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1.
J Immunol ; 207(2): 449-458, 2021 07 15.
Article in English | MEDLINE | ID: mdl-34215657

ABSTRACT

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.


Subject(s)
Antibody-Producing Cells/immunology , Cell Differentiation/immunology , Fas Ligand Protein/immunology , Immunologic Memory/immunology , Interleukins/immunology , Lupus Erythematosus, Systemic/immunology , Animals , Autoimmunity/immunology , B-Lymphocytes/immunology , CD40 Ligand/immunology , Cell Line , HEK293 Cells , Humans , Lymphocyte Activation/immunology , Mice , NIH 3T3 Cells , Plasma Cells/immunology , T-Lymphocytes/immunology
2.
iScience ; 24(2): 102051, 2021 02 19.
Article in English | MEDLINE | ID: mdl-33554062

ABSTRACT

Tumors with an impaired transporter associated with antigen processing (TAP) present several endoplasmic reticulum-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here, we monitored TAP-independent antigen presentation on various healthy cells after establishing a T cell tool recognizing a TAP-independent signal sequence receptor 1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy.

4.
Immunity ; 54(1): 132-150.e9, 2021 01 12.
Article in English | MEDLINE | ID: mdl-33271119

ABSTRACT

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , CD8-Positive T-Lymphocytes/immunology , Glioma/immunology , Glycosphingolipids/metabolism , Glycosyltransferases/metabolism , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Immunotherapy/methods , Antigen Presentation , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Glioma/mortality , Glycosphingolipids/immunology , HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Humans , Lymphocyte Activation , Signal Transduction , Survival Analysis , Tumor Escape
5.
Eur J Immunol ; 51(3): 734-737, 2021 03.
Article in English | MEDLINE | ID: mdl-33275281

ABSTRACT

A single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I antigen presentation pathway. PAKC will accelerate HLA-I research in the fields of oncology, infectiology, and autoimmunity.


Subject(s)
Antigen Presentation/immunology , Histocompatibility Antigens Class I/immunology , Autoimmunity/immunology , Humans , Neoplasms/immunology , Signal Transduction/immunology
6.
Cancer Immunol Res ; 7(5): 797-804, 2019 05.
Article in English | MEDLINE | ID: mdl-30890530

ABSTRACT

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-A*02:01-restricted CD8+ 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.


Subject(s)
Epitopes, T-Lymphocyte/immunology , Leukemia/immunology , T-Lymphocytes, Cytotoxic/immunology , Humans
7.
Biochem J ; 474(5): 667-681, 2017 02 20.
Article in English | MEDLINE | ID: mdl-28008132

ABSTRACT

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME-cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME-cyclase activity in the cyanobacterium SynechocystisSynechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME-cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME-cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.


Subject(s)
Bacterial Proteins/chemistry , Oxygenases/chemistry , Protein Subunits/chemistry , Protochlorophyllide/biosynthesis , Recombinant Proteins/chemistry , Synechocystis/genetics , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , Crystallography, X-Ray , Cyclization , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrogen Bonding , Mutagenesis, Site-Directed , Oxygenases/genetics , Oxygenases/metabolism , Photosynthesis/genetics , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Protein Subunits/genetics , Protein Subunits/metabolism , Protochlorophyllide/chemistry , Protoporphyrins/chemistry , Protoporphyrins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Static Electricity , Synechocystis/enzymology
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