Figure 2
Figure 2. PI3Kγ deficiency or selective blockade of PI3Kγ activity prevents efficient induction of CXCR3 on T cells upon activation. (A,B) T cells from C57BL/6 mice genetically deficient in functional PI3Kγ (PI3Kγ−/−) express less CXCR3 than wild-type C57BL/6 T cells. (A) Flow cytometric analysis of CXCR3 levels on anti-CD3/CD28–activated T cells from wild-type (black hollow peaks) and PI3Kγ−/− (gray hollow peaks) mice. (B) Flow cytometric analysis of lesion-draining LN cells excised from L mexicana–infected wild-type and PI3Kγ−/− C57BL/6 mice. Numbers outside parentheses indicate the percentage of LN cells, whereas numbers in parentheses represent the percentage of CXCR3+ T cells in the CD4+ or CD8+ compartment. Shown are representative results of 3 to 5 independent experiments. (C) Mean fluorescence intensity values (MFI) for CXCR3 staining on CD4+ and CD8+ LN T cells from L mexicana–infected mice. Numbers given are the mean MFI (± SEM) of CXCR3-PE staining on the surface of indicated T cells from 3 independent experiments. *P = .05. (D) Blockade of PI3Kγ activity suppresses efficient induction of CXCR3 on activated T cells. Activated T cells were treated with the PI3Kγ-selective inhibitor AS-605240 (1.25 μM; gray hollow peaks) or vehicle (black hollow peaks) as described before, and expression of CXCR3 was analyzed by flow cytometry. Isotype controls are solid peaks. (E) Analysis of CXCR3mRNA levels in AS-605240–treated versus vehicle-treated T cells by semiquantitative real-time PCR analysis. AS-605240–treated T cells (▭) showed significantly less induction of CXCR3 mRNA than vehicle-treated cells (). (F) The effect of AS-605240 on CXCR3 suppression was dose dependent. (G) AS-605240 treatment did not affect CCR1, CCR5, CCR4, CCR7, CXCR6, and CCR10 mRNA levels. (H) AS-605240–treated T cells (▭) displayed less induction of T-bet mRNA compared with vehicle controls (). Real-time PCR data for each group were normalized to the housekeeping gene gapdh and are expressed as fold induction over nonstimulated cells. (I) Effect of PI3Kγ gene silencing on induction of CXCR3. Activated T cells transfected with PI3Kγ siRNA expressed less CXCR3 compared with controls. Data in panels A-D and I are representative of at least 2 to 5 independent experiments with similar results. Panels F-H represent the mean results (± SEM) of 3 or more independent experiments. A P value from an unpaired Student t test less than .05 (*) was considered significant.

PI3Kγ deficiency or selective blockade of PI3Kγ activity prevents efficient induction of CXCR3 on T cells upon activation. (A,B) T cells from C57BL/6 mice genetically deficient in functional PI3Kγ (PI3Kγ−/−) express less CXCR3 than wild-type C57BL/6 T cells. (A) Flow cytometric analysis of CXCR3 levels on anti-CD3/CD28–activated T cells from wild-type (black hollow peaks) and PI3Kγ−/− (gray hollow peaks) mice. (B) Flow cytometric analysis of lesion-draining LN cells excised from L mexicana–infected wild-type and PI3Kγ−/− C57BL/6 mice. Numbers outside parentheses indicate the percentage of LN cells, whereas numbers in parentheses represent the percentage of CXCR3+ T cells in the CD4+ or CD8+ compartment. Shown are representative results of 3 to 5 independent experiments. (C) Mean fluorescence intensity values (MFI) for CXCR3 staining on CD4+ and CD8+ LN T cells from L mexicana–infected mice. Numbers given are the mean MFI (± SEM) of CXCR3-PE staining on the surface of indicated T cells from 3 independent experiments. *P = .05. (D) Blockade of PI3Kγ activity suppresses efficient induction of CXCR3 on activated T cells. Activated T cells were treated with the PI3Kγ-selective inhibitor AS-605240 (1.25 μM; gray hollow peaks) or vehicle (black hollow peaks) as described before, and expression of CXCR3 was analyzed by flow cytometry. Isotype controls are solid peaks. (E) Analysis of CXCR3mRNA levels in AS-605240–treated versus vehicle-treated T cells by semiquantitative real-time PCR analysis. AS-605240–treated T cells (▭) showed significantly less induction of CXCR3 mRNA than vehicle-treated cells (). (F) The effect of AS-605240 on CXCR3 suppression was dose dependent. (G) AS-605240 treatment did not affect CCR1, CCR5, CCR4, CCR7, CXCR6, and CCR10 mRNA levels. (H) AS-605240–treated T cells (▭) displayed less induction of T-bet mRNA compared with vehicle controls (). Real-time PCR data for each group were normalized to the housekeeping gene gapdh and are expressed as fold induction over nonstimulated cells. (I) Effect of PI3Kγ gene silencing on induction of CXCR3. Activated T cells transfected with PI3Kγ siRNA expressed less CXCR3 compared with controls. Data in panels A-D and I are representative of at least 2 to 5 independent experiments with similar results. Panels F-H represent the mean results (± SEM) of 3 or more independent experiments. A P value from an unpaired Student t test less than .05 (*) was considered significant.

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