PMC

(A) BAFF-triggered NF-κB activation was assessed by immunoblotting using antibodies for p-IKKα/p-IKKβ, p-IκBα, p-p105, the precursor form of p105 and its activated form p50. (B) MAPK activation by assessing the phosphorylation status of ERK1/2 and SAPK/JNK. (C) PI3K/Akt activation assessed using antibody for p-Akt.

(A) Flow cytometry analyses of BAFF and APRIL binding to primary B-ALL cells, on three representative patients. (B) Summary of BAFF () and APRIL (x) binding to primary B-ALL cells (n = 12); solid line represents the mean binding of the respective ligands (% of cells), as assessed by flow cytometry.

(A) Electrophoretic analysis of RT-PCR products of BCMA, TACI and BAFF-R in B-ALL lines (left), primary B-ALL (center) and BCP from normal donors (right). Splenic mature B-cells as positive control (+); PBGD as control transcript. (B) Flow cytometry of surface BCMA, TACI and BAFF-R on representative B-ALL lines, B-ALL patients and normal BCP. (C) Summary of surface BCMA (▪), TACI (▾) and BAFF-R (•) expression (% of cells) in B-ALL patients (n = 23; BCMA+TACI+BAFF-R, 17/23; BCMA+BAFF-R 2/23; TACI+BAFF-R 3/23; only BAFF-R, 1/23; only TACI or BCMA, or BCMA+TACI, 0/23) and lines (n = 6) evaluated by flow cytometry; solid line indicates mean surface expression (% of cells). B-ALL lines: BCMA, mean 2.23%, range 0.6–5.54%; TACI, mean 11.67%, range 1.51–27.2%; BAFF-R, mean 45.96%, range 3.07–99.5%.

(A) Proliferation of primary B-ALL cells (n = 10) cultured in medium alone (Control, x), BAFF-myc (□), CD40L (◊) or BAFF-myc+CD40L (○), as measured by thymidine incorporation. Each point corresponds to mean stimulation index (i.e, proliferation of test condition divided by proliferation in control medium) for each patient tested, per condition; solid line indicates the mean stimulation index per experimental condition. Statistical analysis performed using the Wilcoxon test. (B) Primary B-ALL (n = 8) cultured in control medium (Δ) or with BAFF-myc (□), CD40L (◊) or BAFF-myc+CD40L (○), and evaluated for cellular viability. Each point corresponds to mean survival percentage for each patient tested, per condition; solid lines indicate mean survival percentage per experimental condition. Statistical analysis performed using the Wilcoxon test. (C) Primary B-ALL cells (n = 10) cultured with BCMA-Fc (black bars) or control fusion protein (white bars). Experiments performed in triplicate, and results expressed as mean proliferation index (i.e, proliferation in BCMA-Fc divided by proliferation in control-Fc) for BCMA-Fc vs. control fusion protein.

(A) Electrophoretic analysis of RT-PCR products of BAFF and APRIL in BM-S, BM-EC and MSC. Monocytes and splenic mature B-cells used as positive controls (+) for BAFF-system ligands and receptors, respectively; PBGD as control transcript. (B) Comparison of aminoacid sequence of APRIL-α and APRIL-ε, lacking residues as dashed lines), which results from alternative splicing with exon-1/exon-4 fusion; predicted 90 aminoacid protein lacking the furin convertase motif (in bold) and with early stop codon. (C) Flow cytometry of BAFF, APRIL expression in representative BM-S, MSC and BM-EC cases. (D) Summary of BAFF (Δ) and APRIL (◊) surface expression (%) in BM microenvironmental cells (n = 14; symbols: BM-S, open; MSC, gray; BM-EC, black). Solid line indicates mean surface expression (% of cells), as assessed by flow cytometry. (E) ELISA for soluble BAFF in CM from BM-EC cultures (n = 5 biological replicates).

(A) Electrophoretic analysis of RT-PCR products of BAFF and APRIL in B-ALL lines (left), primary B-ALL (center) and normal BCP (right). Monocytes as positive control (+); PBGD as control transcript. A 940bp band was identified as the new isoform APRIL-δ. (B) Comparison of aminoacid sequences of APRIL-α and the predicted for APRIL-δ, which results from alternative splicing with exon-1/exon-3 fusion (lacking residues as dashed lines); this results in a 104 predicted aminoacid protein lacking the motif for furin convertase (in bold). (C) Flow cytometry of BAFF, APRIL expression on representative cases of B-ALL patients, B-ALL lines and normal BCP. (D) Summary of BAFF (▴) and APRIL (♦) surface expression in B-ALL patients (n = 23; BAFF+APRIL, 22/23; only BAFF, 1/23; only APRIL, 0/23) or lines (n = 6) evaluated by flow cytometry; solid line indicates mean surface expression (% of cells). (E) ELISA for soluble BAFF in plasma from B-ALL patients (n = 39) or age-matched controls (n = 21); solid lines indicate mean levels of BAFF for each group. Statistical analysis performed using Mann-Whitney test.