KLF2 represses CXCR3 expression and function.

<p>(A) Data show CXCR3 mRNA expression quantified by qRT-PCR in the indicated activated CD8 T cell populations normalised to GFP^neg. (B) Data show flow cytometric analysis of CXCR3 surface expression and GFP expression in GFP^pos or GFP-KLF2^pos activated CD8 T cells. (C) GFP-KLF2^neg and GFP-KLF2^pos 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.</p

KLF2 expression is regulated by the strength and duration of TCR and cytokine stimulation.

<p>(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.</p

Low KLF2 expression can control trafficking molecules but high KLF2 expression is required to inhibit proliferation.

<p>(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 GFP^pos, GFP-KLF2^low and GFP-KLF2^high 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-KLF2^neg CD8 T cells was quantified for GFP-KLF2^low, GFP-KLF2^mid and GFP-KLF2^high 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 GFP^neg population from 3 independent experiments.</p

MEK1 and PI3K/PKB activity are required for maximal TCR mediated downregulation of KLF2.

<p>(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.</p