Local and Genome-wide Gene Conversion contributes to secondary somatic diversification of antibody repertoires in Humans and Mice Open Access

Abstract

The ability of the humoral immune system to respond to staggering number of antigens is critically dependent on the generation of somatic diversity in B cells. During B lymphopoiesis, discrete gene segments are somatically recombined through the process of V(D)J recombination, establishing the first stage of somatic diversification. In the periphery, antigen binding and cognate T cell help, license B cells to undergo further somatic diversification through the Darwinian microcosm of the germinal center, where antigen specific B cells undergo rounds of proliferation and mutation followed by selection for antigen binding.

Secondary somatic diversification is critically dependent of the master regulator activation-induced cytidine deaminase. The activity of this enzyme results in intrinsic mutability at cytosine residues, with further processing resulting in either the accumulation of untemplated point mutation through the canonical processes of somatic hypermutation or the templated mutations derived from gene conversion. It is widely held that somatic hypermutation results in untemplated point mutations in mice and humans, where as other species such as rabbits and chickens preferentially utilize templated mutations via gene conversion.

Here we demonstrate that somatic mutations observed in sequences at the antibody loci are derived from both local templated mutagenesis (i.e. donors within the antibody loci) as well as global templated mutagenesis (i.e. donors scattered in the genome). We demonstrate in two contexts the contribution of gene conversion to mutation processes during the germinal center reaction. First, we analyze micro-clusters of mutations (≥2 mutations in 8 bp) to demonstrate that mutations in close proximity exhibit significant linkage disequilibrium. We also demonstrate that non-immunoglobulin genes at the antibody locus share templates for micro-clustered mutations as do somatically mutated antibody sequences. Second, we analyzed large templated events at the heavy chain locus and demonstrated that templated mutagenesis results in templating from inter- and intra-chromosomal locations. Together, these studies provide evidence for the role of gene conversion in the somatic diversification of murine and human B cells.

Table of Contents

Preface1

Chapter 1: Introduction3

B cell diversification3

Primary diversification and antibody structure3

B cell activation and secondary diversification7

Mechanisms of somatic hypermutation12

Figure 1: Neuberger model of somatic hypermutation15

Mechanisms of gene conversion outside of B cells17

Gene conversion and somatic hypermutation during evolution21

Evolution of somatic hypermutation and gene conversion paradigms22

Figure 2: Primary evidence of the state of the field circa 199126

Works cited28

Chapter 2: Clustered mutations at the murine and human IgH locus exhibit significant linkage consistent with templated mutagenesis 37

Abstract38

Introduction39

Results40

Templated mutations occur in murine IgM plasma cells40

Templated mutations occur in murine germinal center B cells43

Templated donor tracts primarily originate from 5’ upstream V gene segments but can also originate from the trans allele 45

                       Templated mutation occurs in human plasmablasts48

                       Templated mutations occur in non-Ig genes inserted into the IgH locus49

        Discussion52

        Methods56

        Figure 1: Somatic hypermutation motifs in IgM plasma cells are shared between individual clones in individual mice 62

        Figure 2: IgM+ and IgG+ germinal center B cells exhibit gene conversion tracts during   the germinal center reaction64

        Figure 3: Germinal center B cells from CB6F1/J mice show templated mutagenesis primarily occurs in cis66

        Figure 4: Human plasmablast sequences demonstrate templated mutagenesis and preferential use of 5’ donors68

        Figure 5: Non-immunoglobulin sequences exhibit templated mutagenesis and preferentially utilize a limited number of IgHV-specific motifs as donors 70

        Supplemental Figure 1: Schematic of sequence analysis by PolyMotifFinder73

        Supplemental Figure 2: Schematic of sequence analysis via RandomCheck 75

        Supplemental Figure 3: IgHV genes from F1 germinal centers are enriched for motifs occurring in the IgHV repertoire and the subset of the repertoire that somatically-mutated    GPT and β-globin match to 77

        Works Cited79

Chapter 3: Somatic diversification of rearranged antibody gene segments by genome-wide templated mutagenesis82

        Abstract83

        Results84

        Methods93

        Figure 1: Somatically-mutated LAIR1 inserts have regions of clustered mutations that match distant genomic regions 101

        Figure 2: TRACE identified regions of the genome contribute to somatic mutagenesis of IgHV/LAIR1 sequences 103

        Figure 3: Somatically-mutated populations of switched memory B cells template somatic mutations from intra- and inter-chromosomal regions105

        Figure 4: TRACE hits correlate with multiple aspects of germinal center B cell biology 107

        Supplemental Figure 1: Workflow of TRACE110

        Supplemental Figure 2: TRACE donor regions cluster between and within human donor samples and correlate with areas of transcription113

        Supplemental Figure 3: Read-number of Hi-C data correlates with distance from TRACE-identified template115

        Supplemental Figure 4: Location of TRACE hits in IgHV 3-15116

        Supplemental Figure 5: Location of TRACE hits in IgHV 5-51117

        Supplemental Figure 6: Location of TRACE hits in LAIR1 insertions118

        Supplemental Figure 7: Location of TRACE hits in unselected passenger transgenes119

        Supplemental Table 1: TRACE donors in upregulated and downregulated fractions of differentially regulated genes in germinal center B cells120

        Works Cited121

Chapter 4: Discussion123

        Gene conversion as an active contributor to somatic hypermutation123

        A model for gene conversion in SHM128

                    Figure 1: Updated model of somatic hypermutation and gene conversion128

        Gene conversion and potential for vaccine design129

                    Figure 2: Ectopic gene conversion in CDR1 of HIV-specific antibody 131

           Works Cited132

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Statistics Computer Science Health Sciences, Immunology
Keyword	Affinity maturation B cell Somatic hypermutation Gene conversion Germinal center
Committee Chair / Thesis Advisor	Joshy Jacob, Emory University
Committee Members	David Steinhauer, Emory University John Altman, Emory University Ignacio Sanz, Emory University Max Cooper, Emory University

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