Induction of d.a.MKK6 in immature LCs results in UCM and enhanced T-cell stimulatory capacity. (A) Bars represent percentages (mean ± SD of 3 independent experiments) of gated CD1a⁺NGFR⁻ LCs (mock infected or grown without DOX) or CD1a⁺NGFR⁺ LCs (d.a.MKK6 or control transduced grown with DOX) expressing CD86, CD83, and CD40. The effect of the specific p38 MAPK inhibitor SB230580 (15 μM/mL) was determined. P values (*P < .05, **P < .01, ***P < .001) were determined for control (ctrl) + DOX versus MKK6 + DOX or MKK6 + DOX versus MKK6 + DOX/SB. (B) Representative dot plots show CD1a versus MHC II expression of TA/d.a.MKK6-NGFR–transduced day 10 LCs grown with or without 2 μg/mL DOX (upper panel) compared with mock-infected LCs stimulated with either 1 μg/mL LPS or 500 ng/mL CD40L for 48 hours (lower panel). DOX-stimulated cells were gated for NGFR expression. (C) Allo-T-cell–stimulatory capacity of sorted CD1a⁺NGFR⁺ LCs expressing either control vector (ctrl) or d.a.MKK6 (MKK6) was determined. In parallel, control-transduced LCs were stimulated with 1 μg/mL LPS. One representative of 4 experiments is shown. (D) Total-cell lysates of CD1a⁺ LCs (mock, ctrl, or d.a.MKK6 infected) stimulated with or without DOX were analyzed by Western blot for MKK6 or actin expression. DOX-stimulated cultures were separated into CD1a⁺NGFR⁺ and CD1a⁺NGFR⁻ LCs by FACS sorting prior to preparing cell lysates (1 representative of 3 independent experiments). For panels A and C, results are presented as the mean ± SD obtained from triplicate cultures.