Resumen de Analysis of Activation Activation-Induced Deaminase target specificity in human memory B lymphocytes
B lymphocytes are responsible for orchestrating the humoral immune response through the generation of antibodies. B lymphocytes that are activate activated by antigen can engage in the germinal center (GC) reaction an generate high affinity plasma cells, which secrete antibodies, and long long- lived memory B cells (MBCs), which provide a reservoir of B cells for subsequent infections. In the GC B cells remodel their immunoglobulin ( genes by two mechanisms of secondary diversification, somatic hypermutation (SHM) and class switch recombination (CSR). Activation Activation- induced deaminase (AID) initiates SHM and CSR by deaminating cytosines in Igs. However, AID can al so deaminate at low frequency non non-Ig regions, known as Off Off-targets, causing mutations and chromosomal translocations with oncogenic potential. Indeed, t he mechanism mechanisms governing AID target specificity ha have not been fully resolved yet , because detection of AI AID-induced mutations, which occur very infrequently, is extremely challenging. Specifically, knowledge of AID offoff-target mutations in healthy human is very limited. To address this question, in this thesis we have analyzed AIDAID-induced mutations in hum an MBCs from peripheral blood and have compared switched MBCs (swMBCs), which represent cells with an extended GC experience, with unswitched MBCs (usMBCs), i.e. extrafollicular MBCs or early GC emigrants. We have developed a methodology that couples a lib rary capture for enrichment of regions, with an Error Error-Corrected Sequencing strategy (ECS) and ad ad-hoc bioinformatics tools to analyze the sequencing data. This led to the development of a publicly available web application to assist in the experimental design of ECS experimentsexperiments. Using this ECS approach we have sequencsequenced 20,262 regions and ha have identified 49 OffOff-target genesgenes, which are more mutated in swMBCs than in usMBCs usMBCs. OffOff-targets show hallmarks of AID activity, including transcriptional activity and presence H 3K36me3 mark. In addition, we found that mutations at Igs and AID Off Off-targets are enriched in different sequence motifs, with Off Off-target genes showing a specific contribution of the SBS15 COSMIC signature, which is associated with a defective MMR repair pathway. Finally, we found that Off Off-targets show marks of genomic instability, are associated with lymphoma genes, and can functionally impact on lymphoma cell cells development and survival. All in all, the results presented in this thesis contribute methodologically to improve the detection of mutations at very low frequencies and strengthen and extend the link between mutations in MBCs of healthy individuals and the development of GC GC-derived lym phomas
