Spatially Resolved CRISPR Screen Sequencing via Perturb-DBiT.

Perturb-seq enabled the profiling of transcriptional effects of genetic perturbations in single cells but lacks the ability to examine the impact on tissue environments. We present Perturb-DBiT for simultaneous co-sequencing of spatial transcriptome and guide RNAs (gRNAs) on the same tissue section for in vivo CRISPR screen with genome-scale gRNA libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture. This platform supports a variety of delivery vectors, gRNA library sizes, and tissue preparations, along with two distinct gRNA capture methods, making it adaptable to a wide range of experimental setups.

DRAK2 regulates myosin light chain phosphorylation in T cells.

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2; also known as STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear.

CRISPR screens unveil nutrient-dependent lysosomal and mitochondrial nodes impacting intestinal tissue-resident memory CD8 T cell formation.

Nutrient availability and organelle biology direct tissue homeostasis and cell fate, but how these processes orchestrate tissue immunity remains poorly defined. Here, using in vivo CRISPR-Cas9 screens, we uncovered organelle signaling and metabolic processes shaping CD8 tissue-resident memory T (T) cell development. T cells depended on mitochondrial translation and respiration.

Antitumor progenitor exhausted CD8 T cells are sustained by TCR engagement.

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8 T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure.

DNAJB1-PRKACA fusion neoantigens elicit rare endogenous T cell responses that potentiate cell therapy for fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a liver tumor with a high mortality burden and few treatment options. A promising therapeutic vulnerability in FLC is its driver mutation, a conserved DNAJB1-PRKACA gene fusion that could be an ideal target neoantigen for immunotherapy. In this study, we aim to define endogenous CD8 T cell responses to this fusion in FLC patients and evaluate fusion-specific T cell receptors (TCRs) for use in cellular immunotherapies.

c-Abl Phosphorylates MFN2 to Regulate Mitochondrial Morphology in Cells under Endoplasmic Reticulum and Oxidative Stress, Impacting Cell Survival and Neurodegeneration.

The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria.

CXCR6 orchestrates brain CD8 T cell residency and limits mouse Alzheimer's disease pathology.

Neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by innate immune-mediated inflammation, but functional and mechanistic effects of the adaptive immune system remain unclear. Here we identify brain-resident CD8 T cells that coexpress CXCR6 and PD-1 and are in proximity to plaque-associated microglia in human and mouse AD brains. We also establish that CD8 T cells restrict AD pathologies, including β-amyloid deposition and cognitive decline.

SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours. DC functions are orchestrated by pattern recognition receptors, although other signals involved remain incompletely defined.

CAR T-cell design dependent remodeling of the brain tumor immune microenvironment identify macrophages as key players that inhibit or promote anti-tumor activity.

Understanding interactions between adoptively transferred immune cells and the tumor immune microenvironment (TIME) is critical for developing successful T-cell based immunotherapies. Here we investigated the impact of the TIME and chimeric antigen receptor (CAR) design on anti-glioma activity of B7-H3-specific CAR T-cells. We show that five out of six B7-H3 CARs with varying transmembrane, co-stimulatory, and activation domains, exhibit robust functionality .

Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.

Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9-dependent apoptosis.

Bacterial TLR2/6 Ligands Block Ciliogenesis, Derepress Hedgehog Signaling, and Expand the Neocortex.

Microbial components have a range of direct effects on the fetal brain. However, little is known about the cellular targets and molecular mechanisms that mediate these effects. Neural progenitor cells (NPCs) control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders.

Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death.

Ca-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak.

The ability of mitochondria to buffer a rapid rise in cytosolic Ca is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca.

Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions.

Invariant natural killer T (iNKT) cells acquire effector functions during development by mechanisms that remain poorly understood. Here, we show that the Hippo kinases Mst1 and Mst2 act as molecular rheostats for the terminal maturation and effector differentiation programs of iNKT cells. Loss of Mst1 alone or together with Mst2 impedes iNKT cell development, associated with defective IL-15-dependent cell survival.

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.

Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8 T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8 T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia.

Mevalonate metabolism-dependent protein geranylgeranylation regulates thymocyte egress.

Thymocyte egress is a critical determinant of T cell homeostasis and adaptive immunity. Despite the roles of G protein-coupled receptors in thymocyte emigration, the downstream signaling mechanism remains poorly defined. Here, we report the discrete roles for the two branches of mevalonate metabolism-fueled protein prenylation pathway in thymocyte egress and immune homeostasis.

Amino Acids License Kinase mTORC1 Activity and Treg Cell Function via Small G Proteins Rag and Rheb.

Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes.

Patrolling monocytes promote the pathogenesis of early lupus-like glomerulonephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with genetic and environmental contributions. Hallmarks of the disease are the appearance of immune complexes (IC) containing autoreactive Abs and TLR-activating nucleic acids, whose deposition in kidney glomeruli is suspected to promote tissue injury and glomerulonephritis (GN). Here, using a mouse model based on the human SLE susceptibility locus TNFAIP3-interacting protein 1 (TNIP1, also known as ABIN1), we investigated the pathogenesis of GN.

LKB1 orchestrates dendritic cell metabolic quiescence and anti-tumor immunity.

Dendritic cells (DCs) play a pivotal role in priming adaptive immunity. However, the involvement of DCs in controlling excessive and deleterious T cell responses remains poorly defined. Moreover, the metabolic dependence and regulation of DC function are unclear.

Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory Activity.

Interleukin-2 (IL-2) and downstream transcription factor STAT5 are important for maintaining regulatory T (Treg) cell homeostasis and function. Treg cells can respond to low IL-2 levels, but the mechanisms of STAT5 activation during partial IL-2 deficiency remain uncertain. We identified the serine-threonine kinase Mst1 as a signal-dependent amplifier of IL-2-STAT5 activity in Treg cells.

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis.

Hippo/Mst signalling couples metabolic state and immune function of CD8α dendritic cells.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α dendritic cells present antigens to CD8 T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours . Although lineage-specific transcriptional regulators of CD8α dendritic cell development have been identified , the molecular pathways that selectively orchestrate CD8α dendritic cell function remain elusive.

Early TCR Signaling Induces Rapid Aerobic Glycolysis Enabling Distinct Acute T Cell Effector Functions.

To fulfill bioenergetic demands of activation, T cells perform aerobic glycolysis, a process common to highly proliferative cells in which glucose is fermented into lactate rather than oxidized in mitochondria. However, the signaling events that initiate aerobic glycolysis in T cells remain unclear. We show T cell activation rapidly induces glycolysis independent of transcription, translation, CD28, and Akt and not involving increased glucose uptake or activity of glycolytic enzymes.