Figure 2. IL-17A production by a subset of DETC requires TCR stimulation.

(A) DETC upregulate Il17a upon TCR stimulation in vitro. Sorted Vγ3⁺ DETC express Il17a following activation by plate-bound anti-CD3ε (10 μg/ml). (B) Freshly isolated and sorted Vγ3⁺ DETC were stimulated with plate-bound anti-CD3ε (10 μg/ml), and IL-17A, IL-17F, and IL-22 protein was measured by ELISA. Data are shown as mean ± SEM from quadruplicate measurements and are representative of 3–4 independent experiments. ***P ≤ 0.001. (C) CsA inhibits anti-CD3ε–mediated IL-17A production in DETC. Cells are gated on Vγ3⁺Thy1.2⁺. (D) Stimulation with IL-1β and IL-23 alone does not induce IL-17A in DETC. Sorted Vγ3⁺ DETC were stimulated with IL-1β, IL-23, or the combination thereof in the presence or absence of suboptimal anti-CD3ε, and IL-17A secretion was assayed by ELISA. Data are shown as mean ± SEM from duplicates. (E) JAML costimulation increases IL-17A in DETC. ELISA analysis for IL-17A from culture supernatants of DETC stimulated with antibody against suboptimal doses of CD3 alone, CD3 and JAML, or JAML alone. Data are shown as mean ± SEM from duplicates. (F) DETC upregulate RORγt upon TCR stimulation. Freshly isolated DETC were stimulated with anti-CD3ε (10 μg/ml) for 3 hours, and RORγt levels were measured by flow cytometry. (G) A subset of RORγt⁺ DETC produces IL-17A. RORγt and IL-17A expression were assessed by flow cytometry in freshly isolated epidermal cell suspensions that were stimulated with anti-CD3ε (10 μg/ml). Cells are gated on Vγ3⁺Thy1.2⁺.