A Practical Approach to T-Cell Receptor Cloning and Expression

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5′-RACE amplification. We here present an improved 5′-RACE protocol that represents a fast and reliable way to identify a TcR from 105 cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality

List of primer sequences and their use and/or specificity.

a<p>I is desoxyinosine.</p>b<p>These primers are designed when clone sequence is known.</p>c<p>Sequence in italic is V-chain specific.</p

Validation of the recombination-system.

<p>(a) SupT1 cells were transduced with the indicated retroviral supernatant or mock-transduced (grey) by spinoculation and cultured for 2 days. They were then washed and stained with anti-TcR-PE and analyzed by flowcytometry. DMF5 inserted into MSGV or MSGV-G vectors resulted in comparable expression. (b) Three days post-transduction, SupT1 were co-stained with anti-CD3 PB and HLA-A2/MART-1 multimer-APC. SupT1 transduced with an irrelevant TcR were used as a multimer negative control. The percentage of cells is shown in each quadrant.</p

TcR composition.

#<p>: frameshift.</p><p>nd: not determined.</p

Electroporation of mRNA encoding MART-1 specific TcRs.

<p>(a) SupT1 were electroporated with or without 20 µg mRNA encoding the indicated TcR_2A. Twelve hours later, the cells were stained with HLA-A2/MART-1 multimer-PE and anti-CD3 Alexa Fluor 647. (b) Human PBMC were electroporated with 20 µg mRNA (same constructs as in (a)) and cultured for 4.5 hours. Following the addition of T2 cells pre-loaded or not (grey) with MART-1 peptide (10 µM final concentration), the cells were co-incubated for an additional 5 hours in the presence of anti-CD107a/b Alexa Fluor 647 antibodies, monensin and brefeldin A. Prior to analysis, cells were stained with anti-CD8 PE. The percentage of CD107a/b positive cells from the CD8 positive population is plotted. PHA was used to control for similar maximal degranulation levels regardless of the mRNA used for electroporation. Each bar represents the mean values of duplicates.</p

TcR structure and cloning strategy.

<p>(a) TcRα and β mRNA structures are similar: The 5′-situated Variable (V) domain encodes for the N-terminal part of the extracellular domain (EC) of the TcR. It starts with the signal sequence (L) upstream of the V-region. It ends at the recombination site with the D domain (only in TcRβ) and Joining domain (J), representing the hypervariable CDR3 domain. On the 3′-side of the messenger lies the Constant (C) region, which encodes for the carboxy-terminal part of the EC, the transmembrane region (TM) and the short intracellular domain (IC). (b) The 5′-RACE of the TcR is performed by reverse-transcription of mRNA into cDNA using an oligo dT primer. The cDNA is then polyC-tailed by TdT and this reaction is followed by two sequential amplifications using nested primers (pC1 and pC2) together with a polyC annealing primer (pGI). The final product is cloned into pTA and sequenced. The sequenced portion results in the full length V-domain and the CDR3 if the tailing has occurred on the 5′-UTR.</p

Retroviral delivery of MART-1 specific TcRs.

<p>(a) B44, B60 and DMF5 TcR_2A were expressed in the pMP71-G vector and transduced into SupT1 cells. After 3 days, cells were co-stained with anti-CD3 PB and HLA-A2/MART-1 multimer-PE. (b) Jurkat cells were transduced with the same constructs as in (a) or with a GFP pMP71-vector (Mock) and incubated for 24 hours with T2 cells loaded with or without MART-1 peptide (10 µM final concentration). IL-2 release was monitored by ELISA assay, and plotted as pg of IL-2 per mL of medium. Each bar represents the mean values of duplicates. Similar results were observed in two separate experiments.</p