Friday, August 21, 2015

Pair socks from a pile: reconstructing TCRβ and TCRα pairs through sequencing

T cell receptor (TCR) β and α chains are generated in the thymus (some in the gut) via randomly rearranged V, J, D, C segments (unlike TCRβ chains, TCRα chains lack D segments). After rearrangement, some TCRβ and TCRα chains form productive pairs, we call TCRs. Note, not every TCRβ and TCRα chains can form productive pair. Afterwards, T cells expressing surface TCR undergo positive and negative selection and enter blood circulation to start patrolling the body.

Advances in personalized medicine created a niche to study TCR specificity in clinical setting. This is especially true for cancer therapy where adoptive transfer of ex vivo expanded tumor antigen specific T cells show protection against various lymphoid malignancies.

Ordinarily TCR specificity are elucidated by tetramer or hybridoma technologies, followed, if necessary, by TCR sequencing. These two, separate steps ensure selective expansion of T cell clones of interests. However, these methods are (1) technically difficult to accomplish (few scientists have "good" hands for in vitro experiments), (2) time consuming and (3) limited in scale (10-100 specificity). 

Another method is a direct TCRβ and TCRα massive-parallel sequencing. In this regard, sequencing technologies developed by Adaptive biotechnologies from Seattle has been cited by many high profile academic papers. 

However, TCR sequencing (typically only TCRβ sequencing) per se cannot provide an answer about TCR specificity, i.e. which TCRβ chain pairs with what TCRα chain. The knowledge of genetic structure of both chains in TCR, however, would allow reconstruction of 3D structure of TCR and it's specificity determination with algorithms that work similar to MHC+peptide algorithms.

The methods itself is based on simple idea, if I understood it correctly. It requires some statistical analysis but principle works the following way: 

1. T cells are collected and distributed among 96 wells. Number of T cells per well can very based on statistical analysis. 
2. In 96 well plate, each well contains an unique small oligo DNA barcode that will be amplified along side with T cells derived cDNA. 
3. T cell derived cDNA is amplified with primers specific for TCR V and C regions and then sequenced.

Afterwards, if every time a particular Vβ and Vα genes are detected together in the same randomly barcoded wells, it is assumed that those two are pairs. This is in principle. Since there are potentially hundreds of of thousands unique TCR pairs, validation of experimental design is critical. In my view, strict validation of this type of readout would require actual determination of pairing using tetramer catch or hybridoma based expansion of T cells and then specific sequencing of recovered clones. 

Validation step the authors suggested is not entirely clear from paper description and may not be sufficiently robust to be accept as a gold standard for pairing. Another weakness is amplification step since it is not clear whether every V and C primer could amplify the target region with the same optimal rate.

David Usharauli

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