Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic non–AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNAiMet) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism allows non–AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.

Crosstalk between tumor cells and other cells within the tumor microenvironment (TME) plays a crucial role in tumor progression, metastases, and therapy resistance. We present iTALK, a computational approach to characterize and illustrate intercellular communication signals in the multicellular tumor ecosystem using single-cell RNA sequencing data. iTALK can in principle be used to dissect the complexity, diversity, and dynamics of cell-cell communication from a wide range of cellular processes.The TME has emerged as a key modulator of tumor progression, immune evasion, and emergence of the anti-tumor therapy resistance mechanisms 1, 2 . The TME includes a diversity of cell types such as tumor cells, a heterogeneous group of immune cells, and the nonimmune stromal components. Tumor cells orchestrate and interact dynamically with these non-tumor components, and the crosstalk between them is thought to provide key signals that can direct and promote tumor cell growth and migration. Through this intercellular communication, tumor cells can elicit profound phenotypic changes in other TME cells such as tumor-associated fibroblasts, macrophages and T cells, and reprogram the TME, in order to escape from immune surveillance to facilitate survival. Therefore, a better understanding of the cell-cell communication signals may help identify novel modulating therapeutic strategies for better patient advantage. However, this has been hampered by the lack of bioinformatics tools for efficient data analysis and visualization.Here, we present iTALK (identifying and illustrating alterations in intercellular signaling network; https://github.com/Coolgenome/iTALK), an open source R package designed to profile and visualize the ligand-receptor mediated intercellular cross-talk signals from singlecell RNA sequencing data (scRNA-seq) ( Fig. 1 and Online Methods). We demonstrated that iTALK can be successfully applied to scRNA-seq data to capture highly abundant ligandreceptor gene (or transcript) pairs, identify gains or losses of cellular interactions by comparative analysis, and track the dynamic changes of intercellular communication signals in longitudinal samples. Notably, functional annotation of ligand-receptor genes is automatically added with our curated iTALK ligand-receptor database, and the output can be visualized in different formats with our efficient data visualization tool, which is implemented as part of iTALK. This approach can be applied to data sets ranging from hundreds to hundreds of thousands of cells and is not limited by sequencing platforms. It is also noteworthy that, in addition to studying the TME, iTALK can also be applied to a wide range of biomedical research fields that involve cell-cell communication.

The adaptor protein CARD9 links detection of fungi by surface receptors to the activation of the NF-κB pathway. Mice deficient in CARD9 exhibit dysbiosis and are more susceptible to colitis. Here we examined the impact of Card9 deficiency in the development of colitis-associated colon cancer (CAC). Treatment of Card9 mice with AOM-DSS resulted in increased tumor loads as compared to WT mice and in the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissue. The impaired fungicidal functions of Card9 macrophages led to increased fungal loads and variation in the overall composition of the intestinal mycobiota, with a notable increase in C. tropicalis. Bone marrow cells incubated with C. tropicalis exhibited MDSC features and suppressive functions. Fluconazole treatment suppressed CAC in Card9 mice and was associated with decreased MDSC accumulation. The frequency of MDSCs in tumor tissues of colon cancer patients correlated positively with fungal burden, pointing to the relevance of this regulatory axis in human disease.

Opportunistic fungal infections are a leading cause of death for immune-compromised patients and there is pressing need to develop new anti-fungal therapeutic agents because of toxicity and resistance to current anti-fungal drugs. Although C-type lectin receptor- and Toll-like receptor-induced signaling pathways are key activators of host anti-fungal immunity, little is known about the negative regulation of these immune responses. Here, we found that JNK1 activation suppresses anti-fungal immunity in mice. We showed that JNK1-deficient mice had significantly higher survival rate in response to Candida albicans infection, and JNK1 expressed in hematopoietic innate immune cells is critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 promoter. Blocking CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced anti-fungal effect in JNK1-deficient mice. Notably, JNK inhibitors exerted potent anti-fungal therapeutic effects in Candida albicans-infected mouse and human cells, indicating that JNK1 can be a therapeutic target for treating fungal infection.

Summary:  Scaffold proteins play pivotal roles in the regulation of signal transduction pathways by connecting upstream receptors to downstream effector molecules. During the last decade, many scaffold proteins that contain caspase‐recruitment domains (CARD) have been identified. Investigating the roles of CARD proteins has revealed that many of them play crucial roles in signaling cascades leading to activation of nuclear factor‐κB (NF‐κB). In this review, we discuss the contributions of CARD proteins to NF‐κB activation in various signaling cascades. In particular, we share some of our personal experiences during the initial investigation of the functions of the CARMA family of CARD proteins and then summarize the roles of these proteins in signaling pathways induced by antigen receptors, G protein‐coupled receptors, receptor tyrosine kinase, and C‐type lectin receptors in the context of recent progress in these field.

Interactions between commensal fungi and gut immune system are critical for establishing colonic homeostasis. Here we found that mice deficient in Dectin-3 (Clec4d -/-), a C-type lectin receptor that senses fungal infection, were more susceptible to dextran sodium sulfate (DSS)-induced colitis compared with wild-type mice. The specific fungal burden of Candida (C.) tropicalis was markedly increased in the gut after DSS treatment in Clec4d -/- mice, and supplementation with C. tropicalis aggravated colitis only in Clec4d -/- mice, but not in wild-type controls. Mechanistically, Dectin-3 deficiency impairs phagocytic and fungicidal abilities of macrophages, and C. tropicalis-induced NF-κB activation and cytokine production. The conditioned media derived from Dectin-3-deficient macrophages were defective in promoting tissue repairing in colonic epithelial cells. Finally, anti-fungal therapy was effective in treating colitis in Clec4d -/- mice. These studies identified the role of Dectin-3 and its functional interaction with commensal fungi in intestinal immune system and regulation of colonic homeostasis.

Background: Recent studies suggest that Mincle expression is induced by Dectin-3-mediated signaling in response to TDM stimulation. Results: Deficiency in Dectin-3 and the CARD9-Bcl10-Malt1 complex are defective for TDM-induced NF-B activation and Mincle. Conclusion: Dectin-3-and CARD9/Bcl10/Malt1-dependent NF-B activation plays an essential role for TDM-induced Mincle expression. Significance: This study provides the molecular insight for designing adjuvants that stimulate the immune system.

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14-to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.Replicative DNA polymerases are highly processive enzymes that synthesize long stretches of DNA without dissociating from the template. Many replicative polymerases depend on accessory proteins called processivity factors, which confer processivity on their cognate polymerases by slowing their dissociation from DNA. Among these are the "sliding clamp" processivity factors (18), exemplified by proliferating cell nuclear antigen (PCNA), the processivity factor for eukaryotic replicative DNA polymerases. All sliding clamps adopt a common structure, in which a dimer or trimer of the sliding clamp protein forms a ring that encircles DNA (20,21,23,28). The association of the sliding clamps with DNA requires the activity of accessory proteins known as clamp loaders (19), which open the clamp ring and reclose it on DNA in an ATP-dependent reaction. The sliding clamp then can tether the catalytic polymerase core to DNA, thus ensuring processivity without impeding the movement of the polymerase.The DNA polymerase that replicates herpes simplex virus (HSV) is a heterodimer of a 137-kDa catalytic subunit (UL30, also called Pol) and a 60-kDa processivity subunit, UL42 (15,24,25). The extreme C terminus of Pol is necessary for interaction with UL42, long-chain DNA synthesis, and viral re...