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biotinylated peptide biot-DSEAGSSSSSNMTWATTILVPDNSSAS- GQSGQEKPR, encompassing the AE2-specific third extracellular loop of AE2, was immobilized into a NLC-neutravidin chip (Biorad). Individual peptide solutions (from 0.15 to 2.5 μM) were injected by triplicate in running buffer (phosphate buffered saline, 0.005% (v/v) Tween 20, pH 7.4) at a flow of 30 mL/min. The interspot signal (obtained in the chip surface not immobilized with protein) was used as reference.
Cell lines and patient samples
The human lymphoma, leukemia and myeloma cell lines used in the study were cultured in RPMI or IMDM media supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin (P- S) as described.40,41 A series of ten fresh peripheral blood cells obtained from untreated patients with B-cell lymphoma with peripheral blood dissemination were included in the study. Primary B cells were isolated from human peripheral blood using a CD19+ isolation kit (Miltenyi Biotec). To determine cell viability
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Peptides specifically targeting AE2 induce apoptosis in tumor
and apoptosis, cells were cultured in IMDM medium with 20% fetal bovine serum and 1% P-S. The research protocol on human subjects was approved by the University of Navarra Institutional Review Board. Informed consent was obtained from patients and healthy donors, in accordance with the Helsinki Declaration.
Cell viability and apoptosis assays
Both primary cells and established cell lines were cultured in flat bottom M96 wells, at a density of 50,000 cells/well, and incubated in 100 mL of cell medium supplemented with the corresponding isoform of the peptide at a final concentration of 50 mg/mL for 24 hours. To determine cell viability, 10 mL of MTS (Promega) were added to each well and plates were read at a wavelength of 450/690 nm. Cell viability was normalized to control group for each experiment. Apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit I (BD), as reported.41,42 Briefly, cell were stained with Annexin V-FITC and ToPro3 and analyzed in a FACS Calibur cytometer (BD).
Figure 2. p17AE2 peptide induces prolifera- tion of effector T cells and apoptosis of reg- ulatory T lymphocytes. Effect of p17AE2 on cell viability, IL-2 secretion and apoptosis in murine (A) and human (B) effector T lympho- cytes. (C) Effect of p17AE2 on cell prolifera- tion, IL-10 secretion and apoptosis in murine regulatory T lymphocytes. (D) Cell viability of human Jurkat (T effector) and Karpas299 (Treg) cell lines upon incubation with p17AE2 peptide. (E) pHi of murine effector and regulatory T cells after p17AE2 treatment. *P<0.05; **P<0.01; ***P<0.001.
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