2 MATERIALS AND METHODS 33
2.12 Analysis of human TCR V β CDR3 repertoire 53
RNA from reactive TILs and PBMC was extracted using the RNeasy Micro Kit (Qiagen), following the manufacturer’s instructions. Briefly, samples were first lysed and then homogenized. Ethanol was added to the lysates to provide ideal binding conditions. The lysates were then loaded onto a RNeasy silica membrane, to which RNA binds while all contaminants are efficiently washed away. Pure, concentrated RNA were eluted in 14 µL water. Isolation of total RNA was performed shortly after cell sorting, in order to minimize loss of RNA. Standard RNA sample handling precautions (i.e. filtered tips and certified RNAase free reagents) were used to avoid RNA degradation. All RNA isolation, cDNA synthesis and PCR preparation steps were carried out in a clean RNAase free room to prevent contamination. RNA samples were stored at -‐80 °C.
2.12.2 SMARTer™ RACE cDNA amplification
SMARTer™ (Switching Mechanism At 5' end of RNA Transcript) RACE (Rapid Amplification of cDNA Ends) kit (Clontech) was used for generating full-‐length cDNAs from TCR-‐β RNA (Zhu et al., 2001). Figure 2.5 provides a schematic representation of the experimental workflow described in this section.
2.12.2.1 First strand cDNA synthesis
Samples and reagents were kept on ice at all times. RNA samples were thawed at room temperature. The following mix was prepared for each sample:
Reagent Amount
RNA 10 µL
Oligo-‐dT 1 µL
Final volume 11 µL
The reaction tubes were placed in a thermal cycler and incubated at 72 °C for 3 min, then at 42 °C for 2 min to anneal the synthesis primer. After incubation, tubes were spun briefly. The following master mix for n reactions was prepared and 8 μL added to each tube:
Reagent Amount
5X First Strand buffer 4 µL
DTT (100 mM) 0.5 µL
dNTP (20 mM) 1 µL
RNase Inhibitor (20 U) 0.5 µL
SMARTScribe RT (100 U) 2 µL
Final volume 8 µL
1 μl of SMARTer II oligo A was added to each tube. In a thermal cycler, the tubes were incubated at 42 °C for 90 min, then 70 °C for 10 min cDNA samples were directly stored at -‐20 °C or used for the following PCR amplification step.
A
B
Figure 2.5. Experimental workflow of the SMARTer RACE approach to TCR-‐β profiling
Abbreviations: Ss, single-‐stranded; ds, double-‐stranded; F, forward primer; R, reverse primer; RT, reverse transcriptase.
(A) Schematic diagram of the reverse transcription and PCR amplification of TCR-‐β chain mRNA sequences (adapted from
Clontech Laboratories Inc. website). Single-‐stranded cDNA from a mRNA template was generated by reverse transcription using an oligo dT primer (located in the constant gene of the TCR-‐β chain) and a MMLV-‐derived RT. When the RT reaches the end of the mRNA template, it adds several non-‐templated nucleotides (indicated as “xxx”). The universal primer anneals to the tail of the cDNA and serves as an extended template for the RT. The “universal anchor” appended to the target during cDNA synthesis, allows subsequent PCR amplification steps (PCR 1 and 2) using a 5’universal primer (forward) and a 3’ Cβ -‐ specific primer. The final product was cloned into a commercial vector and transformed into bacterial cells. (B) Semi-‐nested PCR approach for amplification of TCR-‐β subunits. The primer pair (orange) used for the first PCR captures the entire variable region and some of the constant region of the TCR-‐β cDNA. The primer pair (green) used in the second PCR retains the entire TCR-‐β cDNA variable region and a smaller portion of the constant region. The expected size of final TCR-‐β library cDNA (which include the inserts and the universal adapters) is of approximately 600 bp.
variable constant mRNA ss cDNA ds3cDNA Molecular)cloning)and)bacterial)transformation trb sequencing RT3reaction RACE3reaction Oligo (dT))primer) +)universal3primer +)RT) 1stproduct Final)product XXX universal3anchor XXX XXX XXX XXX XXX 5’ 3’ 3’ 5’ 5’ 3’ TCR$β V D J C PCR$1 PCR$2 Region+required+for+the+identification+of+CDR3+ and+the+V+and+J+gene+segments Primer$ short$(F2) Cβ$R1 Cβ$R2 Universal$primer$A$(F1)$
2.12.2.2 First PCR amplification
The first PCR amplification captures the entire variable region and some of the constant region of the TCR-‐β cDNA. A PCR mastermix was prepared as shown below:
Reagent Amount
Phusion® 5X Green buffer 10 µL
DMSO (100 mM) 0.5 µL
dNTPs (20 mM) 1 µL
10X Universal Primer A (F) 5 µL
Primer Cβ-‐R1 (R) 1 µL
Phusion® HF DNA polymerase 0.25 µL
H2O 29.75 µL
cDNA sample 2.5 µL
Final volume 50 µL
The following cycling conditions were used:
4°C; initial denaturation 94°C; 30 s 30 cycles 63°C; 30 s 72°C; 3 m 72°C; final extension 2.12.2.3 Second PCR amplification
The second PCR amplification used the DNA from the first round of amplification as template. It captures the entire variable region and a smaller region of the constant TCR-‐β cDNA. A PCR mastermix was prepared as shown below:
Reagent Amount
Phusion® 5X Green buffer 10 µL
DMSO (100 mM) 0.5 µL
dNTPs (20 mM) 1 µL
Primer A Short (F) 1 µL
Primer Cβ-‐R2 (R) 1 µL
Phusion® HF DNA polymerase 0.25 µL
H2O 33.75 µL
DNA sample 2.5 µL
Final volume 50 µL
The following cycling conditions were used:
94°C; initial denaturation 94°C; 30 s 30 cycles 66°C; 30 s 72°C; 3 m 72°C; final extension
2.12.2.4 Agarose gel electrophoresis
Electrophoresis gels were prepared with 1% agarose powder (Invitrogen) dissolved in (Tris-‐acetate EDTA)
TAE buffer. For DNA visualisation, Midori Green nucleic acid staining solution (GeneFlow) was added before allowing the gels to set. Samples were allowed to run at 80V for 45 min on the gel, along with 5 μL of 1 Kb DNA HyperLadder™ (Bioline). Gels were visualised under a LED-‐based illuminator (FastGene) and bands cut out using a disposable scalpel (Figure 2.6).
Figure 2.6. Agarose gel analysis of SMARTer RACE cDNA products
Representative gel of PCR amplified TCR-‐β cDNA products from PBMC samples of three melanoma patients (plus and minus stands for the tumour-‐reactive or non-‐tumour reactive population, respectively). Lane 1: molecular weight ladder (MW). An empty lane was kept between samples to avoid cross-‐ contamination during gel loading and electrophoretic run. Water was used as a negative control. Arrow heads indicate the expected TCR-‐β band of approx. 600 bp.
2.12.2.5 DNA extraction from gel bands and purification for cloning
Amplified products were extracted from the agarose gel and purified for subsequent cloning steps. The NucleoSpin® Gel and PCR Clean-‐up kit (Clontech) was used to extract DNA from gel bands, following manufacturer’s instructions. Briefly, the cut-‐out gel band was mixed with Binding Buffer NTI (200 µL/100 mg) and heated at 50 °C for 10 min to dissolve the agarose. In the presence of chaotropic salt, the DNA sample was bound to the silica membrane of a NucleoSpin® column. Contaminations were removed by washing steps with ethanolic Wash Buffer NT3. Finally, pure DNA was eluted under low salt conditions with slightly alkaline Elution Buffer NE (5 mM Tris/HCl, pH 8.5).
2.12.3 Molecular cloning and bacterial transformation
2.12.3.1 Zero Blunt® TOPO® PCR Cloning
Blunt-‐end PCR products were cloned into a pCR™-‐Blunt II-‐TOPO® vector using a Zero Blunt® TOPO® PCR Cloning Kit (Invitrogen), following manufacturer’s instructions. Briefly, a topoisomerase I–based 10-‐minute ligation was performed in order to directly insert the blunt-‐ended PCR products into a plasmid vector. MW 1$Kb 1$Kb
The following 6 µL TOPO® Cloning reaction was set up for each sample: Reagent Amount Fresh PCR product 4 µL Salt solution 1 µL pCR™-‐Blunt II-‐TOPO® 1 µL Final volume 6 µL
The tubes were incubated for 10 min at room temperature, then placed on ice to stop the reaction. TOPO® Cloning reactions were then transformed into chemically competent One Shot® TOP10 E. coli cells (Invitrogen).
2.12.3.2 Transformation of One Shot® TOP10
Chemically competent E. coli cells (One Shot® TOP10) were transformed following the manufacturer’s instructions (Invitrogen). Briefly, one vial of One Shot® E. coli cells was thawed on ice for each transformation. 2 µL of the TOPO® Cloning reaction were added to each vial of cells to be transformed, and mixed gently. The vials were incubated on ice for 30 min and then heat-‐shocked for 30 sec at 42 °C without shaking. 250 µL of room temperature S.O.C. medium (supplied with the kit, Invitrogen) were added to the cells. The tubes were capped and shaken at 37 °C for 1h. 150 µL from each transformation were spread on pre-‐warmed LB-‐Agar plates containing 50 µg/mL kanamycin selective antibiotic. Plates were incubated overnight at 37 °C. An efficient TOPO® Cloning reaction would produce several hundred colonies. Approximately 96 colonies were picked for analysis of transformants. Each colony consists of bacteria transformed with one (and only one) tr sequence.
2.12.3.3 Colony PCR
Colonies containing the insert were screened and selected for amplification and sequencing. Single bacterial colony plasmid inserts were amplified in a sealed 96-‐well plate format (Applied Biosystems) using primers flanking the insertion site (M13F: 5'-‐TTTTCCCAGTCACGAC-‐3'; M13R: 5'-‐ CAGGAAACAGCTATGAC-‐3’). Care was taken in order to pick only single colonies and to avoid cross-‐ contamination between wells. Colony PCR reactions were prepared as follows:
Reagent Amount
Primer M13F 1 µL
Primer M13R 1 µL
DreemTaq Green master mix 23 µL
Final volume 25 µL
Colony PCR reactions were run in a thermocycler as follows:
5 µl of several individual products from plate were run on an agarose gel. Positive colonies were sent for sequencing (Eurofins Genomics, Ebersburg, Germany).
2.12.4 Sequence analysis
TCR-‐β sequences were visualized in the analysis software BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999) and analysed using IMGT/V-‐QUEST (http://www.imgt.org/IMGT_vquest), in order to identify the V, D and J segments for human TCR-‐β (Giudicelli et al., 2011). Grouped sequences for TIL and PBMC samples were ranked in a Microsoft OfficeTM Excel spreadsheet according to their frequency. Unless otherwise stated, all tr and gene segments are described using the ImMunoGeneTics (IMGT) nomenclature (Lefranc et al., 1999). Accordingly, TCR α-‐ and β-‐ chain segments are designated TRA and TRB respectively, followed by the letter V, D or J.