Elucidating Influences of Substrate DNA Sequences on AID Targeting and Outcome During Antibody Maturation

Abstract

During antibody maturation, Activation Induced Cytidine Deaminase (AID) initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class switch recombination (CSR). Mechanisms that target AID and influence point mutational versus DSB outcome have not been fully elucidated. To elucidate influences of V exon and S region substrate sequences on AID targeting and outcome we developed a "passenger" allele approach that measures AID activity in vivo on a fixed "productive" IgH VDJ allele that encodes an IgH chain, versus an array of non-productive VDJ, S region, or non-Ig “passenger” sequences driven by the same promoter on the other allele. Lack of cellular selection for or against mutations or DSBs within passengers enables us to assay intrinsic capacity and preferences of AID to induce SHMs and DSBs within test sequences. We show that in GC B cells, non-Ig passengers target as robust SHM as V exons, definitively establishing the V exon location as "privileged" for SHM. Contrary to dogma in the field, V exon and S region hotspots undergo SHM similarly in CSR-activated B cells. As we discuss based on passenger S region SHM patterns, sequence properties of S regions make them highly sensitive substrates for generating CSR-initiating DSBs. In addition, productive and passenger VB1-8 SHM patterns in GC B cells of Peyer’s Patches (PP) show that PP GC B cell antibody repertoires can be diversified by SHM before affinity maturation. Finally, we assay how sequence-intrinsic AID-targeting preferences influence SHM accumulation in VRC01-class anti-HIV broadly neutralizing antibodies (bnAbs), which contain extraordinarily high SHM levels despite only a fraction of the SHMs being required for function. By assaying passenger substrates representing VRC01-class bnAb precursors, we find that extensive SHM of a V exon subregion does not influence SHM of the rest of the V exon, identify SHMs that are potentially rate-limiting in the maturation process, and find that new SHM hotspots, some of which might influence affinity maturation, arise in a VRC01-class bnAb affinity maturation intermediate. We discuss implications of our findings for the generation of antibody responses.