Antigen structure affects cellular routing through DC-SIGN.

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure-longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens.

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases.

The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/ß, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes.

HIV-1 Gag, Envelope, and Extracellular Determinants Cooperate To Regulate the Stability and Turnover of Virological Synapses.

UNLABELLED: Retroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high-avidity Env-CD4 linkages are resolved over time is unknown. We describe here a tractable two-color, long-term (>24 h) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-to-cell transmission wherein (i) Env-receptor interactions anchor target cells to infected cells, (ii) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and (iii) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes. IMPORTANCE: HIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses (VSs). The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals. We describe here a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people.

Pulsed low-dose-rate radiation (PLDR) reduces the tumor-promoting responses induced by conventional chemoradiation in pancreatic cancer-associated fibroblasts.

Pancreatic cancer is becoming increasingly deadly, with treatment options limited due to, among others, the complex tumor microenvironment (TME). This short communications study investigates pulsed low-dose-rate radiation (PLDR) as a potential alternative to conventional radiotherapy for pancreatic cancer neoadjuvant treatment. Our ex vivo research demonstrates that PLDR, in combination with chemotherapy, promotes a shift from tumor-promoting to tumor-suppressing properties in a key component of the pancreatic cancer microenvironment we called CAFu (cancer-associated fibroblasts and selfgenerated extracellular matrix functional units). This beneficial effect translates to reduced desmoplasia (fibrous tumor expansion) and suggests PLDR's potential to improve total neoadjuvant therapy effectiveness. To comprehensively assess this functional shift, we developed the HOST-Factor, a single score integrating multiple biomarkers. This tool provides a more accurate picture of CAFu function compared to individual biomarkers and could be valuable for guiding and monitoring future therapeutic strategies. Our findings support the ongoing NCT04452357 clinical trial testing PLDR safety and TME normalization potential in pancreatic cancer patients. The HOST-Factor will be used in samples collected from this trial to validate its potential as a key tool for personalized medicine in this aggressive disease.

Netrin G1 Ligand is a new stromal immunomodulator that promotes pancreatic cancer.

Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-ß-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-ß pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer. Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-ß, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.

Trogocytosis of cancer-associated fibroblasts promotes pancreatic cancer growth and immune suppression via phospholipid scramblase anoctamin 6 (ANO6).

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

Meaningful connections: Interrogating the role of physical fibroblast cell-cell communication in cancer.

As part of the connective tissue, activated fibroblasts play an important role in development and disease pathogenesis, while quiescent resident fibroblasts are responsible for sustaining tissue homeostasis. Fibroblastic activation is particularly evident in the tumor microenvironment where fibroblasts transition into tumor-supporting cancer-associated fibroblasts (CAFs), with some CAFs maintaining tumor-suppressive functions. While the tumor-supporting features of CAFs and their fibroblast-like precursors predominantly function through paracrine chemical communication (e.g., secretion of cytokine, chemokine, and more), the direct cell-cell communication that occurs between fibroblasts and other cells, and the effect that the remodeled CAF-generated interstitial extracellular matrix has in these types of cellular communications, remain poorly understood. Here, we explore the reported roles fibroblastic cell-cell communication play within the cancer stroma context and highlight insights we can gain from other disciplines.

NetrinG1+ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5ß1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5ß1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5ß1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.