H2A- and H2E-Derived CD4+CD25+ Regulatory T Cells: A Potential Role in Reciprocal Inhibition by Class II Genes in Autoimmune Thyroiditis
http://www.100md.com
免疫学杂志 2005年第5期
Abstract
We recently described a novel H2E class II-transgenic model (A–E+) of experimental autoimmune thyroiditis (EAT) that permits disease induction with heterologous thyroglobulin (Tg), but unlike conventional susceptible strains, precludes self-reactivity to autologous mouse Tg. In transgenic E+B10 (A+E+) mice, the presence of endogenous H2A genes is protective against H2E-mediated thyroiditis, inhibiting EAT development. The suppressive effect of H2A genes on H2E-mediated thyroiditis mirrors previous reports of H2E suppression on H2A-mediated autoimmune diseases, including EAT. The mechanism of the reciprocal-suppressive effect between class II genes is unclear, although the involvement of regulatory T cells has been proposed. We have recently reported that CD4+CD25+ regulatory T cells mediate peripheral tolerance induced with mouse Tg in CBA mice. To determine whether these cells play a role in our E+-transgenic model, we first confirmed the existence of CD4+CD25+ T cells regulating thyroiditis in E+B10.Ab0 (A–E+) and B10 (A+E–) mice by i.v. administration of CD25 mAb before EAT induction. The depletion of CD4+CD25+ T cells enhanced thyroiditis induction in the context of either H2E or H2A. Moreover, reconstitution of CD4+CD25+ T cells from naive B10 mice restored resistance to EAT. E+B10 (A+E+) mice were also depleted of CD4+CD25+ T cells before the challenge to determine their role in thyroiditis in the presence of both H2A and H2E genes. Depletion of CD4+CD25+ regulatory T cells offset the suppression of H2E-mediated thyroiditis by H2A. Thus, these regulatory T cells may be involved in the reciprocal-suppressive effect between class II genes.
Introduction
Experimental autoimmune thyroiditis (EAT)3, the murine model for Hashimoto’s thyroiditis, is an organ-specific autoimmune disease characterized by mononuclear infiltrate in the thyroid, T cell-proliferative response, autoantibody production, and subsequent destruction of the thyroid follicles. EAT is inducible in susceptible mice by administration of mouse thyroglobulin (mTg), either in repeated doses (1) or in conjunction with an adjuvant such as CFA or LPS (2, 3). Susceptibility to EAT has long been linked to the H2 haplotype (4), paralleled by subsequent HLA associations with autoimmune thyroid disease (5). In particular, susceptibility to EAT has been mapped to the H2A locus (6). For example, mice bearing the Ak haplotype are susceptible to EAT induction, whereas mice bearing the Ab haplotype are resistant (6). The critical role of H2A genes in encoding susceptibility has been verified by the introduction of the AakAbk transgene into a resistant strain; the resultant expression of Ak molecules conferred EAT susceptibility (7). In contrast, involvement of the other murine MHC class II locus, H2E, in susceptibility to EAT is less clear; it is not genomic to every strain, and its presence is not required for susceptibility. However, functional H2E molecules arising from the presence of a H2Ea transgene have been shown to play a suppressive role in autoimmunity because induction of the Eb molecule suppressed the severity of myasthenia gravis in otherwise susceptible B10 mice (8) and inhibited diabetes development in NOD mice (9). Similarly, in EAT, H2Es expression decreased thyroiditis severity after mTg immunization in otherwise susceptible B10.S (A+E–) mice (7).
Interestingly, we have recently discovered a novel H2E-transgenic model that differs in EAT susceptibility from conventional strains. In this model, the conserved Eak transgene was introduced into traditionally resistant B10 (H2Ab) mice lacking expression of endogenous Ab molecules due to targeted deletion of the Ab gene (10). Unlike conventional strains, EAT induction in E+B10.Ab0 (A–E+) mice is limited to heterologous thyroglobulin (Tg), such as human or porcine, and restricted against induction with self-mTg (11). Moreover, when the endogenous Ab molecules were retained, a protective effect was observed. E+B10 (A+E+) mice demonstrated significantly decreased thyroiditis severity following EAT induction with human Tg (hTg) as compared with E+B10.Ab0 mice (11), mirroring the suppressive effect of Es molecules in the susceptible B10.S strain (7). The mechanism of the reciprocal-suppressive effect of H2E and H2A molecules remains to be characterized. Other investigations into the suppressive effect of H2E molecules on H2A-mediated responses indicate that it is an active mechanism of suppression that can be abrogated in vitro by the presence of blocking H2E mAbs, suggesting the possible involvement of suppressor T cells (12, 13, 14).
Investigations of suppressor T cells in other models of autoimmunity have characterized a small group of CD4+ T cells expressing CD25 (IL-2R) (10%) that exhibit regulatory activity. These CD4+CD25+ T cells inhibited the development of autoimmunity, including thyroiditis, following reconstitution of nude mice with CD4+CD25– T cells (15), and in vivo depletion of CD4+CD25+ T cells rendered autoimmune-resistant mouse strains susceptible to induction of autoimmunity (16). We have also reported the influence of CD4+CD25+ regulatory T cells in susceptibility to EAT in their role as mediators of induced peripheral tolerance in CBA mice bearing the susceptible k haplotype (17). Here, we examined the possibility that the suppressive effect of H2Ab or H2Eb molecules on the induction of EAT was mediated through CD4+CD25+ regulatory T cells by using mouse strains expressing these molecules, either singly or together. Here, we report that CD4+CD25+ regulatory T cells are generated in the context of either H2Eb or H2Ab, and these regulatory T cells inhibit the development of autoimmunity in the same context. We also demonstrate that reciprocal suppression of H2Eb- or H2Ab-mediated autoimmunity is reduced by depletion of CD4+CD25+ T cells, suggesting that regulatory T cells may explain the suppressive effect observed by the addition of class II genes.
Materials and Methods
Mice
All mice were bred at the Immunogenetics Mouse Core Facility at the Mayo Clinic. The generation of congenic H2E+-transgenic mice in B10 and B10.Ab0 mice was detailed previously (11). Briefly, MHC class II-deficient mice (Ab0) were derived by targeted deletion of the H2Ab gene (10) in B10 mice (lacking an endogenous E molecule) and screened by flow cytometry. Expression of the E molecule was introduced by intercrosses of B10 or B10.Ab0 mice with Eak-transgenic mice and screened by flow cytometry (18). Progenies were backcrossed to obtain congenic E+ mice on the B10 or B10.Ab0 background (11). Mice were kept on acidified, chlorinated water and used at 10–16 wk of age.
Thyroglobulin
mTg and hTg were obtained from frozen thyroids fractionated on a Sephadex G-200 column as described previously (19). Tg was checked for the presence of LPS by Limulus amebocyte assay (a 100-μg dose contained <1 ng of LPS) and diluted in nonpyrogenic saline.
mAbs and in vivo depletion of CD25+ T cells
CD25 mAb was produced by culture of 1 x 108 PC61 (rat IgG1) hybridoma cells (American Type Culture Collection; Ref.20) in a Cell Max module (Cellco) and harvested according to manufacturer’s instructions. Harvested mAb was twice purified by ammonium sulfate precipitation, and specific Ab concentration was determined as described previously (17). For in vivo depletion, two 1-mg doses of CD25 mAb (containing 10–20% active Ab) or two 1-mg doses of polyclonal rat IgG control (Sigma-Aldrich) were given i.v. 4 days apart. Depletion was monitored in PBL by flow cytometry with PE-7D4 (rat IgM) (Southern Biotechnology Associates) 6 days later, in conjunction with anti-CD4 (YTA 3.1 and YTS 191.1, rat IgG2b) ascites fluid twice purified by ammonium sulfate precipitation and labeled with FITC-NORIG 7.16 (mouse anti-rat IgG2b; Refs.21 and 22). Anti-CD25 mAb treatment resulted in a 70–90% reduction of CD4+CD25+ T cells as indicated in the figure legends.
Adoptive transfer of CD4+CD25+ T cells
As previously described (17), CD4+CD25– and CD4+CD25+ T cell populations were enriched for adoptive transfer studies by magnetic separation of splenocytes from B10 mice first depleted in vivo of CD8+ T cells by administration of two 320-μg CD8 mAb (YTS 156.7 and YTS 169.4, rat IgG2b) doses (23, 24). CD4+ T cell populations were obtained by panning over anti-B cell mAb (187.1, rat IgG; American Type Culture Collection) and anti-CD11b (YBM 6.1.10, rat IgM)-coated petri dishes to remove B cells and macrophages. CD4+CD25+ T cells were selected positively from the remaining cells by labeling with PE-7D4 and anti-PE magnetic microbeads (StemCell Technologies). Viability and purity of CD4+CD25+ and CD4+CD25– fractions were assessed by trypan blue staining and flow cytometric analysis (average viability > 95% at 75–90% purity). CD25 mAb-treated B10 mice were reconstituted i.v. with 1.5–3.0 x 107 enriched cells/mouse 12 days before challenge with 40 μg of mTg and 20 μg of LPS.
Induction of EAT
EAT was induced by injecting i.v. 40 μg of Tg, followed 3 h later with 20 μg of Salmonella enteritidis LPS on days 0 and 7. Animals were sacrificed on day 28, and EAT was assessed by histologic examination of thyroids, in vitro lymphocyte proliferation to Ag, and anti-Tg production.
Histologic evaluation of EAT
Thyroid specimens were sectioned vertically through both thyroid lobes (50–60 sections from 10–15 step levels). Mononuclear cell infiltration was scored on an index of 0–4.0: 0, no infiltration; 0.5, >0–10% thyroid involvement; 1.0, >10–20% involvement with follicular destruction; 2.0, >20–40% involvement; 3.0, >40–80% involvement; and 4.0, >80% involvement (25).
Measurement of autoantibody production
Mice were bled from the tail artery on day 28, and the sera was stored at –20°C. Anti-Tg titers were determined by ELISA using Tg-coated (1 μg/well) Immulon I microtiter plates and alkaline phosphatase-labeled goat anti-mouse IgG (Sigma-Aldrich) as described previously (26).
In vitro proliferation
Splenocytes were cultured in triplicate in RPMI 1640 + 1% normal mouse serum in flat-bottom 96-well plates at 6 x 105 cells/well, either with or without 40 μg of Tg/ml, for 5 days at 37°C, 6% CO2 (27). Cells were pulsed with 1.0 μCi/well of [3H]thymidine 18–20 h before harvest onto glass fiber filter paper (Tomtec Mach3Man Cell Harvester; UKB-Wallac). [3H]Thymidine uptake was assessed using a Microbeta Plus 1450 liquid scintillation counter (UKB-Wallac). Stimulation index was calculated as mean cpm ± SE with Ags divided by mean cpm without Ags.
Statistical analysis
Individual pathology indices between groups were analyzed nonparametrically using the Mann-Whitney U test. A value of p < 0.05 was considered significant statistically.
Results
Depletion of CD4+CD25+ T cells in E+B10.Ab0 (A–E+) mice enhances EAT induced with hTg without altering the restriction against mTg.
We have recently reported that CD4+CD25+ regulatory T cells are responsible for mediating induced tolerance to Tg. After depletion of CD4+CD25+ T cells, tolerance induced by mTg pretreatment in CBA mice was abrogated, and their susceptibility to EAT induction was restored (17). To determine whether CD4+CD25+ T cells also play a role in influencing thyroiditis development in the unique A–E+-transgenic model that is permissive for thyroiditis induction only with heterologous Tg, but not self-mTg (11), CD4+CD25+ T cells in E+B10.Ab0 mice were depleted in vivo. Two 1-mg doses of a depleting CD25 mAb or a polyclonal rat IgG control Ab were administered i.v. 4 days apart (days –14 and –10) as described previously (17). A reduction of 70–90% of CD4+CD25+ T cells was observed by analysis of PBL on day –4, 6 days after the second dose of anti-CD25 (data not shown). Mice were then challenged on days 0 and 7 with the EAT induction protocol of 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS. As illustrated in Fig. 1A, depletion of CD4+CD25+ T cells in E+B10.Ab0 (A–E+) mice resulted in greatly increased mononuclear infiltration of the thyroid following immunization with hTg; 69% (9 of 13) of the CD25-depleted mice exhibited 40–80% thyroid involvement, compared with 23% (3 of 13) in control rat IgG-treated mice. However, the depletion of CD4+CD25+ T cells did not enable induction of thyroiditis with mTg in E+B10.Ab0 mice because none displayed thyroid destruction, and only 8% (1 of 12) showed mild infiltration.
FIGURE 1. In vivo depletion of CD4+CD25+ T cells enhances EAT induction with hTg but does not alter restriction against mTg in B10.E+Ab0 (A–E+) mice. B10.E+Ab0 mice were given two 1-mg doses of anti-CD25 or rat IgG i.v. on days –14 and –10. Reduction of 75–90% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged with 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS on days 0 and 7, and the mice were killed on day 28. A, The graph represents mononuclear cell infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, day. B, Representative thyroid histology from experimental groups (original magnification, x100): normal thyroid architecture (a); infiltration of mononuclear cells 20% following induction of EAT with hTg (b); lack of thyroiditis induction and mononuclear cell infiltration after mTg immunization (c); and CD4+CD25+ T cells depleted before immunization with hTg, infiltration, and destruction 60% (d).
Fig. 1B illustrates representative thyroid pathology from each group. Fig. 1Ba shows normal thyroid histology. Fig. 1Bb is a thyroid section from a hTg-immunized E+B10.Ab0 mouse, with mononuclear cell infiltration involving 20% of the thyroid. In vivo depletion of CD4+CD25+ T cells enhanced thyroiditis induced with hTg, resulting in more severe inflammation involving 60% of the thyroid (Fig. 1Bd). In contrast, the resistance of E+B10.Ab0 mice to thyroiditis induction with mTg was unaltered by depleting CD4+CD25+ T cells; the thyroid section from a CD4+CD25+ T cell-depleted, mTg-challenged mouse shown in Fig. 1Bc demonstrates no mononuclear cell infiltration, similar to that from a normal mouse. No differences were observed in autoantibody production or in vitro-proliferative responses to Ags in hTg-immunized mice pretreated with anti-CD25 compared with mice pretreated with rat IgG, because both remained strongly positive (data not shown). As previously reported (11), splenocytes from hTg-immunized mice did not respond to mTg (data not shown). The increase in thyroiditis severity following anti-CD25 administration indicates that CD4+CD25+ regulatory T cells influencing thyroiditis development can be generated in the context of H2E. The inability of regulatory T cell depletion to alter the resistance to mTg-induced EAT shows that the unique property of the E+B10.Ab0 mouse to respond only to hTg is a function of restricted presentation of autoantigen by the Eb molecule and is not a result of CD25+ T cell regulation.
Depletion of CD4+CD25+ T cells enables susceptibility to EAT induction with mTg in traditionally resistant B10 (H2b) mice
Previous investigation of this unique E+-transgenic mouse model of EAT revealed a suppressive effect of the endogenous Ab genes; E+B10 (A+E+) mice developed less severe thyroiditis following immunization with hTg, compared with E+B10.Ab0 (A–E+) mice (11). This suppressive effect of the endogenous Ab genes might be mediated through CD4+CD25+ regulatory T cells, particularly those activated by self-peptides presented by the endogenous Ab molecule. To determine whether CD4+CD25+ T cells exert a regulatory influence on thyroiditis development by self-Tg epitopes in the context of Ab, we selected traditionally EAT-resistant congenic B10 (A+E–) mice devoid of H2E molecules. Their CD4+CD25+ T cells were depleted before challenge with the EAT induction protocol. As illustrated in Fig. 2, depletion of CD4+CD25+ T cells enabled the induction of thyroiditis with mTg because eight of nine (89%) CD25-depleted mice developed mononuclear cell infiltration of the thyroid, compared with two of eight (25%) controls. Thus, EA
T resistance in B10 mice is mediated by CD4+CD25+ regulatory T cells in the normal context of Ab. However, depletion of CD4+CD25+ T cells did not enable thyroiditis induction with 40-μg doses of hTg (Fig. 2), a dose sufficient to induce EAT in A–E+ mice (Fig. 1A).
FIGURE 2. In vivo depletion of CD4+CD25+ T cells enables EAT induction with mTg, but not hTg, in B10 (A+E–) mice. Two 1-mg doses of anti-CD25 or polyclonal rat IgG i.v. were administered to B10 mice on days –14 and –10. Reduction of 75–85% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged on days 0 and 7 with 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS and killed on day 28. The graph represents mononuclear cell infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Reconstitution of CD4+CD25+ T cells restores EAT resistance in CD25 mAb-treated B10 mice
To determine whether adoptive transfer of CD4+CD25+ T cells would restore EAT resistance in B10 mice, B10 mice were depleted of CD4+CD25+ T cells with two 1-mg doses of anti-CD25 mAb on days –26 and –22 (75–90% reduction as measured by flow cytometric analysis of PBL 6 days later). On day –12, 1.5–3.0 x 107 CD4+CD25+ or CD4+CD25– T cells from naive B10 mice were transferred. Recipient mice were then challenged with 40 μg of mTg and 20 μg of LPS on days 0 and 7, and thyroiditis severity was assessed on day 28. Fig. 3 illustrates that 70% of CD25+ T cell-depleted mice receiving CD4+CD25+ T cells had no thyroiditis; the remaining 3 of 10 (30%) mice had mononuclear cell infiltration of 5–20%. In contrast, more consistent and extensive mononuclear cell infiltration and destruction of up to 40% of the thyroid was observed in CD4+CD25– T cell-reconstituted mice. The specific requirement for CD4+CD25+ T cells to restore resistance to thyroiditis induction in CD25 mAb-treated B10 mice reinforces the role of CD4+CD25+ T cells in regulating susceptibility.
FIGURE 3. Adoptive transfer of CD4+CD25+ T cells restores resistance to EAT induction in CD25+ T cell-depleted B10 mice. B10 mice were given two 1-mg doses of CD25 mAb on days –26 and –22 for depletion of CD25+ cells (75–80% reduction in CD4+CD25+ T cells was observed by FACS analysis of PBL 6 days later). CD4+CD25– and CD4+CD25+ T cells were isolated by magnetic separation of splenocytes from CD8-depleted B10 mice, and 1.5–3.0 x 107 cells/mouse were transferred i.v. on day –12. All mice were challenged with 40 μg of mTg, followed 3 h later by 20 μg of LPS on days 0 and 7. Thyroiditis severity was assessed on day 28. The graph represents mononuclear infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Depletion of CD4+CD25+ T cells reverses the suppressive effect of endogenous Ab molecule in E+B10 (A+E+) mice
The findings presented above demonstrate that CD4+CD25+ T cells are generated in the presence of either H2A or H2E molecules because their removal enhanced thyroiditis severity in both A–E+ and A+E– mice. It was then of interest to examine the role of CD4+CD25+ T cells in E+B10 (A+E+) mice, in which the presence of Ab genes resulted in reduced thyroiditis severity after hTg induction (11). After depleting CD4+CD25+ T cells, E+B10 mice were challenged with either hTg or mTg. As shown in Fig. 4, both hTg- and mTg-induced EAT were increased significantly (p < 0.01) by the elimination of CD4+CD25+ T cells. E+B10 mice immunized with hTg demonstrated strong autoantibody production and in vitro-proliferative response, irrespective of CD4+CD25+ T cell depletion (data not shown). Mice depleted of CD4+CD25+ T cells demonstrated a slight increase in anti-mTg Ab production but no difference in in vitro-proliferative response of splenocytes, compared with polyclonal rat IgG-treated controls (data not shown). The increase in the severity of thyroiditis induced with either hTg or mTg seen in anti-CD25-treated mice indicates that CD4+CD25+ T cells also regulate thyroiditis development in E+B10 (A+E+) mice.
FIGURE 4. In vivo depletion of CD4+CD25+ T cells enhances EAT induced with either hTg or mTg in B10.E+ (A+E+) mice. Mice were given two 1-mg doses of anti-CD25 or polyclonal rat IgG i.v. on days –14 and –10. Reduction of 70–80% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged on days 0 and 7 with EAT induction protocol of 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS and killed on day 28. The graph represents mononuclear infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Discussion
We have previously observed reciprocal-suppressive effects of both H2E and H2A genes on thyroiditis. H2Es genes reduced the severity of thyroiditis encoded by H2As in B10.S mice (7), and H2Ab genes suppressed H2E-mediated thyroiditis in E+B10.Ab0 mice (11). Although such suppressive effects of H2E on other autoimmune responses encoded by H2A molecules has also been previously described (8, 9), the reciprocal effect of H2A genes on H2E-mediated thyroiditis, to our knowledge, has not been demonstrated in other systems. The generation of regulatory T cells responding to Ags presented in the context of H2E has been proposed as a potential suppressor mechanism (8, 9, 12, 13, 14).
CD4+CD25+ regulatory T cells, a subset of T lymphocytes essential in maintaining peripheral immunoregulation inhibiting the development of autoimmunity (15, 28, 29), are derived from high-affinity interactions with self-peptide, both during ontogeny (30, 31, 32) and in the periphery (33). We have recently described a regulatory role for peripherally amplified CD4+CD25+ T cells in EAT-susceptible CBA mice, which mediate mTg-induced tolerance to EAT (17). The influence of CD4+CD25+ T cells in regulating susceptibility to EAT and the potential role of class II molecules in generating these cells led to an examination of their potential influence in class II-transgenic strains.
Our first step to identify a potential role for CD4+CD25+ regulatory T cells in mediating the reciprocal inhibition by H2Eb and H2Ab was to verify their existence in each individual MHC context. For CD4+CD25+ T cells derived from the Eb molecule in E+B10.Ab0 (A–E+) mice, we depleted them in vivo before EAT induction. This transgenic mouse strain is susceptible to thyroiditis induced with heterologous Tg but uniquely resistant to thyroiditis induction with self mTg (11). We observed an increase in thyroiditis severity after hTg immunization (Fig. 1), indicating that CD4+CD25+ regulatory T cells are generated in the context of Eb and the absence of Ab. Of interest is the observation that depletion of CD4+CD25+ T cells did not enable B10.E+Ab0 (A–E+) mice to shed the restriction against mTg-induced thyroiditis (Fig. 1). Because mTg, possessing both unique and shared epitopes (34), is more thyroiditogenic than hTg, the inability of A–E+ mice to develop thyroiditis following immunization with mTg reinforces the
absolute requirement for the correct class II presentation of Tg in EAT induction and indicates that the removal of peripheral inhibition of autoimmunity does not supersede this requirement.
Having demonstrated the existence of CD4+CD25+ T cells that inhibit EAT in the context of Eb, we next examined the development of such cells in the context of Ab, a traditionally EAT-resistant haplotype. After depleting CD4+CD25+ T cells in B10 (A+E–) mice, we immunized the mice with either mTg or hTg. The elimination of resistance to EAT induction with mTg in B10 mice following CD4+CD25+ T cell depletion (Fig. 2) indicates that CD4+CD25+ regulatory T cells that influence thyroiditis development also exist in the context of Ab, and their removal leads to an increase in susceptibility. These data are reminiscent of an earlier report describing increased susceptibility to mTg-induced thyroiditis in EAT-resistant C57BL/6 (H2Ab) mice after administration of cyclophosphamide, an agent known to inactivate preferentially regulatory T cells (35). The data also agree with the finding that elimination of CD4+CD25+ regulatory T cells reduced resistance against the induction of gastritis in traditionally resistant mice (16). The specific role of CD4+CD25+ T cells in influencing susceptibility in traditionally EAT-resistant mice is reinforced additionally by the restoration of resistance to EAT induction following the transfer of CD4+CD25+ T cells in CD25 mAb-treated B10 mice (Fig. 3). Thus, resistance to autoimmunity may, at least in some instances, reflect greater peripheral regulation rather than a poor response to autoantigen. The ability of class II molecules to generate regulatory T cells effective in inhibiting autoimmunity likely varies between haplotypes and may explain the genetic predisposition of certain haplotypes to the development of autoimmunity. In contrast, the inability of hTg to induce thyroiditis in B10 mice, even after depletion of CD4+CD25+ regulatory T cells (Fig. 2), may be related to its poor thyroiditogenicity in traditionally susceptible strains (36, 37) or to its involvement of regulatory T cells other than those expressing CD25 (38). It may also indicate the existence of Ab specificity for epitopes not shared between mTg and hTg, similar to the Eb molecule for nonconserved hTg epitopes (Fig. 1; Ref.11).
The existence of CD4+CD25+ T cells influencing thyroiditis induction in both the context of Ab and Eb supports the idea that these cells may be responsible for the reciprocal-suppressive effect between the two molecules observed previously (8, 11). Suppression of H2A-dependent autoimmune responses by introducing H2E molecules has been described in other models (8, 9, 39). Using our unique Ag-restricted, E+-transgenic model, we were able to examine the influence of CD4+CD25+ regulatory T cells on Ab- and Eb-mediated thyroiditis when both Eb and Ab genes were present. The increase in thyroiditis severity in CD25+ T cell-depleted E+B10 (A+E+) mice immunized with either hTg (mediated by Eb) or mTg (mediated by Ab; Fig. 4) indicates that CD4+CD25+ regulatory T cells influencing thyroiditis exist when both H2A and H2E molecules are present. The reduction in the suppressive effect of the Ab genes on Eb-mediated thyroiditis after depleting CD4+CD25+ T cells suggests that these cells may be responsible for the inhibitory effect due to the presence of the second class II allele.
We cannot point definitively to CD4+CD25+ regulatory T cells as mediators of the suppressive effect of H2A genes on H2E-mediated EAT observed in E+B10 mice at present because we demonstrated the existence of CD4+CD25+ regulatory T cells acting independently in the context of both Eb and Ab (Figs. 1 and 2). We have not discriminated between regulatory T cells generated by either H2E or H2A when eliminating regulatory T cells in vivo, and the observed increase in thyroiditis severity following CD4+CD25+ T cell depletion may be a result of decreased function of Eb- or Ab-derived regulatory T cells that act independently to inhibit thyroiditis mediated in the same context. Moreover, several other possibilities exist to explain the suppressive effect of the H2A genes in this model, such as alteration of the T cell repertoire, potentially including the deletion of autoreactive cells, and qualitative changes in T cell responses due to a combination of T cell epitopes presented in the context of Ab and Eb. Another interesting possibility arises from previous observations that presentation of an epitope from the E-chain by H2A molecules can inhibit collagen-induced arthritis in mice (39), and presentation of a HLA-DR-derived peptide by HLA-DQ can suppress collagen-induced arthritis in transgenic mice (40). A similar event may be occurring in the E+B10 mice, although we have not examined whether the Eb molecule can present Ab-derived peptides, nor whether As molecules can present Es-derived peptides in B10.S mice (7). Nevertheless, our data demonstrate the existence of CD4+CD25+ regulatory T cells generated by both Ab and Eb molecules and that these regulatory T cells may reciprocally suppress the development of autoimmune thyroiditis induced in either context.
The increased susceptibility of E+B10 mice to mTg-induced EAT as compared with congenic B10 mice (Figs. 2 and 4) is also of interest and may relate to regulatory T cell function. The loss of resistance to mTg-induced thyroiditis cannot be attributed to the induction of a responding class II molecule because the resultant Eb is not able to support mTg-induced EAT (11, 41). Possibly, this increase in susceptibility results from the alteration of the T cell repertoire because expression of the H2E molecule results in deletion of T cells expressing TCR V5 and V11 (42). Although the protective effect of Ed genes in NOD mice cannot be associated with the deletion of commonly used TCR V subsets (43), qualitative changes in regulatory T cells could potentially affect the T cell repertoire, and their deletion could lead to increased susceptibility of E+B10 mice to mTg-induced EAT.
Here, our findings indicate that CD4+CD25+ regulatory T cells generated in both the context of H2A and H2E play an important role in influencing the development of thyroiditis. These data also suggest that CD4+CD25+ regulatory T cells may be involved in the reciprocal-suppressive effect exerted between H2A and H2E. Susceptibility to autoimmunity has long been described as a function of class II genes in animal models (4), an association mirrored by human studies (5). More recently, human autoimmune disease has been linked to CTLA-4 (44) and FOXP3 (45), genes associated with CD4+CD25+ regulatory T cell activity (46, 47, 48, 49, 50). Our data suggest that the influence of class II genes and regulatory T cell-associated genes may not be independent and that class II genes may influence the development of autoimmunity both by their presentation of autoantigens as well as by their generation of CD4+CD25+ regulatory T cells.
Disclosures
Acknowledgments
We thank Julie Hanson and staff for breeding and maintaining the transgenic mice, A. M. Mazurco for preparation of histology sections, and Dr. A. A. Giraldo for assistance in evaluation of thyroid pathology. We also thank Dr. C. Jeffries for providing S. enteritidis LPS and Dr. H. Waldmann for the CD4, CD8, and CD11b mAbs.
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 Institute of Health Grant DK45960.
2 Address correspondence and reprint requests to Dr. Yi-chi M. Kong, Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. E-mail address: ykong{at}med.wayne.edu
3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; Tg, thyroglobulin; mTg, mouse Tg; hTg, human Tg.
Received for publication February 24, 2004. Accepted for publication December 23, 2004.
References
ElRehewy, M., Y. M. Kong, A. A. Giraldo, N. R. Rose. 1981. Syngeneic thyroglobulin is immunogenic in good responder mice. Eur. J. Immunol. 11:146.
Rose, N. R., F. J. Twarog, A. J. Crowle. 1971. Murine thyroiditis: importance of adjuvant and mouse strain for the induction of thyroid lesions. J. Immunol. 106:698.
Esquivel, P. S., N. R. Rose, Y. M. Kong. 1977. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. J. Exp. Med. 145:1250
Vladutiu, A. O., N. R. Rose. 1971. Autoimmune murine thyroiditis: relation to histocompatibility (H-2) type. Science 174:1137.
Vaidya, B., P. Kendall-Taylor, S. H. S. Pearce. 2002. Genetics of endocrine disease: the genetics of autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 87:5385.
Beisel, K. W., C. S. David, A. A. Giraldo, Y. M. Kong, N. R. Rose. 1982. Regulation of experimental autoimmune thyroiditis: mapping of susceptibility to the I-A subregion of the mouse H-2. Immunogenetics 15:427.
Kong, Y. M., C. S. David, L. C. Lomo, B. E. Fuller, R. W. Motte, A. A. Giraldo. 1997. Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune thyroiditis. Immunogenetics 46:312.
Christadoss, P., C. S. David, M. Shenoy, S. Keve. 1990. Ek transgene in B10 mice suppresses the development of myasthenia gravis. Immunogenetics 31:241.
Nishimoto, H., H. Kikutani, K. Yamamura, T. Kishimoto. 1987. Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature 328:432.
Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.
Wan, Q., R. Shah, D. J. McCormick, L. C. Lomo, A. A. Giraldo, C. S. David, Y. M. Kong. 1999. H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis. Immunogenetics 50:22.
Baxevanis, C. N., Z. A. Nagy, J. Klein. 1981. A novel type of T-T cell interaction removes the requirement for I-B region in the H-2 complex. Proc. Natl. Acad. Sci. USA 78:3809.
Oliveira, D. B. G., N. Blackwell, A. E. Virchis, R. A. Axelrod. 1985. T helper and T suppressor cells are restricted by the A and E molecules, respectively, in the F antigen system. Immunogenetics 22:169.
Lowenthal, J. W., P. Corthésy, C. Tougne, R. Lees, H. R. MacDonald, M. Nabholz. 1985. High and low affinity IL-2 receptors: analysis by IL-2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J. Immunol. 135:3988.
Aqel, N. M., M. Clark, S. P. Cobbold, H. Waldmann. 1984. Immunohistological screening in the selection of monoclonal antibodies: the use of isotype-specific antiglobulins. J. Immunol. Methods 69:207
Kong, Y. M., A. A. Giraldo, H. Waldmann, S. P. Cobbold, B. E. Fuller. 1989. Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression. Clin. Immunol. Immunopathol. 51:38.
Qin, S., M. Wise, S. P. Cobbold, L. Leong, Y. M. Kong, J. R. Parnes, H. Waldmann. 1990. Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur. J. Immunol. 20:2737.
Fuller, B. E., A. A. Giraldo, H. Waldmann, S. P. Cobbold, Y. M. Kong. 1994. Depletion of CD4+ and CD8+ cells eliminates immunologic memory of thyroiditogenicity in murine experimental autoimmune thyroiditis. Autoimmunity 19:161
Kong, Y. M.. 1996. Experimental autoimmune thyroiditis in the mouse. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 15.7.1. John Wiley & Sons, Inc., New York.
Zhang, W., J. C. Flynn, Y. M. Kong. 2001. IL-12 prevents tolerance induction with mouse thyroglobulin by priming pathogenic T cells in experimental autoimmune thyroiditis: role of IFN- and the costimulatory molecules CD40L and CD28. Cell. Immunol. 208:52.
Wan, Q., M. Kita, J. C. Flynn, J. C. Panos, R. W. Motte, T. F. Davies, A. A. Giraldo, C. S. David, Y. M. Kong. 2001. Participation of V13+ and V1+ T cells in transfer thyroiditis after activation of mouse thyroglobulin-primed T cells by superantigen staphylococcal enterotoxin A. Cell. Immunol. 213:149.
Sakaguchi, S., K. Fukuma, K. Kuribayashi, T. Masuda. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161:72.
Hurwitz, A. A., T. J. Sullivan, R. A. Sobel, J. P. Allison. 2002. Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice. Proc. Natl. Acad. Sci. USA 99:3013
Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, S. Sakaguchi. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.
Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301.
Jonuleit, H., E. Schmitt. 2003. The regulatory T cell family: distinct subsets and their interrelations. J. Immunol. 171:6323.
Gonzalez-Gay, M. A., E. Zanelli, C. J. Krco, G. H. Nabozny, J. Hanson, M. M. Griffiths, H. S. Luthra, C. S. David. 1995. Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis. Immunogenetics 42:35.
Das, P., D. S. Bradley, A. Geluk, M. M. Griffiths, H. S. Luthra, C. S. David. 1999. An HLA-DRB1*0402 derived peptide (HV3 65-79) prevents collagen-induced arthritis in HLA-DQ8 transgenic mice. Hum. Immunol. 60:575.
Kong, Y. M., L. C. Lomo, R. W. Motte, A. A. Giraldo, J. Baisch, G. Strauss, G. J. H?mmerling, C. S. David. 1996. HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: definitive association with HLA-DRB1*0301 (DR3) gene. J. Exp. Med. 184:1167.
Taneja, V., J. Hansen, M. Smart, M. Griffiths, H. Luthra, C. S. David. 1997. Expression of the H2-E molecule mediates protection to collagen-induced arthritis in HLA-DQ8 transgenic mice: role of cytokines. Int. Immunol. 9:1213.
Lund, T., L. O’Reilly, P. Hutchings, O. Kanagawa, E. Simpson, R. Gravely, P. Chandler, J. Dyson, J. K. Picard, A. Edwards, et al 1990. Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A -chain or normal I-E -chain. Nature 345:727
Ueda, H., J. M. M. Howson, L. Esposito, J. Heward, H. Snook, G. Chamberlain, D. B. Rainbow, K. M. D. Hunter, A. N. Smith, G. Di Genova, et al 2003. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506
Bassuny, W. M., K. Ihara, Y. Sasaki, R. Kuromaru, H. Kohno, N. Matsuura, T. Hara. 2003. A functional polymorphism in the promoter/enhancer region of the FOXP3/Scurfin gene associated with type 1 diabetes. Immunogenetics 55:149.
Read, S., V. Malmstr?m, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.
Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.
Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.
Schubert, L. A., E. Jeffery, Y. Zhang, F. Ramsdell, S. F. Ziegler. 2001. Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation. J. Biol. Chem. 276:37672.
Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.(Gerald P. Morris, Yan Yan)
信息仅供参考，不构成任何之建议、推荐或指引。文章版权属于原著作权人，若您认为此文不宜被收录供大家免费阅读，请邮件或电话通知我们，我们收到通知后，会立即将您的作品从本网站删除。We recently described a novel H2E class II-transgenic model (A–E+) of experimental autoimmune thyroiditis (EAT) that permits disease induction with heterologous thyroglobulin (Tg), but unlike conventional susceptible strains, precludes self-reactivity to autologous mouse Tg. In transgenic E+B10 (A+E+) mice, the presence of endogenous H2A genes is protective against H2E-mediated thyroiditis, inhibiting EAT development. The suppressive effect of H2A genes on H2E-mediated thyroiditis mirrors previous reports of H2E suppression on H2A-mediated autoimmune diseases, including EAT. The mechanism of the reciprocal-suppressive effect between class II genes is unclear, although the involvement of regulatory T cells has been proposed. We have recently reported that CD4+CD25+ regulatory T cells mediate peripheral tolerance induced with mouse Tg in CBA mice. To determine whether these cells play a role in our E+-transgenic model, we first confirmed the existence of CD4+CD25+ T cells regulating thyroiditis in E+B10.Ab0 (A–E+) and B10 (A+E–) mice by i.v. administration of CD25 mAb before EAT induction. The depletion of CD4+CD25+ T cells enhanced thyroiditis induction in the context of either H2E or H2A. Moreover, reconstitution of CD4+CD25+ T cells from naive B10 mice restored resistance to EAT. E+B10 (A+E+) mice were also depleted of CD4+CD25+ T cells before the challenge to determine their role in thyroiditis in the presence of both H2A and H2E genes. Depletion of CD4+CD25+ regulatory T cells offset the suppression of H2E-mediated thyroiditis by H2A. Thus, these regulatory T cells may be involved in the reciprocal-suppressive effect between class II genes.
Introduction
Experimental autoimmune thyroiditis (EAT)3, the murine model for Hashimoto’s thyroiditis, is an organ-specific autoimmune disease characterized by mononuclear infiltrate in the thyroid, T cell-proliferative response, autoantibody production, and subsequent destruction of the thyroid follicles. EAT is inducible in susceptible mice by administration of mouse thyroglobulin (mTg), either in repeated doses (1) or in conjunction with an adjuvant such as CFA or LPS (2, 3). Susceptibility to EAT has long been linked to the H2 haplotype (4), paralleled by subsequent HLA associations with autoimmune thyroid disease (5). In particular, susceptibility to EAT has been mapped to the H2A locus (6). For example, mice bearing the Ak haplotype are susceptible to EAT induction, whereas mice bearing the Ab haplotype are resistant (6). The critical role of H2A genes in encoding susceptibility has been verified by the introduction of the AakAbk transgene into a resistant strain; the resultant expression of Ak molecules conferred EAT susceptibility (7). In contrast, involvement of the other murine MHC class II locus, H2E, in susceptibility to EAT is less clear; it is not genomic to every strain, and its presence is not required for susceptibility. However, functional H2E molecules arising from the presence of a H2Ea transgene have been shown to play a suppressive role in autoimmunity because induction of the Eb molecule suppressed the severity of myasthenia gravis in otherwise susceptible B10 mice (8) and inhibited diabetes development in NOD mice (9). Similarly, in EAT, H2Es expression decreased thyroiditis severity after mTg immunization in otherwise susceptible B10.S (A+E–) mice (7).
Interestingly, we have recently discovered a novel H2E-transgenic model that differs in EAT susceptibility from conventional strains. In this model, the conserved Eak transgene was introduced into traditionally resistant B10 (H2Ab) mice lacking expression of endogenous Ab molecules due to targeted deletion of the Ab gene (10). Unlike conventional strains, EAT induction in E+B10.Ab0 (A–E+) mice is limited to heterologous thyroglobulin (Tg), such as human or porcine, and restricted against induction with self-mTg (11). Moreover, when the endogenous Ab molecules were retained, a protective effect was observed. E+B10 (A+E+) mice demonstrated significantly decreased thyroiditis severity following EAT induction with human Tg (hTg) as compared with E+B10.Ab0 mice (11), mirroring the suppressive effect of Es molecules in the susceptible B10.S strain (7). The mechanism of the reciprocal-suppressive effect of H2E and H2A molecules remains to be characterized. Other investigations into the suppressive effect of H2E molecules on H2A-mediated responses indicate that it is an active mechanism of suppression that can be abrogated in vitro by the presence of blocking H2E mAbs, suggesting the possible involvement of suppressor T cells (12, 13, 14).
Investigations of suppressor T cells in other models of autoimmunity have characterized a small group of CD4+ T cells expressing CD25 (IL-2R) (10%) that exhibit regulatory activity. These CD4+CD25+ T cells inhibited the development of autoimmunity, including thyroiditis, following reconstitution of nude mice with CD4+CD25– T cells (15), and in vivo depletion of CD4+CD25+ T cells rendered autoimmune-resistant mouse strains susceptible to induction of autoimmunity (16). We have also reported the influence of CD4+CD25+ regulatory T cells in susceptibility to EAT in their role as mediators of induced peripheral tolerance in CBA mice bearing the susceptible k haplotype (17). Here, we examined the possibility that the suppressive effect of H2Ab or H2Eb molecules on the induction of EAT was mediated through CD4+CD25+ regulatory T cells by using mouse strains expressing these molecules, either singly or together. Here, we report that CD4+CD25+ regulatory T cells are generated in the context of either H2Eb or H2Ab, and these regulatory T cells inhibit the development of autoimmunity in the same context. We also demonstrate that reciprocal suppression of H2Eb- or H2Ab-mediated autoimmunity is reduced by depletion of CD4+CD25+ T cells, suggesting that regulatory T cells may explain the suppressive effect observed by the addition of class II genes.
Materials and Methods
Mice
All mice were bred at the Immunogenetics Mouse Core Facility at the Mayo Clinic. The generation of congenic H2E+-transgenic mice in B10 and B10.Ab0 mice was detailed previously (11). Briefly, MHC class II-deficient mice (Ab0) were derived by targeted deletion of the H2Ab gene (10) in B10 mice (lacking an endogenous E molecule) and screened by flow cytometry. Expression of the E molecule was introduced by intercrosses of B10 or B10.Ab0 mice with Eak-transgenic mice and screened by flow cytometry (18). Progenies were backcrossed to obtain congenic E+ mice on the B10 or B10.Ab0 background (11). Mice were kept on acidified, chlorinated water and used at 10–16 wk of age.
Thyroglobulin
mTg and hTg were obtained from frozen thyroids fractionated on a Sephadex G-200 column as described previously (19). Tg was checked for the presence of LPS by Limulus amebocyte assay (a 100-μg dose contained <1 ng of LPS) and diluted in nonpyrogenic saline.
mAbs and in vivo depletion of CD25+ T cells
CD25 mAb was produced by culture of 1 x 108 PC61 (rat IgG1) hybridoma cells (American Type Culture Collection; Ref.20) in a Cell Max module (Cellco) and harvested according to manufacturer’s instructions. Harvested mAb was twice purified by ammonium sulfate precipitation, and specific Ab concentration was determined as described previously (17). For in vivo depletion, two 1-mg doses of CD25 mAb (containing 10–20% active Ab) or two 1-mg doses of polyclonal rat IgG control (Sigma-Aldrich) were given i.v. 4 days apart. Depletion was monitored in PBL by flow cytometry with PE-7D4 (rat IgM) (Southern Biotechnology Associates) 6 days later, in conjunction with anti-CD4 (YTA 3.1 and YTS 191.1, rat IgG2b) ascites fluid twice purified by ammonium sulfate precipitation and labeled with FITC-NORIG 7.16 (mouse anti-rat IgG2b; Refs.21 and 22). Anti-CD25 mAb treatment resulted in a 70–90% reduction of CD4+CD25+ T cells as indicated in the figure legends.
Adoptive transfer of CD4+CD25+ T cells
As previously described (17), CD4+CD25– and CD4+CD25+ T cell populations were enriched for adoptive transfer studies by magnetic separation of splenocytes from B10 mice first depleted in vivo of CD8+ T cells by administration of two 320-μg CD8 mAb (YTS 156.7 and YTS 169.4, rat IgG2b) doses (23, 24). CD4+ T cell populations were obtained by panning over anti-B cell mAb (187.1, rat IgG; American Type Culture Collection) and anti-CD11b (YBM 6.1.10, rat IgM)-coated petri dishes to remove B cells and macrophages. CD4+CD25+ T cells were selected positively from the remaining cells by labeling with PE-7D4 and anti-PE magnetic microbeads (StemCell Technologies). Viability and purity of CD4+CD25+ and CD4+CD25– fractions were assessed by trypan blue staining and flow cytometric analysis (average viability > 95% at 75–90% purity). CD25 mAb-treated B10 mice were reconstituted i.v. with 1.5–3.0 x 107 enriched cells/mouse 12 days before challenge with 40 μg of mTg and 20 μg of LPS.
Induction of EAT
EAT was induced by injecting i.v. 40 μg of Tg, followed 3 h later with 20 μg of Salmonella enteritidis LPS on days 0 and 7. Animals were sacrificed on day 28, and EAT was assessed by histologic examination of thyroids, in vitro lymphocyte proliferation to Ag, and anti-Tg production.
Histologic evaluation of EAT
Thyroid specimens were sectioned vertically through both thyroid lobes (50–60 sections from 10–15 step levels). Mononuclear cell infiltration was scored on an index of 0–4.0: 0, no infiltration; 0.5, >0–10% thyroid involvement; 1.0, >10–20% involvement with follicular destruction; 2.0, >20–40% involvement; 3.0, >40–80% involvement; and 4.0, >80% involvement (25).
Measurement of autoantibody production
Mice were bled from the tail artery on day 28, and the sera was stored at –20°C. Anti-Tg titers were determined by ELISA using Tg-coated (1 μg/well) Immulon I microtiter plates and alkaline phosphatase-labeled goat anti-mouse IgG (Sigma-Aldrich) as described previously (26).
In vitro proliferation
Splenocytes were cultured in triplicate in RPMI 1640 + 1% normal mouse serum in flat-bottom 96-well plates at 6 x 105 cells/well, either with or without 40 μg of Tg/ml, for 5 days at 37°C, 6% CO2 (27). Cells were pulsed with 1.0 μCi/well of [3H]thymidine 18–20 h before harvest onto glass fiber filter paper (Tomtec Mach3Man Cell Harvester; UKB-Wallac). [3H]Thymidine uptake was assessed using a Microbeta Plus 1450 liquid scintillation counter (UKB-Wallac). Stimulation index was calculated as mean cpm ± SE with Ags divided by mean cpm without Ags.
Statistical analysis
Individual pathology indices between groups were analyzed nonparametrically using the Mann-Whitney U test. A value of p < 0.05 was considered significant statistically.
Results
Depletion of CD4+CD25+ T cells in E+B10.Ab0 (A–E+) mice enhances EAT induced with hTg without altering the restriction against mTg.
We have recently reported that CD4+CD25+ regulatory T cells are responsible for mediating induced tolerance to Tg. After depletion of CD4+CD25+ T cells, tolerance induced by mTg pretreatment in CBA mice was abrogated, and their susceptibility to EAT induction was restored (17). To determine whether CD4+CD25+ T cells also play a role in influencing thyroiditis development in the unique A–E+-transgenic model that is permissive for thyroiditis induction only with heterologous Tg, but not self-mTg (11), CD4+CD25+ T cells in E+B10.Ab0 mice were depleted in vivo. Two 1-mg doses of a depleting CD25 mAb or a polyclonal rat IgG control Ab were administered i.v. 4 days apart (days –14 and –10) as described previously (17). A reduction of 70–90% of CD4+CD25+ T cells was observed by analysis of PBL on day –4, 6 days after the second dose of anti-CD25 (data not shown). Mice were then challenged on days 0 and 7 with the EAT induction protocol of 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS. As illustrated in Fig. 1A, depletion of CD4+CD25+ T cells in E+B10.Ab0 (A–E+) mice resulted in greatly increased mononuclear infiltration of the thyroid following immunization with hTg; 69% (9 of 13) of the CD25-depleted mice exhibited 40–80% thyroid involvement, compared with 23% (3 of 13) in control rat IgG-treated mice. However, the depletion of CD4+CD25+ T cells did not enable induction of thyroiditis with mTg in E+B10.Ab0 mice because none displayed thyroid destruction, and only 8% (1 of 12) showed mild infiltration.
FIGURE 1. In vivo depletion of CD4+CD25+ T cells enhances EAT induction with hTg but does not alter restriction against mTg in B10.E+Ab0 (A–E+) mice. B10.E+Ab0 mice were given two 1-mg doses of anti-CD25 or rat IgG i.v. on days –14 and –10. Reduction of 75–90% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged with 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS on days 0 and 7, and the mice were killed on day 28. A, The graph represents mononuclear cell infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, day. B, Representative thyroid histology from experimental groups (original magnification, x100): normal thyroid architecture (a); infiltration of mononuclear cells 20% following induction of EAT with hTg (b); lack of thyroiditis induction and mononuclear cell infiltration after mTg immunization (c); and CD4+CD25+ T cells depleted before immunization with hTg, infiltration, and destruction 60% (d).
Fig. 1B illustrates representative thyroid pathology from each group. Fig. 1Ba shows normal thyroid histology. Fig. 1Bb is a thyroid section from a hTg-immunized E+B10.Ab0 mouse, with mononuclear cell infiltration involving 20% of the thyroid. In vivo depletion of CD4+CD25+ T cells enhanced thyroiditis induced with hTg, resulting in more severe inflammation involving 60% of the thyroid (Fig. 1Bd). In contrast, the resistance of E+B10.Ab0 mice to thyroiditis induction with mTg was unaltered by depleting CD4+CD25+ T cells; the thyroid section from a CD4+CD25+ T cell-depleted, mTg-challenged mouse shown in Fig. 1Bc demonstrates no mononuclear cell infiltration, similar to that from a normal mouse. No differences were observed in autoantibody production or in vitro-proliferative responses to Ags in hTg-immunized mice pretreated with anti-CD25 compared with mice pretreated with rat IgG, because both remained strongly positive (data not shown). As previously reported (11), splenocytes from hTg-immunized mice did not respond to mTg (data not shown). The increase in thyroiditis severity following anti-CD25 administration indicates that CD4+CD25+ regulatory T cells influencing thyroiditis development can be generated in the context of H2E. The inability of regulatory T cell depletion to alter the resistance to mTg-induced EAT shows that the unique property of the E+B10.Ab0 mouse to respond only to hTg is a function of restricted presentation of autoantigen by the Eb molecule and is not a result of CD25+ T cell regulation.
Depletion of CD4+CD25+ T cells enables susceptibility to EAT induction with mTg in traditionally resistant B10 (H2b) mice
Previous investigation of this unique E+-transgenic mouse model of EAT revealed a suppressive effect of the endogenous Ab genes; E+B10 (A+E+) mice developed less severe thyroiditis following immunization with hTg, compared with E+B10.Ab0 (A–E+) mice (11). This suppressive effect of the endogenous Ab genes might be mediated through CD4+CD25+ regulatory T cells, particularly those activated by self-peptides presented by the endogenous Ab molecule. To determine whether CD4+CD25+ T cells exert a regulatory influence on thyroiditis development by self-Tg epitopes in the context of Ab, we selected traditionally EAT-resistant congenic B10 (A+E–) mice devoid of H2E molecules. Their CD4+CD25+ T cells were depleted before challenge with the EAT induction protocol. As illustrated in Fig. 2, depletion of CD4+CD25+ T cells enabled the induction of thyroiditis with mTg because eight of nine (89%) CD25-depleted mice developed mononuclear cell infiltration of the thyroid, compared with two of eight (25%) controls. Thus, EA
T resistance in B10 mice is mediated by CD4+CD25+ regulatory T cells in the normal context of Ab. However, depletion of CD4+CD25+ T cells did not enable thyroiditis induction with 40-μg doses of hTg (Fig. 2), a dose sufficient to induce EAT in A–E+ mice (Fig. 1A).
FIGURE 2. In vivo depletion of CD4+CD25+ T cells enables EAT induction with mTg, but not hTg, in B10 (A+E–) mice. Two 1-mg doses of anti-CD25 or polyclonal rat IgG i.v. were administered to B10 mice on days –14 and –10. Reduction of 75–85% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged on days 0 and 7 with 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS and killed on day 28. The graph represents mononuclear cell infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Reconstitution of CD4+CD25+ T cells restores EAT resistance in CD25 mAb-treated B10 mice
To determine whether adoptive transfer of CD4+CD25+ T cells would restore EAT resistance in B10 mice, B10 mice were depleted of CD4+CD25+ T cells with two 1-mg doses of anti-CD25 mAb on days –26 and –22 (75–90% reduction as measured by flow cytometric analysis of PBL 6 days later). On day –12, 1.5–3.0 x 107 CD4+CD25+ or CD4+CD25– T cells from naive B10 mice were transferred. Recipient mice were then challenged with 40 μg of mTg and 20 μg of LPS on days 0 and 7, and thyroiditis severity was assessed on day 28. Fig. 3 illustrates that 70% of CD25+ T cell-depleted mice receiving CD4+CD25+ T cells had no thyroiditis; the remaining 3 of 10 (30%) mice had mononuclear cell infiltration of 5–20%. In contrast, more consistent and extensive mononuclear cell infiltration and destruction of up to 40% of the thyroid was observed in CD4+CD25– T cell-reconstituted mice. The specific requirement for CD4+CD25+ T cells to restore resistance to thyroiditis induction in CD25 mAb-treated B10 mice reinforces the role of CD4+CD25+ T cells in regulating susceptibility.
FIGURE 3. Adoptive transfer of CD4+CD25+ T cells restores resistance to EAT induction in CD25+ T cell-depleted B10 mice. B10 mice were given two 1-mg doses of CD25 mAb on days –26 and –22 for depletion of CD25+ cells (75–80% reduction in CD4+CD25+ T cells was observed by FACS analysis of PBL 6 days later). CD4+CD25– and CD4+CD25+ T cells were isolated by magnetic separation of splenocytes from CD8-depleted B10 mice, and 1.5–3.0 x 107 cells/mouse were transferred i.v. on day –12. All mice were challenged with 40 μg of mTg, followed 3 h later by 20 μg of LPS on days 0 and 7. Thyroiditis severity was assessed on day 28. The graph represents mononuclear infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Depletion of CD4+CD25+ T cells reverses the suppressive effect of endogenous Ab molecule in E+B10 (A+E+) mice
The findings presented above demonstrate that CD4+CD25+ T cells are generated in the presence of either H2A or H2E molecules because their removal enhanced thyroiditis severity in both A–E+ and A+E– mice. It was then of interest to examine the role of CD4+CD25+ T cells in E+B10 (A+E+) mice, in which the presence of Ab genes resulted in reduced thyroiditis severity after hTg induction (11). After depleting CD4+CD25+ T cells, E+B10 mice were challenged with either hTg or mTg. As shown in Fig. 4, both hTg- and mTg-induced EAT were increased significantly (p < 0.01) by the elimination of CD4+CD25+ T cells. E+B10 mice immunized with hTg demonstrated strong autoantibody production and in vitro-proliferative response, irrespective of CD4+CD25+ T cell depletion (data not shown). Mice depleted of CD4+CD25+ T cells demonstrated a slight increase in anti-mTg Ab production but no difference in in vitro-proliferative response of splenocytes, compared with polyclonal rat IgG-treated controls (data not shown). The increase in the severity of thyroiditis induced with either hTg or mTg seen in anti-CD25-treated mice indicates that CD4+CD25+ T cells also regulate thyroiditis development in E+B10 (A+E+) mice.
FIGURE 4. In vivo depletion of CD4+CD25+ T cells enhances EAT induced with either hTg or mTg in B10.E+ (A+E+) mice. Mice were given two 1-mg doses of anti-CD25 or polyclonal rat IgG i.v. on days –14 and –10. Reduction of 70–80% of CD4+CD25+ T cells was observed by FACS analysis of PBL on day –4. Mice were challenged on days 0 and 7 with EAT induction protocol of 40 μg of either hTg or mTg, followed 3 h later by 20 μg of LPS and killed on day 28. The graph represents mononuclear infiltration of thyroids of individual mice; results are pooled from two independent experiments. D, Day.
Discussion
We have previously observed reciprocal-suppressive effects of both H2E and H2A genes on thyroiditis. H2Es genes reduced the severity of thyroiditis encoded by H2As in B10.S mice (7), and H2Ab genes suppressed H2E-mediated thyroiditis in E+B10.Ab0 mice (11). Although such suppressive effects of H2E on other autoimmune responses encoded by H2A molecules has also been previously described (8, 9), the reciprocal effect of H2A genes on H2E-mediated thyroiditis, to our knowledge, has not been demonstrated in other systems. The generation of regulatory T cells responding to Ags presented in the context of H2E has been proposed as a potential suppressor mechanism (8, 9, 12, 13, 14).
CD4+CD25+ regulatory T cells, a subset of T lymphocytes essential in maintaining peripheral immunoregulation inhibiting the development of autoimmunity (15, 28, 29), are derived from high-affinity interactions with self-peptide, both during ontogeny (30, 31, 32) and in the periphery (33). We have recently described a regulatory role for peripherally amplified CD4+CD25+ T cells in EAT-susceptible CBA mice, which mediate mTg-induced tolerance to EAT (17). The influence of CD4+CD25+ T cells in regulating susceptibility to EAT and the potential role of class II molecules in generating these cells led to an examination of their potential influence in class II-transgenic strains.
Our first step to identify a potential role for CD4+CD25+ regulatory T cells in mediating the reciprocal inhibition by H2Eb and H2Ab was to verify their existence in each individual MHC context. For CD4+CD25+ T cells derived from the Eb molecule in E+B10.Ab0 (A–E+) mice, we depleted them in vivo before EAT induction. This transgenic mouse strain is susceptible to thyroiditis induced with heterologous Tg but uniquely resistant to thyroiditis induction with self mTg (11). We observed an increase in thyroiditis severity after hTg immunization (Fig. 1), indicating that CD4+CD25+ regulatory T cells are generated in the context of Eb and the absence of Ab. Of interest is the observation that depletion of CD4+CD25+ T cells did not enable B10.E+Ab0 (A–E+) mice to shed the restriction against mTg-induced thyroiditis (Fig. 1). Because mTg, possessing both unique and shared epitopes (34), is more thyroiditogenic than hTg, the inability of A–E+ mice to develop thyroiditis following immunization with mTg reinforces the
absolute requirement for the correct class II presentation of Tg in EAT induction and indicates that the removal of peripheral inhibition of autoimmunity does not supersede this requirement.
Having demonstrated the existence of CD4+CD25+ T cells that inhibit EAT in the context of Eb, we next examined the development of such cells in the context of Ab, a traditionally EAT-resistant haplotype. After depleting CD4+CD25+ T cells in B10 (A+E–) mice, we immunized the mice with either mTg or hTg. The elimination of resistance to EAT induction with mTg in B10 mice following CD4+CD25+ T cell depletion (Fig. 2) indicates that CD4+CD25+ regulatory T cells that influence thyroiditis development also exist in the context of Ab, and their removal leads to an increase in susceptibility. These data are reminiscent of an earlier report describing increased susceptibility to mTg-induced thyroiditis in EAT-resistant C57BL/6 (H2Ab) mice after administration of cyclophosphamide, an agent known to inactivate preferentially regulatory T cells (35). The data also agree with the finding that elimination of CD4+CD25+ regulatory T cells reduced resistance against the induction of gastritis in traditionally resistant mice (16). The specific role of CD4+CD25+ T cells in influencing susceptibility in traditionally EAT-resistant mice is reinforced additionally by the restoration of resistance to EAT induction following the transfer of CD4+CD25+ T cells in CD25 mAb-treated B10 mice (Fig. 3). Thus, resistance to autoimmunity may, at least in some instances, reflect greater peripheral regulation rather than a poor response to autoantigen. The ability of class II molecules to generate regulatory T cells effective in inhibiting autoimmunity likely varies between haplotypes and may explain the genetic predisposition of certain haplotypes to the development of autoimmunity. In contrast, the inability of hTg to induce thyroiditis in B10 mice, even after depletion of CD4+CD25+ regulatory T cells (Fig. 2), may be related to its poor thyroiditogenicity in traditionally susceptible strains (36, 37) or to its involvement of regulatory T cells other than those expressing CD25 (38). It may also indicate the existence of Ab specificity for epitopes not shared between mTg and hTg, similar to the Eb molecule for nonconserved hTg epitopes (Fig. 1; Ref.11).
The existence of CD4+CD25+ T cells influencing thyroiditis induction in both the context of Ab and Eb supports the idea that these cells may be responsible for the reciprocal-suppressive effect between the two molecules observed previously (8, 11). Suppression of H2A-dependent autoimmune responses by introducing H2E molecules has been described in other models (8, 9, 39). Using our unique Ag-restricted, E+-transgenic model, we were able to examine the influence of CD4+CD25+ regulatory T cells on Ab- and Eb-mediated thyroiditis when both Eb and Ab genes were present. The increase in thyroiditis severity in CD25+ T cell-depleted E+B10 (A+E+) mice immunized with either hTg (mediated by Eb) or mTg (mediated by Ab; Fig. 4) indicates that CD4+CD25+ regulatory T cells influencing thyroiditis exist when both H2A and H2E molecules are present. The reduction in the suppressive effect of the Ab genes on Eb-mediated thyroiditis after depleting CD4+CD25+ T cells suggests that these cells may be responsible for the inhibitory effect due to the presence of the second class II allele.
We cannot point definitively to CD4+CD25+ regulatory T cells as mediators of the suppressive effect of H2A genes on H2E-mediated EAT observed in E+B10 mice at present because we demonstrated the existence of CD4+CD25+ regulatory T cells acting independently in the context of both Eb and Ab (Figs. 1 and 2). We have not discriminated between regulatory T cells generated by either H2E or H2A when eliminating regulatory T cells in vivo, and the observed increase in thyroiditis severity following CD4+CD25+ T cell depletion may be a result of decreased function of Eb- or Ab-derived regulatory T cells that act independently to inhibit thyroiditis mediated in the same context. Moreover, several other possibilities exist to explain the suppressive effect of the H2A genes in this model, such as alteration of the T cell repertoire, potentially including the deletion of autoreactive cells, and qualitative changes in T cell responses due to a combination of T cell epitopes presented in the context of Ab and Eb. Another interesting possibility arises from previous observations that presentation of an epitope from the E-chain by H2A molecules can inhibit collagen-induced arthritis in mice (39), and presentation of a HLA-DR-derived peptide by HLA-DQ can suppress collagen-induced arthritis in transgenic mice (40). A similar event may be occurring in the E+B10 mice, although we have not examined whether the Eb molecule can present Ab-derived peptides, nor whether As molecules can present Es-derived peptides in B10.S mice (7). Nevertheless, our data demonstrate the existence of CD4+CD25+ regulatory T cells generated by both Ab and Eb molecules and that these regulatory T cells may reciprocally suppress the development of autoimmune thyroiditis induced in either context.
The increased susceptibility of E+B10 mice to mTg-induced EAT as compared with congenic B10 mice (Figs. 2 and 4) is also of interest and may relate to regulatory T cell function. The loss of resistance to mTg-induced thyroiditis cannot be attributed to the induction of a responding class II molecule because the resultant Eb is not able to support mTg-induced EAT (11, 41). Possibly, this increase in susceptibility results from the alteration of the T cell repertoire because expression of the H2E molecule results in deletion of T cells expressing TCR V5 and V11 (42). Although the protective effect of Ed genes in NOD mice cannot be associated with the deletion of commonly used TCR V subsets (43), qualitative changes in regulatory T cells could potentially affect the T cell repertoire, and their deletion could lead to increased susceptibility of E+B10 mice to mTg-induced EAT.
Here, our findings indicate that CD4+CD25+ regulatory T cells generated in both the context of H2A and H2E play an important role in influencing the development of thyroiditis. These data also suggest that CD4+CD25+ regulatory T cells may be involved in the reciprocal-suppressive effect exerted between H2A and H2E. Susceptibility to autoimmunity has long been described as a function of class II genes in animal models (4), an association mirrored by human studies (5). More recently, human autoimmune disease has been linked to CTLA-4 (44) and FOXP3 (45), genes associated with CD4+CD25+ regulatory T cell activity (46, 47, 48, 49, 50). Our data suggest that the influence of class II genes and regulatory T cell-associated genes may not be independent and that class II genes may influence the development of autoimmunity both by their presentation of autoantigens as well as by their generation of CD4+CD25+ regulatory T cells.
Disclosures
Acknowledgments
We thank Julie Hanson and staff for breeding and maintaining the transgenic mice, A. M. Mazurco for preparation of histology sections, and Dr. A. A. Giraldo for assistance in evaluation of thyroid pathology. We also thank Dr. C. Jeffries for providing S. enteritidis LPS and Dr. H. Waldmann for the CD4, CD8, and CD11b mAbs.
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 Institute of Health Grant DK45960.
2 Address correspondence and reprint requests to Dr. Yi-chi M. Kong, Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. E-mail address: ykong{at}med.wayne.edu
3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; Tg, thyroglobulin; mTg, mouse Tg; hTg, human Tg.
Received for publication February 24, 2004. Accepted for publication December 23, 2004.
References
ElRehewy, M., Y. M. Kong, A. A. Giraldo, N. R. Rose. 1981. Syngeneic thyroglobulin is immunogenic in good responder mice. Eur. J. Immunol. 11:146.
Rose, N. R., F. J. Twarog, A. J. Crowle. 1971. Murine thyroiditis: importance of adjuvant and mouse strain for the induction of thyroid lesions. J. Immunol. 106:698.
Esquivel, P. S., N. R. Rose, Y. M. Kong. 1977. Induction of autoimmunity in good and poor responder mice with mouse thyroglobulin and lipopolysaccharide. J. Exp. Med. 145:1250
Vladutiu, A. O., N. R. Rose. 1971. Autoimmune murine thyroiditis: relation to histocompatibility (H-2) type. Science 174:1137.
Vaidya, B., P. Kendall-Taylor, S. H. S. Pearce. 2002. Genetics of endocrine disease: the genetics of autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 87:5385.
Beisel, K. W., C. S. David, A. A. Giraldo, Y. M. Kong, N. R. Rose. 1982. Regulation of experimental autoimmune thyroiditis: mapping of susceptibility to the I-A subregion of the mouse H-2. Immunogenetics 15:427.
Kong, Y. M., C. S. David, L. C. Lomo, B. E. Fuller, R. W. Motte, A. A. Giraldo. 1997. Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune thyroiditis. Immunogenetics 46:312.
Christadoss, P., C. S. David, M. Shenoy, S. Keve. 1990. Ek transgene in B10 mice suppresses the development of myasthenia gravis. Immunogenetics 31:241.
Nishimoto, H., H. Kikutani, K. Yamamura, T. Kishimoto. 1987. Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature 328:432.
Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis. 1991. Mice lacking MHC class II molecules. Cell 66:1051.
Wan, Q., R. Shah, D. J. McCormick, L. C. Lomo, A. A. Giraldo, C. S. David, Y. M. Kong. 1999. H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis. Immunogenetics 50:22.
Baxevanis, C. N., Z. A. Nagy, J. Klein. 1981. A novel type of T-T cell interaction removes the requirement for I-B region in the H-2 complex. Proc. Natl. Acad. Sci. USA 78:3809.
Oliveira, D. B. G., N. Blackwell, A. E. Virchis, R. A. Axelrod. 1985. T helper and T suppressor cells are restricted by the A and E molecules, respectively, in the F antigen system. Immunogenetics 22:169.
Lowenthal, J. W., P. Corthésy, C. Tougne, R. Lees, H. R. MacDonald, M. Nabholz. 1985. High and low affinity IL-2 receptors: analysis by IL-2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J. Immunol. 135:3988.
Aqel, N. M., M. Clark, S. P. Cobbold, H. Waldmann. 1984. Immunohistological screening in the selection of monoclonal antibodies: the use of isotype-specific antiglobulins. J. Immunol. Methods 69:207
Kong, Y. M., A. A. Giraldo, H. Waldmann, S. P. Cobbold, B. E. Fuller. 1989. Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression. Clin. Immunol. Immunopathol. 51:38.
Qin, S., M. Wise, S. P. Cobbold, L. Leong, Y. M. Kong, J. R. Parnes, H. Waldmann. 1990. Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur. J. Immunol. 20:2737.
Fuller, B. E., A. A. Giraldo, H. Waldmann, S. P. Cobbold, Y. M. Kong. 1994. Depletion of CD4+ and CD8+ cells eliminates immunologic memory of thyroiditogenicity in murine experimental autoimmune thyroiditis. Autoimmunity 19:161
Kong, Y. M.. 1996. Experimental autoimmune thyroiditis in the mouse. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 15.7.1. John Wiley & Sons, Inc., New York.
Zhang, W., J. C. Flynn, Y. M. Kong. 2001. IL-12 prevents tolerance induction with mouse thyroglobulin by priming pathogenic T cells in experimental autoimmune thyroiditis: role of IFN- and the costimulatory molecules CD40L and CD28. Cell. Immunol. 208:52.
Wan, Q., M. Kita, J. C. Flynn, J. C. Panos, R. W. Motte, T. F. Davies, A. A. Giraldo, C. S. David, Y. M. Kong. 2001. Participation of V13+ and V1+ T cells in transfer thyroiditis after activation of mouse thyroglobulin-primed T cells by superantigen staphylococcal enterotoxin A. Cell. Immunol. 213:149.
Sakaguchi, S., K. Fukuma, K. Kuribayashi, T. Masuda. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161:72.
Hurwitz, A. A., T. J. Sullivan, R. A. Sobel, J. P. Allison. 2002. Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice. Proc. Natl. Acad. Sci. USA 99:3013
Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, S. Sakaguchi. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.
Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301.
Jonuleit, H., E. Schmitt. 2003. The regulatory T cell family: distinct subsets and their interrelations. J. Immunol. 171:6323.
Gonzalez-Gay, M. A., E. Zanelli, C. J. Krco, G. H. Nabozny, J. Hanson, M. M. Griffiths, H. S. Luthra, C. S. David. 1995. Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis. Immunogenetics 42:35.
Das, P., D. S. Bradley, A. Geluk, M. M. Griffiths, H. S. Luthra, C. S. David. 1999. An HLA-DRB1*0402 derived peptide (HV3 65-79) prevents collagen-induced arthritis in HLA-DQ8 transgenic mice. Hum. Immunol. 60:575.
Kong, Y. M., L. C. Lomo, R. W. Motte, A. A. Giraldo, J. Baisch, G. Strauss, G. J. H?mmerling, C. S. David. 1996. HLA-DRB1 polymorphism determines susceptibility to autoimmune thyroiditis in transgenic mice: definitive association with HLA-DRB1*0301 (DR3) gene. J. Exp. Med. 184:1167.
Taneja, V., J. Hansen, M. Smart, M. Griffiths, H. Luthra, C. S. David. 1997. Expression of the H2-E molecule mediates protection to collagen-induced arthritis in HLA-DQ8 transgenic mice: role of cytokines. Int. Immunol. 9:1213.
Lund, T., L. O’Reilly, P. Hutchings, O. Kanagawa, E. Simpson, R. Gravely, P. Chandler, J. Dyson, J. K. Picard, A. Edwards, et al 1990. Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A -chain or normal I-E -chain. Nature 345:727
Ueda, H., J. M. M. Howson, L. Esposito, J. Heward, H. Snook, G. Chamberlain, D. B. Rainbow, K. M. D. Hunter, A. N. Smith, G. Di Genova, et al 2003. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506
Bassuny, W. M., K. Ihara, Y. Sasaki, R. Kuromaru, H. Kohno, N. Matsuura, T. Hara. 2003. A functional polymorphism in the promoter/enhancer region of the FOXP3/Scurfin gene associated with type 1 diabetes. Immunogenetics 55:149.
Read, S., V. Malmstr?m, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.
Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.
Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.
Schubert, L. A., E. Jeffery, Y. Zhang, F. Ramsdell, S. F. Ziegler. 2001. Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation. J. Biol. Chem. 276:37672.
Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.(Gerald P. Morris, Yan Yan)
|
微信文章
关注百拇
评论几句
搜索更多
推存给朋友
加入收藏
|