Cd137 41bb

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ cells per well in RPMI-10 FBS supplemented with 10 ng ml⁻¹ IL-4 (Peprotech) and 800 IU ml⁻¹ GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml⁻¹ IL-4 and 1,600 IU ml⁻¹ GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml⁻¹ IL-4, 800 IU ml⁻¹ GM-CSF, 10 ng ml⁻¹ lipopolysaccharide (Sigma-Aldrich), and 100 IU ml⁻¹ IFN-γ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.
On day 5, normal donor-matched CD8+ T cells were enriched using protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers, and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described^{50 (link)}. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. The day 5 protocol was repeated on days 8 and 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).

Normal donor monocytes were plated on day 1 in 6-well plates at 5 × 10⁶ cells per well in RPMI-10 FBS supplemented with 10 ng ml⁻¹ IL-4 (Peprotech) and 800 IU ml⁻¹ GM-CSF (Peprotech) and incubated at 37 °C overnight. On day 2, fresh medium supplemented with 10 ng ml⁻¹ IL-4 and 1,600 IU ml⁻¹ GM-CSF was added to the monocytes and incubated at 37 °C for another 48 h. On day 4, non-adherent cells were removed, and immature dendritic cells washed and pulsed with 5 µM peptide in AIM-V-10% FBS supplemented with 10 ng ml⁻¹ IL-4, 800 IU ml⁻¹ GM-CSF, 10 ng ml⁻¹ lipopolysaccharide (Sigma-Aldrich), and 100 IU ml⁻¹ IFN-γ (Peprotech) at 37 °C overnight. Day 1 was repeated on days 4 and 8 to generate dendritic cells for the second and third stimulations on days 8 and 12, respectively.
On day 5, normal donor-matched CD8+ T cells were enriched using protein kinase inhibitor dasatinib (Sigma-Aldrich), dextramers, and anti-PE or anti-APC beads (Miltenyi Biotec) as previously described^{50 (link)}. Enriched T cells were co-incubated with the appropriate pulsed dendritic cells in AIM-V-10% FBS. The day 5 protocol was repeated on days 8 and 12 using dendritic cells generated on days 4 and 8 for the second and third stimulation, respectively. Expanded T cells were validated for antigen-specificity by staining with the appropriate dextramers and for activation marker 41BB/CD137 (BioLegend).

Cryopreserved and characterized PBMCs were purchased from Cellular Technology Limited (CTL; Shaker Heights, OH). IFN-γ ELISPOT analyses with a subset of peptide antigens were performed independently by Cellular Technology Limited (Shaker Heights, OH). MHC Class I HLA-A*02-restricted dextramers (Immudex) were used according to manufacturer’s instructions. Prior to mixing different combinations of individual dextramers, D-biotin (Life Technologies) was added to the tube, according to manufacturer’s instructions. Fluorescently conjugated antibodies (BioLegend) to the following cell surface markers were used: CD3 (SK7), CD4 (OKT4) CD8 (SK1) and CD137 (4-1BB). Peptides were synthesized by CPC Scientific (Sunnyvale, CA). Mixtures of reconstituted peptides were prepared by adding an equal volume of each peptide to the mixture (note the final concentration of each peptide during stimulation was 1ug/ml).