Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

SUMMARY As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.

Summary The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of ‘orphan’ T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A*02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens, which enables the facile identification of tumor antigens through unbiased screening.

The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping.