Cross-presentation of PbA antigen by MBECs occurs by the cytosolic pathway.

<p>(A) MBEC cultures were established from WT mice as well as TAP1-deficient mice and stimulated with IFNγ. PbA mature iRBCs were added to some wells in triplicate. After 24 h, the wells were washed and cross-presentation of Pb1 was detected using LR-BSL8.4a cells and X-gal staining. ****<i>P</i><0.0001, ns not significant, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (B) PbA mature iRBCs were added (or not) to IFNγ-stimulated MBECs. Lactacystin (10 μM) was added to some wells 6 h later. After another 6 h, all wells were washed and assayed for Pb1 cross-presentation using LR-BSL8.4a cells. <i>n</i> = 3, *<i>P</i><0.05, **<i>P</i><0.01, ANOVA with Bonferroni’s post test on log-transformed data. Results are representative of 2 independent experiments. (C, D) PbA mature iRBCs were added to IFNγ-stimulated MBECs 1 h after either 10 μg/ml chloroquine diphosphate (C) or 10 μM cytochalasin D (D) were added to some wells. The MBECs were washed and co-incubated with LR-BSL8.4a cells 24 h later to measure Pb1 cross-presentation. <i>n</i> = 3, ***<i>P</i><0.001, ****<i>P</i><0.0001, unpaired t-test on log-transformed spot counts.