Exhaustive T-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes

Abstract

Massively parallel sequencing offers a useful approach to characterizing TCR diversity. However, immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and may differ legitimately by only a single nucleotide. As such, the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy. We obtained peripheral blood TCRB mRNA from a healthy donor, at two timepoints 1 wk apart, and generated from multiple libraries a total of 1.7 billion paired sequence reads. The sequencing error rate was determined empirically, by analyzing the portion of each read encoding one of a small number of possible J segments, and used to inform a high stringency data filtering procedure. From the error filtered data, we obtained 1,061,522 distinct TCRB nucleotide sequences. This figure establishes a new, directly measured lower limit on individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. Analysis of naïve (CD45RA+/CD45RO−) and memory (CD45RA−CD45RO+) T-cell fractions highlighted T-cell plasticity, whereby sequences that were highly represented in both subsets at week 1 had transitioned preferentially to the CD45RA−/CD45RO+ subset at week 2. TCRB nucleotide sequences obtained from two additional donors were compared with those from the first donor and revealed highly similar V and J gene usage frequencies among individuals, but only a very small proportion (<1.1%) of shared nucleotide sequences. Analysis of in silico translated sequences indicated, however, that at the amino acid level as many as 14.2% of distinct sequences from one donor were shared with those from another donor. For each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unshared amino acid sequences. We also observed a highly statistically significant association between numbers of shared sequences and shared HLA class I alleles.