Tumor necrosis factor-related apoptosis-inducing ligand inhibits experimental autoimmune thyroiditis by the expansion of CD4+CD25+ regulatory T cells

Abstract

There have been several reports that TNF-related apoptosis-inducing ligand (TRAIL) has the ability to suppress the development of experimental autoimmune diseases, including a mouse model of experimental autoimmune encephalomyelitis, a rabbit model of rheumatoid arthritis, type 1 diabetes mellitus, in mice and experimental autoimmune thyroiditis (EAT) in mice. However, the mechanism underlying TRAIL effect is not well defined. In the present study, we specifically examined TRAIL effects on CD4(+)CD25(+) regulatory T cells. CD4(+)CD25(+) T cells prepared from mouse thyroglobulin (mTg)-immunized CBA/J mice proliferate in the presence of TRAIL and dendritic cells in vitro. These CD4(+)CD25(+) T cells included both CD4(+)CD25(+)CD45RB(Low) (regulatory) and CD4(+)CD25(+)CD45RB(High) (effector) T cells. Our results demonstrated that mTg-immunized mice treated with TRAIL showed significant increases in the number of CD4(+)CD25(+)CD45RB(Low) T cells compared with mice immunized with mTg alone. CD4(+)CD25(+)CD45RB(Low) T cells expressed much higher levels of the forkhead family transcription factor, IL-10, and TGFbeta1 than CD4(+)CD25(+)CD45RB(High) T cells, and these cells can completely suppress the proliferation of the mTg-primed splenocytes in lower concentrations than the unfractionated CD4(+)CD25(+) T cells. Furthermore, transfer of these cells into CBA/J mice prior to mTg-primed splenocyte injection could markedly reduce the frequency and severity of EAT development. CD4(+)CD25(+)CD45RB(Low) T cells were more effective at suppressing histological thyroiditis than unfractionated cells. These results indicated that TRAIL can increase the number of mTg-specific CD4(+)CD25(+)CD45RB(Low) T cells, inhibiting autoimmune responses and preventing the progression of EAT. These findings reveal a novel mechanism by which TRAIL could inhibit autoimmune disease.

Figure 1

Cytokine secretion profile of CD4⁺ T cells. CD4⁺ T cells isolated from mTg-immunized mice treated with TRAIL or BSA were activated with anti-CD3 and anti-CD28 monoclonal antibodies for 48 h in vitro. Their culture supernatants were collected and measured for IL-4, IL-10, and IFN-γ productions. The data are expressed as mean ± sd.

Figure 2

TRAIL effect on splenic T cell or CD4⁺CD25⁺ T cell proliferation in vitro. Splenic T cells isolated from mTg-immunized mice were cultured with mTg, TRAIL, and mTg/TRAIL in the presence of DC. The proliferation of splenic T cells to mTg was determined by ³H-thymidine uptake. **, P < 0.01, compared with all three other groups (A). CD4⁺CD25⁺ T cells isolated from the splenocytes of mTg-immunized mice were cultured with mTg, DC, and TRAIL in the presence of IL-2 for 4 d. The proliferation of CD4⁺CD25⁺ T cells was determined by ³H-thymidine uptake. *, P < 0.05, compared with mTg group; **, P < 0.01, compared with mTg+DC or mTg+TRAIL groups (B).

Figure 3

Inhibitory effect of CD4⁺CD25⁺ T cells. mTg-primed splenocytes or CD4⁺CD25⁻ T cells/DC were cocultured with CD4⁺CD25⁺ T cells isolated from mTg-immunized mice in the presence of mTg. The proliferation of splenocytes (A) or CD4⁺CD25⁻ T cells (B) was determined by ³H-thymidine uptake. *, P < 0.05, compared with mTg or mTg+DC groups.

Figure 4

Increase of CD4⁺CD25⁺CD45RB^Low T cell number by TRAIL treatment in vivo. CD4⁺ T cells were prepared from mTg-immunized mice with or without TRAIL treatment. The CD4⁺ T cells were stained with PE-anti-CD25 and FITC-anti-CD45RB antibodies. The number of CD4⁺CD25⁺CD45RB^Low T cells was determined by flow cytometry (A). The ratio of CD4⁺CD25⁺CD45RB^Low T cells to CD4⁺CD25⁺CD45RB^High T cells was calculated (B).

Figure 5

Expression of Foxp3, IL-10, and TGFβ1 in CD4⁺CD25⁺CD45RB^Low T cells and CD4⁺CD25⁺CD45RB^High T cells. A, RNA was extracted from CD4⁺CD25⁺CD45RB^Low T cells or CD4⁺CD25⁺ CD45RB^High T cells isolated from mTg-immunized mice. The expression of Foxp3, IL-10, and TGFβ1 mRNA was measured by RT-PCR. **, P < 0.01, compared with CD4⁺CD25⁺D45RB^Low T cell group. B, mTg-primed splenocytes were stained with the CD4, CD25, and CD45RB surface markers. Under gating on the CD4 and CD25 markers (I), the expression of Foxp3 protein in CD45RB^Low and CD45RB^High T cells (III) was analyzed by flow cytometry. The staining with rat IgG2a, an isotype control, was also shown (II). Data are representative of three independent experiments.

Figure 6

Suppressive effect of CD4⁺CD25⁺CD45RB^Low T cells. Using flow cytometry, CD4⁺CD25⁺CD45RB^Low T cells were sorted from CD4⁺ T cells isolated mTg-primed splenocytes. MTg-primed splenocytes were cocultured with CD4⁺CD25⁺ CD45RB^Low T cells in the presence of mTg. The proliferation of splenocytes was determined by ³H-thymidine uptake. *, P < 0.05, **P < 0.01, compared with mTg + splenocyte group (A). CD4⁺CD25⁺CD45RB^Low T cells (3 × 10⁵⁾ were transferred to an irradiated CBA/J recipient before injection of the mTg-primed splenocytes (1.5 × 10⁷). After 21 d of CD4⁺CD25⁺CD45RB^Low transfer, all mice were killed and the thyroid sections were analyzed for mononuclear cell infiltration (B). The index of infiltration was determined as previously described (1).