Philosophiae Doctor - PhD / Sequencing an HIV quasispecies with next generation sequencing technologies yields a dataset with significant amplification bias and errors resulting from both the PCR and sequencing steps. Both the amplification bias and sequencing error can be reduced by labelling each cDNA (generated during the reverse transcription of the viral RNA to DNA prior to PCR) with a random sequence tag called a Primer ID (PID). Processing PID data requires additional computational steps, presenting a barrier to the uptake of this method. MotifBinner is an R package designed to handle PID data with a focus on resolving potential problems in the dataset. MotifBinner groups sequences into bins by their PID tags, identifies and removes false unique bins, produced from sequencing errors in the PID tags, as well as removing outlier sequences from within a bin. MotifBinner produces a consensus sequence for each bin, as well as a detailed report for the dataset, detailing the number of sequences per bin, the number of outlying sequences per bin, rates of chimerism, the number of degenerate letters in the final consensus sequences and the most divergent consensus sequences (potential contaminants). We characterized the ability of the PID approach to reduce the effect of sequencing error, to detect minority variants in viral quasispecies and to reduce the rates of PCR induced recombination. We produced reference samples with known variants at known frequencies to study the effectiveness of increasing PCR elongation time, decreasing the number of PCR cycles, and sample partitioning, by means of dPCR (droplet PCR), on PCR induced recombination. After sequencing these artificial samples with the PID approach, each consensus sequence was compared to the known variants. There are complex relationships between the sample preparation protocol and the characteristics of the resulting dataset. We produce a set of recommendations that can be used to inform sample preparation that is the most useful the particular study. The AMP trial infuses HIV-negative patients with the VRC01 antibody and monitors for HIV infections. Accurately timing the infection event and reconstructing the founder viruses of these infections are critical for relating infection risk to antibody titer and homology between the founder virus and antibody binding sites. Dr. Paul Edlefsen at the Fred Hutch Cancer Research Institute developed a pipeline that performs infection timing and founder reconstruction. Here, we document a portion of the pipeline, produce detailed tests for that portion of the pipeline and investigate the robustness of some of the tools used in the pipeline to violations of their assumptions.

En introduisant fréquemment des mutations ponctuelles dans les régions variables des gènes d'immunoglobulines (Ig), le processus d'hypermutation somatique (SHM, initié par la déaminase AID) est essentiel pour augmenter l'affinité des anticorps. En marge de ses cibles physiologiques (les gênes d'Ig), AID peut induire des "dommages collatéraux" au niveau de cibles "illégitimes" qui sont appelées "off targets" (dont certains oncogènes, tel que Bcl6 fréquemment muté dans les lymphomes B). Le risque élevé de dommages collatéraux dans le génome des cellules B implique que les remaniements géniques soient précisément surveillés. Parmi les éléments cis-régulateurs impliqués dans cette surveillance, on compte l'activateur cEμ au locus des chaînes lourdes des Ig (IgH) et ses régions flanquantes d'attachement à la matrice nucléaire MARsEμ (étudiés en détails dans nos modèles de souris KO). Nous montrons que la délétion des régions MARsEμ diminue non seulement les mutations au locus des chaines lourdes des Ig (effet physiologique en cis) mais également au locus des chaines légères Ig situé sur un chromosome différent (effet de trans). A l'aide d'une outil bioinformatique (DeMinEr) que nous avons développé dans le but d'identifier des mutations rares, nous montrons également que les régions MARsEμ sont impliquées dans les dommages collatéraux infligés aux "off targets" des cellules B. Grâce à la technique de FISH 3D, nous proposons que les régions MARsEμ participent à la régulation de la SHM en influençant la position des cibles de AID dans le noyau des cellules B. Notre étude met en évidence un niveau de régulation spatiale du processus de SHM médié par les régions MARsEμ du locus IgH. / By introducing frequent point mutations into the variable regions of immunoglobulin (Ig) genes, somatic hypermutation (SHM, initiated by the AID deaminase) is a driving force for antibody affinity maturation. It is now admitted that AID-induces mutations in germinal centre B cells could affect in parallel to their Ig genes physiological targets, illegitimates targets (including oncogenes) so calles "off targets" (such as Bcl6 with frequent point mutation in B lymphomas). The high risk of "collateral damage" in the B cell genome implies that remodeling events are precisely surveyed. Among cisregulatory elements involved (transcriptional enhancers and chromatin isolators and anchors...), one best candidate is the intronic region including the cEμ enhancer and iths flanking MARsEμ regions that we have been studying extensively using mouse KO model. We recently showed that MARsEμ deletion decreases SHM not only at Ig Heavy chain locus IgH (physiological cis effect) but surprisingly also at the Ig Light chain Kappa locus Ig, located on a different chromosome (trans effect). To extend the study of this intriguing trans effect, we developed a bioinformatic tool called DeMinEr that unveiled that MARsEμ regions were also involved in AID-induced collateral damages to "off-targets". Using FISH 3D, we show that MARsEμ regions harboured the potential not only to locally recruit SHM but also to cause dynamic changes of nuclear structures. The surprising cis and trans effect of MARsEμ deletion, impacting simultaneously nuclear positioning and SHM, revealed an additional level of regulation for targeting mutations to Ig and "off-targets" genes.

Durant l’ontogénie B, le locus des chaines lourdes d’immunoglobulines (IgH) subit trois processus de réarrangements géniques. Lors des phases précoces du développement B, indépendamment de la rencontre avec un antigène, les réarrangements VDJ permettent l’obtention d’un répertoire d’Ig fonctionnelles. Lors des phases tardives, l’hypermutationsomatique (SHM) permet l’augmentation de l’affinité de l’Ig pour son antigène tandis que larecombinaison isotypique (CSR) modifie ses fonctions effectrices. Ces évènements impliquent l’induction de lésions de l’ADN potentiellement oncogéniques, ce qui impose unerégulation très stricte. Cette régulation est assurée par divers éléments cis-régulateurs répartis tout au long du locus IgH, dont la région régulatrice en 3’ (3’RR). La 3’RR s’étend sur 30 kb et contient quatre activateurs transcriptionnels, les trois premiers formant une structure palindromique. Lors de ma thèse, j’ai utilisé plusieurs modèles murins porteurs de délétions de tout ou partie de la 3’RR pour étudier son rôle, ainsi que celui des différents éléments qui la compose lors des diverses étapes de l’ontogénie B. Nous avons pu déterminer comment la 3’RR régule précisément la CSR en ciblant spécifiquement la région switch acceptrice et caractériser le phénomène encore peu connu de CSR vers IgD. D’autre part, nous avons démontré l’importance de la 3’RR lors de la SHM et dans le développement des différentes sous populations lymphocytaires B. Enfin, la comparaison des résultats obtenus lors de l’analyse des différents modèles nous a permis de déterminer que la structure palindromique de la 3’RR est importante pour une SHM efficace, mais relativement dispensable lors de la CSR. / During B-cell development, the heavy chains locus (IgH) undergoes three genic rearrangement events. During the early stages, before encountering the antigen, VDJ rearrangements allow the generation of a functional Ig repertoire. During the late stages, somatic hypermutation (SHM) increases the affinity of the Ig for its antigen, while class switch recombination (CSR) modifies its effector functions. These events imply thegeneration of potentially oncogenic DNA lesions, and thus require a strict regulation. This regulation is assured by several cis-regulatory elements spread along the IgH locus, including the 3’ regulatory region (3’RR). The 3’RR extends on more than 30kb and contains four transcriptional enhancers, the first three displaying a palindromic conformation. During my PhD, I investigated several mouse models carrying deletion of part or totality of the 3’RR to investigate its role during B cell development. We demonstrated how she precisely regulates CSR by specifically targeting the acceptor switch region, and described the poorly known mechanism of CSR toward IgD. Otherwise, we have demonstrated its importance during SHM and in the correct development of the different B cell subpopulations. Finally, by comparing the results obtained during the analysis of the various mouse models, we have demonstrated that the palindromic structure of the 3’RR is required for optimal SHM, but not for CSR.

Durant l’ontogénie B, le locus des chaînes lourdes d’immunoglobulines (IgH) subit trois processus majeurs de réarrangements géniques. Lors de la phase précoce du développement B, indépendamment de la rencontre avec un antigène (Ag), les recombinaisons VHDJH donnent le répertoire diversifié des Ig fonctionnelles. Durant la phase tardive, suite à une activation par l’Ag, l’hypermutation somatique (SHM) permet l’augmentation de l’affinité de l’Ig à son Ag et la recombinaison isotypique (CSR) va modifier ses fonctions effectrices. Tous ces processus sont strictement régulés par différents éléments cis-régulateurs repartis tout au long du locus IgH. La région régulatrice en 3’ (3’RR) en est un. Elle s’étend sur environ 30 Kb et est constituée de quatre activateurs transcriptionnels, dont les trois premiers forment une structure palindromique. La 3’RR contrôle, chez le lymphocyte B-2 (LB-2), la transcription du locus IgH, le devenir de la cellule B, la SHM et la CSR mais elle n’a aucun effet sur les recombinaisons VHDJH et la diversité du répertoire antigénique. Les LB-1 représentent un petit pourcentage des LB totaux. Ils diffèrent des LB-2 par leur origine, développement, fonctions, marqueurs de surface et distribution tissulaire. Les LB-1 maintiennent l'homéostasie dans l'organisme et sont la source principale des Ig naturelles (NIgM et NIgA) au cours des premières phases d'une réponse immunitaire. Lors de ma thèse, nous avons étudié le rôle de la 3’RR sur le développement des LB-1. D’une façon identique aux LB-2, la 3’RR contrôle la transcription du locus IgH, le devenir des cellules B et la SHM dans les LB-1. A l’inverse des LB-2, la 3’RR joue un rôle indirect sur la diversité du répertoire antigénique dans les LB-1 et n’a aucun effet sur la CSR vers IgA. Ces résultats mettent en évidence, pour la première fois, la contribution de la 3'RR dans le développement d’une population cellulaire B à l’interface entre l’immunité innée et acquise. Ils renforcent nos connaissances sur le rôle des éléments cis-régulateurs du locus IgH dans le développement de ces deux immunités. / During B-cell development, the immunoglobulin heavy chain locus (IgH) undergoes three major genic rearrangements. During the early stages, before encountering the antigen (Ag), VHDJH rearrangements allow the generation of the Ig repertoire. During the late stages, after encountering the Ag, somatic hypermutation (SHM) increases the affinity of the Ig for its Ag, while class switch recombination (CSR) modifies its effector functions. All these genetic events are strictly regulated by cis-regulatory elements spread along the IgH locus, including the 3’ regulatory region (3’RR). The 3’RR extends over more than 30kb and contains four transcriptional enhancers, the first three displaying a palindromic conformation. The 3'RR controls B2 B-cell IgH transcription, cell fate, SHM and CSR but not repertoire diversity. B1 B-cells represent a small percentage of total B-cells differing from B2 B-cells by several points such as precursors, development, functions, surface markers and tissue distribution. B1 B-cells act at the steady state to maintain homeostasis and during the earliest phases of an immune response by secreting natural Ig (NIgM and NIgA). During my PhD, we investigated the role of the 3'RR on B1 B-cells. Similarly to B2 B-cells, the 3'RR controls IgH transcription, cell fate and SHM in B1 B-cells. In contrast to B2 B-cells, 3'RR deletion indirectly affects B1 B-cell repertoire diversity and has no effect on their CSR towards IgA. These results highlight, for the first time, the contribution of the 3'RR in the development of a B-cell population at the interface between innate and acquired immunity. Moreover, these results strengthen our knowledge of the role of the cis-regulatory elements of the IgH locus in the development of these two immune responses.

Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.

B cells produce a diverse array of antibody specificities that are of low affinity during the initial phase of a humoral immune response. However, somatic hypermutation of the rearranged V region in the immunoglobulin locus generates new antibody affinities, accompanied by the selection of B cells that produce superior antibody affinities. Somatic hypermutation is initiated by the conversion of G:C base pairs to G:U lesions by the enzyme activation induced cytosine deaminase. Left unrepaired, G:U lesions will give rise to transition mutations at G:C base pairs, but are converted to transition and transversion mutations at G:C and A:T base pairs by the paradoxical participation of the base excision repair and mismatch repair pathways. The mismatch repair pathway, which evolved to correct errors produced during DNA replication, is co-opted by hypermutating B cells to produce A:T mutations via the processing of G:U lesions. This process requires the mismatch repair components Msh2, Msh6, and Exo1, but is additionally dependent upon the translesional DNA polymerase eta, a known A:T mutator, and on ubiquitinated PCNA, an initiator of translesion synthesis. The presence of certain types of lesions in the template strand during DNA replication leads to the activation of translesion synthesis. I propose that a similar mechanism operates during somatic hypermutation to activate translesion synthesis and recruit DNA polymerase eta. Our model suggests that mismatch repair-generated single-stranded DNA tracts contain abasic sites produced as a result of uracil excision by uracil-N-glycosylase. Synthesis opposite abasic sites activates translesion synthesis and results in the recruitment of polymerase eta and the subsequent production of A:T mutations. In this thesis, I present data from hypermutating murine B cells and the B cell line Ramos to support this model, demonstrating that the base excision repair and mismatch repair pathways cooperate during somatic hypermutation to generate A:T mutations. In addition, I explore the role of the Mre11-Rad50-Nbs1 complex in its contribution to A:T mutations in Ramos cells. Taken together, these studies demonstrate that conversion of classical DNA repair pathways into mutation-generating processes is driven by the unique environment of the V region in hypermutating B cells.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

B cells produce a diverse array of antibody specificities that are of low affinity during the initial phase of a humoral immune response. However, somatic hypermutation of the rearranged V region in the immunoglobulin locus generates new antibody affinities, accompanied by the selection of B cells that produce superior antibody affinities. Somatic hypermutation is initiated by the conversion of G:C base pairs to G:U lesions by the enzyme activation induced cytosine deaminase. Left unrepaired, G:U lesions will give rise to transition mutations at G:C base pairs, but are converted to transition and transversion mutations at G:C and A:T base pairs by the paradoxical participation of the base excision repair and mismatch repair pathways. The mismatch repair pathway, which evolved to correct errors produced during DNA replication, is co-opted by hypermutating B cells to produce A:T mutations via the processing of G:U lesions. This process requires the mismatch repair components Msh2, Msh6, and Exo1, but is additionally dependent upon the translesional DNA polymerase eta, a known A:T mutator, and on ubiquitinated PCNA, an initiator of translesion synthesis. The presence of certain types of lesions in the template strand during DNA replication leads to the activation of translesion synthesis. I propose that a similar mechanism operates during somatic hypermutation to activate translesion synthesis and recruit DNA polymerase eta. Our model suggests that mismatch repair-generated single-stranded DNA tracts contain abasic sites produced as a result of uracil excision by uracil-N-glycosylase. Synthesis opposite abasic sites activates translesion synthesis and results in the recruitment of polymerase eta and the subsequent production of A:T mutations. In this thesis, I present data from hypermutating murine B cells and the B cell line Ramos to support this model, demonstrating that the base excision repair and mismatch repair pathways cooperate during somatic hypermutation to generate A:T mutations. In addition, I explore the role of the Mre11-Rad50-Nbs1 complex in its contribution to A:T mutations in Ramos cells. Taken together, these studies demonstrate that conversion of classical DNA repair pathways into mutation-generating processes is driven by the unique environment of the V region in hypermutating B cells.

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.