Figure 5
Figure 5. FOXP1 is dependent upon and cooperates with (constitutive) NF-κB activity to promote expansion and survival of human B cells. Memory B cells were sorted from human peripheral blood and cotransduced with FOXP1-IRES-YFP and CA-IKK2-IRES-GFP. Transduced B cells were cultured with IL-21, IL-2, and CD40L-L cells for the first 3 days and subsequently with IL-21 and IL-2, either in the absence (A-C,E-F) or presence (D) of CD40L-L cells. (A) Cells were analyzed by flow cytometry, 3 (left) and 13 (right) days after transduction. Four populations can be identified: YFP single positive (FOXP1 overexpression; p2), GFP single positive (CA-IKK2 overexpression; p3), YFP/GFP double positive (FOXP1 and CA-IKK2 overexpression; p4), and double negative (p1). (B,D) The percentages of the 4 populations within a single unsorted culture, cultured with IL-21 and IL-2 only (B), or with IL21, IL-2, and CD40L-L cells (D), were followed over time by FACS analysis and normalized to the percentage of each population at day 3 after transduction. Mean ± SEM of 3 independent experiments are shown. (B) Significant differences in relative expansion were observed at day 10 after transduction between the FOXP1+CA-IKK2 double-transduced population vs the CA-IKK2 single-transduced population (*P < .05) and the FOXP1 single-transduced population (**P < .01) by repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. (D) Significant difference in relative expansion was observed between the FOXP1+CA-IKK2 double-transduced population vs the CA-IKK2 single-transduced population (*P < .05) by repeated-measures ANOVA followed by Bonferroni’s multiple comparison test. (C) Six days after transduction, GFP single-positive and GFP/YFP double-positive cells, cultured in the absence of CD40L (as in panel B), were sorted. Gene expression levels of the proapoptotic genes were analyzed by qRT-PCR. Expression levels were normalized to expression levels in CA-IKK2 single-transduced cells. Mean ± SEM of 3 independent experiments are shown. (1-sample t test, *P < .05, **P < .01, ***P < .001). (E) Six days after transduction, live cells of the 4 separate populations of cotransduced cells were sorted and cultured for 4 to 5 more days, after which the percentage of cells in the forward-scatter/side-scatter live gate was determined by flow cytometry and normalized to the percentage of living cells in the nontransduced population. Mean ± SEM of 3 independent experiments are shown (repeated-measures ANOVA followed by Bonferroni’s multiple comparison test; **P < .01, ***P < .001). (F) Seven days after transduction, cells were labeled with eFluor 670 and cultured for 4 days, after which the eFluor 670 intensity was determined by flow cytometry. Representative graphs of 2 independent experiments are shown.

FOXP1 is dependent upon and cooperates with (constitutive) NF-κB activity to promote expansion and survival of human B cells. Memory B cells were sorted from human peripheral blood and cotransduced with FOXP1-IRES-YFP and CA-IKK2-IRES-GFP. Transduced B cells were cultured with IL-21, IL-2, and CD40L-L cells for the first 3 days and subsequently with IL-21 and IL-2, either in the absence (A-C,E-F) or presence (D) of CD40L-L cells. (A) Cells were analyzed by flow cytometry, 3 (left) and 13 (right) days after transduction. Four populations can be identified: YFP single positive (FOXP1 overexpression; p2), GFP single positive (CA-IKK2 overexpression; p3), YFP/GFP double positive (FOXP1 and CA-IKK2 overexpression; p4), and double negative (p1). (B,D) The percentages of the 4 populations within a single unsorted culture, cultured with IL-21 and IL-2 only (B), or with IL21, IL-2, and CD40L-L cells (D), were followed over time by FACS analysis and normalized to the percentage of each population at day 3 after transduction. Mean ± SEM of 3 independent experiments are shown. (B) Significant differences in relative expansion were observed at day 10 after transduction between the FOXP1+CA-IKK2 double-transduced population vs the CA-IKK2 single-transduced population (*P < .05) and the FOXP1 single-transduced population (**P < .01) by repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. (D) Significant difference in relative expansion was observed between the FOXP1+CA-IKK2 double-transduced population vs the CA-IKK2 single-transduced population (*P < .05) by repeated-measures ANOVA followed by Bonferroni’s multiple comparison test. (C) Six days after transduction, GFP single-positive and GFP/YFP double-positive cells, cultured in the absence of CD40L (as in panel B), were sorted. Gene expression levels of the proapoptotic genes were analyzed by qRT-PCR. Expression levels were normalized to expression levels in CA-IKK2 single-transduced cells. Mean ± SEM of 3 independent experiments are shown. (1-sample t test, *P < .05, **P < .01, ***P < .001). (E) Six days after transduction, live cells of the 4 separate populations of cotransduced cells were sorted and cultured for 4 to 5 more days, after which the percentage of cells in the forward-scatter/side-scatter live gate was determined by flow cytometry and normalized to the percentage of living cells in the nontransduced population. Mean ± SEM of 3 independent experiments are shown (repeated-measures ANOVA followed by Bonferroni’s multiple comparison test; **P < .01, ***P < .001). (F) Seven days after transduction, cells were labeled with eFluor 670 and cultured for 4 days, after which the eFluor 670 intensity was determined by flow cytometry. Representative graphs of 2 independent experiments are shown.

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