Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells

Abstract

The antigenic targets recognized by naturally occurring CD4(+) CD25(+) regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4(+) CD25(+) T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4(+) CD25(-) T cells and CD8(+) T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4(+) CD25(+) T cells from immunized mice was 5-10 times greater than CD4(+) CD25(+) T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4(+) CD25(+) T reg cells.

Figure 1.

Immunization with SEREX-defined self-antigens elicits active CD4⁺ CD25⁺ T reg cells. (a) BALB/c mice were immunized with plasmids encoding SEREX-defined self-antigens, SEREX-unrelated molecules, heterologous molecules, or control vector. 1 wk after the second immunization, 5 × 10⁴ CD4⁺ CD25⁺ T cells or 5 × 10⁴ CD4⁺ CD25⁻ T cells from spleens were added to cultures of 5 × 10⁴ CD4⁺ CD25⁻ T cells from DO11.10 mice or 5 × 10⁴ CD8⁺ T cells from DUC18 mice with 5 × 10⁴ MMC-treated BALB/c splenic Thy-1⁻ APC and cognate peptides. (b) 5 × 10⁴ CD4⁺ CD25⁻ T cells from DO11.10 mice were cultured with 5 × 10⁴ MMC-treated BALB/c splenic Thy-1⁻ APC and cognate peptides in the presence of FACSVantage-sorted 5 × 10⁴ CD4⁺ CD25⁺ or CD4⁺ CD25⁻ T cells from naive or Dna J–like 2–immunized BALB/c mice. (c) 5 × 10⁴ CD8⁺ T cells from DUC18 mice were cultured with 5 × 10⁴ CD4⁺ CD25⁺ T cells from naive or Dna J–like 2–immunized BALB/c mice with 5 × 10⁴ MMC-treated BALB/c splenic Thy-1⁻ APC and cognate peptides with or without anti–MHC class II mAb. Proliferation was assessed as described in Materials and methods. These experiments were repeated three (a) or two (c) times with similar results. Data are expressed as mean ± SD.

Figure 2.

Expression of Foxp3 mRNA is enhanced in CD4⁺ CD25⁺ T cells from mice immunized with SEREX-defined self-antigens. BALB/c mice were immunized with plasmids encoding SEREX-defined self-antigens, SEREX-unrelated molecules, heterologous molecules, or control vector. 1 wk after the second immunization, CD4⁺ CD25⁺ T cells and CD4⁺ CD25⁻ T cells were purified from spleens and their levels of Foxp3 mRNA were assessed by real-time quantitative RT-PCR. Normalized Foxp3 mRNA expression values were calculated as the ratio of Foxp3 mRNA expression to HPRT mRNA expression. These experiments were repeated three times with similar results. Data are expressed as mean ± SD.

Figure 3.

Changes in CD4⁺ CD25⁺ T cell suppressive activity after immunization are correlated with Foxp3 mRNA expression levels. (a) 1, 4, or 8 wk after the second immunization with plasmids encoding Dna J–like 2, CD4⁺ CD25⁺ T cells were obtained from spleens. Graded doses of these cells were added to cultures of 5 × 10⁴ CD4⁺ CD25⁻ T cells from DO11.10 mice or 5 × 10⁴ CD8⁺ T cells from DUC18 mice with 5 × 10⁴ MMC-treated BALB/c splenic Thy-1⁻ APC and cognate peptides. Proliferation was assessed as described in Materials and methods. (b) cDNA was prepared from CD4⁺ CD25⁺ T cells at various time intervals after immunization with Dna J–like 2. The expression of Foxp3 mRNA was analyzed as described in the legend of Fig. 2. These experiments were repeated three times with similar results. Data are expressed as mean ± SD.

Figure 4.

Antigen-specific stimulation restores suppressive activity of CD4⁺ CD25⁺ T cells from Dna J–like 2–immunized mice. (a) 1 or 8 wk after the second immunization with plasmids encoding Dna J–like 2, 5 × 10⁴ CD4⁺ CD25⁺ T cells were obtained from spleens. The suppressive activity was analyzed as described in the legend of Fig. 1 a. (b) 5 × 10⁴ CD8⁺ T cells from DUC18 mice were cultured with 5 × 10⁴ CD4⁺ CD25⁺ T cells from mice 8 wk after the second immunization with 5 × 10⁴ MMC-treated BALB/c splenic Thy-1⁻ APC and cognate peptides, with or without anti–MHC class II mAb, and with or without in vitro stimulation as indicated. Proliferation was assessed as described in Materials and methods. These experiments were repeated three (a) or two (b) times with similar results. Data are expressed as mean ± SD.