| The lymphocytes of an organism harbor a diverse collection or repertoire of antigen receptors (antibodies (Ab) on B cells and T cell receptors (TCR) on T cells). This diversity of the adaptive immune repertoire is important for effective immune defense. In my thesis, I have developed computational methods to study the diversity and landscape of the immune repertoire using data from next-generation sequencing experiments and have applied these tools to the study human B cells in the blood and in different tissues. The first tool I developed was an efficient and accurate means of identifying the nearest germline-encoded variable (V) region gene in Ab and TCR rearrangements using anchoring sequences. I used this method to demonstrate that the length of the V gene sequence and its level of somatic mutation influenced the reliability of V gene assignment. Only with adequate V gene assignment can closely related sequences be reliably grouped together into clones (i.e. sequences from lymphocytes that likely derive from a common progenitor cell). With this method, I contributed to a computational pipeline that can process millions of antibody or TCR sequences in hours to days, unlike earlier methods. I used this method to identify and track expanded B-cell clones through the human body, creating an atlas of clonal connections between the blood, bone marrow, spleen, lung, mesenteric lymph node, jejunum, ileum and colon. To power the analysis to study clonal overlap between the tissues, I performed rarefaction analysis on biological replicates to determine how many replicates and how large the clones needed to be to have confidence in clones being present or absent in the different tissue sites. To quantify the level of overlap between two-tissue sites, I used the cosine statistic and showed that my analysis was robust to different measures of clones and clone size. To visualize the tissue distribution of large clones, I created line-circle plots, in which clones are displayed as lines with circles corresponding to the tissues. The sizes of the tissue circles were proportional to clone copy numbers and the colored wedges within the circles indicated the fraction of sequencing libraries that contained sequences from the clone. My analysis revealed the clones tended to partition into two large networks in the human body, one in blood-rich tissues such as the bone marrow, spleen and lung, and another network that was more separated from the blood, in the gastrointestinal tract. I also used methods to visualize and characterize the diversification within B-cell clones due to somatic hypermutation, including lineage tree analysis, the analysis of four-fold silent mutations (mutations that do not change the amino acid sequence), methods for studying intermingling of lineage tree branches (clumpiness) and the use of Bayesian modeling approaches. I used these methods to show that GI tract clones in the human body harbor higher levels of somatic hypermutation and that there is extensive sharing of sequence variants within individual clonal lineages between different sites within the GI tract. Finally, I studied selection of mutations within clonal lineages over time in patients with an autoimmune disease (Sjogren's syndrome) and showed that large clones that were resistant to B-cell depletion therapy were under negative selection. Together, my analysis tools provide a useful means to systematically and quantitatively characterize diversity at the population (repertoire) level and at the clonal level. These tools can be applied to future immune repertoire profiling to study immune responses to vaccines, cancer and infectious disease. |
