Abstract
Motivation: 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 T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition.Results: To track the changes induced by immunization within this 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 antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification.Availability and implementation: The analysis was implemented in R and Python, and source code can be found in Supplementary Data.Contact: b.chain@ucl.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.
Highlights
Adaptive immunity is carried out by populations of B and T lymphocytes, which collectively express a large set of different antigen-specific receptors created during haemopoesis by a unique process of somatic cell gene rearrangements
Previous studies have distinguished between private T-cell receptors (TcRs), found in one or a few individuals, and public TcRs found within the responding repertoire of a majority of individuals
The results demonstrate a coherent but highly distributed set of responses, emerging from the background of the remarkable diversity and plasticity of the TcR-generating system
Summary
Adaptive immunity is carried out by populations of B and T lymphocytes, which collectively express a large set of different antigen-specific receptors created during haemopoesis by a unique process of somatic cell gene rearrangements. The clonal theory of immunity (Burnet, 1959) proposes that lymphocytes carrying receptors that bind an antigen to which the immune system is exposed, for example, during infection or vaccination, respond by proliferating and differentiating. A prediction of this theory is that the frequency of lymphocytes that have been exposed to antigen (i.e. memory or effector cells) will be greater than the frequency of those that have not (i.e. naive) This prediction has been verified for T cells in a wide variety of models, using antigen-specific readouts such as cytokine responses, and Major Histocompatibility Complex (MHC) multimer binding to identify expanded lymphocyte clones (Catron et al, 2004; Hataye et al, 2006; Moon et al, 2007). The response to many antigens seems to consist of a mixture of public and private specificities (Cibotti et al, 1994; Clute et al, 2010; Day et al, 2011; Menezes et al, 2007)
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