A, Dose dependent effect of ZAP-70 kinase activity in thymic selection. Thymic lobes from OT-I Rag−/−β2m−/− mice at gestational day 15.5 were incubated with 0.2 μM OVA in presence or absence of 150 nM, 300nM or 1 μM of the ZAP-70 kinase inhibitor NIBR001. After 7 days of culture, thymocytes were stained with fluorescently labeled antibodies against TCRβ, CD4 and CD8β and subjected to flow cytometry analysis. The percentage of TCRβ-positive, CD8αβ SP cells were determined using the FlowJo software.
B, ZAP-70 kinase inhibition at the negative selection threshold. FTOCs were incubated with 2 μM negative-selecting Q4R7 or 20 μM positive selecting Q4H7 in presence or absence of 1 μM ZAP-70 kinase inhibitor NIBR001. Thymocytes were processed and analyzed as in A.