Peter William Zandstra

T-cells use molecular reactions with nonequilibrium error correction, i.e., proofreading, to discriminate between nearly identical antigens with high specificity and sensitivity. These receptor binding events are known to be force sensitive, yet traditional schemes of proofreading focus on reaction kinetics alone and do not consider the role of force dependent catch/slip bond behavior or interactions with mechanically engaged coreceptors such as adhesion molecules. To address this, we propose a minimal framework for proofreading of ligand discrimination by T-cell receptors (TCRs) that uses endogenous TCR mechanosensation and substrate-mediated mechanical interactions with adhesive proteins (load sharing) to improve recognition fidelity. We leverage the catch bond behavior of cognate antigens to delay decision making and amplify TCR signaling while discarding noncognate slip bond ligands in the presence of a force. By integrating our model with existing structural and molecular data, we show that substrate mechanics regulates the transmission of active cytoskeletal forces through a molecular clutch and controls the energization of bound TCRs needed for optimal proofreading. Our work demonstrates how mechanical forces and substrate properties can augment kinetic proofreading in T-cells, suggesting biomaterial design strategies for immunotherapies that tune the mechanical microenvironment of T-cells to achieve high fidelity TCR-ligand discrimination, antigen recognition, and activation.

Gene syntax—the order and arrangement of genes and their regulatory elements—shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus perturbs the transcription of adjacent genes, but the molecular basis of this effect remains poorly understood. In this work, we show that supercoiling-mediated feedback arises from transcription and regulates expression of adjacent genes in a syntax-specific manner. Using a suite of assays, we measured syntax- and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter, we built and improved compact gene circuits, tuning the expression mean, noise, and stoichiometry across delivery methods and cell types. Integrating supercoiling mediated feedback into models of gene regulation will expand our understanding of native and synthetic systems.

Gene syntax—the order and arrangement of genes and their regulatory elements—shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that the reciprocal effects of transcription and DNA supercoiling, which we term supercoiling-mediated feedback, regulates expression of adjacent genes in a syntax-specific manner. Using a suite of chromatin state assays, we measure syntax-and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter and without altering sequence or copy number, we built compact gene circuits, tuning the expression mean, noise, and stoichiometry across diverse delivery methods and cell types. Integrating supercoiling-mediated feedback into models of gene regulation will expand our understanding of native systems and enhance the design of synthetic gene circuits.

Allogeneic T cell therapies are a highly desirable option to circumvent the cost and complexity of using autologous T cells to treat diseases. Allogeneic CD8⁺ T cells can be made from pluripotent stem cells (PSCs), but deriving CD4⁺ T cells from PSCs remained a significant challenge. Using feeder-and serum-free conditions, we found that CD4⁺ versus CD8⁺ T cell commitment from PSCs can be controlled by fine-tuning the dynamics of Notch and T cell receptor signaling delivered to CD4⁺CD8⁺ double positive T cells. Notch signaling negatively impacts CD4⁺ T cell commitment, and its timed removal allows generation of clonally-diverse and expandable CD4⁺ T cells from PSCs. The resulting CD4⁺ T cells respond to cytokine-mediated polarization by differentiating into Th1, Th2, or Th17 cells, recapitulating canonical helper cell function. These findings represent a significant step towards using PSC-derived CD4⁺ T cells as a low cost, off-the-shelf, cell therapy.

SUMMARY: The hypoblast is an essential extra-embryonic tissue set aside within the inner cell mass early in mammalian embryo development, in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naïve PSC to hypoblast differentiation proceeds via reversion to a transitional ICM-like state, from which hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signalling is critical for hypoblast specification. Revisiting FGF signalling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro , the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mouse and human. Overall, this study demonstrates the utility of human naïve PSC-based models in elucidating mechanistic features of early human embryogenesis.