A: Jurkat cells stably transduced with GFP-containing retroviral constructs expressing CD28 WT or mutants were stimulated with beads ligated with anti-CD3+CD86 for the indicated time, and specific transcripts were quantitated using the Nanostring nCounter. The heat map was constructed from comparative expression profiling of the indicated genes. B–C: Two transcripts, CD226 and TNFA, showed significantly increased expression in cells expressing the CD28 T195P mutant. *: p<0.05 between T195P and WT; ***: p<0.005 between T195P and WT. There is no significant difference between the D124V and WT. D: Luciferase reporters of NF-κB activation were transduced into Jurkat cells expressing the indicated CD28 transgene and stimulated with the indicated beads for four hours. Diagram averages three replicates ± standard deviation. *, p<0.05; **, p<0.01; ns, not significant. Both CD28 mutants activated NF-κB more strongly than WT upon ligation of CD80 or CD86 with CD3 stimulation.