The FLRT3-UNC5B checkpoint pathway inhibits T cell-based cancer immunotherapies.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.

CrebA increases secretory capacity through direct transcriptional regulation of the secretory machinery, a subset of secretory cargo, and other key regulators.

Specialization of many cells, including the acinar cells of the salivary glands and pancreas, milk-producing cells of mammary glands, mucus-secreting goblet cells, antibody-producing plasma cells, and cells that generate the dense extracellular matrices of bone and cartilage, requires scaling up both secretory machinery and cell-type specific secretory cargo. Using tissue-specific genome-scale analyses, we determine how increases in secretory capacity are coordinated with increases in secretory load in the Drosophila salivary gland (SG), an ideal model for gaining mechanistic insight into the functional specialization of secretory organs. Our findings show that CrebA, a bZIP transcription factor, directly binds genes encoding the core secretory machinery, including protein components of the signal recognition particle and receptor, ER cargo translocators, Cop I and Cop II vesicles, as well as the structural proteins and enzymes of these organelles. CrebA directly binds a subset of SG cargo genes and CrebA binds and boosts expression of Sage, a SG-specific transcription factor essential for cargo expression. To further enhance secretory output, CrebA binds and activates Xbp1 and Tudor-SN. Thus, CrebA directly upregulates the machinery of secretion and additional factors to increase overall secretory capacity in professional secretory cells; concomitant increases in cargo are achieved both directly and indirectly.

mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis.

Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

Role of tbc1 in Drosophila embryonic salivary glands.

BACKGROUND: CG4552/tbc1 was identified as a downstream target of Fork head (Fkh), the single Drosophila member of the FoxA family of transcription factors and a major player in salivary gland formation and homeostasis. Tbc1 and its orthologues have been implicated in phagocytosis, the innate immune response, border cell migration, cancer and an autosomal recessive form of non-degenerative Pontocerebellar hypoplasia. Recently, the mammalian Tbc1 orthologue, Tbc1d23, has been shown to bind both the conserved N-terminal domains of two Golgins (Golgin-97 and Golgin-245) and the WASH complex on endosome vesicles. Through this activity, Tbc1d23 has been proposed to link endosomally-derived vesicles to their appropriate target membrane in the trans Golgi (TGN). RESULTS: In this paper, we provide an initial characterization of Drosophila orthologue, we call tbc1. We show that, like its mammalian orthologue, Tbc1 localizes to the trans Golgi. We show that it also colocalizes with a subset of Rabs associated with both early and recycling endosomes. Animals completely missing tbc1 survive, but females have fertility defects. Consistent with the human disease, loss of tbc1 reduces optic lobe size and increases response time to mechanical perturbation. Loss and overexpression of tbc1 in the embryonic salivary glands leads to secretion defects and apical membrane irregularities. CONCLUSIONS: These findings support a role for tbc1 in endocytic/membrane trafficking, consistent with its activities in other systems.

Independent recruitments of a translational regulator in the evolution of self-fertile nematodes.

Pleiotropic developmental regulators have been repeatedly linked to the evolution of anatomical novelties. Known mechanisms include cis-regulatory DNA changes that alter regulator transcription patterns or modify target-gene linkages. Here, we examine the role of another form of regulation, translational control, in the repeated evolution of self-fertile hermaphroditism in Caenorhabditis nematodes. Caenorhabditis elegans hermaphrodites initiate spermatogenesis in an otherwise female body through translational repression of the gene tra-2. This repression is mediated by GLD-1, an RNA-binding protein also required for oocyte meiosis and differentiation. By contrast, we show that in the convergently hermaphroditic Caenorhabditis briggsae, GLD-1 acts to promote oogenesis. The opposite functions of gld-1 in these species are not gene-intrinsic, but instead result from the unique contexts for its action that evolved in each. In C. elegans, GLD-1 became essential for promoting XX spermatogenesis via changes in the tra-2 mRNA and evolution of the species-specific protein FOG-2. C. briggsae GLD-1 became an essential repressor of sperm-promoting genes, including Cbr-puf-8, and did not evolve a strong association with tra-2. Despite its variable roles in sex determination, the function of gld-1 in female meiotic progression is ancient and conserved. This conserved role may explain why gld-1 is repeatedly recruited to regulate hermaphroditism. We conclude that, as with transcription factors, spatially localized translational regulators play important roles in the evolution of anatomical novelties.