Different relative scalings between transient forces and thermal fluctuations tune regimes of chromatin organization.

Within the nucleus, structural maintenance of chromosome protein complexes, namely condensin and cohesin, create an architecture to facilitate the organization and proper function of the genome. Condensin, in addition to performing loop extrusion, creates localized clusters of chromatin in the nucleolus through transient crosslinks. Large-scale simulations revealed three different dynamic behaviors as a function of timescale: slow crosslinking leads to no clusters, fast crosslinking produces rigid slowly changing clusters, while intermediate timescales produce flexible clusters that mediate gene interaction. By mathematically analyzing different relative scalings of the two sources of stochasticity, thermal fluctuations and the force induced by the transient crosslinks, we predict these three distinct regimes of cluster behavior. Standard time-averaging that takes the fluctuations of the transient crosslink force to zero predicts the existence of rigid clusters. Accounting for the interaction of both fluctuations from the crosslinks and thermal noise with an effective energy landscape predicts the timescale-dependent lifetimes of flexible clusters. No clusters are predicted when the fluctuations of the transient crosslink force are taken to be large relative to thermal fluctuations. This mathematical perturbation analysis illuminates the importance of accounting for stochasticity in local incoherent transient forces to predict emergent complex biological behavior.

Interruption of the intratumor CD8+ T cell:Treg crosstalk improves the efficacy of PD-1 immunotherapy.

PD-1 blockade unleashes potent antitumor activity in CD8+ T cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen the response to immunotherapy. Tumor-Treg inhibition is a promising strategy to improve the efficacy of checkpoint blockade immunotherapy; however, our understanding of the mechanisms supporting tumor-Tregs during PD-1 immunotherapy is incomplete. Here, we show that PD-1 blockade increases tumor-Tregs in mouse models of melanoma and metastatic melanoma patients. Mechanistically, Treg accumulation is not caused by Treg-intrinsic inhibition of PD-1 signaling but depends on an indirect effect of activated CD8+ T cells. CD8+ T cells produce IL-2 and colocalize with Tregs in mouse and human melanomas. IL-2 upregulates the anti-apoptotic protein ICOS on tumor-Tregs, promoting their accumulation. Inhibition of ICOS signaling before PD-1 immunotherapy improves control over immunogenic melanoma. Thus, interrupting the intratumor CD8+ T cell:Treg crosstalk represents a strategy to enhance the therapeutic efficacy of PD-1 immunotherapy.

Polymer Modeling Reveals Interplay between Physical Properties of Chromosomal DNA and the Size and Distribution of Condensin-Based Chromatin Loops.

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead-spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain.

NeST: nested hierarchical structure identification in spatial transcriptomic data.

Spatial gene expression in tissue is characterized by regions in which particular genes are enriched or depleted. Frequently, these regions contain nested inside them subregions with distinct expression patterns. Segmentation methods in spatial transcriptomic (ST) data extract disjoint regions maximizing similarity over the greatest number of genes, typically on a particular spatial scale, thus lacking the ability to find region-within-region structure. We present NeST, which extracts spatial structure through coexpression hotspots-regions exhibiting localized spatial coexpression of some set of genes. Coexpression hotspots identify structure on any spatial scale, over any possible subset of genes, and are highly explainable. NeST also performs spatial analysis of cell-cell interactions via ligand-receptor, identifying active areas de novo without restriction of cell type or other groupings, in both two and three dimensions. Through application on ST datasets of varying type and resolution, we demonstrate the ability of NeST to reveal a new level of biological structure.

Interruption of the Intratumor CD8:Treg Crosstalk Improves the Efficacy of PD-1 Immunotherapy.

PD-1 blockade unleashes the potent antitumor activity of CD8 cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen response to immunotherapy. Tumor Treg inhibition is a promising strategy to overcome therapeutic resistance; however, the mechanisms supporting tumor Tregs during PD-1 immunotherapy are largely unexplored. Here, we report that PD-1 blockade increases tumor Tregs in mouse models of immunogenic tumors, including melanoma, and metastatic melanoma patients. Unexpectedly, Treg accumulation was not caused by Treg-intrinsic inhibition of PD-1 signaling but instead depended on an indirect effect of activated CD8 cells. CD8 cells colocalized with Tregs within tumors and produced IL-2, especially after PD-1 immunotherapy. IL-2 upregulated the anti-apoptotic protein ICOS on tumor Tregs, causing their accumulation. ICOS signaling inhibition before PD-1 immunotherapy resulted in increased control of immunogenic melanoma. Thus, interrupting the intratumor CD8:Treg crosstalk is a novel strategy that may enhance the efficacy of immunotherapy in patients.

Identifying multicellular spatiotemporal organization of cells with SpaceFlow.

One major challenge in analyzing spatial transcriptomic datasets is to simultaneously incorporate the cell transcriptome similarity and their spatial locations. Here, we introduce SpaceFlow, which generates spatially-consistent low-dimensional embeddings by incorporating both expression similarity and spatial information using spatially regularized deep graph networks. Based on the embedding, we introduce a pseudo-Spatiotemporal Map that integrates the pseudotime concept with spatial locations of the cells to unravel spatiotemporal patterns of cells. By comparing with multiple existing methods on several spatial transcriptomic datasets at both spot and single-cell resolutions, SpaceFlow is shown to produce a robust domain segmentation and identify biologically meaningful spatiotemporal patterns. Applications of SpaceFlow reveal evolving lineage in heart developmental data and tumor-immune interactions in human breast cancer data. Our study provides a flexible deep learning framework to incorporate spatiotemporal information in analyzing spatial transcriptomic data.

Numerical computation of effective thermal equilibria in stochastically switching Langevin systems.

Stochastically switching force terms appear frequently in models of biological systems under the action of active agents such as proteins. The interaction of switching forces and Brownian motion can create an "effective thermal equilibrium," even though the system does not obey a potential function. In order to extend the field of energy landscape analysis to understand stability and transitions in switching systems, we derive the quasipotential that defines this effective equilibrium for a general overdamped Langevin system with a force switching according to a continuous-time Markov chain process. Combined with the string method for computing most-probable transition paths, we apply our method to an idealized system and show the appearance of previously unreported numerical challenges. We present modifications to the algorithms to overcome these challenges and show validity by demonstrating agreement between our computed quasipotential barrier and asymptotic Monte Carlo transition times in the system.

Deciphering tissue structure and function using spatial transcriptomics.

The rapid development of spatial transcriptomics (ST) techniques has allowed the measurement of transcriptional levels across many genes together with the spatial positions of cells. This has led to an explosion of interest in computational methods and techniques for harnessing both spatial and transcriptional information in analysis of ST datasets. The wide diversity of approaches in aim, methodology and technology for ST provides great challenges in dissecting cellular functions in spatial contexts. Here, we synthesize and review the key problems in analysis of ST data and methods that are currently applied, while also expanding on open questions and areas of future development.

Inferring information flow in spike-train data sets using a trial-shuffle method.

Understanding information processing in the brain requires the ability to determine the functional connectivity between the different regions of the brain. We present a method using transfer entropy to extract this flow of information between brain regions from spike-train data commonly obtained in neurological experiments. Transfer entropy is a statistical measure based in information theory that attempts to quantify the information flow from one process to another, and has been applied to find connectivity in simulated spike-train data. Due to statistical error in the estimator, inferring functional connectivity requires a method for determining significance in the transfer entropy values. We discuss the issues with numerical estimation of transfer entropy and resulting challenges in determining significance before presenting the trial-shuffle method as a viable option. The trial-shuffle method, for spike-train data that is split into multiple trials, determines significant transfer entropy values independently for each individual pair of neurons by comparing to a created baseline distribution using a rigorous statistical test. This is in contrast to either globally comparing all neuron transfer entropy values or comparing pairwise values to a single baseline value. In establishing the viability of this method by comparison to several alternative approaches in the literature, we find evidence that preserving the inter-spike-interval timing is important. We then use the trial-shuffle method to investigate information flow within a model network as we vary model parameters. This includes investigating the global flow of information within a connectivity network divided into two well-connected subnetworks, going beyond local transfer of information between pairs of neurons.