Figure 1.
KLF2 expression is regulated by the strength and duration of TCR and cytokine stimulation.
(A) Data show relative KLF2 mRNA expression quantified by qRT-PCR in purified naïve CD8 T cells incubated with gp33-41 peptide for the times shown. Results are normalised to untreated naïve cells. (B) KLF2 mRNA in purified naïve OT-1 CD8 T cells incubated with SIIQFEHL (Q4H7) or SIIQFERL (Q4R7), SIIQFEKL (Q4) or SIINFEKL (N4) peptide for 4 hours. Results are normalised to untreated naïve cells. (C) CD62L and S1P1 mRNA in purified naïve OT-1 CD8 T cells incubated with Q4H7, Q4R7, Q4 or N4 peptide for 4 hours. Results are normalised to untreated naïve cells. (D) KLF2 mRNA (normalised to time 0 sample) in T cells activated with gp33-41 peptide for 2 days, cultured in IL-2 for 5 days and then cultured without cytokine for the times shown. (E) KLF2 mRNA (normalised to CTL cultured with 20ng/ml IL-2) in T cells activated with gp33-41 peptide for 2 days, cultured in IL-2 for 5 days and then cultured with the indicated concentrations of IL-2 for 18 hours. All data show mean + SEM of at least 3 independent experiments.
Figure 2.
MEK1 and PI3K/PKB activity are required for maximal TCR mediated downregulation of KLF2.
(A) Schematic diagram of KLF2 gene indicating binding sites of primers used. (B) Data show binding of RNA polymerase II to the indicated site in the KLF2 gene measured by ChIP assay. Black bars indicate binding in purified naïve OT-1 CD8 T cells, grey bars indicate OT-1 CD8 T cells stimulated with N4 peptide for 6 hours. (C) Spliced and unspliced KLF2 mRNA in purified naïve OT-1 CD8 T cells incubated with Q4H7, Q4R7, Q4 or N4 peptide for 4 hours. Results are normalised to untreated naïve cells. (D) Western blot of lysates from purified naïve OT-1 CD8 T cells incubated with N4 peptide and inhibitors for 4 hours as indicated. (E) KLF2 mRNA in purified naïve P14 CD8 T cells incubated with gp33-41 peptide and inhibitors for 4 hours as indicated. (F) KLF2, CD62L and S1P1 mRNA in FACS purified GFP positive P14 CD8 T cells transduced with control or FoxO3AAA constructs as shown and treated or not with gp33-41 peptide for 4 hours as indicated. (G) CD62L, spliced and unspliced KLF2 mRNA in purified naïve P14 CD8 T cells incubated with gp33 peptide and inhibitors as indicated for 4 hours. Results for each mRNA are normalised to that particular mRNA level in untreated naïve cells – note that comparison of the amount of unspliced or spliced RNA cannot be made. Data in B, C, E, F and G show mean + SEM of at least 3 independent experiments. Data in D are representative of 3 independent experiments.
Figure 3.
The impact of failing to downregulate KLF2 on the transcriptional programme of TCR activated CD8 T cells.
(A) Data show flow cytometric analysis of activated CD8 T cells transduced with either vector (evGFP) or a GFP-KLF2 fusion construct (data representative of 10 independent experiments). In all experiments using these constructs, CD8 T cells were transduced at 18 hours post-activation, washed out of peptide stimulation at 48 hours and then cultured with IL-2 for a further 48-72 hours prior to experimental procedures. (B) Data show KLF2 mRNA expression in purified naïve P14 CD8 T cells, P14 CD8 T cells treated with gp33-41 peptide for 4 hours, GFP positive activated CD8 T cells purified by FACS from activated CD8 T cells transduced with either evGFP or a GFP-KLF2 fusion protein. After reverse transcription, KLF2 mRNA was quantified by qRT-PCR against a standard curve of a KLF2 cDNA and is expressed as mRNA copy number. Data show mean + SEM of 3 independent experiments. (C) Heat map showing gene expression patterns of genes encoding trafficking molecules in GFPpos and GFP-KLF2pos activated CD8 T cells, (D) CD8 T cell effector molecules, (E) cytokine receptors and (F) transcription factors. Heat maps are normalised to depict a 2-fold regulation as significant. Fold changes where shown are statistically significant (p<0.05) and are relative to the GFPpos control.
Figure 4.
KLF2 represses CXCR3 expression and function.
(A) Data show CXCR3 mRNA expression quantified by qRT-PCR in the indicated activated CD8 T cell populations normalised to GFPneg. (B) Data show flow cytometric analysis of CXCR3 surface expression and GFP expression in GFPpos or GFP-KLF2pos activated CD8 T cells. (C) GFP-KLF2neg and GFP-KLF2pos activated CD8 T cells were competitively assayed for their ability to migrate to CXCL10. Data shown are the percentage of cells migrating (relative to input controls) at the given CXCL10 concentrations. (D) MFI of CXCR3 quantified by flow cytometry in naïve OT-1 CD8 T cells activated with N4, Q4, Q4R7 or Q4H7 for 24 hours. (E) CXCR3 expression measured by flow cytometry in naïve OT-1 CD8 T cells incubated with N4 peptide and PD184352 for 24 hours as indicated. Data in A, C & D show mean + SEM of 3 independent experiments. Data in B and E representative of 3 independent experiments.
Figure 5.
KLF2 re-expression inhibits proliferation but not via c-myc repression.
(A) Data show the cellular DNA content of GFP-KLF2neg and GFP-KLF2pos activated CD8 T cells. Data are representative of 4 independent experiments. (B) Expression of c-myc mRNA in FACS purified activated CD8 T cells quantified by qRT-PCR (data normalised to GFPneg and show mean + SEM of 3 independent experiments). (C) DNA synthesis measured by EdU uptake in the T cell populations shown (data show mean percentage EdU uptake + SEM, n=3). (D) Cell counts of GFPpos and KLF2pos activated CD8 T cells; data shown are mean ± SEM of 3 independent experiments.
Figure 6.
Rapamycin treatment controls trafficking molecules but not DNA synthesis in activated CD8 T cells.
(A) Data show expression of KLF2 mRNA determined by qRT-PCR in purified naïve CD8 T cells and T cells activated with gp33-41 peptide for 2 days then cultured with IL-2 plus DMSO or 20nM rapamycin for 5 days. Data are normalised to expression in control T cells and show mean + SEM of 3 independent experiments. (B) CD62L surface expression in CD8 T cells treated as in (A) measured by flow cytometry (data representative of 5 independent experiments). (C) CXCR3 mRNA in CD8 T cells treated as in (A) quantified by qRT-PCR. Data normalised to control T cells and show mean + SEM of 3 independent experiments. (D) CXCR3 surface expression in activated CD8 T cells treated as in (A) measured by flow cytometry, data representative of 3 independent experiments. (E) DNA synthesis measured by EdU uptake in T cells activated with gp33-41 peptide for 2 days then cultured with IL-2 plus either DMSO or rapamycin for 2 days. Data show mean percentage EdU uptake + SEM of 3 independent experiments.
Figure 7.
Low KLF2 expression can control trafficking molecules but high KLF2 expression is required to inhibit proliferation.
(A) Data show the gating strategy used to identify populations with differential KLF2 expression in GFP-KLF2 transduced activated CD8 T cells. For all experiments P14 CD8 T cells were activated for with gp33-41 peptide and retroviral transduction was performed 18 hours after activation. At 48 hours after the initial activation cells were washed and cultured for a further 3 days with 20ng/ml IL-2. (B) Population expression of CD62L measured by flow cytometry in GFPpos, GFP-KLF2low and GFP-KLF2high activated CD8 T cells. (C) CD62L median fluorescence intensities (MFIs) in CD8 T cell populations as indicated. (D) Inhibition of DNA synthesis relative to control GFP-KLF2neg CD8 T cells was quantified for GFP-KLF2low, GFP-KLF2mid and GFP-KLF2high CD8 T cells (data show mean + SEM of 3 independent experiments). (E) CXCR3 MFIs in indicated CD8 T cells populations. Data shown in C & E are the mean + SEM of MFIs normalised to GFPneg population from 3 independent experiments.