Inhibins regulate peripheral regulatory T cell induction through modulation of dendritic cell function

Abstract

We have previously reported that the absence of inhibins results in impaired dendritic cell (DC) maturation and function, leading to decreased T cell activation and diminished delayed-type hypersensitivity responses. Here, we investigated the role of inhibins in peripheral regulatory T cell (Treg) induction in vitro and in vivo. Inhibin deficient (Inhα^-/-) mice showed an increased percentage of peripherally induced Tregs in colonic lamina propria and mesenteric lymph nodes, compared to Inhα^+/+ mice, which correlated with increased expression of PD-L1 in CD103⁺ and CD8α⁺ DCs. Lipopolysaccharide-stimulated bone marrow-derived and ex vivo spleen- and lymph node-purified CD11c⁺ Inhα^-/- DCs induced higher Tregs in vitro. Moreover, in vivo anti-DEC205-ovalbumin (OVA) DC targeting of mice with adoptively transferred OVA-specific T cells showed enhanced induced peripheral Treg conversion in Inhα^-/- mice. These data identify inhibins as key regulators of peripheral T cell tolerance.

Keywords: Tregs; dendritic cells; inhibins; peripheral tolerance.

Figure 1

Tregs are incremented in the periphery in the absence of inhibin. Inhα^+/+ and Inhα^−/− mice were analyzed for Tregs (CD4⁺CD25⁺FoxP3⁺), thymic (Helios⁺) or peripheral (Helios⁻). (A) Gate strategy for Treg analysis. (B) Frequency (top) and number (bottom) in colonic lamina propria (LP) (left), mesenteric lymph node (MLN) (center), and peripheral lymph node (PLN) (right). Mean ± SEM, n = 3–5 mice. Statistical significance was determined by two‐tailed unpaired Student's t test. *P ≤ 0.05.

Figure 2

Inhα^−/− DC subsets have differential expression of MHC‐II, CD80 and PD‐L1 in MLN compared to Inhα^−/−. Inhα^+/+ and Inhα^−/− mice were analyzed for cDC subpopulations in MLN. (A) MHC‐II, CD80, CD86 and PD‐L1 expression within the resident DC (Lin⁻CD11c^hiMHC‐II⁺CD8⁺, Lin⁻CD11c^hiMHC‐II⁺CD8⁻) (left) and migratory DC (Lin⁻CD11c⁺MHC‐II^hiCD103⁺CD11b⁻, Lin⁻CD11c⁺MHC‐II^hiCD103⁺CD11b⁺, Lin⁻CD11c⁺MHC‐II^hiCD103⁻CD11b⁺, Lin⁻CD11c⁺MHC‐II^hiCD103⁺CD11b⁻) (right) subpopulations. (B) Analysis of MHC‐II, CD80 and CD86 in LPS‐stimulated splenic CD11c⁺ DC. Bar graphs represent relative expression of mean fluorescence intensity (MFI) compared to unstimulated splenic CD11c⁺ Inhα^+/+ DCs. Relative expression was calculated as the ratio: MFI of LPS‐stimulated DCs/MFI of unstimulated DCs, for both Inhα^+/+ and Inhα^−/− DCs. Mean ± SEM, n = 5 mice. Statistical significance was determined by the two‐tailed unpaired Student's t test. *P ≤ 0.05, **P ≤ 0.01.

Figure 3

Inhibin controls DC‐dependent Treg cell induction in vitro. BMDC (A,B) or splenic and PLN CD11c⁺ DCs (C,D) were cocultivated with naïve T cells in presence of anti‐CD3 (0.1 μg·mL⁻¹) and TGFβ (0.25 ng·mL⁻¹). After 5 days, induction of Tre (CD4⁺CD25⁺FoxP3⁺) was evaluated. (A) Treg conversion from naïve T cells in the presence of wild‐type (WT) or Inhα^−/− iBMDCs or mBMDCs at different ratios. Graphs represent frequency (left) and total numbers (right) of Treg population. (B) CD25 (left) and FoxP3 (right) expression at induced Treg population are shown. (C) Treg conversion from naïve T cells in the presence of splenic and PLN iCD11c⁺ or mCD11c⁺ DCs, WT or Inhα^−/−, at 1 : 5 ratio. Graphs represent frequencies (left) and total numbers (right) of Treg population. (D) CD25 (left) and FoxP3 (right) expression at induced Treg population as in (B). Bar graphs of CD25 and FoxP3 represent relative expression of MFI compared to Inhα^+/+ mice. Mean ± SEM, n = 3. Statistical significance was determined by the two‐tailed paired Student's t test. *P ≤ 0.05, **P ≤ 0.01.

Figure 4

Antigen target of Inhα^−/− DCs through anti‐DEC205‐OVA induces an increased number of peripherally induced Tregs (pTregs) in vivo. OT‐II⁺CD45.1⁺ naïve T cells were transferred into Inhα^+/+ or Inhα^−/− mice, and 24 h later mice were immunized intradermally in the ear, with anti‐DEC205‐OVA (α‐DEC‐OVA) or OVA, either with or without CT as adjuvant. Evaluation of pTregs was performed 7 days after immunization. (A) Representative dot plots of transferred cells (CD45.1⁺Vβ5⁺; top) and pTregs (CD25⁺FoxP3⁺; bottom) are shown for Inhα^+/+ and Inhα^−/− receptor mice. (B) Percentage (left) and number (right) of CD4⁺ T cells (top), transferred cells (middle), and pTregs (bottom). Mean ± SEM, n = 3. Statistical significance was determined by two‐tailed unpaired Student's t test. *P ≤ 0.05, **P ≤ 0.01.