Quantitative analysis of the T cell receptor repertoire

The T cell receptor repertoire provides a window into the cellular adaptive immune response. In the context of cancer, determining the repertoire within a tumor can give important insights into the evolution of the T cell anti-cancer response, and has the potential to identify specific personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing and analyzing T cell receptors which is economical, robust, sensitive and versatile. The key experimental step is the ligation of a single stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. We describe a detailed protocol describing this method to create libraries of T cell receptors from in vitro T cell cultures, blood or tissue samples. We combine this with a computational pipeline, which incorporates sample multiplexing, T cell receptor annotation and error correction to provide accurate counts of individual T cell receptor sequences within samples. The integrated experimental and computational pipeline should be of value to researchers interested in documenting and understanding the T cell immune response to cancer, and in manipulating it for therapeutic purposes.