A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Abstract: Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export

CRISPR/Cas9 generated human CD46, CD55 and CD59 knockout cell lines as a tool for complement research

To prevent unwanted complement activation and subsequent damage, complement activation must be tightly regulated on healthy host cells. Dysregulation of the complement system contributes to the pathology of diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. To investigate complement regulator deficiencies, primary patient cells may be used, but access to patient cells may be limited and cells are heterogeneous between different patients. To inhibit regulator function on healthy host cells, blocking antibodies can be used, though it may be difficult to exclude antibody-mediated effects. To circumvent these issues, we created single and combined complement regulator human knockout cells to be able to in vitro investigate complement activation and regulation on human cells. CRISPR/Cas9 was used to knockout (KO) complement regulatory proteins CD46, CD55 and/or CD59 in human HAP1 cells. Single cell derived cell lines were profiled by Sanger sequencing and flow cytometry. To confirm the lack of complement regulatory function, the cells were exposed to complement in normal human serum and subsequently C3 and C4 deposition on the cell surface were detected by using flow cytometry. We created single KO cell lines that completely lacked CD46, CD55 or CD59. We additionally generated double CD46/CD55, CD46/CD59 and CD55/CD59 KOs and triple CD46/CD55/CD59 KOs. Upon classical pathway activation, deletion of CD46 resulted in increased C3 and C4 deposition, while deleting CD55 mainly resulted to increased C3 deposition, confirming their reported function in complement regulation. Upon alternative pathway activation, C3 deposition was only observed on the triple CD46/CD55/CD59 KO cells and not on any of the other cell lines, suggesting that human cells are resistant to spontaneous complement activation and suggesting a role for CD59 in C3 regulation. The generation of complement regulator KO cell lines provides a relevant tool for future in vitro investigations of complement activation and regulation on human cells. Furthermore, these cell lines may also be helpful to evaluate therapeutic complement inhibitors and may shed light on novel roles of complement regulatory proteins as we here observed for CD5

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Abstract: Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export

Dendritic cells can prime anti-tumor CD8+ T cell responses through major histocompatibility complex cross-dressing

Antigen cross-presentation, wherein dendritic cells (DCs) present exogenous antigen on major histocompatibility class I (MHC-I) molecules, is considered the primary mechanism by which DCs initiate tumor-specific CD8+ T cell responses. Here, we demonstrate that MHC-I cross-dressing, an antigen presentation pathway in which DCs acquire and display intact tumor-derived peptide:MHC-I molecules, is also important in orchestrating anti-tumor immunity. Cancer cell MHC-I expression was required for optimal CD8+ T cell activation in two subcutaneous tumor models. In vivo acquisition of tumor-derived peptide:MHC-I molecules by DCs was sufficient to induce antigen-specific CD8+ T cell priming. Transfer of tumor-derived human leukocyte antigen (HLA) molecules to myeloid cells was detected in vitro and in human tumor xenografts. In conclusion, MHC-I cross-dressing is crucial for anti-tumor CD8+ T cell priming by DCs. In addition to quantitatively enhancing tumor antigen presentation, MHC cross-dressing might also enable DCs to more faithfully and efficiently mirror the cancer cell peptidome

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

OBJECTIVES: The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody-mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). METHODS: We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D. RESULTS: We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane-bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody-mediated diseases. CONCLUSION: The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement-mediated killing

A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation

MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunit

T Cells Specific for an Unconventional Natural Antigen Fail to Recognize Leukemic Cells

MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A02:01–restricted CD8 þ T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation

Abstract Objectives The complement system is an important component of innate immunity. The alternative pathway (AP) amplification loop is considered an essential feed forward mechanism for complement activation. However, the role of the AP in classical pathway (CP) activation has only been studied in ELISA settings. Here, we investigated its contribution on physiologically relevant surfaces of human cells and bacterial pathogens and in antibody‐mediated complement activation, including in autoimmune haemolytic anaemia (AIHA) setting with autoantibodies against red blood cells (RBCs). Methods We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP‐depleted sera or antibodies against factor B and factor D. Results We show that the amplification loop is redundant when efficient CP activation takes place. This is independent of the presence of membrane‐bound complement regulators. The role of the AP may become significant when insufficient CP complement activation occurs, but this depends on antibody levels and (sub)class. Our data indicate that therapeutic intervention in the amplification loop will most likely not be effective to treat antibody‐mediated diseases. Conclusion The AP can be bypassed through efficient CP activation. The AP amplification loop has a role in complement activation during conditions of modest activation via the CP, when it can allow for efficient complement‐mediated killing

The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation.

We evaluated the contribution of the AP to complement responses initiated through the CP on human RBCs by serum of AIHA patients and recombinant antibodies. Moreover, we studied complement activation on Neisseria meningitidis and Escherichia coli. The effect of the AP was examined using either AP-depleted sera or antibodies against factor B and factor D