Discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening.

T cell receptor (TCR) gene therapy is a potent form of cellular immunotherapy in which patient T cells are genetically engineered to express TCRs with defined tumor reactivity. However, the isolation of therapeutic TCRs is complicated by both the general scarcity of tumor-specific T cells among patient T cell repertoires and the patient-specific nature of T cell epitopes expressed on tumors. Here we describe a high-throughput, personalized TCR discovery pipeline that enables the assembly of complex synthetic TCR libraries in a one-pot reaction, followed by pooled expression in reporter T cells and functional genetic screening against patient-derived tumor or antigen-presenting cells. We applied the method to screen thousands of tumor-infiltrating lymphocyte (TIL)-derived TCRs from multiple patients and identified dozens of CD4+ and CD8+ T-cell-derived TCRs with potent tumor reactivity, including TCRs that recognized patient-specific neoantigens.

Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces.

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin.

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells.

The spatial organization of chromatin is pivotal for regulating genome functions. We report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. Our imaging data revealed TAD-like structures with globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring with nonzero probabilities at all genomic positions but preferentially at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not diminish TAD-like structures in single cells but eliminated preferential domain boundary positions. Moreover, we observed widespread, cooperative, multiway chromatin interactions, which remained after cohesin depletion. These results provide critical insight into the mechanisms underlying chromatin domain and hub formation.