FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells

Abstract

Peripheral T-cell tolerance is thought to significantly contribute to the prevention of autoimmunity, and it has been shown that antigen-presenting steady-state dendritic cells efficiently induce peripheral tolerance. We previously showed that dendritic-cell-induced tolerance is a T-cell-intrinsic process that depends on coinhibitory molecules such as programmed death-1. Here we specifically analyze the involvement of FoxP3(+) regulatory T cells, which are known to be important for maintenance of self-tolerance. We show that antigen presentation by steady-state dendritic cells failed to induce peripheral tolerance in the absence of FoxP3(+) regulatory T cells but induced protective CD8(+) T-cell-mediated immunity instead. Regulatory T-cell-depleted mice had massively increased numbers of dendritic cells in lymph nodes. Dendritic cells isolated from mice without regulatory T cells had up-regulated costimulatory molecules and showed stronger T-cell stimulatory capacity ex vivo, suggesting that regulatory T cells contribute to peripheral tolerance by keeping the dendritic cells in an immature state. Using blocking antibodies, we demonstrate that CTLA-4 but not IL-10 is necessary for control of dendritic cells by regulatory T cells.

Fig. 1.

Antigen presentation by steady-state DCs induces protective immunity instead of tolerance in the absence of FoxP3⁺ Treg cells (A) DIETER (D) and DEREG/DIETER (DD) mice were injected i.p. with 1 μg DT or PBS on days −1, 1, 3, and 5 and with 2 mg TAM on day 0. LCMV GP_33–41/D^b- and β-Gal_497–505/K^b-specific CD8⁺ T cells were quantified in the blood on day 8 by staining with MHC class I tetramers. Each point in the graph represents an individual mouse and a representative experiment of three is shown. Student’s t test: tetramer GP_33–41/D^b: DD vs. DD/DT, P = 0.0017; β-Gal_497–505/K^b: DD vs. DD/DT, P = 0.0003; all other comparisons: not significant (NS). (B) Mice depicted in A were challenged on day 8 with 200 pfu LCMV–WE and the splenic virus titers were determined on day 13 using a focus-forming assay. Each symbol represents an individual mouse. One representative experiment of two is shown. Student’s t test: DD vs. DD/DT, P = 0.0148; all other comparisons: NS.

Fig. 2.

Depletion of FoxP3⁺ Treg cells changes the phenotype, numbers, and T-cell stimulatory capacity of DCs in vivo. Age- and sex-matched DEREG mice and wild-type littermates (six mice per group) were injected i.p. with 1 μg DT or with PBS on days 0 and 2 and spleens, peripheral lymph nodes (pooled inguinal, brachial, axillary, and submandibular), and mesenteric lymph nodes were removed on day 5, digested with collagenase and DNaseI, stained with appropriate antibodies, analyzed by FACS or purified by magnetic sorting, and used as stimulator cells in an allogenic T-cell proliferation assay. (A) Median fluorescence intensities (MFI) for CD80, CD86, CD70, CD40, PD-L1, and PD-L2 of CD11c^highI-A^b+ DCs from peripheral lymph nodes (Upper), mesenteric lymph nodes (Middle), and spleens (Lower) of DT-treated DEREG (solid bars) and wild-type littermates (open bars). (B) The frequency (Left) and total number (Right) of I-A^b+ CD11c^high in peripheral lymph nodes (Upper), mesenteric lymph nodes (Middle), and spleen (Lower) were determined. (C) Purified T cells from BALB/c mice were stimulated with titrated numbers of DCs purified from DT-treated DEREG mice (solid bars) or wild-type littermates (open bars) at a T:DC cell ratio of 4. P values (Student’s t test): ***P < 0.0005, **P < 0.005, *P < 0.05. One of three independent experiments is shown.

Fig. 3.

CTLA-4 but not IL-10 is involved in Treg cell-mediated suppression of DC activation and CTL priming in the immunological steady state. DIETER mice were injected with 2 mg TAM plus blocking antibodies against CTLA-4 or IL-10R or respective control antibodies. (A) LCMV GP_33–41/D^b-specific CD8⁺ T cells were quantified in the blood on day 8 by staining with MHC class I tetramers. (B) Mice depicted in A were challenged on day 8 with 200 pfu LCMV–WE and the splenic virus titers were determined on day 13 using a focus-forming assay. (C) To determine whether the blockade of CTLA-4 or the IL-10R resulted in DC activation, C57BL/6 mice were injected i.v. with anti-CTLA-4 or anti-IL-10R or the respective control antibodies. Three days later, activation state and numbers of DC were assessed by flow cytometry. Student’s t test (A and C) or Mann–Whitney test (B): *P < 0.05. One representative of three independent experiments is shown.