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. 2009 Jul 17;31(1):122-30.
doi: 10.1016/j.immuni.2009.05.011.

KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors

Michael A Weinreich  1 , Kensuke Takada, Cara Skon, Steven L Reiner, Stephen C Jameson, Kristin A Hogquist
Affiliations

KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors

Michael A Weinreich et al. Immunity. .

Abstract

The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid-binding receptor S1P(1) and the selectin CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirmed the upregulation of the chemokine receptor CXCR3 on KLF2-deficient T cells. However, we showed that this was a cell-nonautonomous effect, as revealed by CXCR3 upregulation on wild-type bystander cells in mixed bone-marrow chimeras with KLF2-deficient cells. Furthermore, KLF2-deficient T cells overproduced IL-4, leading to the upregulation of CXCR3 through an IL-4-receptor- and eomesodermin-dependent pathway. Consistent with the increased IL-4 production, we found high concentrations of serum IgE in mice with T cell-specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P(1) and CD62L and restrains spontaneous cytokine production in naive T cells.

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Figures

Figure 1
Figure 1. CXCR3 is upregulated on KLF2 deficient SP thymocytes
A) Relative chemokine receptor gene expression from sorted WT and CD4Cre/KLF2fl/fl (KLF2KO) thymocytes. CD4 or CD8 SP, TCRβ+ cells were sorted using a “dump strategy” to exclude CD25, TCRγ, NK1.1, CD1d/aGal-cer tetramer binding cells. RNA was prepared from cells of 2 independent sorts from 2 animals and 2 littermate controls. Two independent cDNA preparations were made from each RNA sample. B) Flow cytometry histograms of dump negative CD4 or CD8 SP thymocytes. Black line indicates KLF2 KO and gray shaded histogram indicates the WT. In S1P1 histograms, the dark gray, broken line represents staining with a control, anti-GST antibody. “nd” indicates not detected. Data in B are representative of independent analysis from at least 3 animals of each genotype.
Figure 2
Figure 2. CXCR3 is expressed on wild type thymocytes in KLF2 deficient environment
Mixed bone marrow chimeras were created by mixing bone marrow from Thy1.1+/CD45.2+ WT mice with that from Thy1.2+/CD45.2+ KLF2 KO mice at unequal ratios and reconstituting CD45.1+ lethally irradiated recipients. A) Schematic for unequal mixed bone marrow chimera set up. B) Flow cytometry for surface expression of CD62L and CXCR3 on “dump negative” CD4 or CD8 SP thymocytes, as defined in figure 1 and gated as indicated. The observed chimerism ratio in SP thymocytes for these particular 8 week post-transplant animals is shown. Similar results were observed in over 10 chimeric animals in multiple experiments.
Figure 3
Figure 3. Cell-nonautonomous regulation of CXCR3 occurs at the transcriptional level
Single positive thymocytes were sorted from either WT (Thy1.1+) from a WT thymus (left bar), WT (Thy1.1+) cells from a KLF2 deficient majority mixed chimera thymus (middle bar), and KLF2 deficient from a KLF2 deficient thymus (right bar). All values are shown relative to the value from WT cells sorted on the same day. Graph is of the mean +/− SD of 4 PCR experiments and 2 separate sorts. Y-axis is on a logarithmic scale. “nd” indicates not detected. # indicates detection in only one PCR reaction. * p<0.05, ** p<0.0001.
Figure 4
Figure 4. A KLF2-GFP reporter mouse shows no correlation between KLF2 and CXCR3 in CD4 or CD8 memory T cells
Top panel: CD44 expression on “dump negative” CD4 and CD8 T cells from spleen of KLF2-GFP reporter mice (see Supplementary Figure 2). Middle and bottom panels show flow cytometry dot plots from the gated CD44 high “memory phenotype” CD4 (left) and CD8 (right) populations. Similar results were observed in two other KLF2-GFP reporter mice
Figure 5
Figure 5. KLF2 deficient cells are defective in thymic emigration
Recent thymic emigrants (RTE) were measured in mixed bone marrow chimeras by analysis of peripheral tissues 48 hours after intrathymic injection of a biotinylating agent. The graph displays the ratio of KLF2 deficient to WT cells in various populations. To allow comparison between different experiments, the ratio in DP was normalized to one. Thus a ratio greater than one signifies an increase in KLF2 deficient cells relative to WT, and less than one means relatively more WT cells. Error bars indicate the standard deviation. N=8 chimeras for LN and spleen, and N=3 for liver. * p < 0.05, ** p < 0.001.
Figure 6
Figure 6. Bystander upregulation of CXCR3 is dependent on the IL-4 receptor
A) Quantitative RT PCR analysis of IL-4 mRNA from sorted “dump negative” CD4SP thymocytes from WT and KLF2 KO mice. Graph is of the mean +/− SD of 4 PCR experiments and 2 separate sorts. B) Thymocytes were stimulated ex vivo with PMA and ionomycin for 5 hours, and then intracellular staining was done for IL-4. “Dump negative” CD4SP and CD8SP are shown in the dot plots. The bar graph on the right shows the average percent of IL-4 secretion from 4 paired sets of animals in one experiment. The experiment was repeated 3 times with similar results. * p < 0.05. C) Flow cytometry histograms of CD124 surface expression on SP thymocytes from mixed chimeras, gated as in figure 2b. D) Serum IgE levels. Serum was collected from mice 40–100 days old. WT: N=12, KLF2 KO: N=22. E) Flow cytometry from the indicated mixed chimeras for surface expression of CXCR3 on “dump negative“ SP thymocytes. Data are representative of greater than 5 experiments. Percentages indicate the proportion in “dump negative” CD4SP gate. F) CXCR3 and CCR3 expression on mature (Qa-2 high) CD8 SP thymocytes after 4 days of in vitro culture with IL-7 (filled gray) or IL-4 (black line). Results were representative of 5 independent experiments.
Figure 7
Figure 7. CXCR3 upregulation is dependent on Eomesodermin
A) Quantitative RT PCR on RNA from sorted “dump” negative CD4SP and CD8SP thymocytes from WT and intact KLF2 deficient mice. Displayed are KLF2 deficient mRNA levels relative to WT. Graphs represent the mean +/− SD of 4 PCR experiments and 2 separate sorts. B) Flow cytometry from indicated mixed chimeras for surface expression of CXCR3 on “dump negative” SP thymocytes. Results are representative of 3 experiments.
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Comment in

  • How to be naive.
    Stein JV. Stein JV. Immunity. 2009 Jul 17;31(1):9-11. doi: 10.1016/j.immuni.2009.06.010. Immunity. 2009. PMID: 19604488

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