CCR4 is required for efficient negative selection and shifts the cell fate decision to negative selection from T reg cell generation in response to rare autoantigens. (A) Ccr4^+/+ OT-II and Ccr4^−/− OT-II thymocytes were incubated for 24 h on thymic slices in the presence of the indicated concentration of OVA_323-339 peptide, after which the normalized cellularity of the indicated subsets was quantified. The quantity of thymocytes initially loaded onto the slices was calculated as Input. (B) The percentage of OT-II CD4SP thymocytes (left) responding (undergoing negative selection or up-regulating CD25), (middle) surviving, or (right) up-regulating CD25 in response to the indicated concentration of OVA_323-339 relative to 0nM peptide was calculated from data in A. Means ± SEM compiled from three independent experiments with three technical replicates per experiment. *, P < 0.05 (unpaired Student’s t test).