CD8
T cells mediate antigen-specific immune responses that can induce rejection of solid tumors. In this process, dendritic cells (DCs) are thought to take up tumor antigens, which are processed into ...peptides and loaded onto MHC-I molecules, a process called "cross-presentation." Neither the actual contribution of cross-presentation to antitumor immune responses nor the intracellular pathways involved in vivo are clearly established because of the lack of experimental tools to manipulate this process. To develop such tools, we generated mice bearing a conditional DC-specific mutation in the
gene, a critical regulator of endoplasmic reticulum-phagosome traffic required for cross-presentation. DCs from these mice show impaired cross-presentation ex vivo and defective cross-priming of CD8
T cell responses in vivo. These mice are also defective for antitumor immune responses and are resistant to treatment with anti-PD-1. We conclude that Sec22b-dependent cross-presentation in DCs is required to initiate CD8
T cell responses to dead cells and to induce effective antitumor immune responses during anti-PD-1 treatment in mice.
Pore-forming proteins (PFPs) are present in all domains of life, and play an important role in host-pathogen warfare and in the elimination of cancers. They can be employed to deliver specific ...effectors across membranes, to disrupt membrane integrity interfering with cell homeostasis, and to lyse membranes either destroying intracellular organelles or entire cells. Considering the destructive potential of PFPs, it is perhaps not surprising that mechanisms controlling their activity are remarkably complex, especially in multicellular organisms. Mammalian PFPs discovered to date include the complement membrane attack complex (MAC), perforins, as well as gasdermins. While the primary function of perforin-1 and gasdermins is to eliminate infected or cancerous host cells, perforin-2 and MAC can target pathogens directly. Yet, all mammalian PFPs are in principle capable of generating pores in membranes of healthy host cells which-if uncontrolled-could have dire, and potentially lethal consequences. In this review, we will highlight the strategies employed to protect the host from destruction by endogenous PFPs, while enabling timely and efficient elimination of target cells.
Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still ...unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8
T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain ...incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers.
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•Quinazolinamine derivatives are potent enhancers of antigen import into the cytosol•Antigen import enhancement occurs as a consequence of lysosomal trapping•Antigen import enhancers facilitate cross-presentation and synergize with anti-PD-L1•Proteomic organellar maps can be used as a tool to detect biological effects of drugs
Kozik et al. apply a screening strategy to identify drugs that facilitate endosome-to-cytosol import of antigens in dendritic cells during cross-presentation. They characterize selected compounds using proteomic organellar mapping, live-cell imaging, in vitro cross-presentation assays, and tumor models and demonstrate that enhancing import facilitates cross-presentation and synergizes with checkpoint-based immunotherapy.
Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated ...vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.
Adenovirus has enormous potential as a gene-therapy vector, but preexisting immunity limits its widespread application. What is responsible for this immune block is unclear because antibodies ...potently inhibit transgene expression without impeding gene transfer into target cells. Here we show that antibody prevention of adenoviral gene delivery in vivo is mediated by the cytosolic antibody receptor TRIM21. Genetic KO of TRIM21 or a single-antibody point mutation is sufficient to restore transgene expression to near-naïve immune levels. TRIM21 is also responsible for blocking cytotoxic T cell induction by vaccine vectors, preventing a protective response against subsequent influenza infection and an engrafted tumor. Furthermore, adenoviral preexisting immunity can lead to an augmented immune response upon i.v. administration of the vector. Transcriptomic analysis of vector-transduced tissue reveals that TRIM21 is responsible for the specific up-regulation of hundreds of immune genes, the majority of which are components of the intrinsic or innate response. Together, these data define a major mechanism underlying the preimmune block to adenovirus gene therapy and demonstrate that TRIM21 efficiently blocks gene delivery in vivo while simultaneously inducing a rapid program of immune transcription.
Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated ...endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase-knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.
The sensing of pathogenic stimuli by dendritic cells (DCs) promotes antigen processing for cross-presentation, whereby the activation of DCs is followed by their migration to lymph nodes and spleen, ...where the cross-presented antigens can prime naïve T-cells for an adaptive immune response. DCs can also cross-present antigens in the absence of infection, but endogenous signals that promote antigen processing and presentation remain poorly understood.
We observed that DCs efficiently cross-present cell-associated antigens from fibroblasts (3T3), even in the absence of pathogenic stimulus. We hypothesized that signals that promote DC activation might also promote processing of cell-associated antigens for presentation. In order to determine the molecular components responsible for DC activation, we performed a CRISPR knockout screen targeting a total of 704 known or predicted regulators involved in sensing of pathogens/danger signals. DCs were co-cultured with 3T3 and sorted into two populations for high and low expression of activation markers on DCs and the guides enriched were determined by sequencing.
Prostaglandin E receptor 2 (PTGER2) was found to be the major hit. The results were validated in cDC1-like cell line, MutuDC, where direct activation of PTGER2 using its ligand prostaglandin E2 (PGE2) resulted in increased expression of activation markers (CD83 and CD86). Inhibition of the PGE2 production or of its receptor using small molecule inhibitors, resulted in significantly reduced activation of dendritic cells when co-cultured with 3T3 cells. The DC activation via PTGER2 was found to be mediated via cAMP production upon adenylyl cyclase activation. In addition, the inhibition of PTGER2 and PGE2 production via COX1/COX2 also resulted in reduced cross-presentation of cell-associated antigens.
CRISPR screen found PTGER2 as the molecular component in dendritic cells that can regulate both DC activation and cross-presentation of cell-associated antigens in the absence of infection.
The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef's functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins ...clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV.
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