Naturally-existing CD4(+)CD25(+)Foxp3(+) regulatory T cells are required for tolerance to experimental autoimmune thyroiditis induced by either exogenous or endogenous autoantigen

Abstract

Murine experimental autoimmune thyroiditis (EAT) is a model for Hashimoto's thyroiditis, an organ-specific autoimmune disease characterized by mononuclear cell infiltration and destruction of the thyroid gland. Susceptibility to EAT is MHC-linked, and influenced by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Treg depletion enables thyroiditis induction with mouse thyroglobulin (mTg) in traditionally-resistant mice and mTg-induced, Treg-mediated tolerance protects against EAT induction in genetically-susceptible mice. Here, we demonstrate the existence of naturally-existing CD4(+)CD25(+)Foxp3(+) Tregs (nTregs) influencing thyroiditis development in naive susceptible mice and that induction of thyroiditis in these mice involves overcoming peripheral homeostatic immune suppression by nTregs. Additionally we demonstrate that nTregs are required for induction of antigen-specific tolerance, indicating that induced EAT tolerance is a result of activation of naturally-existing nTregs rather than de novo generation of induced Tregs (iTregs). Examination of several potential costimulatory molecules previously described as involved in peripheral activation of Tregs demonstrates a critical role indeed for CTLA-4 in the activation of nTregs leading to development of EAT tolerance and providing a mechanism for mTg-induced Treg activation during tolerance induction. Together, these data reinforce the important role of Tregs in mediating self-tolerance, and illuminate a potential mechanism for their therapeutic expansion in induced tolerance.

Fig. 1

In vivo depletion of CD4⁺Foxp3⁺CD25⁺ T cells abrogates tolerance induced by endogenous mTg released via TSH infusion. Composite data from two independent experiments are shown. Mice were first implanted i.p. with osmotic pumps secreting TSH or saline control. Groups of mice were then injected i.v. with two 1-mg doses of CD25 mAb or control rat IgG, 4 days apart, 8 or 12 days after pump implantation. Depletion of CD4⁺Foxp3⁺CD25⁺ cells was monitored by FACS 6 days after anti-CD25 treatment. After 4 more days, mice were challenged i.v. with 40 µg mTg and 20 µg LPS (days 0, 7) and killed on day 28. (A) FACS analysis of PBL from rat IgG or anti-CD25 treated mice 6 days after Ab treatment. Labeling for CD4, CD25 and intracellular Foxp3 expression showed depletion of the CD25⁺ subset in CD4⁺Foxp3⁺ cells (right panel). (B) Thyroid pathology of individual mice 28 days after immunization. (C) mTg Ab levels in the groups.

Fig. 2

CD4⁺CD25⁺ T cells from naive or tolerized mice have similar Foxp3 expression and in vitro suppressive capabilities. (A) Pooled splenocytes from naive mice or from mice with established tolerance (3 animals each) were labeled for extracellular expression of CD4 and CD25, and intracellular Foxp3 expression. Cells were analyzed by flow cytometry, gating on CD4⁺ T cells (shown in inset), and expression of CD25 and Foxp3 on these CD4-gated cells were compared between groups. (B and C) CD4⁺CD25⁺ and CD4⁺CD25⁻T cell-enriched populations were obtained from CD8-depleted naive or tolerized mice by in vitro cell separation. (B) Fractionated cells were co-cultured at graded doses of 3–9×10⁴ cells/well with 4×10⁵ mTg-primed splenocytes/well. Cultures were stimulated with 40 µg/ml mTg for 5 days, and proliferative response was assessed by [³H]thymidine uptake. (C) 2×10⁵ fractionated cells/well were co-cultured with 4×10⁵ mTg-primed splenocytes/well and stimulated with 40 µg/ml mTg or mTg epitope T₄(2553) for 5 days, and proliferative response was assessed by [³H]thymidine uptake; comparisons within mTg- or T4(2553)-stimulated groups. *, P<0.01; **, P<0.01.

Fig. 3

In vivo depletion of CD4⁺CD25⁺ T cells lowers the threshold for EAT induction in naive mice. Mice were given i.v. 1 mg CD25 mAb or control rat IgG on days −14 and −10. Depletion of CD4⁺ CD25⁺ T cells was verified by flow cytometric analysis of PBL on day −4. Mice were subsequently challenged with an adjuvantless EAT induction protocol of i.v. 20 µg mTg 4 times/week for 4 weeks beginning on day 0; thyroid pathology was evaluated on day 35. (A) Mononuclear cell infiltration of thyroid of individual mice; results pooled from two independent experiments. (B) Representative thyroid histology from experimental groups (original, 100x) demonstrating normal thyroid architecture. (C) Infiltration of mononuclear cells involving approximately 10% of the thyroid, observed in about 50% of IgG-treated control mice challenged with repeated doses of mTg without adjuvant. (D) Mononuclear cell infiltration involving ∼25% of the thyroid observed in mice depleted of CD4⁺CD25⁺ T cells prior to challenge. (E) Anti-mTg IgG from day 35 sera determined by ELISA with serial dilutions up to 1:1600; positive O.D. readings at 1:100 and 1:800 are shown. (F) In vitro proliferative response of pooled splenocytes to stimulation with mTg or mTg epitope T₄(2553). Comparisons between rat IgG- or anti-CD25-treated groups: *, P<0.01; **, P<0.01.

Fig. 3

In vivo depletion of CD4⁺CD25⁺ T cells lowers the threshold for EAT induction in naive mice. Mice were given i.v. 1 mg CD25 mAb or control rat IgG on days −14 and −10. Depletion of CD4⁺ CD25⁺ T cells was verified by flow cytometric analysis of PBL on day −4. Mice were subsequently challenged with an adjuvantless EAT induction protocol of i.v. 20 µg mTg 4 times/week for 4 weeks beginning on day 0; thyroid pathology was evaluated on day 35. (A) Mononuclear cell infiltration of thyroid of individual mice; results pooled from two independent experiments. (B) Representative thyroid histology from experimental groups (original, 100x) demonstrating normal thyroid architecture. (C) Infiltration of mononuclear cells involving approximately 10% of the thyroid, observed in about 50% of IgG-treated control mice challenged with repeated doses of mTg without adjuvant. (D) Mononuclear cell infiltration involving ∼25% of the thyroid observed in mice depleted of CD4⁺CD25⁺ T cells prior to challenge. (E) Anti-mTg IgG from day 35 sera determined by ELISA with serial dilutions up to 1:1600; positive O.D. readings at 1:100 and 1:800 are shown. (F) In vitro proliferative response of pooled splenocytes to stimulation with mTg or mTg epitope T₄(2553). Comparisons between rat IgG- or anti-CD25-treated groups: *, P<0.01; **, P<0.01.

Fig. 4

In vivo depletion of CD4⁺CD25⁺ T cells prevents subsequent induction of EAT tolerance. Mice were given i.v. 1 mg CD25 mAb or control rat IgG on days −24 and −20; CD4⁺CD25⁺ T cell depletion was monitored by flow cytometric analysis of PBL on day −14. All mice were subsequently tolerized to EAT with 100 µg dmTg i.v. on days −10 and −3, and challenged with an EAT induction protocol of 40 µg mTg and 20 µg LPS on days 0 and 7. Mice were sacrificed on day 28 to examine thyroiditis development. Graph illustrates extent of mononuclear cell infiltration of thyroids of individual mice pooled from two independent experiments.

Fig. 5

Tolerance induction is inhibited specifically by in vivo administration of CTLA-4 mAb. mAbs to CD28 (250 µg), CD40L (250 µg), and CTLA-4 (1 mg) were given i.v. on days −16, −15, −14, −9, −8, and −7 to cover the window of tolerance induction by tolerizing doses of 100 µg dmTg on days −15 and −8. Mice were subsequently challenged with an EAT induction protocol of 40 µg mTg and 20 µg LPS on days 0 and 7, and sacrificed on day 28 to examine thyroiditis development. Graph illustrates extent of mononuclear cell infiltration in thyroids of individual mice pooled from two independent experiments.