Minimally activated CD8 autoreactive T cells specific for IRBP express a high level of Foxp3 and are functionally suppressive

Abstract

Purpose: Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide.

Methods: Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity.

Results: CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 microg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-beta1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function.

Conclusions: Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells.

Figure 1

IFN-γ production and Foxp3 expression by CD8 IRBP-specific T cells correlated inversely with their degree of activation. (A, B) MACS column–purified CD8 T cells (4 × 10⁶/well) were stimulated for 48 hours in 12-well plates with IRBP1-20 (10, 0.1, or 0 μg/mL) and tested for IFN-γ production (A) or Foxp3 expression (B; P < 0.01). (C–F) MACS column-purified CD4 (C, E) or CD8 (D, F) T cells (4 × 10⁶/well) were stimulated for 48 hours in 12-well plates with IRBP1-20 (10, 1, 0.1, or 0 μg/mL) (C, D) or SEE (E, F; 1000, 30, 1, or 0 ng/mL; P < 0.01). The data are the mean ± SD of results in three separate experiments. Note that the CD8 IRBP-specific T cells were only partially activated by immunizing autoantigen (D), but were fully capable of producing proinflammatory cytokines if properly activated (F).

Figure 2

In vitro suppressive effect of Foxp3^high CD8 T cells. (A, B) Foxp3^high CD8 T cells have a strong inhibitory effect on IFN-γ production by responder T cells and are more inhibitory for CD8 than CD4 responder T cells. Responder CD8 (A) or CD4 (B) T cells were separated from IRBP1-20-immunized B6 mice using flow cytometry, then incubated for 48 hours in 12-well plates (4 × 10⁶ cells/well) with immunizing peptide (10 μg/mL) and APCs in the absence or presence of Foxp3^high CD8 T cells at a regulatory T to effector T cell ratio of 1:4, and then IL-2 and IFN-γ levels in the culture supernatants were measured by ELISA. The result shown is representative of those obtained in more than five experiments (P < 0.01). (C) Regulatory T cells isolated from IRBP-induced mice were more suppressive of the uveitogenic T cells than the encephalitogenic T cells. Foxp3^high CD8 T cells were isolated from IRBP-immunized B6 mice. Nylon wool column–enriched responder T cells (4 × 10⁶) isolated either from IRBP-or from MOG-immunized B6 mice were incubated for 48 hours in 12-well plates with immunizing peptide (10 μg/mL) and APCs in the absence or presence of Foxp3^high CD8 T cells at a regulatory T to effector T cell ratio of 1:4, and IFN-γ levels in the culture supernatants were measured by ELISA.

Figure 3

TGF-β1 activated CD8 and CD4 suppressor T cells. (A, B) Responder CD4 (A) or CD8 (B) T cells were separated from IRBP1-20-immunized B6 mice by flow cy-tometry and were stimulated in vitro with antigen (10 μg/mL) and APCs in the absence or presence of 1 ng/mL of TGF-β1 for 3 days. The T cells were then separated by single-density gradient centrifugation, and cultured in IL-2 (10 ng/mL) medium for another 24 hours before the real-time PCR assessment of Foxp3 expression. The x-fold increase was then calculated (P < 0.01). (C, D) After the 3-day in vitro stimulation with antigen and TGF-β1, the CD8 (3C) or CD4 (D) T cells were assessed for suppressor activity of CD8 (C) and CD4 (D) T-cell responses. The responder T cells were freshly prepared CD8⁺ CD122⁻ or CD4⁺ CD25⁻ T cells from immunized B6 mice. After coculture of responder (4 × 10⁶ cells/well) and suppressor T cells (ratio 4:1) for 48 hours in 12-well plates with immunizing peptide (10 μg/mL) and APCs, the culture supernatants were sampled for measurement of IL-2 and IFN-γ by ELISA. The result shown is representative of those obtained in more than five experiments. TGF-CD4 T_reg: TGFβ1-induced regulatory CD4 cells; TGF-CD8 T_reg: TGFβ1-induced regulatory CD8 cells (P < 0.01).

Figure 4

Suppressor effect of the CD8⁺CD122⁺ T cell subset. (A–D) Unfractionated T cells from IRBP1-20-immuned mice were double-stained with PE-conjugated anti-CD25 or anti-CD122 antibody and FITC-conjugated anti-CD4 or anti-CD8 antibody. Some of the CD8 T cells express CD122, but not CD25, whereas some of the CD4 T cells express CD25, but not CD122. (E–G) Separation of CD8⁺CD122⁺ T cells from CD8⁺ CD122⁻ T cells on a magnetic column (auto-MACS; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). (H) Unfractionated CD8 T cells (12% CD122⁺), CD122⁺-enriched CD8 T cells (93%), and CD8 T cells partially depleted of CD122⁺ cells (6% CD122⁺) were stimulated with 10 μg/mL of IRBP 1-20 in the presence of APCs and assessed for thymidine incorporation. CD8 IRBP-specific T cells showed increased proliferation when the CD122⁺ subset was partially depleted (P < 0.01). (I–J) Isolated CD8⁺CD122⁺ and CD4⁺ CD25⁺ T cells were suppressive of the CD4 (I) and CD8 (J) response. Magnetic bead-separated CD8⁺CD122⁻ or CD4⁺ CD25⁻ responder T cells were incubated for 48 hours in 96-well plates (4 × 10⁵ cells/well) with immunizing peptide (10 μg/mL) and APCs in the absence or presence of the indicated freshly prepared CD8⁺CD122+ or CD4⁺CD25+ T_reg cells (1 × 10⁵ cells/well), and [³H] thymidine incorporation during the last 8 hours was assessed (P < 0.01).

Figure 5

CD8⁺CD122⁻ T cells gained suppressor activity after stimulation with low-dose antigen or high-dose antigen in the presence of TGF-β1. (A) CD8⁺CD122⁻ T cells were isolated from IRBP1-20-immunized B6 mice, and stimulated for 48 hours in 12-well plates with 10 or 0.1 μg/mL IRBP1-20 10 μg/mL of IRBP1-20 plus 1 ng/mL TGF-β1. After an in vitro stimulation by low-dose antigen or high-dose antigen plus TGF-β1, the CD8⁺CD122⁻ T cells expressed increased levels of Foxp3 (P < 0.01). (B, C) Freshly prepared CD8⁺CD122⁻ (B) or CD4⁺CD25⁻ (C) responder cells were incubated for 48 hours in 12-well plates (4 × 10⁶ cells/well) with immunizing peptide (10 μg/mL) and APCs, in the absence or presence of the indicated Foxp3^high CD8⁺CD122⁻ T_reg cells at a regulatory T cell to responder T cell ratio of 1:4, then IL-2 and IFN-γ in the culture supernatants were measured by ELISA. The results are representative of more than five experiments. LDA-converted-T_reg: CD8CD122⁻ T cells converted by low-dose antigen; TGF-β-converted T_reg: CD8CD122⁻ T cells converted by TGFβ1 (P < 0.01).