Effect of AF1q on B-cell differentiation. (A,C) AF1q antagonizes the emergence of CD7⁻CD10⁺ pre-pro-B cells in serum-free cultures. CD34⁺Lin⁻ HPCs were transduced with AF1q, A2M, or the empty vector (Ctl), sorted based on GFP expression, and cultured for 5-11 days with SCF, FLT3L, TPO, and IL-7 under serum-free conditions. (A) Flow cytometry dot-plots of GFP⁺-gated cells. Numbers in quadrants indicate the percentage of each population. Data are from 1 of 4 independent experiments. (C) Relative quantification of the effect exerted by AF1q or A2M on CD7⁻CD10⁺ pre-pro-B-cell differentiation or overall cell proliferation. To limit experimental variability due to donor effect, results are normalized relative to CD10⁺ cell percentages and absolute cell numbers in cultures of empty vector–transduced cells (Ctl). Data are means ± SD percentages of 4 independent experiments. Statistically significant differences (P < .05, Student t test) in percentages or absolute numbers between AF1q/A2M and the control conditions are marked by asterisks. (B,D) AF1q antagonizes B-cell development in cocultures onto MS5 cells. (B) CD34⁺ HPCs transduced as above with AF1q, A2M, or the empty vector were seeded onto MS5 cells and cultured for 2 weeks in the absence of exogenous cytokines before absolute cell numbers and CD19⁺GFP⁺ B-cell percentages were determined. Data are representative of 1 of 4 independent experiments. (D) Relative quantification of the effect exerted by AF1q and A2M on B-cell development (CD19⁺ B-cell percentages) or global cell expansion (total cell numbers); results are normalized relative to percentages of CD19⁺ cells and absolute cell numbers in cultures of empty vector–transduced cells (Ctl). Irr indicates irrelevant antibody. Data are means ± SD percentages of 4 independent experiments. Statistically significant differences are indicated. (E) Limiting-dilution analysis. CD34⁺ HPCs were transduced and cultured as above. Positive wells were scored on day 14 and analyzed by FACS as indicated above. Empty circles represent CD19⁺ B-cell percentages. FACS analysis was restricted to cell-containing wells. Medians and P values are indicated. Statistical significance was determined by the Wilcoxon test. (F) AF1q does not affect NK-cell development. CD34⁺ HPCs transduced with AF1q, A2M, or the empty vector and sorted as above were seeded onto MS5 cells and cultured for 3 weeks with SCF, FLT3L, IL-2, IL-7, and IL-15 before absolute cell numbers and CD56⁺GFP⁺ NK-cell percentages were determined. Results are normalized relative to CD56⁺ cell percentages and absolute cell numbers in cultures of empty vector–transduced cells (Ctl). Means ± SD percentages of 4 independent experiments are shown. (G-H) AF1q deficiency enhances B-cell differentiation. CD34⁺ HPCs transduced with lentiviral vectors driving the expression of shRNA₅₄ (shAF1q) or scrambled shRNAs (shCtl) were seeded onto MS5 cells and cultured under conditions that promote B-cell (black bars) or NK-cell (empty bars) development. CD19⁺ and CD56⁺ cell percentages (G) and absolute numbers (H) were determined by FACS. Results are normalized relative to the control condition (shCtl). Means ± SD of 4 independent experiments are shown. Statistically significant differences are indicated.