Figure 2.
Figure 2. In vivo leukemogenesis and increased response to IL-7 as a result of putative electrostatic interactions between the EJM regions of IL7R-RRI and γc. (A) Ba/F3 ectopically expressing IL7R-WT or IL7R-RRI were cultured with different concentrations of mIL-7, and cell viability (GFP+ cells per 1000 beads) was evaluated after 48 hours by flow cytometry. (B) Unsorted Ba/F3 cells ectopically expressing IL7R-WT, IL7R-RRI, IL7R-RI (p.T244>RI), or IL7R-IH (p.I241>IH) were cultured with different concentrations of human IL-7 (hIL-7), and numbers of GFP+ cells per 1000 beads were evaluated after 96 hours by flow cytometry. (C) Percentage of GFP+ cells in peripheral blood lymphocytes and (D) Kaplan-Meier curves (GFP+ cells >0.1% in peripheral blood lymphocytes being the event) of C3H/HePas isogenic mice injected with Ba/F3 cells expressing IL7R-WT or IL7R-RRI. Animals injected with IL7R-WT Ba/F3 cells were observed for 110 days with no sign of engraftment. The IL7R-P2 was not tested in C3H/HePas mice, but in Balb/c and NOD/SCID mice, engraftment occured in about 2 weeks. Curves in (D) were compared by the log-rank test. (E) Infiltration of GFP+ cells in different organs of representative mice injected with IL7R-RRI Ba/F3 cells. SSC, side scatter. (F) Molecular dynamics simulations of the IL7RA and the γc transmembrane peptides interacting in a lipid bilayer. ns, nanoseconds. Peptide associations were stable (upper panel). In the wild-type pair the electrostatic interactions were characteristic of nonpolar bonds. Much stronger electrostatic interactions were observed for the IL7R-RRI/γc pair (lower panel) as a result of bonds between arginine (R243) and γc E261 of IL7R-RRI (supplemental Figure 8). (G) STAT3/5-luciferase transactivation assay performed in HEK293T cells as above, using different combinations of IL7RA (in pMIG) and γc constructs (in pcDNA3.1+-C-DYK). Data are representative of at least 2 independent experiments. Results are the mean ± SD of triplicate samples. Data were analyzed by ANOVA and Tukey post hoc test. ***P < .001; ****P < .0001.

In vivo leukemogenesis and increased response to IL-7 as a result of putative electrostatic interactions between the EJM regions of IL7R-RRI and γc. (A) Ba/F3 ectopically expressing IL7R-WT or IL7R-RRI were cultured with different concentrations of mIL-7, and cell viability (GFP+ cells per 1000 beads) was evaluated after 48 hours by flow cytometry. (B) Unsorted Ba/F3 cells ectopically expressing IL7R-WT, IL7R-RRI, IL7R-RI (p.T244>RI), or IL7R-IH (p.I241>IH) were cultured with different concentrations of human IL-7 (hIL-7), and numbers of GFP+ cells per 1000 beads were evaluated after 96 hours by flow cytometry. (C) Percentage of GFP+ cells in peripheral blood lymphocytes and (D) Kaplan-Meier curves (GFP+ cells >0.1% in peripheral blood lymphocytes being the event) of C3H/HePas isogenic mice injected with Ba/F3 cells expressing IL7R-WT or IL7R-RRI. Animals injected with IL7R-WT Ba/F3 cells were observed for 110 days with no sign of engraftment. The IL7R-P2 was not tested in C3H/HePas mice, but in Balb/c and NOD/SCID mice, engraftment occured in about 2 weeks. Curves in (D) were compared by the log-rank test. (E) Infiltration of GFP+ cells in different organs of representative mice injected with IL7R-RRI Ba/F3 cells. SSC, side scatter. (F) Molecular dynamics simulations of the IL7RA and the γc transmembrane peptides interacting in a lipid bilayer. ns, nanoseconds. Peptide associations were stable (upper panel). In the wild-type pair the electrostatic interactions were characteristic of nonpolar bonds. Much stronger electrostatic interactions were observed for the IL7R-RRI/γc pair (lower panel) as a result of bonds between arginine (R243) and γc E261 of IL7R-RRI (supplemental Figure 8). (G) STAT3/5-luciferase transactivation assay performed in HEK293T cells as above, using different combinations of IL7RA (in pMIG) and γc constructs (in pcDNA3.1+-C-DYK). Data are representative of at least 2 independent experiments. Results are the mean ± SD of triplicate samples. Data were analyzed by ANOVA and Tukey post hoc test. ***P < .001; ****P < .0001.

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