T cell co-cultures with lung macrophages and DC

Naïve CD4⁺DO11.10⁺ T cells were isolated from spleens of DO11.10⁺RAG2^−/− Il4ra^R576Foxp3^EGFP mice by fluorescein-activated cell sorting (FACS). AM and IM were isolated by FACS and were aliquoted at 2×10⁴ cells in 48 well plates, then either sham treated or treated overnight with UFP at 10 μg/ml. The UFP treatment did not induce increased apoptosis as compared to sham treatment, as assessed by Annexin V staining (data not shown). The APC were washed twice with PBS to remove residual UFP, and the T cells were then added at 4×10⁵ cells/well in a final volume of 0.5 ml 10% fetal calf serum/RPMI culture medium. Cultures were treated with the OVA_323–339 peptide at 1μM, as indicated. Anti-murine Notch Ab were added at 10 μg/ml each, as indicated.