Ibrutinib drives Th1-mediated L major immunity in an in vivo model of Th2-dominant cutaneous leishmaniasis. (A) Schematic representation of the L major mouse experiment time course. Mice were initiated on ibrutinib (25 mg/kg/day) or vehicle 2 days prior to being infected with 2 × 106 stationary-phase L major promastigotes. Lesion size was tracked for 9 weeks and immune correlates were collected upon sacrifice at week 9. (B) Lymphocytes isolated from draining lymph nodes were stimulated with L major antigens for 72 hours and culture supernatant was analyzed by ELISA for IL-4 and IL-10. Error bars represent SEM. (C) Lymphocytes isolated from draining lymph nodes were stimulated with L major antigens for 72hr and culture supernatant was analyzed by ELISA for IFN-γ. IFN-γ responses are displayed as a ratio with IL-4 (left panel) or IL-10 (right panel) to compare relative Th1 and Th2 immunity in ibrutinib- or vehicle-treated groups. (D) Whole-mount gross histologic preparations of vehicle- and ibrutinib-treated L major–infected footpads are depicted along with a centimeter ruler for size comparison. Cutaneous lesions are visible on the underside of the footpad as indicated by arrows. (E) Log dilution of parasites obtained from footpad lesions are displayed. Error bars represent SEM. (F) Time course analysis of cutaneous lesion size over the 9-week period of L major infection. Measurements were taken at weekly intervals. Error bars represent SEM.
