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Background and Aims As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease. Methods To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo. Results Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries. Conclusions We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.

Numerous studies have shown viral antigens can persist in lymph nodes after resolution of the infection. We showed that lymphatic endothelial cells (LECs), which comprise the lymphatic vasculature necessary for antigen drainage from the tissue, is the predominant cell type required for the persistence of antigen within the lymph node. We termed this process antigen archiving due the ability of LECs to actively archive antigens to which an immune response has occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells (DC), resulting in presentation of the archived antigens to CD8+ memory T cells and improving effector function. In more recent data, we demonstrated that LEC death causes the release of archived antigens during LN contraction. The objective of this study was to determine if during a secondary unrelated infection whether LEC death would occur and cause archived antigens to be released. As expected, we found that a second and unrelated viral infection causes LEC death within two to three weeks following infection. Within the same time frame that we observed LEC death, we observed a significant increase in archived antigen-specific endogenous memory T cells. This increase only occurred in mice that received an unrelated viral infection and not in those mice that did not receive the unrelated viral infection. In conclusion, archived antigen release during a secondary unrelated infection potentially boosts the archived antigen specific memory CD8+ T cell population. Understanding the mechanism of antigen release during multiple infections could ultimately lead to better strategies for vaccination and improve our understanding of how LECs influence immunity.

While the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding signaling via the intracellular domain of PD-L1, termed PD-L1 reverse signaling. The objective of this study is to determine how PD-L1 reverse signaling impacts dendritic cell (DC) function during immune responses. We outline a major role for PD-L1 reverse signaling in the control of DC migration from the skin to the draining lymph node (dLN). We find that total loss of PD-L1 on DCs impairs migration to the dLN following an innate immune stimulus. Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. This TSS motif proves critical for promoting chemokine mediated DC migration to the dLN during inflammation. The loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. We found that the TSS motif is required for chemokine receptor signaling, and disruption of this motif impairs signaling downstream of the Gα subunit of the heterotrimeric G protein complex, leading to impairment in ERK phosphorylation and actin polymerization in DCs. Finally, we demonstrate that ligation of PD-L1 by one or more of the known binding partners of PD-L1, CD80 and PD-1, appears to support migration of DCs to the dLN. These studies identify a novel function for PD-L1 reverse signaling in supporting DC migration during initiation of immune responses, and raises questions about the potential impact of PD-L1 antibody blockade on DC function in patients with cancer.

// Agnieszka A. Kendrick 1 , Johnathon Schafer 1 , Monika Dzieciatkowska 1 , Travis Nemkov 1 , Angelo D’Alessandro 1 , Deepika Neelakantan 2 , Heide L. Ford 2 , Chad G. Pearson 3 , Colin D. Weekes 4 , Kirk C. Hansen 1 , Elan Z. Eisenmesser 1 1 Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, CO, USA 2 Department of Pharmacology, School of Medicine, University of Colorado Denver, CO, USA 3 Department of Cell and Developmental Biology, School of Medicine, University of Colorado Denver, CO, USA 4 Division of Oncology, Department of Medicine, University of Colorado Denver, CO, USA Correspondence to: Elan Z. Eisenmesser, email: elan.eisenmesser@ucdenver.edu Keywords: metabolism, ancillary protein, PDAC, transmembrane, tumor microenvironment Received: September 20, 2016 Accepted: November 30, 2016 Published: December 27, 2016 ABSTRACT Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner’s proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.

Once thought to be a remnant of cell division, the midbody (MB) has recently been shown to have roles beyond its primary function of orchestrating abscission. Despite the emerging roles of post-abscission MBs, how MBs accumulate in the cytoplasm and signal to regulate cellular functions remains unknown. Here, we show that extracellular post-abscission MBs can be internalized by interphase cells, where they reside in the cytoplasm as a membrane-bound signaling structure that we have named the MBsome. We demonstrate that MBsomes stimulate cell proliferation and that MBsome formation is a phagocytosis-like process that depends on a phosphatidylserine/integrin complex, driven by actin-rich membrane protrusions. Finally, we show that MBsomes rely on dynamic actin coats to slow lysosomal degradation and propagate their signaling function. In summary, MBsomes may sometimes serve as intracellular organelles that signal via integrin and EGFR-dependent pathways to promote cell proliferation and anchorage-independent growth and survival., The midbody is a structure found between the two dividing daughter cells that recruits regulators during cell division, but its role after cell division is poorly understood. Here, the authors find that midbodies can be internalized and send out intracellular signals to stimulate cell proliferation.

SUMMARY Although the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding the intracellular domain of PD-L1. Here, we outline a major role for PD-L1 intracellular signaling in the control of dendritic cell (DC) migration from the skin to the draining lymph node (dLN). Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. The TSS motif proves critical for chemokine-mediated DC migration to the dLN during inflammation. This loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. Finally, the TSS motif is required for chemokine receptor signaling downstream of the Gα subunit of the heterotrimeric G protein complex, ERK phosphorylation, and actin polymerization in DCs., Graphical Abstract, In Brief Lucas et al. define three residues within the cytoplasmic tail of PD-L1 that are required for proper dendritic cell migration from the skin to the lymph node. These three-amino-acid residues promote chemokine signaling in dendritic cells and productive T cell responses to skin infections.

CD147 is a highly glycosylated type I transmembrane protein upregulated in pancreatic cancer where it has received significant attention as a potential therapeutic target due to its regulation of several proteins that drive cancer progression. While CD147 was initially thought to act as a ligand on the cell surface to directly stimulate the secretion of several proteins, recent findings have suggested that CD147 may contribute to cancer progression through a much more complex set of interactions. The present study identified CD147 as having a unique chaperone role for multiple transmembrane proteins important for signaling events related to cancer metastasis and metabolic maintenance in pancreatic cancer. A robust combination of cellular, biochemical, proteomic and metabolomic studies showed that CD147 depletion leads to a significant decline in cell proliferation and migration, an increase in epithelial phenotype, and a striking reprogramming of cellular metabolism. Xenograft mouse models further confirmed these findings where tumor growth and metabolism were dependent on CD147 expression. In order to determine the underlying mechanism of CD147's role in pancreatic cancer, we combined crosslinking methods with mass spectrometry to identify the specific interactions and discovered that CD147 is responsible for targeting multiple transmembrane proteins to the cell surface. Furthermore, CD147 depletion caused a down-regulation of a subset of these proteins, which could then be reversed with proteosomal inhibitor treatment indicating that CD147 is a chaperone. Together, our studies illustrate the functional consequence of CD147 expression and chaperone activity in relation to regulating pancreatic cancer progression.