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Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ∼25% of expressed genes in germinal center B cells. Observations in Ung−/− Msh2−/− mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung−/− Msh2−/− mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-β binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID.

Summary: Driven by dramatic technological improvements, large-scale characterization of lymphocyte receptor repertoires via high-throughput sequencing is now feasible. Although promising, the high germline and somatic diversity, especially of B-cell immunoglobulin repertoires, presents challenges for analysis requiring the development of specialized computational pipelines. We developed the REpertoire Sequencing TOolkit (pRESTO) for processing reads from high-throughput lymphocyte receptor studies. pRESTO processes raw sequences to produce error-corrected, sorted and annotated sequence sets, along with a wealth of metrics at each step. The toolkit supports multiplexed primer pools, single- or paired-end reads, and emerging technologies that employ single-molecule barcodes. pRESTO has been tested on data generated from Roche and Illumina platforms. It has a built in capacity to parallelize the work between available processors, and is able to efficiently process millions of sequences generated by typical high-throughput projects. As part of ongoing collaborations, pRESTO has been applied to multiple B-cell repertoire studies, employing both Roche 454 and Illumina MiSeq sequencing technologies. Availability: pRESTO is freely available for academic use. The software package and detailed tutorials may be downloaded from http://clip.med.yale.edu/pRESTO.

Quantifying antigen driven selection based on mutation patterns observed in Immunoglobulin (Ig) DNA sequences can provide insights into the basic biology that underlies the adaptive immune response, and may further serve as diagnostic and prognostic markers. High-throughput sequencing approaches make large-scale characterization of B cell Ig repertoires feasible. However, analyzing selection in these large datasets, presents fundamental challenges. Here I will present a new computational framework for Bayesian estimation of Antigen-driven SELectIoN (BASELINe) in Ig sequecnes. BASELINe provides a more intuitive means to analyze selection by shifting the problem from one of detecting selection to one of quantifying it. It allows, for the first time, comparative analysis between sequences derived from different germlines. We have made this method practical for complete repertoire analysis through an online tool (clip.med.yale.edu/baseline) and a highly optimized implementation that can analyze up to 10,000 sequences in under four minutes on a single processor. By testing this method on computer simulations as well as on real sequences from genetically engineered mice, we have shown that indeed there is a significant improvement in our ability to quantify selection strength. We then demonstrate how BASELINe can be applied to high throughput sequencing datasets to arrive at several novel non-trivial observations.

We identify the interfollicular (IF) zone of the lymph node as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, where they participated in long-lived interactions and upregulated Bcl-6, which controls the differentiation of both cell types. During this time, T cells also acquired the Tfh markers CXCR5, PD-1 and GL7. While T cell immigration into the follicle interior occurred three days post immunization, responding B cells remained largely restricted to the IF zone and the area adjacent to the sub capsular sinus at this timepoint. B cell entry into the follicle interior, and formation of nascent germinal centers, did not occur until a day later. Notably, in the absence of cognate B cells, PD-1hi Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 expression was not maintained on T cells that nevertheless persisted within the follicle. Thus, cognate B cells are not required for Tfh cell differentiation in the IF zone, but are instead required for the long-term maintenance of the activated Tfh phenotype.