Linear array genotyping test

HeLa (HPV-18+), SiHa (HPV-16+), and C-33A (HPV–) CCC were kindly provided by Dr. Boukamp (DKFZH, Heidelberg, Germany). The presence of HPV type was confirmed by the Linear array genotyping test (Roche). NK-92 cells were obtained from American Type Culture Collection (ATCC CRL-2407™; Manassas, VA, USA). Blood of healthy donors was obtained with informed consent and NK primary cells were isolated using FACSAria I cell sorting (BD Biosciences, San Jose, CA, USA). The CCC (HeLa, SiHa, and C-33A) were maintained in vitro propagated in Dulbecco’s modified Eagle’s culture medium (DMEM; GIBCO™ Invitrogen Corp., Carlsbad, CA, USA) with 10% heat-inactivated Fetal bovine serum (FBS; GIBCO™ Invitrogen Corp.), and NK-92 cells were maintained in vitro propagated in RPMI-1640 culture medium (GIBCO™ Invitrogen Corp.) with 100 U/mL recombinant human IL-2 (BioLegend) and FBS at a final concentration of 15% (GIBCO™ Invitrogen Corp.); both media were supplemented with 1X L-glutamine (at a 2-mM final concentration; GIBCO™ Invitrogen Corp.) and antibiotics (penicillin/streptomycin; GIBCO™ Invitrogen Corp.). These media will be referred to as DMEM-S and RPMI-S. Cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂.

HPV DNA testing had been previously performed on the baseline ASCUS/LSIL samples from this study population. HPV DNA was extracted (MagNA Pure LC robot, Roche Diagnostics, Pleasanton, California, USA) from a lysed cell pellet of 1 milliliter of the PreservCyt sample. The DNA of 37 HPV types [24] was detected and genotyped by the Linear Array Genotyping Test (Roche Diagnostics) according to the manufacturer’s instructions [21] (link). Beta-globin was included in the test as an internal control to test for sample adequacy and avoid false negatives.