Fig. 6. CD45 is required for affinity discrimination of antigen.

(A) Jurkat T cells were engineered to express the OT1 TCR transgene and the CD8 coreceptor (J.OT1). CD45 was deleted from J.OT1 cells using CRISPR/Cas9 (J.OT1/CD45). (B) Cells were co-cultured with T2-Kb antigen presenting cells pulsed with the indicated peptide antigens (0.05 nM) and upregulation of CD69 was assessed as a readout of T cell activation. Error bars represent the means ± SEM (N=3) and ** P < 0.01, *** P < 0.001 (two-way ANOVA Bonferroni multiple comparisons test). (C to F) CD45-deficient cells (J.OT1/CD45) were transiently transfected with CD45 to generate a broad range of CD45 levels. Cells were then co-cultured with T2-Kb cells and peptide antigen for 16 hours. The percentage of CD69 positive cells was plotted as a moving average versus CD45 expression levels. Differing concentrations of OVA peptide (C) and overlay of OVA concentrations on a log scale (left) or linear (right) axis (D). Black bar denotes approximate WT level of CD45. (E) Altered peptide ligands at a fixed concentration (50 nM) and (F) overlay of altered peptide ligands on a log scale (left) or linear (right) axis. Black bar denotes approximate WT level of CD45. Histograms can be found in supplemental information. All data are representative of three independent experiments.