V(D)J Recombination

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net.

High-throughput immune repertoire sequencing is promising to lead to new statistical diagnostic tools for medicine and biology. Successful implementations of these methods require a correct characterization, analysis, and interpretation of these data sets. We present IGoR (Inference and Generation Of Repertoires)-a comprehensive tool that takes B or T cell receptor sequence reads and quantitatively characterizes the statistics of receptor generation from both cDNA and gDNA. It probabilistically annotates sequences and its modular structure can be used to investigate models of increasing biological complexity for different organisms. For B cells, IGoR returns the hypermutation statistics, which we use to reveal co-localization of hypermutations along the sequence. We demonstrate that IGoR outperforms existing tools in accuracy and estimate the sample sizes needed for reliable repertoire characterization.

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (VH), diversity (DH) and joining (JH) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track VH-DHJH motion in B-lymphocytes. We find that VH and DHJH segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in VH-DHJH motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.

V(D)J recombination-mediated Igκ light chain variable region exon assembly in precursor (pre)-B cells involves recombination activating gene (RAG) endonuclease-orchestrated cleavage between and joining of paired Vκ and Jκ gene segments and flanking RAG-targeting recombination signal sequences (RSSs)1-3. The 3.1-megabase Igk contains 4 Jκs (Jκ1, 2, 4, 5) and 100-plus Vκs in clusters oriented for deletional or inversional joining2. Vκ-to-Jκ joining is ordered, with primary Vκ-to-Jκ1 rearrangements occurring first, followed by secondary rearrangements of upstream Vκs that replace primary VκJκ1s by joining to Jκ2-5 (refs. 4,5). Loop extrusion moves deletional-oriented and inversional-oriented, locus-wide Vκs past the Cer/Sis CTCF-binding element-based diffusion platform for short-range diffusional presentation to Jκ1-bound RAG in the primary recombination centre (RC). To achieve diffusion-mediated Vκ-to-Jκ1 joining, Igk evolved powerful Vκ-associated and Jκ-associated RSSs3. Secondary Igk rearrangements replace non-functional or autoreactive primary VκJκ1 rearrangements, expanding the Igκ repertoire and mediating central tolerance by means of receptor editing4,6-11. Here we describe studies that elucidate the physiologically critical secondary Igk recombination mechanism. Primary deletional and inversional VκJκ1 joins, respectively, delete or displace Cer/Sis, creating a pre-B cell population that harbours secondary VκJκ1-based RCs across the Vκ locus and leaves most unrearranged Vκs immediately upstream of secondary RCs in deletional orientation. High-throughput assays demonstrated that RAG scanning from secondary VκJκ1-based RCs, collectively, extends linearly across the Vκ locus in primary pre-B cell populations. Correspondingly, studies of induced pluripotent stem (iPS) cell-generated mouse models or cell lines with physiological VκJκ-rearrangements further revealed that deletional and, originally, inversional Vκs are mostly captured by Jκ2-5-based secondary RCs in deletional orientation by means of linear RAG scanning. Strong Vκ-RSSs contribute to restricting secondary rearrangements, including potential editing rearrangements, to Vκs immediately upstream of a given secondary RC and support, at a lower level, linear scanning-based inversional Vκ-to-Jκ rearrangements. Our findings implicate Cer/Sis deletion and/or displacement as a developmental switch that converts the two-loop-based diffusional primary Igk rearrangement mechanism into a one-loop-based linear scanning secondary rearrangement mechanism.