Chasing the chains by testing them twice: Immunological insights from TCR repertoire analysis

Abstract

The adaptive immune system employs a large number of T cells, that each have a specificity defined by their T-cell receptor (TCR). Characterizing the TCR repertoire in humans and mice thus provides a way to address outstanding immunological questions. The repertoires can be assessed quantitatively using high-throughput sequencing. Interpretation of the resulting data is challenging due to the vast diversity of the TCR repertoire and the relatively small number of cells that can be analyzed in an experiment. In this thesis we analyze TCR repertoires from humans and mice, by integrating information obtained from multiple samples. In Chapter 2 we devise various models for the distribution of TCR clone sizes in the human naive T cell pool. We characterise the repertoire of TCR α and β chains by repertoire sequencing of various cell subsets. Comparing the model predictions with the experimental outcomes, we find solid evidence for the presence of very large TCRαβ clones in the naive T cell repertoire. These large naive T-cell clones are only partly explained by their generation probability. Chapter 3 aims to identify sequence characteristics of such abundant naive T cell clones. We find that the D segment is missing in a substantial fraction of the abundant TCR β sequences in the naive T cell repertoire of young individuals. Such sequences appear to be mostly generated before birth, to persist over a human lifetime, and, as a result, to be excessively shared between individuals. Chapter 4 is a short unpublished report on quantifying the effects of age on the TCR repertoire. We present example analyses and discuss potential pitfalls of assessing ageing effects on the TCR repertoire. Chapter 5 describes a pilot study on using TCR sequencing to follow the T-cell response upon pneumococcal conjugate vaccination. We define quantitative requirements to classify T-cell expansions but detect these in only a minority of the donors. Our analysis suggests that the vaccine-induced T-cell response is small and/or very broad, and highlights experimental requirements to characterise such responses in future studies. A key limitation of the studies in humans introduced above is the limited number of cells that can be analysed compared to the total size of the T-cell pool. As a result, many of the TCR chains that make up the diversity of the TCR repertoire will be missed in any analysis. Bulk sequencing methods also do not uncover the full TCR diversity present in an individual as the information on the pairing of α and β chains is not obtained. In Chapter 6 we overcome both limitations by studying the TCR repertoire of one-TCRa mice that have a strongly reduced receptor diversity. This allows us to study the nearly complete TCR diversity, and compare the repertoire among individual lymph nodes and mice, revealing a spatial organisation of the TCR repertoire. In summary, this thesis describes how we studied T-cell biology quantitatively by combining simplified models with careful bioinformatics analyses.