Figure 2.
Figure 2. TCR associates with and transactivates CXCR4; this interaction is disrupted by AMD3100. (A) Schematic diagram of FRET between fluorescent fusion proteins of CXCR4 and CD3ζ. (B-H) Jurkat T cells were transfected with the indicated fluorescent fusion proteins and cultured for 16 to 18 hours. (B,E-F) Transfected cells were analyzed via flow cytometry to assess cell surface levels of the indicated receptors or YFP. (C) Graph summarizing the mean fold increase of cell surface levels of the indicated receptor in cells transfected with CXCR4-YFP and CD3ζ-CFP compared with vector control transfected cells, ± SEM (n = 3). (D,G-H) Sixteen to 18 hours posttransfection, the cells were pretreated with 120 μM AMD3100 where indicated for 1 hour and then stimulated with 1 μg/mL OKT3 crosslinked with 0.1 mg/mL goat anti-mouse immunoglobulin G (IgG) for 20 minutes. Spectra of the same cells were obtained before and after stimulation. (D,G) Representative spectra are shown. (H) Summary of 3 to 4 independent experiments. Each bar denotes the percentage change in CFP or YFP in response to OKT3 stimulation. *Significant difference compared with CXCR4-YFP/CD3ζ-CFP, vehicle sample (P < .05). (I) Schematic diagram of a PLA analyzing interactions between CXCR4-YFP and CD3ζ. (J-K) Jurkat T cells were transfected with CXCR4-YFP, cultured for 16 to 18 hours, pretreated with AMD3100 for 1 hour, centrifuged onto fibronectin- and OKT3-coated coverslips, and incubated for 30 minutes at 37°C. Cells were then fixed and stained as described in “Methods.” PLA was visualized using an LSM780 laser-scanning confocal microscope (Carl Zeiss) with a 100×/1.46 oil objective and laser/emission filter: 488/500-554 for CXCR4-YFP to identify transfected cells, 405/411-481 for 4′,6-diamidino-2-phenylindole (DAPI) (blue) and 594/624 for PLA (red). ZEN software was used for acquisition of images. FIJI was used to assess total PLA fluorescence. (J) Representative results are shown (original magnification ×100). (K) Summary of images acquired in 3 independent experiments for a total of 17 to 30 cells per condition, ± SEM (P < .05). (L-M) Jurkat T cells were transfected with CXCR4-YFP, incubated for 16 to 18 hours, treated with 60 μM AMD3100 for 1 hour, stimulated with crosslinked OKT3 as in panel D for 5 minutes, lysed, harvested for immunoprecipitation for CXCR4, and immunoblotted for pS339-CXCR4 and total CXCR4. (L) Representative results are shown. (M) Summary of the mean fold increase in pS339-CXCR4 upon CD3 stimulation compared with unstimulated cells, ± SEM (n = 3; P < .05). DIC, differential interference contrast; Unstim., unstimulated.

TCR associates with and transactivates CXCR4; this interaction is disrupted by AMD3100. (A) Schematic diagram of FRET between fluorescent fusion proteins of CXCR4 and CD3ζ. (B-H) Jurkat T cells were transfected with the indicated fluorescent fusion proteins and cultured for 16 to 18 hours. (B,E-F) Transfected cells were analyzed via flow cytometry to assess cell surface levels of the indicated receptors or YFP. (C) Graph summarizing the mean fold increase of cell surface levels of the indicated receptor in cells transfected with CXCR4-YFP and CD3ζ-CFP compared with vector control transfected cells, ± SEM (n = 3). (D,G-H) Sixteen to 18 hours posttransfection, the cells were pretreated with 120 μM AMD3100 where indicated for 1 hour and then stimulated with 1 μg/mL OKT3 crosslinked with 0.1 mg/mL goat anti-mouse immunoglobulin G (IgG) for 20 minutes. Spectra of the same cells were obtained before and after stimulation. (D,G) Representative spectra are shown. (H) Summary of 3 to 4 independent experiments. Each bar denotes the percentage change in CFP or YFP in response to OKT3 stimulation. *Significant difference compared with CXCR4-YFP/CD3ζ-CFP, vehicle sample (P < .05). (I) Schematic diagram of a PLA analyzing interactions between CXCR4-YFP and CD3ζ. (J-K) Jurkat T cells were transfected with CXCR4-YFP, cultured for 16 to 18 hours, pretreated with AMD3100 for 1 hour, centrifuged onto fibronectin- and OKT3-coated coverslips, and incubated for 30 minutes at 37°C. Cells were then fixed and stained as described in “Methods.” PLA was visualized using an LSM780 laser-scanning confocal microscope (Carl Zeiss) with a 100×/1.46 oil objective and laser/emission filter: 488/500-554 for CXCR4-YFP to identify transfected cells, 405/411-481 for 4′,6-diamidino-2-phenylindole (DAPI) (blue) and 594/624 for PLA (red). ZEN software was used for acquisition of images. FIJI was used to assess total PLA fluorescence. (J) Representative results are shown (original magnification ×100). (K) Summary of images acquired in 3 independent experiments for a total of 17 to 30 cells per condition, ± SEM (P < .05). (L-M) Jurkat T cells were transfected with CXCR4-YFP, incubated for 16 to 18 hours, treated with 60 μM AMD3100 for 1 hour, stimulated with crosslinked OKT3 as in panel D for 5 minutes, lysed, harvested for immunoprecipitation for CXCR4, and immunoblotted for pS339-CXCR4 and total CXCR4. (L) Representative results are shown. (M) Summary of the mean fold increase in pS339-CXCR4 upon CD3 stimulation compared with unstimulated cells, ± SEM (n = 3; P < .05). DIC, differential interference contrast; Unstim., unstimulated.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal