Fig. 3: ZFP36 and ZFP36L1 determine the CD8 effector program early after T cell activation.
From: The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins
a Experimental design: In WT cells or cells transduced with non-targeting guides the ZFP36 and ZFP36L1 proteins are transiently expressed after T cell activation prior to transduction as well as upon restimulation of CTL. Cells which have undergone Cas9 mediated deletion of ZFP36 and ZFP36L1 lack both proteins upon restimulation but not during the initial activation period. CD4cre mediated deletion generates naïve CD8+ T cells which lack ZFP36 and ZFP36L1 during initial activation and re-stimulation. b In vitro killing of N4 peptide loaded EL4 cells after three hours at indicated CTL to EL4 ratios, using CTLs with CD4cre mediated deletion of ZFP36 and ZFP36L1 (n = 6) and WT CTLs (n = 7). c Killing assay using CTLs which were transduced with non-targeting guides (n = 3) or guides targeting both ZFP36 and ZFP36L1 (n = 3) is shown. For (b) and (c) data is represented as mean values ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. d Representative flow cytometry plots showing GzmB and TNF expression in CTLs after three hours of WT and CD4cre dKO CTL exposure to peptide loaded EL4 cells at a 1:1 ratio. e Plots showing geometric mean fluorescence intensity for GzmB and TNF as in (d), gated on the positive population. Each dot represents a biological replicate from four independent experiments. f Representative flow cytometry plots showing GzmB and TNF expression in CTLs treated with non-targeting and ZFP36 and ZFP36L1 specific guides, after three hours of exposure to peptide loaded EL4 cells at a 1:1 ratio. g Plots showing geometric mean fluorescence intensity for GzmB and TNF as in (f), gated on the positive population. For sgRNAs targeting ZFP36 and ZFP36L1 or non-targeting controls the data is representative of two independent experiments, each with three independent pairs of sgRNAs. Statistical significance was determined by a two-tailed unpaired Students t-test. In all panels filled circles represent the respective WT control and open shaded circles the dKO. Source Data are provided as Source Data file.
