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Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4 + T Cells in the Regulation of T Cell Differentiation

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Distinct Roles of α7 nAChRs in Antigen-Presenting Cells and CD4 + T Cells in the Regulation of T Cell Differentiation

Masato Mashimo et al. Front Immunol.

Abstract

It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323-339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

Keywords: DO11. 10 mouse; GTS-21; Th1; Th17; Th2; regulatory T cells; α7 nAChR.

Figures

Figure 1
Figure 1
GTS-21 suppresses CD4+ T cell differentiation in OVA-activated DO.11.10 spleen cells. The cells were cultured with OVA and the indicated concentrations of GTS-21 for 5 days. Cultured cells were examined for surface expression of CD4, CD25 and intracellular expression of FoxP3, IL-4, IL-17, IFN-γ. Plots are gated on CD4+ T cells. (A) Representative flow cytometric plots for CD4+CD25+FoxP3+ T cells (Tregs), CD4+IFN-γ+ T cells (Th1), CD4+IL-4+ T cells (Th2), and CD4+IL-17+ T cells (Th17) at the indicated concentrations of GTS-21. (B) Corresponding percentages of OVA-activated Tregs and Th1, Th2, and Th17 cells of CD4+ T cells detected at the indicated concentrations of GTS-21. The bars represent means ± SEM for at least three samples. Note that GTS-21 suppressed OVA-activated differentiation. C, control (without OVA). ##P < 0.01 vs. C. *P < 0.05, **P < 0.01 vs. GTS-21 at 0 μM. (C) Representative flow cytometric histograms for CFSE-labeled CD4+ cells after 5-day incubation with OVA with or without GTS-21. (D) Corresponding percentages of proliferating CD4+ cells. The bars represent means ± SEM for at least three samples. C, control (without OVA). ##P < 0.01 vs. C. **P < 0.01 vs. GTS-21 at 0 μM. (E) Effect of GTS-21 on Th cytokine synthesis. Levels of IL-2, IFN-γ, IL-4, IL-17, and IL-6 in the conditioned media were determined using ELISAs after culture for 5 days. The bars represent means ± SEM for at least three samples. Note that GTS-21 suppressed OVA-activated Th cytokine synthesis. C, control (without OVA). ##P < 0.01 vs. C. *P < 0.05, **P < 0.01 vs. GTS-21 at 0 μM.
Figure 2
Figure 2
GTS-21 enhances CD4+ T cell differentiation in OVAp-activated DO.11.10 spleen cells. The cells were cultured with OVAp and the indicated concentrations of GTS-21 for 5 days. Cultured cells were examined for surface expression of CD4, CD25 and intracellular expression of FoxP3, IL-4, IL-17, IFN-γ. Plots are gated on CD4+ T cells. (A) Representative flow cytometric plots for Tregs, Th1, Th2, and Th17 at the indicated concentrations of GTS-21. Gates were used to calculate the percentages of positive cells. (B) Corresponding percentages of OVAp-activated Tregs and Th1, Th2, and Th17 cells of CD4+ T cells in the presence of the indicated concentrations of GTS-21. Note that GTS-21 up-regulated OVAp-activated differentiation into all lineages. The bars represent means ± SEM for at least three samples. C, control (without OVAp). #P < 0.05, ##P < 0.01 vs. C. *P < 0.05, **P < 0.01 vs. GTS-21 at 0 μM. (C) Representative flow cytometric histograms for CFSE-labeled CD4+ cells after 5-day incubation with OVAp with or without GTS-21. (D) Corresponding percentages of proliferating CD4+ cells. The bars represent means ± SEM for at least three samples. C, control (without OVA). ##P < 0.01 vs. C. **P < 0.01 vs. GTS-21 at 0 μM. (E) Effect of GTS-21 on Th cytokine synthesis. Levels of IL-2, IFN-γ, IL-4, IL-17, and IL-6 in the conditioned media were determined using ELISAs after culture for 5 days. The bars represent means ± SEM for at least three samples. Note that GTS-21 enhanced OVAp-activated synthesis of Th cytokines, except IL-2. C, control (without OVA). ##P < 0.01 vs. C. *P < 0.05, **P < 0.01 vs. GTS-21 at 0 μM.
Figure 3
Figure 3
Effects of GTS-21 on FITC-OVA endocytosis; expression of MHC class II, CD40, and CD80 molecules in DO.11.10 CD11b+ and CD11c+ cells; and the viability of DO.11.10 spleen cells. (A) Representative micrographs showing FITC-OVA endocytosis in CD11b+ and CD11c+ cells. The spleen cells were cultured with FITC-OVA (50 μg/ml) on poly-d-lysine-coated glass-bottom dishes in the presence or absence of GTS-21 for 4 h. (B) Flow cytometric analysis of OVA-FITC uptake into CD11b+ and CD11c+. Graphs show the percentages of OVA-FITC+CD11b+ (left) or CD11c+ (right) cells in the presence of the indicated concentrations of GTS-21. The bars represent means ± SEM for at least three samples. C, control (without OVA-FITC). ##P < 0.01 vs. C. Note that GTS-21 did not affect FITC-OVA endocytosis in APCs. (C) Mean fluorescence intensity (MFI) of the gated positive population for each of the respective OVA-FITC+CD11b+ (left) or CD11c+ (right) markers. (D) Percentages of CD11b+ and CD11c+ cells showing surface expression of MHC class II, CD40, and CD80. DO.11.10 spleen cells were cultured for 16 h with OVA in the presence or absence of GTS-21 (30 μM). (E) MFI of MHC II, CD40 and CD80 in CD11b+ (left) or CD11c+ (right) markers. Note that GTS-21 did not affect surface expression of MHC class II, CD40 or CD80 in APCs. (F) Viability (7AAD exclusion) of CD4+ T cells, and CD11b+ and CD11c+ cells. C, control (without OVA). ##P < 0.01 vs. C. **P < 0.01 vs. GTS-21 at 0 μM. Note that GTS-21 slightly decreased the viability of CD11b+ cells but had no significant effect on the viability of CD4+ T cells or CD11c+ cells.
Figure 4
Figure 4
GTS-21 enhances APC-independent CD4+ T cell differentiation induced by anti-CD3/CD28 Abs. (A) Naïve CD4+ T cells isolated from the spleens of DO11.10 mice using a naïve CD4+ T cell isolation kit (130-104-453, Miltenyi Biotec) were stimulated with anti-CD3/CD28 and cultured for 5 days under Treg, Th1, Th2, and Th17 polarizing conditions (Table 1) in the presence of the indicated concentrations of GTS-21. (B) Naïve CD4+ T cells isolated from the spleens of WT or α7-KO mice were stimulated with anti-CD3/CD28 Abs and cultured for 5 days under Treg, Th1, Th2, and Th17 polarizing conditions (Table 1) in the absence or presence of 30 μM GTS-21. Note that GTS-21 enhanced T cell differentiation only in CD4+ cells from the WT mice. The bars represent means ± SEM for at least three samples. C, control (without OVA). ##P < 0.01 vs. C. *P < 0.05, **P < 0.01 vs. GTS-21 at 0 μM.

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