Tracking global changes induced in the CD4 T cell receptor repertoire by immunization with a complex antigen using short stretches of CDR3 protein sequence.

The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different TcRs in CD4+ T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcR beta chain. In order to track the changes induced by immunization within this very heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the CDR3 repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analysis of these different distributions of sequence clusters differentiated between immunised and unimmunised mice with 100\% efficiency. The CD4+ T cell receptor repertoires of mice 5 and 14 days post immunisation were clearly different from that of unimmunised mice, but were not distinguishable from each other. However, the repertoires of mice 60 days post immunisation were distinct both from naive mice, and the day 5/14 animals. Our results reinforce the remarkable diversity of the T cell receptor repertoire, resulting in many diverse private TcRs contributing to the T cell response even in genetically identical mice responding to the same antigen. Finally, specific motifs defined by short sequences of amino acids within the CDR3 region may have a major effect on TcR specificity. The results of this study provide new insights into the properties of the CD4+ adaptive T cell response.