedly reduced responses, as compared with the control group (Fig. 3A). H1'22–42-treated mice showed the highest reduction at 1 μg/ml cognate epitope (p < 0.001). The therapy also reduced responses to other epitopes (H416–39, H471–94) cross-reactively (p < 0.05). H471–94 treatment resulted in the highest inhibition of response to cognate epitope at 0.1 μg/ml (p < 0.01), as well as to the other epitopes, H1'22–42 and H416–39 (p < 0.01; Fig. 3A). H416–39 treatment also markedly reduced responses to cognate epitope (optimally at 1 μg/ml, p < 0.001) as well as to H1'22–42 and H471–94 (p < 0.001).
FIGURE 3. Low-dose peptide therapy decreases IFN- responses by lupus T cells in ELISPOT. A, Splenic T cells from saline, H1'22–42, H416–39, or H471–94 peptide-treated SNF1 mice were challenged with tolerizing peptide epitope and other relevant epitopes in various concentrations in vitro. Baseline IFN- spots in lupus T plus APC cultures without Ag were 5 ± 3 spots per 1 x 106 T cells. B, Low-dose treatment with peptide (H471–94-treated group shown here) also inhibited IFN- responses to nucleosomes in vitro as compared with control SNF1 mice. IFN- responses are expressed in mean ± SEM positive spots per 1 x 106 T cells from three experiments (five mice per group).
Because low-dose peptide treatment reduced IFN- responses against histone peptide epitopes cross-reactively (Fig. 3A), T cell responses to whole nucleosomes were also assessed and found to be significantly reduced in H471–94-treated mice (Fig. 3B). Similar results were found in H1'22–42 (p < 0.01) and H416–39-treated mice (p < 0.001, data not shown).
Very low-dose peptide therapy generates CD8+ and CD4+CD25+ Treg cells
Low-dose peptide treatment suppressed autoantibodies without causing Th1/Th2 deviation, indicating the possibility of Treg cell involvement. Using the helper-suppression assay, the ability of CD4+CD25– T cells, CD4+CD25+ T cells, or CD8+ T cells from the peptide-treated mice to suppress nucleosome-stimulated autoantibody production in cocultures of lupus Th and B cells of unmanipulated SNF1 mice was determined. CD4+CD25+ T cells and CD8+ T cells from tolerized mice strongly suppressed the ability of unmanipulated lupus CD4+ T cells to help B cells to produce IgG autoantibodies (Fig. 4A). Because help was already optimal in the nucleosome-stimulated helper assay cultures, the levels of autoantibodies produced by unmanipulated SNF1 lupus Th and B cells cultured by themselves did not change significantly upon their cocultures with the CD4+CD25– T cells from the treated mice. Compared with those levels, suppressions of autoantibodies to dsDNA, ssDNA, and nucleosomes by CD4+CD25+ T cells from H416–39-treated mice were 25, 98.8, and 83%, respectively (p < 0.05, p < 0.001, p < 0.001, respectively); from H471–94-treated were mice 24, 74, and 76% (all p < 0.01–<0.001); but from age-matched unmanipulated SNF1 mice were 7, 6.2, and 17% (p > 0.2), respectively. Similarly, suppressions of autoantibody production to dsDNA, ssDNA, and nucleosome by CD8+ T cells from the same groups, respectively, were 41, 99.8, and 72.6% (all p < 0.001); 55, 78,, and 90% (all p < 0.001); and 15, 17, and 21.2% (p < 0.02). Both sets of Treg cells from peptide-treated mice were effective at up to a 1:10 ratio in inhibiting autoantibody production in the helper-suppression assays (data not shown).
FIGURE 4. Induction of potent CD4+CD25+ and CD8+ Treg cells by low-dose peptide therapy. A, CD4+CD25+T cells and CD8+ T cells from low-dose peptide-tolerized SNF1 mice suppressed anti-dsDNA, anti-ssDNA, and anti-nucleosome autoantibody production by lupus Th and B cells from 5-mo-old, unmanipulated SNF1 mice in the nucleosome-stimulated, helper suppression assay (in these examples, the ratio of Treg:lupus Th was 1:1). Baseline levels of IgG autoantibodies produced by B cells cultured by themselves were: anti-dsDNA, 0.01 ± 0.005; anti-ssDNA, 0.04 ± 0.006; antinucleosome, 0.02 ± 0.001; and antihistone, 0.03 ± 0.002 mg/dl. B, Treg cells induced by peptide treatment also suppressed directly the IFN- responses of unmanipulated SNF1 lupus T cells to nucleosomes presented by APC in the ELISPOT assay (ratio of Treg:lupus Th = 1:10). Results are expressed as percent suppression (mean ± SEM) from three experiments (five mice per group). Baseline number of IFN- spots produced by lupus T cells plus APC cultures without Ag were 10 ± 4 spots per 1 x 106 T cells. The purity of each subset of T cells was >90%.
Direct suppressing ability of the IFN- response to autoantigen was also determined by coculturing Treg cells from treated mice with T cells from 51/2-mo-old unmanipulated SNF1 mice in the presence of nucleosomes (1 μg/ml; Fig. 4B). Both sets of Treg cells from peptide-treated mice were effective at up to a 1:100 ratio (Treg cells:target lupus T cells) in strongly inhibiting autoantigen-specific responses of lupus T cells in ELISPOT assays (p < 0.001, Fig. 4B).
Taken together, CD4+CD25+ or CD8+ T cells from peptide-treated mice showed 3- to 16-fold greater suppressive activity on autoantibody production and nucleosome-specific responses than equivalent numbers of those cells from age-matched control SNF1 mice (Fig. 4, p < 0.01–<0.001). Furthermore, we could not detect any differences in the suppressive ability of CD28+ vs CD28– subsets of CD8+ T cells, as found in other systems (20).
Adoptively transferred Treg cells suppress autoantibody production and nephritis in vivo
We isolated each Treg subset from peptide-treated mice and adoptively transferred them into prenephritic (4-mo-old) SNF1 mice. One day after adoptive cell transfer, recipient SNF1 mice were immunized with pathogenic H1'22–42 in CFA. SNF1 mice immunized with H1'22–42 (100 μg/mouse) in adjuvant (CFA) developed severe nephritis and produced high levels of autoantibodies earlier than age-matched SNF1 mice injected with CFA alone or the nonstimulatory peptide H2B59–73 in CFA, as described previously (6). CD4+CD25– T cell transfer did not affect autoantibody levels in the H1'22–42-immunized mice, since they were maximally immunostimulated by autoantigen immunization (6). In comparison to those levels, suppression of serum autoantibodies to dsDNA, ssDNA, nucleosomes, and histone by CD4+CD25+ Treg cells from H471–94-treated mice was 40, 75, 94, and 97%, respectively (p < 0.025–<0.001) and from H416–39- treated mice was100, 80, 92, and 94%, respectively (all p < 0.001; Fig. 5). Suppression of the same IgG autoantibodies by CD8+ Treg cells from H471–94-treated group was 45, 95, 94, and 97%, respectively (p < 0.025–<0.001) and from H416–39-treated mice was 99, 76, 97, and 97%, respectively (all p < 0.001; Fig. 5). Both types of Treg cells inhibited serum autoantibody levels for up to 2 mo after the one-time adoptive transfer. During this period, 30% of the CD4+CD25– T cell recipient group developed severe nephritis within 6 wk of immunization with H1'22–42 and died a week later (data not shown), whereas the incidence of severe nephritis and death in the Treg cell recipient groups was nil at this time (p < 0.05). Because the lupus-prone mice were maximally stimulated by major autoepitope immunization, incidence of disease and level of autoantibodies in H1'22–42-immunized SNF1 mice without adoptive transfer were not significantly different from that in H1'22–42-immunized SNF1 mice that had received CD4+CD25– T cells from peptide-tolerized mice (Fig. 5). After 21/2 mo posttransfer, all of the mice receiving CD4+CD25+ Treg cells still survived (p < 0.05), but 30% of mice receiving either CD4+CD25– or CD8+ cells were dead. The one-time recipients of CD4+CD25+ Treg cells still had higher survival at 31/2 mo posttransfer, as compared with the latter groups (75% vs 50%).
FIGURE 5. Adoptive transfer of Treg cells suppresses pathogenic autoantibodies in lupus-accelerated SNF1 mice. CD4+CD25– T, CD4+CD25+ T, and CD8+ T cells from H471–94-treated (A) or H416–39-treated (B) SNF1 mice were purified by MACS and immediately injected i.v. into 16-wk-old recipient SNF1 at 1 x 106 cells/mouse. One day after transfer, the recipient mice were immunized with 100 μg of H1'22–42 peptide in 0.1 ml of CFA. After transfer, proteinurea was measured every week. One month after immunization, sera were collected for measuring IgG class autoantibodies to nuclear Ags (five mice per group). Levels of autoantibodies in serum of H1'22–42-immunized SNF1 mice without adoptive transfer were not significantly different from those in H1'22–42-immunized SNF1 mice that had received CD4+CD25– T cells from peptide-tolerized mice (p > 0.05). The purity of each subset of T cells was >90%.
Both sets of Treg produce TGF-, but only CD4+CD25+ Treg cells are partially contact dependent
We found that Ab to IL-10 (10–250 μg/ml) did not abrogate the suppression by the Treg cells in the helper-suppression assay (data not shown). With 10-250 μg/ml anti-TGF- Ab, CD4+CD25+ T cell-mediated suppression of production of autoantibodies to dsDNA was not affected; however, that to ssDNA and nucleosomes was reduced, but not abrogated. However, the suppression by regulatory CD8+ T cells in the same helper assay cultures was almost completely abrogated by anti-TGF- Ab (Fig. 6A). Furthermore, CD8+ Treg cells could suppress autoantibody production across a Transwell membrane barrier (Fig. 6B), indicating that soluble TGF- from these cells mediates the immunosuppression. The CD4+CD25+ T cells showed reduced suppression of autoantibody production through the membrane, indicating their suppression is significantly contact dependent (p < 0.01). We next measured TGF-1 production by Treg cell subsets (Fig. 6C). Both CD4+CD25+ and CD8+ T cells from peptide-treated mice produced increased amounts of total TGF-1 upon stimulation with H471–94 or anti-CD3.
FIGURE 6. Suppression of IgG autoantibody production by CD4+CD25+ T cells is mediated by TGF- and cell contact, but suppression by CD8+ T cells is mediated mainly by TGF-. CD4+CD25+ or CD8+ T cells (5 x 105 each) from H471–94- or H416–39-tolerized mice were cocultured with T and B cells (1 x 106 each) from 3- to 4-mo-old unmanipulated SNF1mice in the presence of nucleosomes and anti-cytokine Abs (five mice per group). A, Representative helper suppression assay in the presence of 250 μg/ml anti-TGF- or isotype control. B, Treg cells were separated by membranes from helper assay mixtures containing nucleosomes plus lupus T and B cells from unmanipulated, 3- to 4-mo-old SNF1 in Transwell plates. It should be noted that the helper assay mixture of lupus T and B cells used here (A and B) came from 1- to 2-mo younger, unmanipulated SNF1 mice than those in Fig. 4A. The purity of each subtype of T cells was >90%. C, TGF-1 production by CD4+CDC25+ or CD8+ T cells (1 x 106 each) from H471–94 peptide-tolerized mice stimulated with H471–94 or soluble anti-CD3 (1 μg/ml) plus APC. Results are expressed in mean ± SEM from three experiments. Baseline values of TGF- production without stimulation were 318 ± 14 pg/ml for CD4+CDC25+ T cells and 217 ± 20 pg/ml for CD8+ T cells from the H471–94 peptide-tolerized mice. D, Percentage of CD4+CD25+ T cells in 1 x 106 splenocytes from low-dose peptide-tolerized mice and control mice are shown. This result is representative of nine separate experiments.
Phenotypes of CD4+CD25+ and CD8+ Treg cell subsets
We analyzed cell surface molecules that are relevant to Treg cells (13, 14, 21). Peptide therapy increased the numbers of CD4+CD25+ T cells up to 1.8-fold more in SNF1 mice than in controls (p < 0.02; Fig. 6D). Total numbers of CD4+CD25+CD62L+ T cells per 1 x 106 splenocytes in peptide-tolerized mice were 2.3 x 104 and those from controls were 1.9-fold less (1.2 x 104). CD4+CD25+ cells, but not CD8+ Treg cells from H471–94- or H416–39-treated mice, showed slightly (1.3-fold) increased Foxp3 expression than controls (p < 0.01, data not shown). The CD8+ T cell population was strongly positive for surface expression of TGF-, CD62L, and GITR, and the CD4+CD25+ T cells were strongly positive for GITR, CD62L, TGF-, LAP, and CTLA-4 (data not shown). Low-dose peptide therapy did not change the overall phenotypes of CD4+CD25+ T or CD8+ T cells, when compared with the same subsets isolated from control SNF1 mice (data not shown), indicating that a small percentage of autoantigen-specific Treg cells are induced.
The autoepitope peptides that are effective in low-dose therapy contain class I epitopes
The nucleosomal histone peptides having MHC class II epitopes stimulate CD4+ autoimmune Th cells of lupus (5, 6, 7, 9). Because CD8+ Treg cells were also induced by these autoepitopes, we looked for MHC class I-binding motifs, as described elsewhere (22, 23). Proteasomal cleavage probability and MHC-peptide-binding scores were assigned by computer prediction (http://www.mpiib-berlin.mpg.de/MAPPP/). We considered motifs with scores >0.5 as class I epitopes. The highest overall score for the sequence (bold, underlined) in the peptide containing the motif for binding to each class I molecule of the H-2d haplotype is shown (the SNF1 mice are H-2d/q in haplotype): H471–94 TYTEHAKRKTVTAMDVVYALKRQG (Kd, and Ld motif); Kd: cleavage probability: 1.0, binding score: 0.42, overall score: 0.71; Ld: cleavage probability:1.0, binding score: 0.32, overall score: 0.66; H416–39 KRHRKVLRDNIQGITKPAIRRLAR (Kd motif); Kd: cleavage probability:1.0, binding score: 0.34, overall score: 0.67; KRHRKVLRDNIQGITKPAIRRLAR (Ld motif); Ld: cleavage probability: 1.0, binding score: 0.30, overall score: 0.64; and H1'22–42 STDHPKYSDMIVAAIQAEKNR (Kd motif); Kd: cleavage probability:1.0, binding score: 0.53, overall score: 0.76.
Low-dose therapy does not cause generalized immunosuppression
SNF1 mice were tolerized with H471–94 peptide or saline as control. Ten days after the third injection, the mice were immunized with HEL in CFA (100 μg/mouse) twice at 2-wk intervals. Seven days after the second immunization, the mice were bled to measure anti-HEL Ab response. Low-dose peptide-tolerized mice actually produced a 2-fold higher titer of anti-HEL Ab than control mice (Fig. 7). Moreover, IFN- or IL-2 response to in vitro rechallenge with HEL was actually increased in low-dose peptide-tolerized mice than in control mice (p < 0.01), but responses to anti-CD3 were similar in both groups (p > 0.2, Fig. 7 and data not shown).
FIGURE 7. Low-dose tolerance therapy does not cause generalized immunosuppression. Control or low-dose peptide (H4 71–94)-tolerized SNF1 mice were immunized with HEL in CFA and then immune responses to HEL in both groups were compared (five mice per group). A,. Anti-HEL Ab responses were analyzed by ELISA. B,. IFN- responses to HEL or anti-CD3 Ab were measured by ELISPOT. Results are expressed as mean ± SEM from three experiments.
Discussion
Our studies indicate that nucleosomal-histone peptide epitopes are suitable for Ag-specific tolerance therapy of lupus. Nucleosome is one of the major immunogens driving lupus autoimmunity in murine and human SLE (2, 5, 7, 24, 25). Critical peptide epitopes from nucleosomal histones are recognized by autoimmune T cells of lupus patients, irrespective of their MHC haplotypes (5, 6, 7). The peptide epitopes are derived from a highly conserved, ubiquitous self-Ag, which is a product of ongoing apoptosis in generative lymphoid organs. Therefore, we have not observed any anaphylactic/allergic reactions with these peptides when used either for immunization or for tolerance therapy in >1000 SNF1 mice. Our peptides, administered s.c. in a very low-dose regimen, generate Treg cells that suppress by TGF- and/or by cell contact rather than causing Th2 deviation with allergic reactions seen in the case of peptide therapy of experimental autoimmune encephalomyelitis/multiple sclerosis and NOD diabetes (26, 27). Beneficial effects of our peptides outlast their short half-life by generating longer-lasting Treg cells.
Thus, only 1 μg of nucleosomal histone peptide (H1'22–42, H416–39, or H471–94) injected s.c. every 2 wk to SNF1 mice with clinically overt lupus could restore the life span to normal (2 year) by markedly delaying death from severe nephritis. This s.c. dosage is 300- to 1000-fold lower than what we have previously used for i.v. nucleosomal peptide therapy (8) and what others have applied with anti-DNA autoantibody V region and related peptides (28, 29, 30). After 3 mo of low-dose H1'22–42, H416–39, or H471–94 peptide treatment, IgG autoantibodies against nuclear Ags were reduced up to 90–100% as compared with controls after 3-mo therapy, indicating impairment of pathogenic T cell help. Interestingly, IgG deposits were observed in kidneys from both tolerized and control mice, which did not correlate with their serum autoantibody levels. However, perivascular and interstitial infiltrations of mononuclear cells containing T, B, and plasma cells were markedly decreased in the peptide-tolerized SNF1 mice in contrast to control mice. Thus, very low-dose peptide therapy especially prevented local inflammatory damage in kidneys possibly by diminishing migration and activation of nephritogenic T and B cells, which might share antigenic specificities with the cells responsible for autoantibody production in the periphery.
Consistent with previous work, we found that pathogenic lupus T cells responding to nucleosomal epitopes are mainly IFN--producing Th1 cells (5, 6). Each peptide treatment cross-reactively suppressed responses by lupus T cells to other peptide epitopes in addition to the tolerizing peptide and to nucleosomes, the major lupus immunogen containing many autoepitopes. A single peptide from a histone in the nucleosome can be recognized by multiple autoimmune T cells with diverse TCRs and, conversely, a single autoimmune T cell can promiscuously recognize multiple nucleosomal peptides that are structurally different (5, 8, 9). Thus, a single epitope may tolerize multiple autoimmune Th cells and tolerizing one set of Th cells would deprive help for a broad spectrum of autoimmune B cells (tolerance spreading). The suppression of the IFN- response to autoantigens was dose dependent, demonstrating autoantigen specificity, but it was overcome at higher doses, as in other systems (31). Increased production of TGF- by Treg cells upon stimulation with nucleosomal peptide and lack of suppression of the immune response to foreign Ag (HEL) immunization, again indicates that the peptide therapy generated autoantigen-specific Treg cells. Moreover, suppression of help in autoantibody production by the Treg cells also required the presence of nucleosomes (autoantigenic stimulation) in the helper-suppression assay cultures.
Thus, the low-dose nucleosomal peptide therapy repairs deficiencies of TGF--producing cells and CD8+ Treg function that has been observed in SLE (32, 33, 34, 35, 36). Unlike the case in organ-specific autoimmunity (15, 31, 37), the role of CD4+CD25+ Treg in spontaneous SLE is controversial (38), but they could be potently induced by our therapy. We found that IL-10 is not involved in suppression of lupus with the low-dose peptide therapy. Like high-dose tolerance i.v. (8), nasal tolerance with one of the autoepitopes, H471–94, also could delay or treat lupus nephritis in SNF1 mice, but by generating IL-10-producing T cells (39). IL-10-producing Treg cells might benefit lupus with some caveats (30, 40).
The CD4+CD25+ Treg population could contain a subset of T cells that were secondarily induced to produce TGF-, which suppresses autoimmunity (13, 32, 35, 41, 42). CD8+ Treg cells induced by low-dose tolerance were not CTL because they suppressed across membranes, even though the autoepitope peptides inducing such Treg cells contained class I-binding motifs. Thus, the CD8+ Treg cells we have induced by nucleosomal peptides are different from the TCR clonotype-specific and cytotoxic suppressor cells in other systems (20, 22, 43, 44, 45). TGF- produced by the CD8+ T cells could have induced some of the CD4+CD25+ Treg cells in our system. Indeed, the suppressive effect of these adaptive Treg cells were similar at 1:1, 1:10, or 1:100 ratios (suppressor: target), suggesting involvement of "infectious tolerance" mechanisms, as in other systems (12, 42). We are in the process of studying how a combination of CD4+CD25+ and CD8+ Treg cells could interact in our low-dose peptide tolerance system, as exemplified in a graft-versus-host disease model (46). Although our long-term studies with spontaneous lupus disease were in progress (see footnote #2), another group induced a CD4+CD25+ subset of Treg cells by continuous infusion of a model Ag in low doses (47).
Thus, although lupus T cells are resistant to classical anergy induction (48, 49, 50), tolerance therapy with select nucleosomal peptides still works by generating suppressive Treg cells that impair T cell help for production of a broad spectrum of pathogenic autoantibodies and especially inhibit inflammatory insults in the lupus kidney. Moreover, these Treg cells induced by very low-dose tolerance could possibly suppress activated dendritic cells and other APC in lupus (51, 52).
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Ethan Shevach (National Institutes of Health, Bethesda, MD) for critically reviewing our work and providing helpful suggestions.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants R37-AR39157 and RO1-AI41985 (to S.K.D.).
2 Preliminary results on low-dose peptide tolerance were presented by us as an abstract at the Annual Meeting of American College of Rheumatology in 2002 (Arthritis Rheum. 46, Supplement: S225, Abstract 526, 2002).
3 Address correspondence and reprint requests to Dr. Syamal K. Datta, Division of Rheumatology, Northwestern University Feinberg School of Medicine, 240 East Huron Street, McGaw 2300, Chicago, IL 60611. E-mail address: skd257{at}northwestern.edu
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; Treg, regulatory T; HEL, hen egg lysozyme; AP, alkaline phosphatase; GITR, glucocorticoid-induced TNFR family-related protein; PAS, periodic acid-Schiff.
Received for publication November 8, 2004. Accepted for publication January 3, 2005.
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We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 μg nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H471–94 was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN- responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90–100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+CD25+ and CD8+ Treg cells produced and required TGF-1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF--producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.
Introduction
Nucleosomes, derived from apoptotic cells (1), are major immunogens for initiating cognate interactions between autoimmune Th and B cells in systemic lupus erythematosus (SLE)4 (2). CD4+ Th cells drive the production of pathogenic anti-DNA autoantibodies in lupus patients and lupus-prone SNF1 mice (3, 4). Only certain peptides in nucleosomal histones are immunodominant, and spontaneous priming to these particular epitopes occurs in preclinical lupus. The five major autoepitopes for lupus nephritis-inducing Th cells in murine and human lupus are H1'22–42, H2B10–33, H385–102, H416–39, and H471–94 (5, 6, 7). These peptide epitopes are cross-reactively recognized by autoimmune Th cells and B cells. The peptides accelerate lupus nephritis upon immunization, but they delay or even reverse disease upon tolerization in high doses (5, 6, 8). These nucleosomal peptides can be promiscuously presented and recognized in the context of diverse MHC class II alleles, behaving like universal epitopes (9, 10). Thus, universally tolerogenic peptides could be developed for therapy of lupus in humans despite their HLA diversity. High-dose tolerance therapy (300 μg i.v.) with the autoepitopes was effective in halting the progression of established lupus nephritis in SNF1 mice (8). However, high-dose peptide given i.v. may not be suitable in humans. Therefore, in this study, we developed therapy with 300-fold lower doses of the epitopes administrated s.c.
Materials and Methods
Mice
NZB and SWR mice were purchased from The Jackson Laboratory. Lupus-prone SNF1 hybrids were bred and females were used, as approved by the Animal Care and Use Committee.
Peptides
All peptides were synthesized by F-moc chemistry and their purity was checked by amino acid analysis by the manufacturer (Chiron Mimotopes).
Tolerance induction with very low doses of peptides
For long-term experiments, serologically autoimmune, but prenephritic, 12-wk-old SNF1 females (nine mice per group) were injected s.c. with either H1'22–42, H2B10–33, H2B59–73, H416–39, or H471–94 peptide (1 μg/mouse) in saline every 2 wk until the animals died. Control group received only saline. The mice were monitored weekly for proteinurea using albustix (VWR Scientific). Sera were collected every 21/2 months for the determination of total IgG and IgG subclasses of antinuclear autoantibodies. A parallel batch of identically treated mice of each group was followed and sacrificed at different time points for evaluation of renal lesions. To test the immunological consequences of the tolerance therapy early on, another batch of 12-wk-old SNF1 mice (five per group) were treated as above, but they received a total of three injections of each peptide at 2-wk intervals. Ten days after the third injection, these short-term batches of mice were sacrificed for analysis of autoimmune T and B cells and regulatory T (Treg) cells.
Autoantibody quantitation
IgG class autoantibodies to ssDNA, dsDNA, histone and nucleosome (histone-DNA complex) were measured by ELISA (2, 6). Subclasses of IgG autoantibodies were detected by ELISA using alkaline phosphatase (AP)-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (Southern Biotechology Associates).
Cell isolation
Total, CD4+ and CD8+ T cells from spleens were purified by appropriate MACS isolation kits using magnetic bead-conjugated Abs specific to each Ag. CD4+CD25+ T cells were purified by a mouse regulatory T cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec). CD28+ and CD28– subsets of CD8+ T cells were separated by using anti-CD28-PE conjugate and anti-PE Microbeads (Miltenyi Biotec). Purity of all isolated cell subsets was >90%.
Adoptive transfer
CD4+CD25–, CD4+CD25+, or CD8+ T cells (1 x 106 cells/mouse) from low-dose peptide (H471–94 or H416–39)-tolerized SNF1 mice were purified by MACS and then immediately injected i.v. into 4-mo-old SNF1 mice. One day after transfer, the mice were immunized i.p. with 100 μg of H1'22–42 peptide in CFA, which accelerates lupus nephritis. Recipient mice were monitored for nephritis and IgG autoantibody levels in serum.
ELISPOT assay
ELISPOT assay plates (Cellular Technology) were coated with capture Abs against IL-2, IL-4, IL-10, or IFN- (BD Pharmingen) in PBS at 4°C overnight. Splenic T cells (1 x 106) from treated mice were cultured with irradiated (3000 rad) splenic APC (B cells, macrophages, and dendritic cells) from 1-mo-old SNF1 mice in the presence of peptides or PBS alone as control. Cells were removed after 24 h of incubation for IFN- and IL-2 or 48 h for IL-4 and IL-10, and the reactions were visualized by addition of the individual anti-cytokine Ab-biotin and subsequent AP-conjugated streptavidin. Cytokine-expressing cells were detected by Immunospot scanning and analysis (Cellular Technology).
Cytokine ELISA
CD4+ T cells (1 x 106) or CD8+ T cells (1 x 106) from low-dose peptide-tolerized or -unmanipulated SNF1 mice were stimulated with H471–94 peptide or Ab to CD3 (1 μg/ml) with splenic APC. Culture supernatants were collected after 48 h (for TGF-1, after 72 h). IL-10 was measured by BD OptEIA ELISA set (BD Pharmingen). For TGF-1, samples were acidified by addition of HCl at 20 mM for 15 min and neutralized by NaOH, and then amounts of TGF-1 were measured by a TGF-1 Emax ImmunoAssay System (Promega).
Helper suppression assay
To detect suppression of autoantibody-inducing help, whole T cells (2.5 x 106/well) or purified CD4+CD25–, CD4+CD25+, or CD8+ T cells (0.3, 0.6, 1.25, or 2.5 x 106/well) from peptide-tolerized or saline-treated mice were cocultured with a helper assay mixture (6) consisting of splenic B cells (5 x 106/well) and Th cells (2.5 x 106/well) from 3- to 5-mo-old, unmanipulated SNF1 mice in 24-well plates (or in 96-well plates with 1/10 cell numbers) for 7 days. The cocultures were performed in the presence of 10 μg/ml cognate peptides or 1 μg/ml nucleosomes. Culture supernatants were collected and assayed for IgG Abs against dsDNA, ssDNA, histones, and nucleosomes as described elsewhere (6). Helper suppression assays were also performed in the presence of anti-IL-10 Ab (10–250 μg/ml), anti-TGF- Ab (10–250 μg/ml), or isotype control for each (R&D Systems) (11).
Transwell experiments
Treg cells (7.5 x 105) from peptide-treated SNF1 mice plus APC (7.5 x 105) were placed in Transwell chambers (12) separated by a 0.4-μm permeable membrane (Corning Costar) from a helper assay coculture of splenic B cells (7.5 x 105/well) from 4- to 5-mo old and T cells (7.5 x 105/well) from 3- to 4-mo-old unmanipulated SNF1 mice. After 7 days of culture, supernatants were assayed for IgG autoantibodies.
Flow cytometry
T cells from tolerized or control mice were stained with PE-conjugated anti-CD62L, CTLA-4, CD69, PD-1, or 4-1BB (BD Pharmingen), TGF-1, latency-associated protein, or glucocorticoid-induced TNFR family related gene (GITR; R&D Systems) at 4°C for 30 min, as described previously (13, 14). Matched PE-conjugated IgG isotype controls were used. Cells were then stained with FITC-conjugated anti-CD25 and CyChrome-conjugated anti-CD4 at 4°C for 20 min. For intracellular CTLA-4 staining, cells were first surface-labeled with FITC-conjugated anti-CD25 and Cy-conjugated anti-CD4 for 20 min at 4°C. Cells were then fixed and permeabilized and then stained with PE-anti-CTLA-4 or PE-IgG isotype control. Cells were analyzed using FACSCalibur (BD Pharmingen).
Real-time RT-PCR
RNA from T cell subsets was isolated by RNeasy kit (Qiagen) and then cDNA synthesized to measure expression of Foxp3, as described previously (15).
Examination of kidneys
Kidney sections were stained with H&E and periodic acid-Schiff and graded 0–4+ for pathologic changes in a blinded fashion, as described elsewhere (6, 8, 16, 17). Immunohistochemistry was done as previously described (6, 17).
ELISA for anti-hen egg lysozyme (HEL) Abs
HEL (10 μg/ml) was coated onto 96-well plates (Nunc). After blocking with 1% BSA in PBS, serially diluted sera were added. Anti-HEL IgG Abs were detected by AP-conjugated anti-mouse IgG (Southern Biotechnology Associates).
Statistical analysis
Chi-square test, log rank test, and the Student two-tailed t test were used. Results are expressed as mean ± SEM.
Results
Very low-dose peptide epitope therapy postpones lupus nephritis and prolongs life span
Twelve-weel-old prenephritic SNF1 females (nine mice per group) were injected s.c. every 2 wk with one of the major autoepitopes, H1'22–42, H2B10–33, H416–39, or H471–91, or a nonstimulatory peptide H2B59–73, each at 1 μg (0.42 nM) of peptide per dose per mouse in saline. A control group received only saline. The control group started developing severe nephritis from 20 wk of age, as documented by persistent proteinurea of >100 mg/dl, and a 3–4+ renal pathology (Fig. 1A). At 31 wk of age, 60% of the saline control group, 40% of the H2B59–73 peptide-injected group, and 33.3% of the H2B10–33-injected group of mice developed severe nephritis (p > 0.2), whereas the H1'22–42, H416–39, or H471–94 peptide-injected mice did not develop disease at this time point (p < 0.01). The largest differences were at 35–38 wk of age, when 80% of the control mice had severe nephritis and the H2B10–33, H2B59–73, or H416–39 group had an incidence of 44.4, 60, or 40%, respectively (p > 0.1–>0.2); whereas in the H1'22–42 or H471–94 group the incidence was nil (p < 0.01). By 47 wk of age, all mice in the saline control group, 88.8% of the H2B10–33 group, 80% of the H2B59–73 group, and 85.7% of the H416–39 group developed severe nephritis, whereas only 40% of the H1'22–42 (p < 0.05) and 20% of the H471–94 peptide-injected groups had severe nephritis (p < 0.01).
FIGURE 1. Beneficial effects of low-dose tolerance therapy. Incidence of severe lupus nephritis (A) and percent survival (B) of lupus-prone SNF1 mice injected with respective nucleosomal histone peptide or saline every 2 wk (nine mice/group). C, Representative kidney sections from peptide-tolerized (left, H471–94 peptide-treated in this example) or control (right, saline-treated) SNF1 mice (H&E; original magnification, x100). The saline control shows marked perivascular and interstitial infiltrate of mononuclear cells, dilated tubules with casts, and hyalinized, sclerotic glomeruli. Lower panels (original magnification, x400) show that in contrast to the peptide-treated mice (left), kidney from control-treated mice (right) shows advanced glomerular lesions with sclerosis, crescent formation, and marked thickening of basement membranes, and perivascular, interstitial infiltrates of mononuclear cells. D, Representative immunohistochemistry (original magnification, x200) shows IgG deposits in glomeruli from both control (right upper panel) and peptide-tolerized (left upper) SNF1 mice. However, marked perivascular cellular infiltrates containing IgG+ plasma cells (upper right panel), as well as CD4+ T (lower left panel), CD8+ T (lower middle panel), and CD138+ plasma cells (lower right panel) were detected only in kidneys of control-treated mice as shown. Positive staining is brown. The results in C and D are representative of five mice per group.
The saline-injected mice rapidly died within 12 mo (Fig. 1B). At this time, 22.2% of the H2B10–33 and 20% of the H2B59–73 peptide-treated animals were alive (p > 0.2), whereas 57.1% of the H416–39-treated (p < 0.05) and 100% of the H1'22–42 and H471–94-treated animals were alive (p < 0.01). At 15 mo, 11.1% of H2B10–33 and 20% of H2B59–73-treated mice were alive (p > 0.2), whereas 42.9% of H416–39 (p < 0.05), 60% of H1'22–42 (p < 0.05), and 100% of H471–94 (p < 0.01)-treated mice were alive. At 21 mo of age, 60% of H471–94 peptide-treated animals were alive (p < 0.05), whereas only 20% of H1'22–42 and 33.3% of H416–39-treated mice were alive; but all other groups were dead. Therefore, very low-dose therapy with H471–94 extended the life span longer than 21 mo, in contrast to the control group that all died within 12 mo. Log rank test for survival was consistent: H1'22–42-treated group (p = 0.000315), H2B10–33-treated group (p = 0.153), H2B59–73-treated group (p = 0.217), H416–39-treated (p = 0.0162), and H471–94-treated group (p = 0.0000182).
Three months after the start of therapy, kidney sections from control mice had an overall score of 3.5 ± 0.5 for nephritis, whereas the H471–94-, H1'22–42-, or H416–39-treated groups showed 1.4 ± 0.8 as the overall score (p < 0.001; Fig. 1C). Glomerular IgG deposits were observed in kidneys from both tolerized and control mice (Fig. 1D), but perivascular and interstitial infiltrations of mononuclear cells containing CD4+ and CD8+ T cells and IgG-producing B cells were markedly reduced in the peptide-treated mice (p < 0.001).
Very low-dose peptide therapy reduces antinuclear autoantibody levels in serum
Sera were first assayed 2 wk after the fifth injection, i.e., 3 mo after starting therapy (at early nephritic age). H471–94 treatment, as compared with H2B59–73 (Fig. 2A), was very effective in reducing levels of autoantibodies pathognomonic of lupus nephritis (18, 19). H471–94 therapy reduced IgG class autoantibodies to dsDNA, ssDNA, nucleosomes, and histones by 41.5, 50, 94.2, and 98.6%, respectively (p < 0.01, p < 0.001, p < 0.001, and p < 0.001, respectively); that of IgG2a subclass autoantibodies by 54, 95.3, 94, and 98%, respectively (p < 0.01, p < 0.001, p < 0.001, and p < 0.001, respectively); that of IgG2b autoantibodies by 82.9, 68, 89, and 88%, respectively (p < 0.01, p < 0.01, p < 0.02, and p < 0.001, respectively); and that of IgG3 autoantibodies by 45, 83.2, 80, and 99%, respectively (all p < 0.001). IgG1 autoantibody levels were already very low in the controls.
FIGURE 2. Low-dose peptide therapy markedly reduces the levels of IgG class autoantibodies (A) and their subclasses (B) in serum. In this sampling, SNF1 mice were bled after 3 mo of treatment (at 6 mo of age) and were assayed for levels of IgG autoantibodies to dsDNA, ssDNA, histone, and nucleosomes. Autoantibody levels (mean ± SEM, mg/dl) are from nine mice per treatment group (key within figure). It should be noted that the commercial Ab reagents used to measure IgG class as a whole vs IgG subclasses were different in sensitivity, thus the standard curves were not comparable.
H416–39 therapy also reduced IgG autoantibodies to nuclear autoantigens as much as H471–94. H1'22–42 therapy reduced the levels of IgG autoantibodies against dsDNA, ssDNA, and nucleosome effectively, but not against histone. H2B10–33 reduced the levels of IgG2a autoantibodies against dsDNA and ssDNA by 25% (p < 0.02) and 85% (p < 0.001), respectively, and that of IgG2b autoantibodies against dsDNA, ssDNA, and nucleosomes by 93, 61, and 82%, respectively (p < 0.001, p < 0.001, and p < 0.05, respectively), but the levels of IgG2a against nucleosome and histone actually increased by 29 and 34%, respectively. The low-dose peptide therapy did not cause IgG1 isotype shift (Th2 deviation), and total polyclonal IgG levels were not significantly different among the groups.
T cell response to autoepitopes was markedly reduced in peptide-treated mice
To test the immunologic consequences of the low-dose peptide therapy early on, a separate set of 12- to 14-wk-old SNF1 mice was injected with the most effective peptide epitope (H416–39 or H471–94), or saline, or H2B59–73 every 2 wk, three times, and then sacrificed. Animals were 18–20 wk old at this time. T cells in unmanipulated SNF1 mice are spontaneously primed to the major nucleosomal peptide epitopes early in life and respond to them in vitro (5, 6). T cells from peptide-treated or control mice in this study were challenged with the epitopes by coculturing with APC in the presence of the peptides or nucleosomes, and their cytokine responses were measured (IL-2, IL-4, IL-10, and IFN-). Only IFN- was detected. T cells from H1'22–42, H416–39, and H471–94-treated mice showed markedly reduced responses, as compared with the control group (Fig. 3A). H1'22–42-treated mice showed the highest reduction at 1 μg/ml cognate epitope (p < 0.001). The therapy also reduced responses to other epitopes (H416–39, H471–94) cross-reactively (p < 0.05). H471–94 treatment resulted in the highest inhibition of response to cognate epitope at 0.1 μg/ml (p < 0.01), as well as to the other epitopes, H1'22–42 and H416–39 (p < 0.01; Fig. 3A). H416–39 treatment also markedly reduced responses to cognate epitope (optimally at 1 μg/ml, p < 0.001) as well as to H1'22–42 and H471–94 (p < 0.001).
FIGURE 3. Low-dose peptide therapy decreases IFN- responses by lupus T cells in ELISPOT. A, Splenic T cells from saline, H1'22–42, H416–39, or H471–94 peptide-treated SNF1 mice were challenged with tolerizing peptide epitope and other relevant epitopes in various concentrations in vitro. Baseline IFN- spots in lupus T plus APC cultures without Ag were 5 ± 3 spots per 1 x 106 T cells. B, Low-dose treatment with peptide (H471–94-treated group shown here) also inhibited IFN- responses to nucleosomes in vitro as compared with control SNF1 mice. IFN- responses are expressed in mean ± SEM positive spots per 1 x 106 T cells from three experiments (five mice per group).
Because low-dose peptide treatment reduced IFN- responses against histone peptide epitopes cross-reactively (Fig. 3A), T cell responses to whole nucleosomes were also assessed and found to be significantly reduced in H471–94-treated mice (Fig. 3B). Similar results were found in H1'22–42 (p < 0.01) and H416–39-treated mice (p < 0.001, data not shown).
Very low-dose peptide therapy generates CD8+ and CD4+CD25+ Treg cells
Low-dose peptide treatment suppressed autoantibodies without causing Th1/Th2 deviation, indicating the possibility of Treg cell involvement. Using the helper-suppression assay, the ability of CD4+CD25– T cells, CD4+CD25+ T cells, or CD8+ T cells from the peptide-treated mice to suppress nucleosome-stimulated autoantibody production in cocultures of lupus Th and B cells of unmanipulated SNF1 mice was determined. CD4+CD25+ T cells and CD8+ T cells from tolerized mice strongly suppressed the ability of unmanipulated lupus CD4+ T cells to help B cells to produce IgG autoantibodies (Fig. 4A). Because help was already optimal in the nucleosome-stimulated helper assay cultures, the levels of autoantibodies produced by unmanipulated SNF1 lupus Th and B cells cultured by themselves did not change significantly upon their cocultures with the CD4+CD25– T cells from the treated mice. Compared with those levels, suppressions of autoantibodies to dsDNA, ssDNA, and nucleosomes by CD4+CD25+ T cells from H416–39-treated mice were 25, 98.8, and 83%, respectively (p < 0.05, p < 0.001, p < 0.001, respectively); from H471–94-treated were mice 24, 74, and 76% (all p < 0.01–<0.001); but from age-matched unmanipulated SNF1 mice were 7, 6.2, and 17% (p > 0.2), respectively. Similarly, suppressions of autoantibody production to dsDNA, ssDNA, and nucleosome by CD8+ T cells from the same groups, respectively, were 41, 99.8, and 72.6% (all p < 0.001); 55, 78,, and 90% (all p < 0.001); and 15, 17, and 21.2% (p < 0.02). Both sets of Treg cells from peptide-treated mice were effective at up to a 1:10 ratio in inhibiting autoantibody production in the helper-suppression assays (data not shown).
FIGURE 4. Induction of potent CD4+CD25+ and CD8+ Treg cells by low-dose peptide therapy. A, CD4+CD25+T cells and CD8+ T cells from low-dose peptide-tolerized SNF1 mice suppressed anti-dsDNA, anti-ssDNA, and anti-nucleosome autoantibody production by lupus Th and B cells from 5-mo-old, unmanipulated SNF1 mice in the nucleosome-stimulated, helper suppression assay (in these examples, the ratio of Treg:lupus Th was 1:1). Baseline levels of IgG autoantibodies produced by B cells cultured by themselves were: anti-dsDNA, 0.01 ± 0.005; anti-ssDNA, 0.04 ± 0.006; antinucleosome, 0.02 ± 0.001; and antihistone, 0.03 ± 0.002 mg/dl. B, Treg cells induced by peptide treatment also suppressed directly the IFN- responses of unmanipulated SNF1 lupus T cells to nucleosomes presented by APC in the ELISPOT assay (ratio of Treg:lupus Th = 1:10). Results are expressed as percent suppression (mean ± SEM) from three experiments (five mice per group). Baseline number of IFN- spots produced by lupus T cells plus APC cultures without Ag were 10 ± 4 spots per 1 x 106 T cells. The purity of each subset of T cells was >90%.
Direct suppressing ability of the IFN- response to autoantigen was also determined by coculturing Treg cells from treated mice with T cells from 51/2-mo-old unmanipulated SNF1 mice in the presence of nucleosomes (1 μg/ml; Fig. 4B). Both sets of Treg cells from peptide-treated mice were effective at up to a 1:100 ratio (Treg cells:target lupus T cells) in strongly inhibiting autoantigen-specific responses of lupus T cells in ELISPOT assays (p < 0.001, Fig. 4B).
Taken together, CD4+CD25+ or CD8+ T cells from peptide-treated mice showed 3- to 16-fold greater suppressive activity on autoantibody production and nucleosome-specific responses than equivalent numbers of those cells from age-matched control SNF1 mice (Fig. 4, p < 0.01–<0.001). Furthermore, we could not detect any differences in the suppressive ability of CD28+ vs CD28– subsets of CD8+ T cells, as found in other systems (20).
Adoptively transferred Treg cells suppress autoantibody production and nephritis in vivo
We isolated each Treg subset from peptide-treated mice and adoptively transferred them into prenephritic (4-mo-old) SNF1 mice. One day after adoptive cell transfer, recipient SNF1 mice were immunized with pathogenic H1'22–42 in CFA. SNF1 mice immunized with H1'22–42 (100 μg/mouse) in adjuvant (CFA) developed severe nephritis and produced high levels of autoantibodies earlier than age-matched SNF1 mice injected with CFA alone or the nonstimulatory peptide H2B59–73 in CFA, as described previously (6). CD4+CD25– T cell transfer did not affect autoantibody levels in the H1'22–42-immunized mice, since they were maximally immunostimulated by autoantigen immunization (6). In comparison to those levels, suppression of serum autoantibodies to dsDNA, ssDNA, nucleosomes, and histone by CD4+CD25+ Treg cells from H471–94-treated mice was 40, 75, 94, and 97%, respectively (p < 0.025–<0.001) and from H416–39- treated mice was100, 80, 92, and 94%, respectively (all p < 0.001; Fig. 5). Suppression of the same IgG autoantibodies by CD8+ Treg cells from H471–94-treated group was 45, 95, 94, and 97%, respectively (p < 0.025–<0.001) and from H416–39-treated mice was 99, 76, 97, and 97%, respectively (all p < 0.001; Fig. 5). Both types of Treg cells inhibited serum autoantibody levels for up to 2 mo after the one-time adoptive transfer. During this period, 30% of the CD4+CD25– T cell recipient group developed severe nephritis within 6 wk of immunization with H1'22–42 and died a week later (data not shown), whereas the incidence of severe nephritis and death in the Treg cell recipient groups was nil at this time (p < 0.05). Because the lupus-prone mice were maximally stimulated by major autoepitope immunization, incidence of disease and level of autoantibodies in H1'22–42-immunized SNF1 mice without adoptive transfer were not significantly different from that in H1'22–42-immunized SNF1 mice that had received CD4+CD25– T cells from peptide-tolerized mice (Fig. 5). After 21/2 mo posttransfer, all of the mice receiving CD4+CD25+ Treg cells still survived (p < 0.05), but 30% of mice receiving either CD4+CD25– or CD8+ cells were dead. The one-time recipients of CD4+CD25+ Treg cells still had higher survival at 31/2 mo posttransfer, as compared with the latter groups (75% vs 50%).
FIGURE 5. Adoptive transfer of Treg cells suppresses pathogenic autoantibodies in lupus-accelerated SNF1 mice. CD4+CD25– T, CD4+CD25+ T, and CD8+ T cells from H471–94-treated (A) or H416–39-treated (B) SNF1 mice were purified by MACS and immediately injected i.v. into 16-wk-old recipient SNF1 at 1 x 106 cells/mouse. One day after transfer, the recipient mice were immunized with 100 μg of H1'22–42 peptide in 0.1 ml of CFA. After transfer, proteinurea was measured every week. One month after immunization, sera were collected for measuring IgG class autoantibodies to nuclear Ags (five mice per group). Levels of autoantibodies in serum of H1'22–42-immunized SNF1 mice without adoptive transfer were not significantly different from those in H1'22–42-immunized SNF1 mice that had received CD4+CD25– T cells from peptide-tolerized mice (p > 0.05). The purity of each subset of T cells was >90%.
Both sets of Treg produce TGF-, but only CD4+CD25+ Treg cells are partially contact dependent
We found that Ab to IL-10 (10–250 μg/ml) did not abrogate the suppression by the Treg cells in the helper-suppression assay (data not shown). With 10-250 μg/ml anti-TGF- Ab, CD4+CD25+ T cell-mediated suppression of production of autoantibodies to dsDNA was not affected; however, that to ssDNA and nucleosomes was reduced, but not abrogated. However, the suppression by regulatory CD8+ T cells in the same helper assay cultures was almost completely abrogated by anti-TGF- Ab (Fig. 6A). Furthermore, CD8+ Treg cells could suppress autoantibody production across a Transwell membrane barrier (Fig. 6B), indicating that soluble TGF- from these cells mediates the immunosuppression. The CD4+CD25+ T cells showed reduced suppression of autoantibody production through the membrane, indicating their suppression is significantly contact dependent (p < 0.01). We next measured TGF-1 production by Treg cell subsets (Fig. 6C). Both CD4+CD25+ and CD8+ T cells from peptide-treated mice produced increased amounts of total TGF-1 upon stimulation with H471–94 or anti-CD3.
FIGURE 6. Suppression of IgG autoantibody production by CD4+CD25+ T cells is mediated by TGF- and cell contact, but suppression by CD8+ T cells is mediated mainly by TGF-. CD4+CD25+ or CD8+ T cells (5 x 105 each) from H471–94- or H416–39-tolerized mice were cocultured with T and B cells (1 x 106 each) from 3- to 4-mo-old unmanipulated SNF1mice in the presence of nucleosomes and anti-cytokine Abs (five mice per group). A, Representative helper suppression assay in the presence of 250 μg/ml anti-TGF- or isotype control. B, Treg cells were separated by membranes from helper assay mixtures containing nucleosomes plus lupus T and B cells from unmanipulated, 3- to 4-mo-old SNF1 in Transwell plates. It should be noted that the helper assay mixture of lupus T and B cells used here (A and B) came from 1- to 2-mo younger, unmanipulated SNF1 mice than those in Fig. 4A. The purity of each subtype of T cells was >90%. C, TGF-1 production by CD4+CDC25+ or CD8+ T cells (1 x 106 each) from H471–94 peptide-tolerized mice stimulated with H471–94 or soluble anti-CD3 (1 μg/ml) plus APC. Results are expressed in mean ± SEM from three experiments. Baseline values of TGF- production without stimulation were 318 ± 14 pg/ml for CD4+CDC25+ T cells and 217 ± 20 pg/ml for CD8+ T cells from the H471–94 peptide-tolerized mice. D, Percentage of CD4+CD25+ T cells in 1 x 106 splenocytes from low-dose peptide-tolerized mice and control mice are shown. This result is representative of nine separate experiments.
Phenotypes of CD4+CD25+ and CD8+ Treg cell subsets
We analyzed cell surface molecules that are relevant to Treg cells (13, 14, 21). Peptide therapy increased the numbers of CD4+CD25+ T cells up to 1.8-fold more in SNF1 mice than in controls (p < 0.02; Fig. 6D). Total numbers of CD4+CD25+CD62L+ T cells per 1 x 106 splenocytes in peptide-tolerized mice were 2.3 x 104 and those from controls were 1.9-fold less (1.2 x 104). CD4+CD25+ cells, but not CD8+ Treg cells from H471–94- or H416–39-treated mice, showed slightly (1.3-fold) increased Foxp3 expression than controls (p < 0.01, data not shown). The CD8+ T cell population was strongly positive for surface expression of TGF-, CD62L, and GITR, and the CD4+CD25+ T cells were strongly positive for GITR, CD62L, TGF-, LAP, and CTLA-4 (data not shown). Low-dose peptide therapy did not change the overall phenotypes of CD4+CD25+ T or CD8+ T cells, when compared with the same subsets isolated from control SNF1 mice (data not shown), indicating that a small percentage of autoantigen-specific Treg cells are induced.
The autoepitope peptides that are effective in low-dose therapy contain class I epitopes
The nucleosomal histone peptides having MHC class II epitopes stimulate CD4+ autoimmune Th cells of lupus (5, 6, 7, 9). Because CD8+ Treg cells were also induced by these autoepitopes, we looked for MHC class I-binding motifs, as described elsewhere (22, 23). Proteasomal cleavage probability and MHC-peptide-binding scores were assigned by computer prediction (http://www.mpiib-berlin.mpg.de/MAPPP/). We considered motifs with scores >0.5 as class I epitopes. The highest overall score for the sequence (bold, underlined) in the peptide containing the motif for binding to each class I molecule of the H-2d haplotype is shown (the SNF1 mice are H-2d/q in haplotype): H471–94 TYTEHAKRKTVTAMDVVYALKRQG (Kd, and Ld motif); Kd: cleavage probability: 1.0, binding score: 0.42, overall score: 0.71; Ld: cleavage probability:1.0, binding score: 0.32, overall score: 0.66; H416–39 KRHRKVLRDNIQGITKPAIRRLAR (Kd motif); Kd: cleavage probability:1.0, binding score: 0.34, overall score: 0.67; KRHRKVLRDNIQGITKPAIRRLAR (Ld motif); Ld: cleavage probability: 1.0, binding score: 0.30, overall score: 0.64; and H1'22–42 STDHPKYSDMIVAAIQAEKNR (Kd motif); Kd: cleavage probability:1.0, binding score: 0.53, overall score: 0.76.
Low-dose therapy does not cause generalized immunosuppression
SNF1 mice were tolerized with H471–94 peptide or saline as control. Ten days after the third injection, the mice were immunized with HEL in CFA (100 μg/mouse) twice at 2-wk intervals. Seven days after the second immunization, the mice were bled to measure anti-HEL Ab response. Low-dose peptide-tolerized mice actually produced a 2-fold higher titer of anti-HEL Ab than control mice (Fig. 7). Moreover, IFN- or IL-2 response to in vitro rechallenge with HEL was actually increased in low-dose peptide-tolerized mice than in control mice (p < 0.01), but responses to anti-CD3 were similar in both groups (p > 0.2, Fig. 7 and data not shown).
FIGURE 7. Low-dose tolerance therapy does not cause generalized immunosuppression. Control or low-dose peptide (H4 71–94)-tolerized SNF1 mice were immunized with HEL in CFA and then immune responses to HEL in both groups were compared (five mice per group). A,. Anti-HEL Ab responses were analyzed by ELISA. B,. IFN- responses to HEL or anti-CD3 Ab were measured by ELISPOT. Results are expressed as mean ± SEM from three experiments.
Discussion
Our studies indicate that nucleosomal-histone peptide epitopes are suitable for Ag-specific tolerance therapy of lupus. Nucleosome is one of the major immunogens driving lupus autoimmunity in murine and human SLE (2, 5, 7, 24, 25). Critical peptide epitopes from nucleosomal histones are recognized by autoimmune T cells of lupus patients, irrespective of their MHC haplotypes (5, 6, 7). The peptide epitopes are derived from a highly conserved, ubiquitous self-Ag, which is a product of ongoing apoptosis in generative lymphoid organs. Therefore, we have not observed any anaphylactic/allergic reactions with these peptides when used either for immunization or for tolerance therapy in >1000 SNF1 mice. Our peptides, administered s.c. in a very low-dose regimen, generate Treg cells that suppress by TGF- and/or by cell contact rather than causing Th2 deviation with allergic reactions seen in the case of peptide therapy of experimental autoimmune encephalomyelitis/multiple sclerosis and NOD diabetes (26, 27). Beneficial effects of our peptides outlast their short half-life by generating longer-lasting Treg cells.
Thus, only 1 μg of nucleosomal histone peptide (H1'22–42, H416–39, or H471–94) injected s.c. every 2 wk to SNF1 mice with clinically overt lupus could restore the life span to normal (2 year) by markedly delaying death from severe nephritis. This s.c. dosage is 300- to 1000-fold lower than what we have previously used for i.v. nucleosomal peptide therapy (8) and what others have applied with anti-DNA autoantibody V region and related peptides (28, 29, 30). After 3 mo of low-dose H1'22–42, H416–39, or H471–94 peptide treatment, IgG autoantibodies against nuclear Ags were reduced up to 90–100% as compared with controls after 3-mo therapy, indicating impairment of pathogenic T cell help. Interestingly, IgG deposits were observed in kidneys from both tolerized and control mice, which did not correlate with their serum autoantibody levels. However, perivascular and interstitial infiltrations of mononuclear cells containing T, B, and plasma cells were markedly decreased in the peptide-tolerized SNF1 mice in contrast to control mice. Thus, very low-dose peptide therapy especially prevented local inflammatory damage in kidneys possibly by diminishing migration and activation of nephritogenic T and B cells, which might share antigenic specificities with the cells responsible for autoantibody production in the periphery.
Consistent with previous work, we found that pathogenic lupus T cells responding to nucleosomal epitopes are mainly IFN--producing Th1 cells (5, 6). Each peptide treatment cross-reactively suppressed responses by lupus T cells to other peptide epitopes in addition to the tolerizing peptide and to nucleosomes, the major lupus immunogen containing many autoepitopes. A single peptide from a histone in the nucleosome can be recognized by multiple autoimmune T cells with diverse TCRs and, conversely, a single autoimmune T cell can promiscuously recognize multiple nucleosomal peptides that are structurally different (5, 8, 9). Thus, a single epitope may tolerize multiple autoimmune Th cells and tolerizing one set of Th cells would deprive help for a broad spectrum of autoimmune B cells (tolerance spreading). The suppression of the IFN- response to autoantigens was dose dependent, demonstrating autoantigen specificity, but it was overcome at higher doses, as in other systems (31). Increased production of TGF- by Treg cells upon stimulation with nucleosomal peptide and lack of suppression of the immune response to foreign Ag (HEL) immunization, again indicates that the peptide therapy generated autoantigen-specific Treg cells. Moreover, suppression of help in autoantibody production by the Treg cells also required the presence of nucleosomes (autoantigenic stimulation) in the helper-suppression assay cultures.
Thus, the low-dose nucleosomal peptide therapy repairs deficiencies of TGF--producing cells and CD8+ Treg function that has been observed in SLE (32, 33, 34, 35, 36). Unlike the case in organ-specific autoimmunity (15, 31, 37), the role of CD4+CD25+ Treg in spontaneous SLE is controversial (38), but they could be potently induced by our therapy. We found that IL-10 is not involved in suppression of lupus with the low-dose peptide therapy. Like high-dose tolerance i.v. (8), nasal tolerance with one of the autoepitopes, H471–94, also could delay or treat lupus nephritis in SNF1 mice, but by generating IL-10-producing T cells (39). IL-10-producing Treg cells might benefit lupus with some caveats (30, 40).
The CD4+CD25+ Treg population could contain a subset of T cells that were secondarily induced to produce TGF-, which suppresses autoimmunity (13, 32, 35, 41, 42). CD8+ Treg cells induced by low-dose tolerance were not CTL because they suppressed across membranes, even though the autoepitope peptides inducing such Treg cells contained class I-binding motifs. Thus, the CD8+ Treg cells we have induced by nucleosomal peptides are different from the TCR clonotype-specific and cytotoxic suppressor cells in other systems (20, 22, 43, 44, 45). TGF- produced by the CD8+ T cells could have induced some of the CD4+CD25+ Treg cells in our system. Indeed, the suppressive effect of these adaptive Treg cells were similar at 1:1, 1:10, or 1:100 ratios (suppressor: target), suggesting involvement of "infectious tolerance" mechanisms, as in other systems (12, 42). We are in the process of studying how a combination of CD4+CD25+ and CD8+ Treg cells could interact in our low-dose peptide tolerance system, as exemplified in a graft-versus-host disease model (46). Although our long-term studies with spontaneous lupus disease were in progress (see footnote #2), another group induced a CD4+CD25+ subset of Treg cells by continuous infusion of a model Ag in low doses (47).
Thus, although lupus T cells are resistant to classical anergy induction (48, 49, 50), tolerance therapy with select nucleosomal peptides still works by generating suppressive Treg cells that impair T cell help for production of a broad spectrum of pathogenic autoantibodies and especially inhibit inflammatory insults in the lupus kidney. Moreover, these Treg cells induced by very low-dose tolerance could possibly suppress activated dendritic cells and other APC in lupus (51, 52).
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Ethan Shevach (National Institutes of Health, Bethesda, MD) for critically reviewing our work and providing helpful suggestions.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants R37-AR39157 and RO1-AI41985 (to S.K.D.).
2 Preliminary results on low-dose peptide tolerance were presented by us as an abstract at the Annual Meeting of American College of Rheumatology in 2002 (Arthritis Rheum. 46, Supplement: S225, Abstract 526, 2002).
3 Address correspondence and reprint requests to Dr. Syamal K. Datta, Division of Rheumatology, Northwestern University Feinberg School of Medicine, 240 East Huron Street, McGaw 2300, Chicago, IL 60611. E-mail address: skd257{at}northwestern.edu
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; Treg, regulatory T; HEL, hen egg lysozyme; AP, alkaline phosphatase; GITR, glucocorticoid-induced TNFR family-related protein; PAS, periodic acid-Schiff.
Received for publication November 8, 2004. Accepted for publication January 3, 2005.
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