A quantitative framework for lymphocyte fate decisions

During an adaptive immune response activated B and T lymphocytes undergo rapid clonal expansion and generate extensive cellular heterogeneity. How lymphocytes guarantee the emergence of functional diversity amongst responding cells is not fully understood. In this thesis, the strategies utilised by the adaptive immune system for the diversification of B and T cells is investigated at the cellular, molecular and clonal levels in a quantitative manner. Activated B cell heterogeneity is predominantly driven by two critical programs. Firstly, the differentiation of antibody-secreting cells (ASCs) and secondly, the diversification of antibody isotype by class switch recombination (CSR). The regulation of these two processes was investigated through combined clonal and molecular analysis using a high-throughput proliferative lineage tracing approach to study ASC differentiation and CSR across thousands of clones. Two distinct fate programs emerged. Firstly, the timing of ASC differentiation within clones was strongly correlated. Diversity in commitment to the ASC lineage is established early and could be traced to the naive founder cell, from where it is transmitted to all progeny during clonal expansion. In striking contrast, isotype switching was highly variable across related cells irrespective of common ancestry, revealing a highly stochastic, cell-autonomous process regulated late within activated single cells. Further analysis demonstrated that single cells faced with a choice of two heavy chain isotypes solve the conflict using stochastic selection that is independent of their clonal lineage. As the principle molecular drivers of CSR are well known, their variation amongst single cell within clonal families was measured. Extensive variation was demonstrated in the expression of both activation-induced cytidine deaminase (AID) and the transcription of the germline noncoding RNAs. Furthermore, there was no correlation between AID expression and germline transcription