How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

Chimeric antigen receptor (CAR) T cell therapy is one of the most promising cancer treatments. However, different hurdles are limiting its application and efficacy. In this context, how aging influences CAR-T cell outcomes is largely unknown. Here we show that CAR-T cells generated from aged female mice present a mitochondrial dysfunction derived from nicotinamide adenine dinucleotide (NAD) depletion that leads to poor stem-like properties and limited functionality in vivo. Moreover, human data analysis revealed that both age and NAD metabolism determine the responsiveness to CAR-T cell therapy. Targeting NAD pathways, we were able to recover the mitochondrial fitness and functionality of CAR-T cells derived from older adults. Altogether, our study demonstrates that aging is a limiting factor to successful CAR-T cell responses. Repairing metabolic and functional obstacles derived from age, such as NAD decline, is a promising strategy to improve current and future CAR-T cell therapies.

In recent years, immunotherapy has become the most promising therapy for a variety of cancer types. The development of immune checkpoint blockade (ICB) therapies, the adoptive transfer of tumor-specific T cells (adoptive cell therapy (ACT)) or the generation of T cells engineered with chimeric antigen receptors (CAR) have been successfully applied to elicit durable immunological responses in cancer patients. However, not all the patients respond to these therapies, leaving a consistent gap of therapeutic improvement that still needs to be filled. The innate immune components of the tumor microenvironment play a pivotal role in the activation and modulation of the adaptive immune response against the tumor. Indeed, several efforts are made to develop strategies aimed to harness innate immune cells in the context of cancer immunotherapy. In this review, we describe the contribution of innate immune cells in T-cell-based cancer immunotherapy and the therapeutic approaches implemented to broaden the efficacy of these therapies in cancer patients.

Naïve T cells are key players in cancer immunosurveillance, even though their function declines during tumor progression. Thus, interventions capable of sustaining the quality and function of naïve T cells are needed to improve cancer immunoprevention. In this context, we studied the capacity of Urolithin-A (UroA), a potent mitophagy inducer, to enhance T cell–mediated cancer immunosurveillance. We discovered that UroA improved the cancer immune response by activating the transcription factor FOXO1 in CD8+ T cell. Sustained FOXO1 activation promoted the expression of the adhesion molecule L-selectin (CD62L) resulting in the expansion of the naïve T cells population. We found that UroA reduces FOXO1 phosphorylation favoring its nuclear localization and transcriptional activity. Overall, our findings determine FOXO1 as a novel molecular target of UroA in CD8+ T cells and indicate UroA as promising immunomodulator to improve cancer immunosurveillance. Significance: Urolithin-A, a potent mitophagy inducer, emerges as a promising tool to enhance cancer immunosurveillance by activating the FOXO1 transcription factor in CD8+ T cells. This activation promotes the expansion of naïve T cells, offering a novel avenue for improving cancer immune response and highlighting UroA as a potential immunomodulator for bolstering our body's defenses against cancer.

Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans-Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediated TGN access. Our study provides a proof in principle that compartment-specific biosensors can be used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.