The blood used in the screening for p37 (HPV16 E6 peptide) responses was collected by the Welsh Blood Transfusion Service (WBTS). All the buffy-coats came from female donors and were tested for HIV, HBV and HCV by the WBTS. Due to the anonymous character o f the donation it was impossible to know the age o f the donors, or whether they had history of cervical dysplasia or neoplasia.
The blood used as feeders was collected from laboratory staff (into 50 ml Falcon tubes containing lOU/ml of heparin) without any restriction o f age or gender. Blood was taken with informed consent. Since donors were healthy volunteers, no ethical approval was required.
10-2-Isolation of PBMC from whole blood
PBMC were enriched from whole blood by density gradient centrifugation.
Briefly, blood was layered upon an equal volume o f Ficoll-Hypaque (Histopaque-1077, Sigma) in a 50ml Falcon tube. Blood was centrifuged with the brake inactivated for 2 0
minutes at 755g. This resulted in the formation of a distinct monolayer, enriched with PBMC and platelets at the surface between Histopaque and plasma. This monolayer was harvested with a pastette (Alpha, Hampshire, UK) into a 50 ml Falcon tube and serum- free RPMI media was added until 50 ml. Enriched PBMC were centrifuged at 612g for 10 minutes and serum-free RPMI media was then used to wash them, with the first 2 washes centrifuged at 425g for 5 minutes and the final spin at 272g for 5 minutes so as to reduce the platelet content of pellet.
10-3-Stocks of peptides
All the peptides were obtained from Mimotopes (Heswall, UK), dissolved in DMSO (Sigma) to give a stock o f lOOmg/ml. From this, aliquots o f lOpg/pl were done by dilution in DMSO (Sigma) and stored at -20°C.
10-4-Expansion of Belxl and Belx2 clones using allogenic feeder system Belxl and belx2 clones, CD4+ T-cells specific for HPV16 E6127-141 peptide, were expanded in a T75 flask. 2xl07 irradiated PBMC feeders mixture (from 3 donors) were cultured in 50ml AB-RPMI medium supplemented with 20U/ml o f IL-2, 0.5pg/ml Purified Phytohaemagglutinin (PHA). 1 x 106 T-cells were added to the flask which was placed into a 37°C incubator tilted at 45° so as to enhance cell-cell contact. On day 5 o f expansion, half o f the medium was removed and replaced with the same volume o f AB- RPMI medium supplemented with 20U/ml o f IL-2. Cells were re-suspended and cultured for further 2 days upright. Cells were then harvested and plated out in fresh AB-RPMI
2-3 days when media turned yellow, half o f the media was replaced by AB-RPMI medium supplemented with 20U/ml of IL-2.
10-5-Expansion of HC3 and HC6 clones using allogenic feeder system
HC3 and HC6 clones, CD4+ T-cells specific for influenza peptide (HA 100 and HA107), were expanded in a T75 flask. 2xl07 irradiated PBMC feeders mixture (from 3 donors) were cultured in 50ml X-VIVO 15 RPMI medium supplemented with 20U/ml of IL-2, 0.5pg/ml PHA. lxlO6 T-cells were added to the flask which was placed into a 37°C incubator tilted at 45° so as to enhance cell-cell contact. On day 5 o f expansion, half o f the medium was removed and replaced with the same volume o f X-VIVO 15 RPMI medium supplemented with 20U/ml o f IL-2. Cells were re-suspended and cultured for further 2 days upright. Cells were then harvested and plated out in fresh X-VIVO 15 RPMI medium supplemented with 20U/ml of IL-2, at 2 x 106 cells/well, in 24 well plates.
Every 2-3 days when medium turned yellow, half of the medium was replaced by X- VIVO 15 RPMI medium supplemented with 20U/ml of IL-2.
11-DETECTION OF PEPTIDE SPECIFIC T-CELLS RESPONSE BY IFNy ELISA ASSAY
To measure the IFNy secretion induced by peptides, some co-cultures o f T-cells in presence o f APC and peptides were done. T-cells were plated out at 5xl04 cells/well in 48 well plates. B-LCL cells were added at a ratio o f 2:1 in a final volume o f 0.5 ml of AB-RPMI. Cells were cultured in the presence o f different concentrations o f peptides (Opg/ml to 10fig/ml). Cells were incubated at 37°C overnight, 1 OOjllI o f supernatant was
collected from each well and used for ELISA and 300|l l1 o f supernatant was harvested and stored at -80°C.
For ELISA, a 96 well flat-bottom Maxisorp plate (Nunc) was coated with 50jnl o f anti-human IFNy antibody 1-D1K (diluted with coating buffer (0.04M Na2C0 3, 0.06M NaHCC>3 pH 9.6) to 2pg/ml, Mabtech, Nacka Strand, Sweden) and incubated overnight at 4°C. The plate was washed twice with 200pl/well o f PBS and lOOpl/well o f blocking buffer (PBS 1 /o FCS) was added and incubated for 1 hour at room temperature (R' 1').
Plate was then washed 5 times with ELISA wash buffer (PBS 0.05% Tween) and the collected supernatants were added in triplicate at 100pl/well. For each experiment the IFNy standard (Mabtech) was used according to manufacturer instructions. Briefly, IFNy standard was reconstituted to give lOpg/ml stock solution, which was then aliquoted and stored at -20°C. One aliquot o f lp l was thawed and diluted 1 in 10,000 with dilution buffer to give a final solution at lOOOpg/ml, 200pl o f the standard solution was added in triplicate to the first wells and lOOpl of this was used to generate V2 serial dilutions of IFNy using dilution buffer. The plate with the supernatants and the standard was incubated at RT for 2 hours and then washed 5 times with 200pl/well ELISA wash buffer. The antibody 7-B6-1-biotin (Mabtech) was diluted in dilution buffer (PBS, 0.1%
BSA) at lpg/ml, added at 50pl/well and incubated for 1 hour at RT. After the incubation the plate was washed 5 times and 50pl/well o f substrate mix (equal volume o f substrate reagent A and substrate reagent B mixed at RT, BD biosciences) were added and incubated in the dark for 10 to 30 minutes, until the standard had clearly developed. The reaction was stopped by adding 50pl/well o f ELISA stop reagent (1M phosphoric acid).
The optical density was then analysed using a Bechenmark Microplate reader (Bio-Rad, Watford, UK) at 450nm. A standard curve was created with Graphpad Prism® using the serially diluted IFNy standard, allowing the determination o f the IFNy concentration in the collected samples.