This method can help answer key questions in the immunology field, such as the TCR alpha and beta repertoire of an antigen's specific population, as well as the in vivo functionality of human TCRs. The main advantages of this technique are the relatively high efficiency of the PCR reaction as well as its compatibility with subsequent TCR cloning. This protocol uses a two-step PCR to amplify the TCR alpha and beta chains from a single cell without prior knowledge of the TCR sequence.
The first PCR is a multiplex reaction using a pool of alpha or beta variable region-specific primers in conjunction with a primer specific for the constant region. The second PCR is a nested reaction that primes off of the adapter sequence incorporated at the five prime end of the PCR product as well as an internal constant region sequence. These primers also incorporate unique restriction sites into the five prime and three prime ends of the PCR product to facilitate subsequent cloning.
Once cloned into the template vector, the encoded TCR sequence is chimeric, consisting of human variable regions with mirroring constant regions. The chimeric TCR construct allows for interaction between the TCR and the mirroring CD3 signaling complex, making this TCR cloning protocol compatible with subsequent functional studies in mirroring cell lines and in humanized mouse models. Set up for the single cell sorting by preparing the reverse transcription reagents in a clean, template-free area.
Generally individuals new to the single-cell PCR method will struggle because of the low levels of template. On the other hand, the sensitivity of the multiplex nested PCR is sensitive to low levels of template contamination. Therefore, it is necessary to thoroughly clean the areas in which you will be setting up the PCR reactions.
A mixture of three reverse primers specific for the constant region of the alpha and beta chains are used in this reaction. Prepare the reverse transcription master-mix by combining reagents in a pipetting reservoir. To account for pipetting error, make enough reaction mixture for ten extra wells.
Use a multichannel to pipette six microliters per well into a 96-well PCR plate. Cover the plate loosely with a seal and keep the plate on ice. Prepare T cells for single cell sort by staining the cells with antibodies to cell surface markers.
Sort T cells at once cell per well while leaving the twelve row empty, which will serve as a negative control. Seal the plate with adhesive film and spin down at 1000g for five minutes at four degrees Celsius. Immediately run the reverse transcription reaction.
If possible, run the first PCR reaction the same day. To run the multiplex reaction, separately mix the V alpha and V beta PCR master mixes in pipetting reservoirs. Keep the plate with cDNA on ice or on a cold block.
Transfer 22.5 microliters of TCR beta reaction into a new 96-well piece PCR plate. Then use a multichannel to transfer 2.5 microliters of cDNA into the PCR plate. Use a multichannel to pipette 22.5 microliters of TCR alpha reaction directly into the plate containing the remainder of the cDNA.
Seal the plates with adhesive, gently vortex, and briefly spin down. Run the first PCR reaction. Plates can be stored at 20 degrees Celsius.
The next day, prepare the second nested PCR reactions. Using a multichannel, pipette the master mixes for the TCR alpha and TCR beta reactions at 22.5 microliters per well into two new 96-well PCR plates. Add the multiplex PCR reaction template at 2.5 microliters per well.
Seal the plates, gently vortex, and briefly spin down. The remaining first PCR reaction should be sealed and stored at 20 degrees Celsius to serve as a source of backup template. Perform the second PCR reaction.
After the second nested PCR reaction is complete, run out five microliters of the reaction on a 1%agarose gel. Include the positive and negative control wells. The expected band should run at around 500 base pairs in size.
Purify the remainder of the second reaction, twenty microliters, using a 96-well format PCR purification kit and perform Sanger sequencing. The obtained TCR sequences can be analyzed using online available immunogenetic software. The sequence information can be used to assess the repertoire and clonality of the T-cell population.
Paired TCR alpha and beta sequences can be used in further functional analysis. Perform consecutive ligations of TCR beta and TCR alpha into the template vector. The template TCR beta and TCR alpha are replaced with the PCR amplified chains.
In the first ligation, the template TCR beta is replaced with the PCR amplified beta chain. After successful insertion of TCR beta has been verified by test-digest insequencing, replace the template TCR alpha with the PCR amplified alpha chain. HEK293T cells are used to test successful TCR chain pairing and cell surface expression.
HEK293T cells are co-transfected with the TCR vector marked by the ametrine fluorescent reporter and a vector encoding the CD3 complex marked by GFP. Surface expression of the chimeric TCR is verified by flow cytometry by staining the cells with anti-mouse CD3 antibody. The level of surface CD3 expression in cells transfected with the chimeric TCR construct should be comparable to those transfected with the fully mirroring control template vector.
In summary, we have described an efficient PCR-based protocol for the identification and cloning of paired TCR chains from single cells. We anticipate that this will be valuable tool to study human T-cell responses in autoimmunity, tumor surveillance, and pathogen elimination. The main advantage of the PCR protocol described here is its compatibility with subsequent in vitro and in vivo studies.
