2 MATERIALS AND METHODS 33
2.11 Flow cytometric analysis 49
Samples were acquired on a BD FACSCanto™ II flow cytometer (BD Biosciences) using the FACSDiva software (BD Biosciences). Data analysis was performed on FlowJo version 5.7 (Tree Star Inc, US). Anti-‐ mouse Igκ antibody capture beads (BD Biosciences) were used to prepare individual compensation tubes for each mAb used in the experiment.
2.11.1 Labelling cells with fluorescence conjugated antibodies
Cells were washed in FACS buffer (700 xg 3 min), counted by trypan blue exclusion and transferred to either a 96 well plate or 5mL FACS tube (Elkay Laboratory Products Ltd, UK) and washed in FACS buffer (700 xg for 3 min). Cells were washed in PBS and stained 5min at room temperature with LIVE/DEAD® Violet (Life Technologies) (diluted 1:40 in PBS) for dead cell exclusion before surface staining (20 min on ice and in the dark) with relevant mouse anti-‐human mAb.
The following mouse-‐anti human mAbs were used depending on each experiment:
mAb specificity Fluorochrome Clone Supplier
CD3 PerCP BW264/56 Miltenyi Biotec
CD8 APC-‐Vio770 APC PE BW135/80 Miltenyi Biotec CD4 PE-‐Vio770 FITC APC
VIT4 Miltenyi Biotec
αβ TCR PE
FITC APC
IP26 Biolegend
INF-‐γ APC 45-‐15 Miltenyi Biotec
CD107a PE H4A3 BD Bioscence
TNF-‐α PE-‐Vio770 MAb11 Bioscence
CD19 Pacific Blue HIB19 Biolegend
The following mouse-‐anti human TCRBV mAbs (Beckman Coulter), PE-‐ or FITC-‐conjugated, were used for TCR β-‐chain scanning: (Arden et al., 1995; Folch and Lefranc, 2000)
Vβ IMGT nomenclature Clone Fluorochrome
1 TRBV9 BL37.2 FITC 2 TRBV20-‐1 MPB2D5 FITC/PE 3 TRBV28 CH92 FITC 4 TRBV29-‐1 WJF24 PE 5.1 TRBV5-‐1 IMMU 157 FITC 5.2 TRBV5-‐6 36213 FITC 5.3 TRBV5-‐5 3D11 PE 7.1 TRBV4-‐1 ZOE FITC
7.2 TRBV4-‐3 ZIZOU4 PE 8 TRBV12 56C5.2 FITC 9 TRBV3 FIN9 PE 11 TRBV25 C21 FITC 12 TRBV10 VER2.32.1 FITC 13.1 TRBV6-‐5 IMMU 222 FITC 13.2 TRBV6-‐2 H132 PE 13.6 TRBV6-‐6 JU74.3 FITC 14 TRBV27 CAS1.1.3 FITC 16 TRBV14 TAMAYA1.2 FITC 17 TRBV19 E17.5F3.15.13 FITC 18 TRBV18 BA62.6 PE 20 TRBV30 ELL1.4 PE 21.3 TRBV11-‐2 IG125 FITC 22 TRBV2 IMMU 546 FITC 23 TRBV13 AF23 PE
Cells were washed twice in FACS buffer and finally resuspended in 100-‐200 µL of PBS. Cells were kept on ice in the dark (or fixed in 2% PFA) until flow cytometric analysis.
2.11.2 Intracellular cytokine staining (ICS) assay
Cells were washed from culture medium and rested overnight in R5 prior to activation. Subsequently, cells were incubated at 37 °C, 5% CO2 for 4 h, with and without target cells, at a 1:1 ratio, in R5 containing GolgiStop™ and GolgiPlug™ (BD Biosciences), according to manufacturer’s instructions. Cells were then stained with LIVE/DEAD® Violet (Life Technologies) and Abs against desired cell surface markers. Cells were prepared for ICS by incubation with Cytofix/Cytoperm™ (BD Biosciences) according to manufacturer’s instructions, before staining for 20 min on ice with mouse anti-‐human IFNγ-‐APC mAb (Miltenyi Biotec). Cells were resuspended in PBS (or fixed with 2% PFA and stored overnight at 4 °C in the dark) before flow cytometry and data analysis.
2.11.3 Blocking antibody assay
HLA-‐restriction of CTL-‐mediated killing was achieved by pre-‐incubating the target cells with HLA specific antibodies for 1 h at 37 °C, 5% CO2 before addition of effector cells. The following monoclonal antibodies were used for blocking assays: anti-‐HLA-‐A, B, C (clone W6/32, Biolegend) and anti-‐HLA-‐DR, DP, DQ (clone Tü39, Biolegend) at a final concentration of 10 µg/mL.
2.11.4 pMHC tetramer staining
Soluble biotinylated pMHC-‐I were produced as previously described in Section 2.1.8. Peptide-‐MHC-‐I tetramers were assembled over five separate 20 min steps with the successive addition of streptavidin, APC or PE conjugates (Life Technologies) to monomeric pMHC at a molar ratio of 1:4. The desired number of cells, typically 0.5–1x105 of a T-‐cell clone or 1–3x106 TILs, was transferred to flow cytometry tubes and cells washed with FACS buffer (700xg 3 min). Cells were treated with the PKI (protein kinase inhibitor) (Dasatinib, Axon Medchem, Reston) at a final concentration of 50 nM for 30 min at 37 °C and then stained with tetramer without washing. Treatment with PKI prevents TCR triggering and internalization of the TCR and any pMHC tetramer bound to it. PKI is unstable when stored at 4 °C, so 1 mM DMSO aliquots of PKI were stored at -‐80 °C and working aliquots of 100 nM were prepared in PBS for each experiment. Tetramer concentrations ranged from 0.02 to 2.4 mg (0.4–48 mg/mL with respect to the monomeric pMHC concentration) per stain in 50 μL FACS buffer, and typically 0.5mg was used. Following tetramer addition, cells were placed on ice and in the dark for 30 min. All subsequent Ab staining of the cells was performed for 20 min on ice and in the dark. Cells were washed first in FACS buffer and then PBS before LIVE/DEAD® Violet stain (Life Technologies). Following a 5 min incubation at room temperature in the dark, mAbs against cell-‐surface markers were added directly without washing and incubated on ice for 20 min in the dark. Samples were prepared for flow cytometry by washing twice in FACS buffer and resuspended in PBS or 2% PFA.
2.11.4.1 “Boosted” pMHC tetramer staining
A “boosted” protocol was used to enhance tetramer staining intensity of T-‐cell populations and T-‐cell clones described in Chapter 4. A schematic diagram is shown in Figure 2.4 (Tungatt et al., 2015).
Figure 2.4. Schematic representation of the ‘boosted’ pMHC tetramer staining protocol used
(adapted from Tungatt et al., 2015).
Alongside a standard pMHC tetramer, the staining protocol includes the binding of a mouse anti-‐fluorochrome unconjugated primary (1 ̊) antibody to the pMHC multimer associated fluorochrome, followed by a goat anti-‐mouse conjugated secondary (2 ̊) Ab.
TCR Fluorochrome Conjugated32 ̊ antibody Unconjugated31 ̊ antibody pMHC3tetramer
Briefly, post–pMHC tetramer staining, the cells were washed in FACS buffer (700 xg 3 min) and labelled with anti-‐fluorochrome unconjugated primary Ab for 20 min on ice in the dark. Following two washes in FACS buffer, the anti-‐Ab conjugated secondary Ab was added and incubated for 20 min on ice in the dark. Primary (1°) unconjugated mAbs were used at a concentration of 10 mg/mL (0.5 mg/test). A goat anti-‐mouse conjugated secondary (2°) Ab was used at 2 mg/mL (0.1 mg/test). The fluorochrome conjugated to the 2° Ab was matched to the one used for the initial pMHC multimer staining. Both anti-‐fluorochrome and anti-‐Ab antibodies were spun at maximum speed in a micro-‐centrifuge for 1 min to remove any aggregates before staining cells.
The following primary and secondary antibodies were used in Chapter 4:
Primary (1˚) unconjugated Fluorochrome Clone Supplier Mouse anti-‐PE Mouse anti-‐APC PE001 APC003 BioLegend BioLegend Secondary (2˚) conjugated
Goat anti-‐mouse PE
APC polyclonal BD Biosciences
2.11.5 Viable sorting of tumour reactive T cells
Autologous tumour cells were plated 1x106/well in a 24 multi-‐well plate in R10 the day before sorting. TIL samples or autologous PBMC were rested overnight in R5 medium. Cells were then stimulated with autologous tumour (1:1) in the presence of 15 µL of anti-‐TNF-‐α PE-‐Cy7 (clone Mab11, BD Biosciences), 15 µL CD107a PE (clone H4A3, BD Biosciences) and 10 µM of TAPI-‐0 (Calbiochem) for 4h at 37 °C, 5% CO2. Note that following the 5-‐hour incubation period with anti-‐TNF-‐α mAb, cells were not re-‐stained with anti-‐TNF-‐α in any subsequent steps. Following incubation, cells were LIVE/DEAD® Violet stained and surface stained with anti-‐CD3 PerCP (clone BW264/56, Miltenyi Biotec), anti-‐CD8 APC Vio770 (clone BW135/80, Miltenyi Biotec), anti-‐αβTCR FITC (clone IP26, Biolegend) and anti-‐γδTCR APC (clone 11F2, Miltenyi Biotec) mAbs. Cells from each experimental condition were washed and sorted directly into microfuge tubes containing 350 µL lysis buffer (Qiagen) and stored at −80 °C until RNA extraction.