Cutting edge: in vitro generated Th17 cells maintain their cytokine expression program in normal but not lymphopenic hosts

Abstract

Upon activation, naive CD4(+) T cells differentiate into effector Th cell subsets. The stability and plasticity of effector Th cells have not been well understood. In this study we used an IL-17F-red fluorescent protein reporter mouse to analyze the plasticity of Th17 cells in vitro and in vivo. We found that in vitro generated Th17 cells poorly maintained their differentiation program in vitro and could be reprogrammed into other T cell lineages. Moreover, upon transfer into lymphopenic hosts, Th17 cells rapidly lost their IL-17 expression and were converted into Th1 cells independently of IL-7 signaling. However, Th17 cells maintained their phenotypes well in normal animals, even in the absence of Ag and inflammation. These results, although suggesting the plasticity of Th17 cells, also indicate active maintenance of their program in vivo.

FIGURE 1

Plasticity of Th17 cells in vitro. A, Naive CD4⁺CD25⁻CD62L^highCD44^low T cells from Il17f^rfp mice were polarized with plate-bound anti-CD3 and anti-CD28 under Th17 conditions (TGFβ, IL-7, anti-IL-4, anti-IFN-γ, and IL-23) for 4 days and RFP⁺ cells were sorted by FACS. After sorting, cells were assessed for IL-17, IFN-γ, and Foxp3 expression by intracellular staining. B–D, The sorted RFP⁺ cells were restimulated under the indicated conditions. IL-17-, IL-4-, IFN-γ-, and Foxp3-expressing cells were then analyzed by intracellular staining (B). CD4⁺ T cells were restimulated with anti-CD3 for 4 h for real-time PCR analysis (C) or for 24 h for cytokine measurement by ELISA (D). The experiment was performed twice with consistent results. p values were calculated with the t test; *, p < 0.05; **,p < 0.005.

FIGURE 2

IL-17-producing T cells become IFN-γ-producing T cells in lymphopenic mice. Naive CD4⁺CD25⁻CD62L^highCD44^low T cells were sorted from splenocytes and lymph node cells of Il17f^rfp mice and stimulated with plate-bound anti-CD3/CD28 Abs in the presence of the indicated cytokine combination (A) or IL-6 and TGF-β (B) with anti-IL-4/IFN-γ neutralization Abs. At day 4, RFP⁺ cells were further sorted and i.v. transferred into Rag1^−/− mice. In B, a total of 500 μg of control Ig (rat IgG) or anti-IL-7RAb was i.p. injected on days 0, 1, 3, and 5. Six days later, splenocytes of recipients were stimulated with PMA/ionomycin for 4 h in the presence of GolgiPlug, stained with FITC-conjugated anti-CD4Ab and PerCP-Cy5.5-conjugated anti-TCRβ Ab followed by intracellular staining of indicated cytokines. CD4⁺TCRβ⁺ cells were gated and analyzed. Numbers in dot-plot quadrants represent the percentages. In A, the percentages (means ± SD) combining three independent experiments (n = 9 mice) are shown. In B, the percentages (means ± SD) combining two independent experiments (control Ig group, n = 5 mice; anti-IL-7R (αIL-7R) Ab group, n = 4 mice) are shown.

FIGURE 3

Stability of TH17 cells in vivo. Naive CD4⁺CD25⁻CD62L^highCD44^low T cells from Il17f^rfp OT-II mice were activated with OVA peptide and irradiated APC under Th17 conditions for 4 days, and RFP⁺ cells were sorted by FACS. Sorted cells (CD45.2⁺) were transferred i.v. into B6.SJL congenic mice (CD45.1⁺). A, The recipient mice were nonimmunized (Non/Immun.) or immunized (Immun.) s.c. with OVA peptide emulsified in CFA (three mice per group were analyzed individually,). Seven days later, spleen cells were restimulated with OVA peptide for 24 h and CD45.1⁺ and CD45.2⁺ CD4 T cells were assessed for IL-17, IFN-γ, and Foxp3 expression by intracellular staining. Numbers in dot-plot quadrants represent the percentages (means ± SD) combining two independent experiments (nonimmunized group, n = 6 mice; immunized group, n = 6 mice). B, The recipient mice were immunized s.c. with OVA peptide emulsified in CFA or IFA (three mice per group were analyzed individually). Seven day later, spleen cells were restimulated with OVA peptide or 24 h, and Ly5.2⁺ CD4 T cells were assessed for IL-17, IL-17F, IFN-γ, and Foxp3 expression by intracellular staining. Numbers in dot-plot quadrants represent the percentages (means ± SD) combining two independent experiments (IFA group, n = 6 mice; CFA group, n = 6 mice).