Following Institutional Review Board (UPCI protocol 99-069) approval and informed consent, blood was obtained from healthy donors (Western PA blood bank) or HNC patients treated with cetuximab (NCI-2011-02479, NCT01218048). Lymphocytes were purified by Ficoll-Paque™ PLUS centrifugation (Amersham Biosciences, Uppsala, Sweden) and stored frozen. DC were generated as described previously (2 (link)). NK cells were purified using EasySep kits (Stem cell technologies, Vancouver, BC, Canada) and purity was >95% CD16+, CD56+, CD3− evaluated with flow cytometry (23 (link)).
The HNC cell lines PCI-15B (HLA-A2−EGFR+) was isolated from patient treated at the University of Pittsburgh Cancer Institute (Pittsburgh, PA) through the explant/culture method. JHU-029 (HLA-A2−EGFR+ and MAGE-3+) cell line was a kind gift from Dr. James Rocco (Harvard Medical School, Boston, MA) in January 2007. All cell lines were authenticated, and validated as unique using STR profiling and HLA genotyping every 6 months (24 (link), 25 (link)). Cell lines were grown in IMDM (Sigma, St. Louis, MO) supplemented with 10% FBS (Cellgro, Manassas, VA), 2% L-glutamine and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2, 95% humidity atmosphere. Adherent tumor cells were detached by warm Trypsin-EDTA (0.25%) solution (Invitrogen, Carlsbad, CA).
The HNC cell lines PCI-15B (HLA-A2−EGFR+) was isolated from patient treated at the University of Pittsburgh Cancer Institute (Pittsburgh, PA) through the explant/culture method. JHU-029 (HLA-A2−EGFR+ and MAGE-3+) cell line was a kind gift from Dr. James Rocco (Harvard Medical School, Boston, MA) in January 2007. All cell lines were authenticated, and validated as unique using STR profiling and HLA genotyping every 6 months (24 (link), 25 (link)). Cell lines were grown in IMDM (Sigma, St. Louis, MO) supplemented with 10% FBS (Cellgro, Manassas, VA), 2% L-glutamine and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2, 95% humidity atmosphere. Adherent tumor cells were detached by warm Trypsin-EDTA (0.25%) solution (Invitrogen, Carlsbad, CA).
