Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1.

Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.

AMBRA1 controls the translation of immune-specific genes in T lymphocytes.

T cell receptor (TCR) engagement causes a global cellular response that entrains signaling pathways, cell cycle regulation, and cell death. The molecular regulation of mRNA translation in these processes is poorly understood. Using a whole-genome CRISPR screen for regulators of CD95 (FAS/APO-1)-mediated T cell death, we identified AMBRA1, a protein previously studied for its roles in autophagy, E3 ubiquitin ligase activity, and cyclin regulation. T cells lacking AMBRA1 resisted FAS-mediated cell death by down-regulating FAS expression at the translational level. We show that AMBRA1 is a vital regulator of ribosome protein biosynthesis and ribosome loading on select mRNAs, whereby it plays a key role in balancing TCR signaling with cell cycle regulation pathways. We also found that AMBRA1 itself is translationally controlled by TCR stimulation via the CD28-PI3K-mTORC1-EIF4F pathway. Together, these findings shed light on the molecular control of translation after T cell activation and implicate AMBRA1 as a translational regulator governing TCR signaling, cell cycle progression, and T cell death.

Epithelial-immune cell crosstalk for intestinal barrier homeostasis.

The intestinal barrier is mainly formed by a monolayer of epithelial cells, which forms a physical barrier to protect the gut tissues from external insults and provides a microenvironment for commensal bacteria to colonize while ensuring immune tolerance. Moreover, various immune cells are known to significantly contribute to intestinal barrier function by either directly interacting with epithelial cells or by producing immune mediators. Fulfilling this function of the gut barrier for mucosal homeostasis requires not only the intrinsic regulation of intestinal epithelial cells (IECs) but also constant communication with immune cells and gut microbes. The reciprocal interactions between IECs and immune cells modulate mucosal barrier integrity. Dysregulation of barrier function could lead to dysbiosis, inflammation, and tumorigenesis. In this overview, we provide an update on the characteristics and functions of IECs, and how they integrate their functions with tissue immune cells and gut microbiota to establish gut homeostasis.

Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation.

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

PD-1 controls differentiation, survival, and TCR affinity evolution of stem-like CD8+ T cells.

Stem-like progenitors are a critical subset of cytotoxic T cells that self-renew and give rise to expanded populations of effector cells critical for successful checkpoint blockade immunotherapy. Emerging evidence suggests that the tumor-draining lymph nodes can support the continuous generation of these stem-like cells that replenish the tumor sites and act as a critical source of expanded effector populations, underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Using advanced 3D multiplex immunofluorescence imaging, here we identified antigen-presentation niches in tumor-draining lymph nodes that support the expansion, maintenance, and affinity evolution of a unique population of TCF-1+PD-1+SLAMF6 hi stem-like CD8+ T cells. Our results show that contrary to the prevailing view that persistent TCR signaling drives terminal effector differentiation, prolonged antigen engagement well beyond the initial priming phase sustained the proliferation and self-renewal of these stem-like T cells in vivo . The inhibitory PD-1 pathway plays a central role in this process by mediating the fine-tuning of TCR and co-stimulatory signal input that enables selective expansion of high affinity TCR stem-like clones, enabling them to act as a renewable source of high affinity effector cells. PD-1 checkpoint blockade disrupts this fine tuning of input signaling, leading to terminal differentiation to the effector state or death of the most avid anti-tumor stem-like cells. Our results thus reveal an unexpected relationship between TCR ligand affinity recognition, a key negative feedback regulatory loop, and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 checkpoint blockade during cancer immunotherapy provides a short-term anti-tumor effect that comes at the cost of diminishing efficacy due to progressive loss of these critical high affinity stem-like precursors.

The IBEX Knowledge-Base: Achieving more together with open science.

Iterative Bleaching Extends multipleXity (IBEX) is a versatile method for highly multiplexed imaging of diverse tissues. Based on open science principles, we created the IBEX Knowledge-Base, a resource for reagents, protocols and more, to empower innovation.

CRISPR/Cas9-Based Genetic Screening to Study T-Cell Function.

T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.

APOBEC3 induces mutations during repair of CRISPR-Cas9-generated DNA breaks.

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR-Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR-Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

Key elements for designing and performing a CRISPR/Cas9-based genetic screen.

Reverse genetic screens are invaluable for uncovering gene functions, but are traditionally hampered by some technical limitations. Over the past few years, since the advent of the revolutionary CRISPR/Cas9 technology, its power in genome editing has been harnessed to overcome the traditional limitations in reverse genetic screens, with successes in various biological contexts. Here, we outline these CRISPR/Cas9-based screens, provide guidance on the design of effective screens and discuss the potential future directions of development of this field.