Antigenic strength controls the generation of antigen-specific IL-10-secreting T regulatory cells

Abstract

Administration of peptides i.n. induces peripheral tolerance in Tg4 myelin basic protein-specific TCR-Tg mice. This is characterized by the generation of anergic, IL-10-secreting CD4+ T cells with regulatory function (IL-10 Treg). Myelin basic protein Ac1-9 peptide analogs, displaying a hierarchy of affinities for H-2 A(u) (Ac1-9[4K]<<[4A]<[4Y]), were used to investigate the mechanisms of tolerance induction, focusing on IL-10 Treg generation. Repeated i.n. administration of the highest affinity peptide, Ac1-9[4Y], provided complete protection against EAE, while i.n. Ac1-9[4A] and Ac1-9[4K] treatment resulted in only partial protection. Ac1-9[4Y] was also the most potent stimulus for IL-10 Treg generation. Although i.n. treatment with Ac1-9[4A] gave rise to IL-10-secreting CD4+ T cells, the population as a whole was also capable of secreting IFN-gamma after an in vitro recall response to Ac1-9[4A] or [4Y]. In addition to IL-10 production, other facets of tolerance, namely, anergy and suppression (both in vitro and in vivo), were affinity dependent, with i.n. Ac1-9[4Y]-, [4A]- or [4K]-treated CD4+ T cells being the most, intermediate and least anergic/suppressive, respectively. These findings demonstrate that the generation of IL-10 Treg in vivo is driven by high signal strength.

Figure 1

Decrease in the susceptibility to EAE induction upon i.n. treatment with MBP peptides of increasing affinity for H-2 A^u. Tg4 mice were treated with ten i.n. doses of MBP Ac1–9[4K], [4A] or [4Y] peptides. EAE was induced 3 days after the last peptide treatment in peptide-treated and untreated mice by s.c. immunization with SCH emulsified in PBS/CFA containing M. tuberculosis on day 0 followed by two i.p. injections of PT administered on days 0 and 2. Individual mice were monitored daily for the development of EAE and scored for 40 days post immunization. Values represent mean EAE clinical scores from five mice per treatment group; this experiment was repeated in Tg4 Rag1^−/− mice with similar results.

Figure 2

Increasing in vivo activation of CD4⁺ T cells by i.n. treatment with Ac1–9 analogs of higher affinity. Splenocytes from naïve Tg4 mice were labeled with CFSE and adoptively transferred into untreated Tg4 recipients by i.p. injection on day 0. On day 1 post cell transfer, the recipient mice were challenged with a single i.n. dose of PBS as a control or MBP Ac1–9[4K], [4A] or [4Y]. Spleens from recipient mice were collected on day 3 and stained with anti-CD4, anti-CD69 mAb and PI prior to flow cytometry. Analysis was performed gating on live CD4⁺CFSE⁺ lymphocytes. (A) CFSE profile of CD4⁺ cells. Numbers represent mean division index values. (B) CSFE versus CD69 profile of CD4⁺ cells. Data are shown for one recipient mouse and are representative of two independent assays with similar results.

Figure 3

The influence of affinity on CD4⁺ T-cell responsiveness and cytokine secretion profile. Tg4 mice were treated with ten i.n. doses of MBP Ac1–9[4K], [4A] or [4Y] peptides. Splenic CD4⁺ cells were positively selected from either untreated Tg4 or peptide-treated Tg4 mice 3 days after the last peptide treatment. In total, 5 × 10⁴ CD4⁺ T cells per well were cultured with 1 × 10⁵ irradiated B10.PL splenocytes as APC in the presence of a tenfold titration of MBP Ac1–9[4K], [4A] or [4Y] ranging from 0.001 to 100 μg/mL. (A) Proliferative responses were measured at 72 h by ³[H]-thymidine incorporation. (B) Cytokine responses of CD4⁺ T cells from the above cultures were measured by ELISA at 24 h (IL–2) and 72 h (IFN-γ and IL-10) after in vitro restimulation. Data show mean ± SEM (n = 3). Data are representative of three independent experiments.

Figure 4

Affinity dependent IL-10 Treg generation during tolerization. Tg4 mice were treated with 14 i.n. doses of MBP Ac1–9 Ac1–9[4K], [4A] or [4Y] peptides. Spleens were isolated from peptide-treated Tg4 mice 2 h after the last peptide treatment. ICCS for IL-2, IFN-γ, IL-4 and IL-10 was performed after an additional 4-h stimulation with PMA and ionomycin. (A) Cells are gated on V8⁺ cells from each peptide treatment group and analyzed for IL-2, IFN-γ and IL-10 expression with cytokine gates based on isotype controls. (B) Dot plots show co-secretion of IFN-γ and IL-10 by Vβ8⁺ T cells from each peptide treatment group. Data are representative of at least three independent experiments.

Figure 5

In vitro suppression of naïve CD4⁺ T-cell proliferation by CD4⁺ T cells from peptide-treated mice. Tg4 mice were treated with 14 i.n. doses of MBP Ac1–9[4K], [4A] or [4Y] peptides. Splenocytes were isolated 3 days after the last treatment and expanded in complete medium containing 10 μg/mL of MBP Ac1–9[4K] and 20 U/mL of rhIL-2 for 5 days. CD4⁺ T cells were positively selected from untreated mouse spleens as well as IL-2-expanded splenocytes from i.n. Ac1–9[4K]-, [4A]- and [4Y]-treated mice. (A) In total, 5 × 10⁴ of each untreated and peptide-treated CD4⁺ T cells were either cultured alone or co-cultured at decreasing ratios from 1:1 to 1:32 of peptide-treated to untreated CD4⁺ T cells in the presence of 1 × 10⁵ irradiated B10.PL splenocytes as APC and 100 μg/mL of MBP Ac1–9[4K]. Proliferative responses were measured at 72 h by ³[H]-thymidine incorporation. (B) IL-2 secretion by CD4⁺ T cells from the above cultures was measured by ELISA at 24 h after in vitro stimulation. Data show mean ± SEM (n = 3). Data are representative of three independent experiments. *p<0.05 versus untreated control.

Figure 6

Peptide affinity associated inhibition of proliferation of Tg4 CD4⁺ T cells in vivo. Tg4 mice were either left untreated or treated with ten i.n. doses of MBP Ac1–9[4K], [4A] or [4Y] peptides. Splenocytes from naïve Tg4 mice were labeled with CFSE and 5 × 10⁷ cells were transferred into untreated or i.n. MBP[4K]-, [4A]- or [4Y]-treated Tg4 recipients by i.p. injection 3 days after the last peptide treatment. One day after cell transfer, recipient mice were challenged with a single dose of MBP Ac1–9[4A]; a separate group of untreated recipient mice was challenged with a single dose of PBS. Spleens from recipient mice were collected on day 3 and stained with anti-CD4 and PI prior to flow cytometry. CFSE profile of CD4⁺ cells: Data show results from one recipient mouse and are representative of four independent experiments. Numbers represent mean division index values. *p<0.05 versus untreated control.