Highlights Deterministic barcoding in tissue enables NGS-based spatial multi-omics mapping. DBiT-seq identified spatial patterning of major tissue types in mouse embryos. DBiT-seq revealed fine features such as retinal pigmented epithelium and microvascular endothelium at the cellular level. Direct integration with scRNA-seq data allows for rapid cell type identification.

Large eddy simulations of turbulent flow over a sphere are conducted at subcritical Reynolds numbers (Re=3700 and 104) based on the freestream velocity and sphere diameter. At Re=3700, the separating shear layer persists downstream to form a cylindrical vortex sheet and its instability becomes manifest at x≈2d. The flow right behind the sphere contains only a few vortices. On the other hand, at Re=104, the shear-layer instability occurs right behind the sphere in a form of vortex rings, and the flow becomes turbulent in the near wake. Therefore, at Re=104, the size of the recirculation region is smaller and the wake recovers more quickly than at Re=3700. At both Reynolds numbers, large-scale waviness of vortical structures is observed in the wake and the plane containing the large-scale waviness changes quasirandomly in time. This waviness is more pronounced at Re=104 than at Re=3700. The mechanism responsible for this large-scale waviness of vortical structures is shown to be closely associated with the temporal evolution of vortices generated by the shear-layer instability.

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 + helper T (T H ) cells and CD8 + cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T H 1 and T H 2 signature cytokines, e.g. , interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3 . However, rather than conforming to stringent T H 1 or T H 2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T H 1/T H 2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T H 1/T H 2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.