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* Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany;
Division of Rheumatology, University of California, Los Angeles, CA 90095;
Department of Nephrology/Transplantation, Charité University Hospital, Humboldt University of Berlin, Berlin, Germany; and
Deutsches Rheumaforschungszentrum, Berlin, Germany
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and IL-4 expression and an increase in TGF
. Increased frequencies of regulatory IFN-+ and IL10+ CD4+ T cells were later detected. Such regulatory IL-10+/IFN-+ type 1 regulatory T cells prevented autoantibody generation and anti-CD3-induced proliferation of naive T cells. In conclusion, these results indicate that SmD183–119 peptide may play a dominant role in the activation of helper and regulatory T cells that influence autoantibody generation and murine lupus. |
Introduction |
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Few lupus-specific T cell epitopes have yet been characterized in self-Ags recognized by CD4+ T cells from lupus patients and lupus-prone mice (4, 5, 6, 7). Among them, peptides derived from histone, a constituent of nucleosomes, have been identified as T cell targets in the (NZBxSWR)F1 murine lupus model as well as in some humans with lupus (8, 9). However, the diversity of autoantibodies found in lupus and the variable clinical expression of the disease suggest that specificities other than those of the antinucleosomal type are involved in the pathogenesis of lupus (10).
We have identified the C-terminal peptide of SmD1 protein (D1 protein of the Sm proteins, part of small nuclear ribonucleoprotein), one of the small nuclear ribonucleoproteins of the splicing machinery, as a major target of the B and T cell autoimmune responses associated with human and murine lupus (3, 6). Approximately 70% of patients with SLE have Abs to SmD183–119, compared with <7% of control patients with other diseases and healthy individuals (3). T cells that recognize SmD183–119 peptide and provide T cell help for both anti-dsDNA and anti-SmD183–119 Abs were recently identified in lupus-prone NZB/W mice, revealing a new model that may help to explain associations between these autoantibodies (7). However, the effect of tolerance to SmD183–119 in reactive T cells on the production of anti-dsDNA Abs and the development of lupus remains unclear.
The induction of tolerance to relevant peptide autoepitopes was found to have a beneficial effect on several organ-specific autoimmune diseases. Unlike these autoimmune diseases, the systemic autoimmunity associated with lupus involves a complex web of polyclonal T and B cell hyperactivity and multiple susceptibility genes (10).
One well-established method for inducing tolerance to prototypic protein Ags in mice involves the i.v. injection of high doses of a soluble Ag (11). The mechanism of this effect remains controversial. In some experiments, i.v. Ag injection resulted in deletion or anergy of the Ag-reactive T cells (12, 13). In other models, i.v. Ag injection was found to ameliorate autoimmune disease through specific T suppressor cells (14) or by alteration of T cell cytokine profiles. The disparity of these findings may partly be due to differences in the model systems. In the (NZBxSWR)F1 lupus-prone mouse model, for example, high dose tolerance to histone peptides reduced the generation of anti-dsDNA Abs and improved survival (9, 10), but the mechanisms of tolerance induction remained unclear (4). In the NZB/W model, high dose tolerance to IgG peptides prolonged survival, delayed the appearance of anti-dsDNA, and prevented the serum levels of cytokines from increasing, all by as yet unknown mechanisms (15, 16).
In the present study the activity of reactive T cells was modulated by high dose application of SmD183–119, resulting in tolerance. In contrast to our previous studies, SmD183–119 was used in this study as a soluble peptide applied at a high dose and without any carrier protein. This treatment modality delayed the production of pathogenic autoantibodies, postponed the onset of nephritis, and resulted in prolonged survival. The identification of regulatory T cells (Tr cells) after high dose i.v. tolerance induction and the characterization of T cells involved in the tolerance process open up new possibilities for future therapeutic strategies.
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Materials and Methods |
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(NZBxNZW)F1 (NZB/W) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or from Harlan Winkelmann (Borchen, Germany). Strains were bred either at the University of California rheumatology vivarium (Los Angeles, CA) or at the Bundesinstitut für Gesundheitlichen Verbraucherschutz und Veterinärmedizin (Berlin-Marienfelde, Germany). Only female mice were used in these experiments, which were conducted following an approved protocol in accordance with institutional and state regulations.
Antigens
SmD183–119 peptide (VEPKVKSKKREAVAGRGRGRGRGRGRGRGRGRGGPRR) was synthesized as described by Atherton et al. (17) and purified by reverse phase chromatography using a C18 column (Vydac, Hesperia, CA). The controls consisted of saline, a randomized peptide of SmD183–119 (CREKGRVGRGRPAVGRRGVGRPGRRGSRARGEGKGRK), a hen egg lysozyme (HEL) peptide (aa 106–116; NAWVAWRNRCK), and, in selected in vitro experiments, an OVA peptide (aa 323–339).
Treatment protocol and parameters to determine the effect on disease
A total of 62 NZB/W females received monthly i.v. injections of SmD183–119 peptide (600–1000 µg/mo; n = 17), a randomized peptide of SmD183–119 (600–1000 µg/mo; n = 15), or saline (n = 30) from the age of 6–10 wk on. Plasma urea was estimated using a commercially available test kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Proteinuria was determined once monthly by testing the morning urine on 3 consecutive days with Albustix (Bayer Diagnostics, Munich, Germany) as described previously (7). Anti-SmD183–119 peptide, anti-dsDNA Abs, and survival were also monitored. For histological analysis, kidney sections were harvested from 6-mo-old NZB/W mice that had been treated monthly with either SmD183–119 peptide (n = 7) or saline (n = 7) starting from the age of 12 wk. To exclude nonspecifically caused immunosuppression by high doses of the SmD183–119 peptide, mice were immunized with HEL and the randomized peptide after high dose SmD183–119 treatment. The Ab levels (for HEL) and the dsDNA-specific ELISPOT data (for the randomized peptide) were compared with those of mice receiving immunizations with these Ags without previous tolerance induction.
Treatment protocol for in vitro studies
The role of T cell help in anti-dsDNA Ab production after SmD183–119 tolerance induction was also investigated in vitro. Briefly, NZB/W mice were treated at the ages specified in the figure legends with either 600-1000 µg of SmD183–119 i.v. (n = 6/age group), the randomized peptide of SmD183–119 (n = 6), or saline (n = 6). Splenic T cells were harvested 1 wk after treatment or the mice were subsequently immunized once or twice s.c. with SmD183–119 peptide or the randomized peptide emulsified with CFA or IFA as specified in the figure legends; those receiving a second immunization 2 wk after the first immunization were inoculated with IFA. Nine days after the last immunization, splenic T cells were harvested and either purified or directly used for experiments.
To analyze cytokine responses to SmD183–119 after high dose tolerance by ELISPOT, 7-wk-old NZB/W mice were given an i.v. injection of 600-1000 µg of SmD183–119. For intracellular cytokine staining, these mice were then s.c. immunized with 10 µg of SmD183–119 peptide emulsified in either CFA or IFA 1 and 3 wk later. Splenic T cells were harvested 9 days after the last immunization. Splenic T cells were also harvested from long term survivors (aged 27–38 wk) in which high dose SmD183–119 tolerance had been induced starting at an age of 2–3 mo (n = 13). The controls consisted of T cells from each age-matched group treated with either 600 µl of saline i.v. (n = 14) or 600 µg of HEL peptide i.v. (n = 9) and from untreated mice of variable age (four mice per group, two spleens pooled). The T cells were stimulated with PMA (10 ng/ml) plus ionomycin (1 µg/ml) for 6 h at 37°C before intracellular cytokine staining.
Transfer experiments
NZB/W mice, aged 6–10 wk, were i.v. injected with 1 mg of SmD183–119 diluted in 300 µl of saline (n = 6) or with 300 µl of saline alone (n = 6). Splenic cells were harvested from each group 1–2 wk later. After purifying CD90+ T cells by MACS (Miltenyi Biotec, Auburn, CA), 7 x 106 T cells from SmD183–119-treated mice and saline-treated mice were i.v. transferred to 14-wk-old NZB/W mice (n = 16/group); 12 other mice were injected with saline.
Anti-dsDNA Abs were detected by ELISA as described previously (18, 19). A monoclonal anti-DNA Ab derived from murine hybridoma (clone MRSS-1/D2D4; American Type Culture Collection, Manassas, VA) was used as the standard. Using sera from 35 12-wk-old NZB/W mice, the cut-off was defined as 4.6 arbitrary units (AU).
Anti-SmD183–119 peptide Abs were detected by an ELISA using polystyrene-coated SmD183–119 peptide as described previously (18). Using a pool of adult MRL/lpr mouse sera, a standard curve measured in AU was established. The cut-off point of this assay was determined using 43 sera from 12-wk-old NZB/W mice. A positive result was defined as a concentration +2 SD higher than mean concentration (147 AU/ml).
Western blot assays were performed using MOLT four-cell extracts according to standard protocols with minor modifications as described previously (18).
Histology of kidney sections derived from treated and untreated mice
Kidney sections derived from 6-mo-old NZB/W mice treated with SmD183–119 or saline were stained with H&E and evaluated by light microscopy.
Purification of T and B cells for dsDNA- and SmD183–119-specific ELISPOT assay
CD90+ T cells derived from the differently treated groups and B220+ B cells derived from sick NZB/W mice showing proteinuria of >100 mg/dl in splenic single-cell suspensions were enriched via the Vario MACS magnetic purification system using appropriate microbead-coated Abs (CD90 and B220; Miltenyi Biotec) as described previously (7). Because B220 is also expressed by other cells, including plasmacytoid dendritic cells, only one B cell donor or two pooled B cell donors were used for each experiment to exclude differences in the number of B cells. As determined by FACS, the purity of the isolated cell populations ranged from 92–99%. In preparation for ELISPOT assay, B and T cells were cultured at 37°C at a ratio of 1:10 in DMEM, 10% FCS, and 20% supernatants of rat splenic cells treated with Con A, on 12-well culture plates (Invitrogen Life Technologies, Paisley, U.K.) with or without the following peptides: SmD183–119 peptide, the randomized peptide of SmD183–119, and the OVA peptide. Five days later, the cells were separated by centrifugation and dissolved in DMEM with 10% FCS for the ELISPOT analysis of T cell help. Supernatants were collected and kept frozen at –20°C until detection of Abs by ELISA.
ELISPOT analysis
B cells (105) and T cells (106) suspended in complete medium (DMEM with 10% FCS) with or without the previously used peptide were added to each well of a 96-well plate coated with dsDNA and blocked as described previously (7). After 8–10 h of incubation at 37°C, the cells were poured off, and the plates were washed 12–16 times. ELISPOT assay was performed as described previously (7). ELISPOT results for anti-dsDNA and anti-SmD183–119 Ab-forming cells (AFC) were confirmed by simultaneous ELISA of the supernatants harvested after 5 days of culture.
Cytokine ELISPOTs
Following the manufacturer’s instructions, purified anti-cytokine Abs (100 µl/well, anti-IFN-, anti-IL-4, anti-IL-10, and anti-TGF- (all from BD Pharmingen, San Diego, CA) were coated onto 96-well plates (Costar, Cambridge, MA), left to stand overnight at 4°C, and blocked with 2% BSA in PBS for 2 h at 37°C. Splenic T cells (2 x 105) harvested from treated or untreated mice at different ages as well as 2 x 104 B cells derived from proteinuric NZB/W mice (>100 mg/dl proteinuria) were purified as described above and suspended in AIM V medium (Invitrogen Life Technologies) and cocultured with or without peptides (5 µg/well) for 72 h at 37°C in 5% CO2. After removing the cells, ELISPOT assays were performed as described previously (7). Using an inverted microscope (Leitz, Rockleigh, NJ), cytokine-expressing cells appearing as blue spots were enumerated as the number of cytokine-expressing cells per 2 x 105 T cells.
Intracellular cytokine detection by FACS
T cells were stimulated with PMA (10 ng/ml) and ionomycin (1 µg/ml), and brefeldin A was added (10 µg/ml; Sigma-Aldrich, Munich, Germany) after 2 h. Cells were fixed, permeabilized, and stained for intracellular cytokines and surface markers as described previously (20). The following mAbs (all from BD Pharmingen) were used for intracellular and surface staining according to the manufacturer’s instructions: anti-mouse CD3-FITC (clone 145-2C11), anti-mouse CD4-R-PE, (clone GK1.5), anti-mouse allophycocyanin-IL-10 (clone JES5-16E3), and anti-mouse R-PE-IFN- Ab (clone XMG1.2). Completely unstained controls and stained isotype controls (AP-IgG2b, clone A95-1; PE-IgG1, clone R3-34) were also used. Samples were analyzed by four-color analysis on FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) using FCS-Express software (version 1.0). T lymphocytes were identified based on their forward and sideward scatter properties and CD3 and CD4 gating.
An SmD183–119-reactive IFN- +/IL-10+ T cell line was established by giving 8-wk-old NZB/W mice two i.p. injections of SmD183–119 (100 and 70 µg) emulsified in either CFA or IFA over the course of 2 wk. Seven days after the last injection, splenic and lymph node CD4+ T cells were harvested by MACS and cultured with irradiated (10 min, 30 Gy) CD90-depleted splenocytes (APC) in the presence of 5 µg/ml SmD183–119 for 3 days as described previously (7). The cells were subsequently stimulated with 50 U IL-2/ml, then restimulated 3–4 days later, followed by a resting period of 4–5 days. The Ag restimulation procedure was repeated every 14–17 days. For functional comparisons, an SmD183–119-specific Th line was generated as described previously (7). Briefly, splenic CD90+ T cells were harvested from untreated 8- to 10-wk-old NZB/W mice by MACS and cultured as described above. T cell characterization was performed after 6 wk or more of culture. T cells were stimulated with the SmD183–119 peptide or the control peptides (OVA and the randomized peptide) at concentrations of 5 and 50 µg/ml. After 4 days, T cells were stimulated with PMA/ionomycin to detect differences in the cytokine expression.
Generation of IFN- +/IL-10+ T cells and detection of their in vitro function on naive T cells
Six 11-wk-old NZB/W mice received an i.v. injection of 1 mg of SmD183–119, followed by s.c. boosters of 20 µg of SmD183–119 emulsified in IFA 7 and 14 days later. Eight mice treated i.v. with peptide-free saline and immunized with either saline (n = 4) or SmD183–119 peptide (n = 4) served as the controls. One week later, CD90+ splenic T cells harvested from each mouse were analyzed separately. T cells were characterized for their cytokine pattern directly ex vivo with and without PMA/ionomycin stimulation. In addition, T cells were cultured for 3 days in a medium containing 5 µg/ml SmD183–119, irradiated APC in RPMI 1640 (ratio 1:3), 10% FCS supplemented with 1 µg/ml anti-mouse CD3 (BD Pharmingen), 1 µg/ml anti-mouse CD28, 4 x 10–4 M vitamin D3, and 1 x 10–1 M dexamethasone as described previously (21). On day 4, a fraction of cells was removed for intracellular cytokine staining and characterization. In the remaining cells, the medium was replaced by 50 U/ml IL-2 in RPMI 1640. On day 8, these T cells were cocultured with a 1-, 3-, or 9-fold greater number of naive CD62L+ labeled with CFSE (Molecular Probes, Eugene, OR) together with 10 µg/ml SmD183–119, irradiated APC, and 1 mg/ml anti-mouse CD3. After 3.5 days, viable T cells were stained and tested for CFDA-SE by FACS.
Statistics
Survival was assessed by log rank and 
2 tests using GraphPad PRISM 3.0 for Windows 95. The nonparametric Mann-Whitney U test was used to statistically analyze the influence of i.v. SmD183–119 on the laboratory parameters (Ab levels, urea, and proteinuria). The two groups evaluated by ELISPOT were compared using Student’s t test. SEM was represented using error bars; p < 0.05 was considered statistically significant.
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Results |
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The 17 mice treated with SmD183–119 peptide (600–1000 µg/mo) lived longer than those treated with saline (n = 30; p = 0.016) or randomized peptide (600–1000 µg/mo; n = 15; p = 0.024). Fifty percent mortality ranged from 35 wk with saline to 42 wk with SmD183–119 (Fig. 1a). Compared with saline, the randomized peptide did not improve survival.
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Tolerance to high dose SmD183–119 ameliorates lupus nephritis
SmD183–119 (n = 17) led to lower urinary protein concentrations than saline (n = 30) at 3 (p = 0.015), 5 (p = 0.032), 6 (p = 0.046), and 7 (p = 0.046) mo of age (Fig. 2a). Compared with saline-treated mice, proteinuria in the randomized peptide group decreased temporarily at 5 mo of age (p = 0.031), but later rose to the same level. Mean plasma urea concentrations were lower in the SmD183–119 group than in the saline group (p = 0.005 at age 6 mo; Fig. 2b). The randomized peptide did not influence plasma urea levels compared with those of saline-treated mice. Light microscope analysis of kidney sections from 6-mo-old, saline-treated, NZB/W mice starting from the age of 12 wk revealed pronounced glomerular changes characterized by collapsing glomerular loops, microaneurysms, and marked glomerular hypercellularity with focal extracapillary proliferation (Fig. 2c). All these morphological signs of lupus nephritis were also present in age-matched NZB/W mice treated with SmD183–119 (n = 7), but to a much lesser extent (Fig. 2d). Other signs, such as interstitial inflammation, fibrosis, and tubular damage, were also less pronounced in the SmD183–119 group than in the saline group (data not shown).
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B cells derived from proteinuric NZB/W mice were cultured with and without SmD183–119 peptide in the presence of T cells from mice that were treated differently (high dose i.v. SmD183–119 or high dose i.v. SmD183–119 plus subsequent s.c. immunization with low dose SmD183–119 emulsified in CFA). T cells derived from saline-treated, 7-wk-old, NZB/W mice provided T cell help for the generation of anti-dsDNA AFC in the presence of B cells and 5 µg/ml SmD183–119 (Fig. 3a; p < 0.01), as shown by dsDNA-specific ELISPOT assay. In contrast, the number of anti-dsDNA-specific AFC obtained using T cells derived from age-matched NZB/W mice 1 wk after high dose i.v. SmD183–119 was less than that observed in T cells from saline-treated mice (p = 0.004). The effect became more and more pronounced with increasing SmD183–119 concentration (p < 0.001) in vitro. The randomized peptide of SmD183–119 did not affect the number of anti-dsDNA-specific AFC in untreated or treated mice (Fig. 3a). The baseline secretion of anti-dsDNA AFC had probably already received T cell help in vivo and did not change in cultures without SmD183–119. Furthermore, high dose tolerance to the randomized peptide did not influence the effect of the SmD183–119 peptide to provide help for the generation of anti-dsDNA Abs (data not shown). In contrast, high dose tolerance to SmD183–119 did not influence the immune response to HEL, and for the randomized peptide, high doses of SmD183–119 did not prevent the slightly increased generation of anti-dsDNA ELISPOTs in mice immunized with the randomized peptide as previously described (7) (data not shown).
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Transfer of T cells from NZB/W mice tolerized with SmD183–119 i.v. reduced the anti-dsDNA Ab response in the recipients
Six- to 10-wk-old NZB/W mice (n = 6) were tolerized with high doses of SmD183–119 to assess the capacity of Tr cells. One week after tolerization, splenic CD90+ T cells were harvested, concentrated by MACS, and transferred into 14-wk-old recipients. At 5 and 6 mo of age, the anti-dsDNA Ab levels in NZB/W mice inoculated with T cells from the SmD183–119-treated mice (n = 16) were lower than those in mice inoculated with T cells from saline-treated mice (n = 16; p = 0.006 and 0.0018, respectively) or in mice treated with saline alone (n = 12; p = 0.002 and 0.0016, respectively; Fig. 4). T cells from untreated, age-matched, NZB/W mice did not decrease the levels of anti-dsDNA Abs in the recipient mice compared with those in untreated mice.
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Compared with the findings in untreated NZB/W mice, tolerance to high dose SmD183–119 led to down-regulation of the expression of IFN- (p < 0.001), IL-2 (p = 0.008), IL-4 (p = 0.0014), and IL-10 (p < 0.001) in 7-wk-old NZB/W mice, as determined by cytokine ELISPOT after in vitro stimulation with the SmD183–119 peptide (Fig. 5). Relative to the untreated 7-wk-old controls, TGF- expression in the high dose SmD183–119 group increased (p = 0.03). Intracellular cytokine staining of CD4+ T cells derived from the draining lymph nodes after high dose tolerization and subsequent immunization with 10 µg of SmD183–119 emulsified in IFA confirmed these results, showing diminished expression of IFN- and IL-10 (data not shown). In contrast to the findings 1–2 wk after i.v. SmD183–119 treatment, CD4+ T cells derived from 29- to 38-wk-old NZB/W mice receiving i.v. SmD183–119 starting from the age of 3 mo showed increased frequencies of IL-10+/IFN-+ T cells, a subgroup of T cells thought to have regulatory capabilities. Mice treated with SmD183–119 had a significantly higher frequency of IFN-+/IL-10+ CD4+ T cells (mean, 6.5%; n = 13) than those treated with the control peptide (0.3%; n = 9; p < 0.001) or saline (0.76%; n = 14; p = 0.019; data not shown). As shown for two pooled 7-mo-old nephritic NZB/W mice, tolerance to high dose SmD183–119 increased the number of IFN-+/IL-10+ CD4+ T cells by up to 19% in some of the long term survivors (Fig. 6d). In the untreated controls, the frequency of IFN-+/IL-10+ CD4+ T cells peaked when the mice developed nephritis at the age of 
25 wk and decreased with progressive nephritis (Fig. 6, a–c).
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Compared with saline-treated mice, up-regulation of IL-10+ and IFN- +/IL-10+ T cells was also detected in T cell cultures from NZB/W mice treated with high dose SmD183–119, followed by immunization with SmD183–119 peptide and administration of supplements such as vitamin D3, dexamethasone, IL-2, anti-CD3, and anti-CD28, which are candidates to increase the number of Tr cells (Fig. 7, a and b) (22). Nearly all T cells in this culture exhibited the CD4+/CD25+ phenotype, whereas the frequencies of CD8+ T cells were no greater than those found in the untreated controls (data not shown).
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, among other factors (data not shown).
We also established a T cell line showing specific proliferation in response to SmD183–119, but not to the control peptides (Fig. 8a). This line consists of CD4+ T cells (98.8%) and CD8+ T cells (1.5%); 83% of the CD4+ T cells were CD45 RB low, and 99.8% were CD25+ (data not shown). This T cell line contained high frequencies of IL-10-expressing T cells, and there was a high percentage (26%) of IFN-+/IL-10+ T cells when stimulated with PMA/ionomycin after a 4-day culture with SmD183–119 (Fig. 8b). In contrast, the frequency of IFN-/IL-10+CD4+ T cells was lower (<8%) after stimulation with the control peptides. Restimulation of this cell line with SmD183–119 in the presence of B cells in vitro led to a decrease in anti-dsDNA and anti-SmD183–119 Ab generation, as demonstrated by both ELISPOT (Fig. 8c) and culture supernatant analysis by ELISA (data not shown). As shown by immunoblot analysis of the supernatants, reactivity to several nuclear Ags decreased after restimulation with SmD183–119, but not with the control peptides (Fig. 8d). In contrast, B and T cell cultures derived from untreated mice or from a SmD183–119-specific Th cell line showed increased frequencies of anti-dsDNA- and anti-SmD183–119-specific plasma cells as well as reactivities to several Ags in the presence of SmD183–119 (Fig. 8c); this was demonstrated by immunoblotting, as described previously (7).
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Discussion |
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In the present study modulation of T cell reactivity in response to high dose SmD183–119 tolerance induction prolonged survival and delayed the onset of nephritis in female NZB/W lupus mice (Figs. 1 and 2). Anti-dsDNA and anti-SmD183–119 Ab levels were lower in mice treated with SmD183–119 than in those treated with saline or control peptides. The fact that tolerance to high dose SmD183–119 did not alter the effect of control peptides on autoantibody generation suggests that specific tolerance mechanisms may play a role. This effect was also detected in short term in vitro cultures of T plus B cells (Fig. 3). These findings underline the importance of SmD183–119, a peptide unrelated to nucleosomes, in the generation of anti-dsDNA Abs.
Our finding of altered cytokine expression in response to treatment with i.v. SmD183–119 and the detection of cytokine changes several months after high dose tolerance induction to SmD183–119 suggest that this is a long-lasting effect.
Furthermore, the present study revealed the adoptive transfer of tolerance by T cells, the diminished capacity of T cells from untreated NZB/W mice to provide T cell help for anti-dsDNA Ab production in the presence of T cells from tolerized mice, and the inhibited proliferation of activated naive T cells by T cells derived from tolerized mice. These findings indicate for the first time that some T cells induced by SmD183–119-derived tolerance fulfill a regulatory function.
As shown in different autoimmune diseases, cytokine changes in Th1 and Th2 cytokines in response to high dose tolerance may reflect either the generation of Tr cells and/or the deletion/anergy of effector T cells (24, 25, 26). Decreased expression of IL-4 and IFN- in response to peptide consensus of stimulatory sequences from the VH regions has been reported to occur in the NZB/W lupus model (15). In the present study, SmD183–119 administration decreased the frequency of T cells expressing IFN-, IL-4, IL-2, and IL-10 despite restimulation with SmD183–119 1 wk after treatment, suggesting that deletion or anergy of the effector cells occurs (Fig. 5). Furthermore, tolerance to i.v. SmD183–119 has been found to up-regulate TGF--secreting T cell expression, which is reportedly decreased in lupus (27). However, by using the ELISPOT assay, we cannot fully exclude that TGF- is produced by non-T cells.
In the present study, the induction of tolerance to high dose SmD183–119 results in the production of type 1 Tr cell (Tr1)-like cells that express both IFN- and IL-10 several months after starting tolerance (20, 28) (Fig. 6). This cell type has not been described in lupus previously, but has been observed to prevent severe or systemic inflammatory immune responses during infections (29) such as human tuberculosis (30) and Lyme disease (31). Nevertheless, our data show that IL-10+/IFN-+ T cells also occur spontaneously when mice with lupus start to develop proteinuria. This probably reflects a compensatory mechanism in NZB/W mice. However, despite mouse-to-mouse variations, the frequencies of these Tr1 cells in untreated mice were much lower than those in SmD183–119-treated mice.
The successful in vitro generation of regulatory IL-10+/IFN-+ T cells (Fig. 7) by making the mice tolerant to high doses of SmD1, repeatedly stimulating them with SmD183–119 in vivo and in vitro, and subsequently culturing them with constituents known to increase the number of regulatory T cells (21, 32) as well as the generation of a T cell line with similar cytokine expression in response to SmD183–119 (Fig. 8) may be useful for developing additional therapeutic strategies based on regulatory autoantigen-specific T cells (33). However, the mechanisms involved in the generation of autoantigen-specific regulatory T cells in lupus, e.g., the role of TGF in Tr1 cell generation, is still under investigation. The suppressive capacity of these IL-10+IFN-+ T cells depends on IL-10 expression, because addition of anti-IL-10, but not of IFN-+ or the isotype control, strongly enhanced the anti-dsDNA Ab levels in T and B cell cultures with SmD183–119 (data not shown). The role of other suppressive mechanisms is still under investigation. IL-10 is a regulatory cytokine that inhibits Th1 response and proliferation of CD4+ T cells (34), but its role in lupus remains controversial. IL-10 is a regulatory cytokine that inhibits Th1 response and proliferation of CD4+ T cells (34). It is produced by B cells and monocytes, and IL-10 produced by non-T cells appears to predominate (35). The effect seems to be dependent on disease activity, because treatment with IL-10 caused PBMCs from patients with active disease to decrease their Ab production, whereas patients with inactive disease showed increase anti-dsDNA Ab levels after IL-10 treatment of PBMC (36). A pathogenic role of IL-10 can be postulated because systemic anti-IL-10 treatment is beneficial in murine and human lupus (37, 38). In another experimental animal lupus model, a protective role of IL-10 has been shown (39). Our hypothetical explanation for this paradox is that IL-10 from Tr1 cells is protective, because APC and Th1 are silenced. If that does not occur, IL-10 from other cells will augment generation of AFC and promote pathogenic Abs. A possible protective role of autoantigen-specific IL-10+ T cells is also suggested by our ongoing work characterizing human T cells after stimulation with SmD183–119 and comparing the frequencies of IL-10+ T cells with disease activity and autoantibody generation (unpublished observations). A reduced IL-10 production by T cells derived from SLE patients has been detected previously (35); therefore, deficiencies of Tr1 cells could participate in lupus pathogenesis. Our ongoing work will address the role of autoantigen-specific IL-10+ Tr1 cells in lupus. The in vivo effect of the T cell line with a high percentage of IL-10+/IFN-+ and IL-10+ T cells would probably answer the question of how autoantigen-specific suppression of murine lupus can occur and whether specificities other than anti-dsDNA or anti-Sm are affected, as our in vitro studies suggest (Fig. 8).
Nevertheless, the fact that despite SmD183–119-derived tolerization our NZB/W mice still developed lupus suggests that incomplete suppression of lupus with emergence of additional pathogenic mechanisms may also play a role. In the NZB/W lupus model, regulation of the immune system is critically influenced by several genes (40). Furthermore, long-lived plasma cells not activated by Th cells may occur during lupus development; these cells might therefore be refractory to tolerance induction (41). This would explain the lower effect of high dose tolerance to SmD183–119 in nephritic mice. In these mice, administration of high SmD183–119 doses starting at 5–7 mo of age temporarily decreased levels of anti-dsDNA Abs and proteinuria, but survival was not changed. Following this assumption, both immunoablation and tolerance induction would be necessary to influence lupus in mice with terminal disease. In contrast, we cannot exclude the possibility that larger numbers of T cell epitopes are probably necessary for long term tolerance.
In conclusion, the fact that treatment of mice with high dose SmD183–119 peptide resulted in improved survival indicates that SmD183–119 is one of the primary targets that initiate the cognate interaction between pathogenic Th and B cells associated with lupus. This finding suggests that the peptide autoepitope may play a dominant role in lupus development. The induction of Tr cells and the occurrence of autoimmunity toward SmD183–119 in murine and human lupus may well pave the way for new therapeutic approaches to autoantigen-specific therapy.
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Acknowledgments |
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1 This work was supported by grants from the Humboldt University Research Foundation, the Deutsche Forschungsgemeinschaft (Ri 1056/2–1, SFB 421, C4, and C6), Merck, Sharp, and Dohme GmbH Grant Arthritis/Arthrosis 2000, National Institutes of Health Grants R37AI46776 and P60AR36834, a grant from the Arthritis Foundation Southern California Chapter, and gifts from the Paxson Family, the Mitchell Family, and the Dorough Foundation. 
2 Address correspondence and reprint requests to Dr. Gabriela Riemekasten, Department of Rheumatology and Clinical Immunology, Charité University Hospital, Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail address: gabriela.riemekasten{at}charite.de
3 G.R. and D.L. contributed equally to this work.
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; AFC, Ab-forming cell; AU, arbitrary unit; HEL, hen egg lysozyme; Sm protein, Smith protein; SmD1, D1 protein of the Sm proteins, part of small nuclear ribonucleoprotein; Tr1, type 1 regulatory T cell.
Received for publication December 1, 2003. Accepted for publication August 20, 2004.
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