Down-regulation of myasthenogenic T cell responses by a dual altered peptide ligand via CD4+CD25+-regulated events leading to apoptosis

Abstract

The myasthenogenic peptides p195-212 and p259-271 are sequences of the human acetylcholine receptor and were shown to induce myasthenia gravis-associated immune responses in mice. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited, in vitro and in vivo, those responses. The aims of this study were to elucidate the events that follow the in vivo treatment with the dual APL and to characterize the cell population that is induced by the latter. We demonstrate here that s.c. administration of the dual APL up-regulates CD4+CD25+ regulatory T cells that are characterized by up-regulated expression of cytotoxic T lymphocyte-associated antigen 4, intracellular and membranal TGF-beta, and Foxp3. Administration of the dual APL to mice concomitant with the immunization with either of the myasthenogenic peptides resulted also in the up-regulation of c-Jun-NH2-terminal kinase activity and of Fas signaling pathway molecules as determined by measuring Fas, Fas ligand, and caspase 8. Thus, our results suggest that the suppression of myasthenia gravis-associated T cell responses exerted by the dual APL is mediated by the CD4+CD25+ immunoregulatory T cell function via TGF-beta or cytotoxic T lymphocyte-associated antigen 4, which further stimulate a cascade of events that up-regulates apoptosis.

Fig. 1.

The dual APL inhibits the in vivo priming of LN cells and down-regulates the secretion of IFN-γ. SJL mice were immunized with p195–212 in CFA (open bars) and concomitantly treated with the dual APL (200 μg per mouse s.c. in PBS; filled bars). Mice were killed at day 10, and LN cells were taken for proliferation assay (A) or for cytokine secretion (B) as described in Materials and Methods. Results are representative of three experiments performed.

Fig. 2.

CTLA-4 expression in LN cells of BALB/c mice that were treated with the dual APL. BALB/c mice were immunized with p259–271 and treated with the dual APL at day 7 after immunization. Mice were killed at day 10, and their LN cells were stained with anti-CTLA-4-PE and analyzed by FACS. The results presented are after reduction of background staining with the matched isotype control, and they represent one experiment of three performed.

Fig. 3.

The effect of treatment with the dual APL on the expression of CD4⁺CD25⁺CTLA4⁺ T cells and on Foxp3 gene expression in BALB/c and SJL mice. BALB/c (A–C) and SJL (D–G) mice were immunized with p259–271 and p195–212, respectively. Seven days after immunization, the mice were treated with the dual APL, and their LN cells were harvested 3 days later. LN cells of BALB/c(A–C) and SJL (D–F) mice were stained for CD4⁺, CD25⁺, and CTLA-4. A–F Left demonstrates LN cell staining of mice that were immunized with the myasthenogenic peptides, and A–F Right shows staining of LN cells of mice that were treated with the dual APL in addition to their immunization. CD4⁺CD25⁺-gated cells that were also stained for CTLA-4 are shown in C and F. (G) Total RNA was isolated, reverse-transcribed as described in Materials and Methods, and further subjected to real-time PCR of the Foxp3 gene. p195–212-immunized mice (open bar) and dual APL-treated mice (filled bar) are presented. Results are of a representative experiment of four performed.

Fig. 4.

The effect of the dual APL on the expression of intracellular and membranal TGF-β in CD4⁺CD25⁺ T cells from LN cells of BALB/c and SJL mice. BALB/c (A–C) and SJL (D–G) mice were immunized with p259–271 and p195–212, respectively, and treated with the dual APL 7 days later. LN cells of BALB/c were stained for CD4⁺, CD25⁺, and intracellular and membranal TGF-β. Left demonstrates LN cell staining of mice that were immunized with the myasthenogenic peptide alone, and Right shows LN cell staining of mice that were immunized and treated with the dual APL. C and F represent CD4⁺CD25⁺-gated cells that were also stained for intracellular TGF-β.(G) CD4⁺CD25⁺-gated cells that were also stained for membranal TGF-β. Results are of one experiment of three performed.

Fig. 5.

The effect of the dual APL on JNK activity in SJL and BALB/c mice. BALB/c(A and B) and SJL (C and D) mice were immunized with p259–271 and p195–212, respectively (open bars in Top), and concomitantly treated with the dual APL (filled bars in Top). LN cells were harvested 10 days later, and lysates were prepared from total LN cell populations (A and C) or from purified T cells (B and D). Samples (100 μg of protein per lane) were separated on SDS/PAGE and transferred to nitrocellulose membranes. Anti-JNK2 (total JNK; Bottom) or anti-pJNK (Middle) antibodies were applied to the membranes. Protein expression was determined by photodensitometry using the nih image program. Data are from a representative experiment of four performed.

Fig. 6.

The effect of the dual APL on Fas/FasL protein expression and on caspase 8 gene expression in BALB/c and SJL mice. BALB/c(A–C) and SJL (D–G) mice were immunized with p259–271 and p195–212, respectively, and concomitantly treated with the dual APL. Mice immunized with PBS in CFA served as controls. Spleen cells were harvested 10 days later and taken for staining of CD4, Fas, and FasL. Alternatively, LN cells were taken for real-time PCR to determine expression of the caspase 8 gene (G). (A and D) Spleen cells of PBS in CFA-injected mice. (B and E) Spleen cells of mice immunized with the myasthenogenic peptide alone. (C and F) Spleen cells of immunized and dual APL-treated mice. (G) Total RNA was isolated and reverse-transcribed to prepare cDNA, which was further subjected to real-time PCR of the caspase 8 gene. p195–212-immunized mice (open bar) and dual APL-treated mice (filled bar) are presented. Results are of a representative experiment of three performed.

Fig. 7.

The cascade of events leading to the down-regulating effect of the dual APL.