2 MATERIALS AND METHODS 33
2.10 Functional T-‐cell assays 45
Crude (50-‐60% purity) and pure (> 90% purity) peptides used throughout this study were synthesized by GL Biochem Ltd. (Shanghai, China) and Peptide Protein Research Ltd. (Hampshire, UK), respectively. Lyophilised peptides (4 mg) were reconstituted in DMSO to a final stock concentration of 20 mg/mL and stored at -‐80 °C. Reconstituted peptides were thawed last minute on ice and a working dilution was prepared in R0 medium. HLA-‐A2-‐restricted viral peptides used as controls in this thesis are: GILGFVFTL (designated as GIL) from Influenza viruses M1 matrix protein (Bednarek et al., 1991) and NLVPMVATV (designated as NLV) from CMV virus (Diamond et al., 1997). The full list of tumour-‐ associated antigens used is provided in the Appendix.
2.10.2 IFN-‐γ Enzyme-‐Linked ImmunoSpot (ELISpot) assay
Mouse anti-‐human IFN-‐γ antibody 1-‐DIK (Mabtech) was diluted to 10 μg/mL with PBS, and 50 μL were added to each well of an ELISpot plate (PVDF-‐backed plate, Millipore). Coated plates were incubated for 4 h at 37 °C wrapped in cling film, washed thoroughly 5 times with 250 μL PBS/well, and blocked with 100 μL of R10 medium for 1 h at RT. Rested T cells (overnight at 37°C in R5) were added to the wells (105 cells) in 100 μL of R5 medium. The peptide was added at the desired concentration (10-‐5 M) to appropriate wells in 20 μL of R0 medium. The final volume (200 μL/well) was made up in R5 medium. Phytohaemagglutinin (PHA) (Sigma Aldrich) 2 pg/mL was added to the positive control wells; T cells only were added to the negative control wells. ELISpot wells were set up in duplicate. Control wells that did not require peptide received media alone, so that the total volume is 200 μL. The plate was then wrapped in silver foil and incubated at 37 °C for 16-‐18 hours. Plates were washed 3 times with 150 μL PBS and incubated with 100 μL sterile water for 10 min at room temperature (therefore lysing the remaining bound cells). The plate was further washed twice with 150 μL/well PBS. Secondary biotinylated antibody 7-‐B6-‐1-‐Biotin (Mabtech) (1:1000 in PBS) was added at 50 μL/well and incubated in the dark at room temperature for 2 hours. The plate was washed 5 times with PBS, and 50 μL of Streptavadin-‐Alkaline phosphatase (1:1000 in PBS) was added to each well. The plates were incubated for 2 hours in the dark at RT, followed by 5 washes with 150 μL PBS. Developing solution was made fresh using 25X AP colour development buffer (Bio-‐Rad), AP-‐conjugate substrate A and B solutions (Bio-‐Rad), and sterile water. The solution was added at 50 μL/well and left to develop in the dark between 10 to 20 min, until spots were clearly visible. Developing reaction was stopped by washing plates with tap water; plates where then air dried in the dark before spot counting using an automated ELISpot counting system (AID ELISpot reader, Cadama Medical).
2.10.3
51Chromium
(
51Cr) release cytotoxicity assay
Functional assays to determine specific T-‐cell killing of target cells were carried out by 51Cr release cytotoxicity assay. Effector T cells were rested overnight in R5 when supernatant was harvested for ELISA the following day. Assays were performed in duplicate for each sample in a 96-‐well round-‐ bottomed plate. Briefly, target cells (2000/well) were labelled for 1 hour at 37 °C with 30 µCi 51Cr (sodium chromate in normal saline, Perkin Elmer) per 1x106 cells, washed with R10 and allowed to leach for a further hour at 37 °C in R10 to remove any excess 51Cr from the cells. After 51Cr labelling, target cells were washed, resuspended in R5, and plated with effector T cells at the desired effector-‐ to-‐target (E/T) ratio in a final volume of 150 µL of R10. Target cells were also incubated with 5% Triton X-‐100 detergent or alone to give the total and spontaneous 51Cr released from the target cells respectively. After a 5-‐ and/or 18-‐hour incubation at 37 °C, 5% CO2, the supernatants were harvested (15 μL), mixed with 150 μL of Optipahse Supermix Scintillation Cocktail (Perkin Elmer) in 96-‐well polyethylene terephthalate plates (Perkin Elmer) and sealed. The amount of released 51Cr was measured indirectly on a 1450-‐MicrobetaTM counter (Perkin Elmer). The percent-‐specific target cell lysis by T cells was calculated according to the following formula: (experimental release – spontaneous release) / (maximal release – spontaneous release) *100 = % specific lysis.
2.10.4 Peptide activation assay by Enzyme Linked Immunosorbent Assay (ELISA)
T-‐cells were washed with R0 medium and rested in R5 medium overnight before being used in an activation assay. In a single well of a 96 round bottom-‐well plate, 3x104 T-‐cells were primed with 6x104
antigen presenting cells per 100 μL of R5 medium and a titration of peptide from 10-‐5 to 10-‐12 M for 13 h at 37 °C, 5%CO2. The plate was centrifuged at 300 xg 3 min to sediment cells to the bottom of the well. 50 μL/well of supernatant was harvested and diluted with 70 μL of R0 medium. Supernatants were analysed by ELISA (described in section 2.10.5).
2.10.5 MIP-‐1β/TNF-‐α ELISA
MIP-‐1β/TNF-‐α ELISA were performed using DuoSet® human ELISA kit following manufacturer’s instructions (R&D Systems). All washes were performed with 190 μL of 0.05% Tween 20-‐PBS (wash buffer) using an automated microplate washer. Briefly, a half-‐well flat bottom ELISA microplate (Corning Costar) was coated with 50 μL of mouse anti-‐human MIP-‐1β/TNF-‐ α capture antibody (1.5 μg/mL) and incubated overnight at RT. The plate was washed 3 times, 150 μL of 1% BSA-‐PBS (reagent diluent) solution was added to each well and the plate was incubated for 1 h at RT. Following 3 washes, 50 μL of cell supernatant collected from an activation assay was added to each well and incubated 1 h and 15 min at RT. A recombinant human MIP-‐1β (or TNF-‐α) standard (R&D Systems) was titrated from
Figure 2.2. Example of MIP-‐1β standard curve
The linear regression line equation (y= m*x) was calculated and used to interpolate MIP-‐1B concentrations for each sample.
The plate was washed 3 times and coated with 50 μL biotinylated goat anti-‐human MIP-‐1β (or TNF-‐α) (50 ng/mL) detection antibody. After 1h and 15 min incubation at RT, the plate was washed 3 times and HRP-‐conjugated streptavidin (50 µL) was added to the wells. The plate was incubated in the dark for 20 min at room temperature followed by 3 final washes. 50 μL of a 1:1 ratio colour reagents A and B (R&D Systems) were added to each well and incubated for a maximum of 15 min at RT. The reaction was blocked by adding 25 μL of stop solution (R&D Systems). The OD450nm of each well was read using a Bio-‐rad iMark microplate reader with correction set to 570 nm.
2.10.6 Combinatorial peptide library (CPL) scans
T cells were challenged with either a 9-‐mer or 10-‐mer combinatorial peptide library (CPL) in positional scanning format (Pepscan Presto Ltd) and their activation towards each mixture was evaluated by the release of MIP-‐1β chemokine by ELISA (described in section 2.10.5). The decamer CPL contains a total of 9.36 x1012 (= (10+19) x199) different decamer peptides and is divided into 200 different peptide mix. In every peptide mixture, one position has a fixed L-‐amino acid residue and all other positions are degenerate, with the possibility of any one of 19 natural L-‐amino acids being incorporated in each individual position. The cysteine residue was omitted from the random positions to avoid formation of disulphide bonds between peptides within the mixture and peptide aggregation. Each peptide mix consists of 3.2 x1011 (199) different decamer peptides in approximately equimolar concentrations. Figure 2.3 depicts a 10-‐mer CPL.
OD MI P -1 β ( pg/m l) 0.0 0.2 0.4 0.6 0.8 0 500 1000
Figure 2.3. Schematic diagram of a 10mer combinatorial peptide library
Each of the 20 natural proteogenic amino acids at each position and all possible combinations of the other amino acids in the other positions. For instance, sub-‐library 1 has an Alanine at P1 and an equimolar mix of amino acids at all the other positions. Sub-‐Library 200 has Tyrosine at P10 and an equimolar mix at other positions. Each sub-‐library contains 3.2 x 1011 different peptides.
CD8+ T cells were washed with R0 medium and rested in R5 medium overnight. Antigen presenting cells were plated in single well of a 96 round bottom-‐well plate at a concentration of 6x104 cells per 45 μL and pulsed with 5 μL of peptide mixture from a decamer peptide library at a final concentration of 100 μM for 1 hour at 37 °C, 5% CO2; 3x104 CD8+ T cells (50 μL) were added to pulsed antigen presenting cells. Plates were incubated overnight at 37 °C, 5% CO2 and centrifuged at 300 xg for 5 min.
Subsequently, the supernatant (50 µL) was harvested and assayed for MIP-‐1β by ELISA according to the manufacturer's instructions (R&D Systems) (Section 2.10.5).
2.10.6.1 Epitope identification
A novel webtool (PI CPL) developed by Dr Barbara Szomolay in Cardiff, was used to link the raw data of CPL scans to the likelihood of the peptide to bind the cognate T-‐cell clone (Szomolay et al., 2016). The webtool is integrated into the WSBC webtools and is accessible at http://wsbc.warwick.ac.uk/wsbcToolsWebpage. Human self databases were compiled on the basis of public available protein sequence databases provided by NCBI (National Center for Biotechnology Information), UniProt (Universal Protein Resource), and PDB (Protein Data Bank).