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The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge. [ABSTRACT FROM AUTHOR]

Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns. [ABSTRACT FROM AUTHOR]

The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level.