Major role of gamma delta T cells in the generation of IL-17+ uveitogenic T cells

Abstract

We show that in vitro activation of interphotoreceptor retinoid-binding protein (IRBP)-specific T cells from C57BL/6 mice immunized with an uveitogenic IRBP peptide (IRBP(1-20)) under TH17-polarizing conditions is associated with increased expansion of T cells expressing the gammadelta TCR. We also show that highly purified alphabeta or gammadelta T cells from C57BL/6 mice immunized with IRBP(1-20) produced only small amounts of IL-17 after exposure to the immunizing Ag in vitro, whereas a mixture of the same T cells produced greatly increased amounts of IL-17. IRBP-induced T cells from IRBP-immunized TCR-delta(-/-) mice on the C57BL/6 genetic background produced significantly lower amounts of IL-17 than did wild-type C57BL/6 mice and had significantly decreased experimental autoimmune uveitis-inducing ability. However, reconstitution of the TCR-delta(-/-) mice before immunization with a small number of gammadelta T cells from IRBP-immunized C57BL/6 mice restored the disease-inducing capability of their IRBP-specific T cells and greatly enhanced the generation of IL-17(+) T cells in the recipient mice. Our study suggests that gammadelta T cells are important in the generation and activation of IL-17-producing autoreactive T cells and play a major role in the pathogenesis of experimental autoimmune uveitis.

FIGURE 1

Activation of IL-17⁺ IRBP-specific T cells is associated with expansion of γδ T cells. T cells prepared from the spleens and draining lymph nodes of IRBP_1–20-immunized B6 mice at day 13 postimmunization. were stimulated in vitro with immunizing Ag (10 μg/ml) and APCs (irradiated spleen cells) for 2 days. The activated T cell blasts were then separated by Ficoll gradient centrifugation and cultured in IL-2 (Th1-polarized)- or IL-23 (Th17-polarized)-containing medium for 5 days. Finally, the cells were stained for intracellular IL-17 and IFN-γ using FITC-anti-IL-17 and PE-anti-IFN-γ Abs (A), for the αβ TCR and CD3 (B), and for the γδ TCR and IL-17 (C). Approximately 15–20% of the Th17-polarized T cells did not express the αβ TCR but expressed the γδ TCR. Results are representative of multiple experiments. FL-1H and FL-2H, fluorescence.

FIGURE 2

γδ T cells isolated from IRBP-immunized B6 mice predominantly express Vγ4Vδ4. Splenic T cells from naive or IRBP_1–20, MOG_35–55, or CFA-immunized B6 mice were stained using a panel of mAbs specific for γδ TCR V segments (Vγ4, Vγ1, Vδ4, Vδ5, and Vδ6.3) and an Ab specific for the mouse pan-TCR δ chain (GL3), followed by FACS analysis. The results shown are representative of those in five experiments. FL-1H, fluorescence.

FIGURE 3

Role of γδ and αβ T cells in the Ag-specific IL-17 response. A–C, Purification of αβ TCR⁺ and γδ TCR⁺ T cells: Purified αβ TCR⁺ and γδ TCR⁺ T cells were prepared from IRBP_1–20-immunized B6 mice using magnetic beads. A, One-step positive selection using GL3 (mAb specific for pan-mouse γδ TCR) resulted in 95% pure γδ T cells. B, Further depletion of αβ T cells resulted in 99% pure γδ T cells. C, 99% pure αβ T cells were prepared similarly. D–F, The partially purified (D) or highly purified (E) γδ T cells or the indicated mixtures (F), with the total number of cells in all 4 × 10⁵ T cells/ well, were incubated with the indicated Ag and APCs in 96-well plates, and the culture supernatants were tested for IL-17. The results shown are representative of those in three experiments.

FIGURE 4

Test of interaction between αβ and γδ T cells using culture inserts. A–C, In a 24-well plate, a total of 2 × 10⁶/well in vivo primed αβ and γδ T cells from IRBP_1–20-immunized B6 mice (99% pure) were tested alone (B and C) or as a 9:1 mixture (A) of αβ and γδ TCR⁺ T cells for production of IL-17 or IFN-γ after in vitro stimulation with IRBP_1–20 and APCs. Only the cells in the lower chambers are cocultured with irradiated splenocytes. D, 90% αβ and 10% γδ TCR⁺ T cells were cultured in a same well but separated by inserts. αβ T cells were in the upper chamber, and γδ T cells were in the lower chamber; E, αβ T cells were seeded in the lower chamber, and γδ T cells were seeded in the upper chamber. Results are representative of those in three experiments.

FIGURE 5

Transfer of γδ T cells into TCR-δ^−/− mice restores their ability to generate IRBP-specific T cells. The IFN-γ- and IL-17-producing abilities of the IRBP-specific T cells of TCR-δ^−/− mice, either untreated or injected with 2 × 10⁵ γδ T cells were determined 2 day after in vitro stimulation with immunizing Ag (A). Intracellular staining of IL-17⁺ T cells were assessed after 2 days of in vitro stimulation with immunizing Ag and another 3-day culture in medium containing IL-23 (10 ng/ml; B). The disease-inducing ability of the IRBP-specific T cells (2 × 10⁶) from the TCR-δ^−/− mice, either untreated or injected with γδ T cells, was also compared after 48 h of in vitro stimulation with the immunizing Ag (C and D). Results are representative of those in two separate experiments. KO, Knockout.