Human anti-C1q autoantibodies bind specifically to solid-phase C1q and enhance phagocytosis but not complement activation.

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology.

Apolipoprotein L genes are novel mediators of inflammation in beta cells.

AIMS/HYPOTHESIS: Inflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage. METHODS: We used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-ßH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells. RESULTS: APOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus. CONCLUSIONS/INTERPRETATION: APOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes. DATA AVAILABILITY: scRNAseq data generated by our laboratory and used in this study are available in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/ ), accession number GSE218316.

OMIP-104: A 30-color spectral flow cytometry panel for comprehensive analysis of immune cell composition and macrophage subsets in mouse metabolic organs.

Obesity-induced chronic low-grade inflammation, also known as metaflammation, results from alterations of the immune response in metabolic organs and contributes to the development of fatty liver diseases and type 2 diabetes. The diversity of tissue-resident leukocytes involved in these metabolic dysfunctions warrants an in-depth immunophenotyping in order to elucidate disease etiology. Here, we present a 30-color, full spectrum flow cytometry panel, designed to (i) identify the major innate and adaptive immune cell subsets in murine liver and white adipose tissues and (ii) discriminate various tissue-specific myeloid subsets known to contribute to the development of metabolic dysfunctions. This panel notably allows for distinguishing embryonically-derived liver-resident Kupffer cells from newly recruited monocyte-derived macrophages and KCs. Furthermore, several adipose tissue macrophage (ATM) subsets, including perivascular macrophages, lipid-associated macrophages, and pro-inflammatory CD11c+ ATMs, can also be identified. Finally, the panel includes cell-surface markers that have been associated with metabolic activation of different macrophage and dendritic cell subsets. Altogether, our spectral flow cytometry panel allows for an extensive immunophenotyping of murine metabolic tissues, with a particular focus on metabolically-relevant myeloid cell subsets, and can easily be adjusted to include various new markers if needed.

IFNÉ£ but not IFNα increases recognition of insulin defective ribosomal product-derived antigen to amplify islet autoimmunity.

AIMS/HYPOTHESIS: The inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNÉ£) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells. METHODS: Single-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-ßH1 was monitored using peptide-specific CD8 T cells. RESULTS: We found that IFNγ induces the expression of the PSMB10 transcript encoding the ß2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. DATA AVAILABILITY: The single-cell RNA-seq dataset is available from the Gene Expression Omnibus (GEO) using the accession number GSE218316 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218316 ).

Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells.

AIMS/HYPOTHESIS: Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. METHODS: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1ß release. RESULTS: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. DATA AVAILABILITY: The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

Diabetes risk loci-associated pathways are shared across metabolic tissues.

AIMS/HYPOTHESIS: Numerous genome-wide association studies have been performed to understand the influence of genetic variation on type 2 diabetes etiology. Many identified risk variants are located in non-coding and intergenic regions, which complicates understanding of how genes and their downstream pathways are influenced. An integrative data approach will help to understand the mechanism and consequences of identified risk variants. METHODS: In the current study we use our previously developed method CONQUER to overlap 403 type 2 diabetes risk variants with regulatory, expression and protein data to identify tissue-shared disease-relevant mechanisms. RESULTS: One SNP rs474513 was found to be an expression-, protein- and metabolite QTL. Rs474513 influenced LPA mRNA and protein levels in the pancreas and plasma, respectively. On the pathway level, in investigated tissues most SNPs linked to metabolism. However, in eleven of the twelve tissues investigated nine SNPs were linked to differential expression of the ribosome pathway. Furthermore, seven SNPs were linked to altered expression of genes linked to the immune system. Among them, rs601945 was found to influence multiple HLA genes, including HLA-DQA2, in all twelve tissues investigated. CONCLUSION: Our results show that in addition to the classical metabolism pathways, other pathways may be important to type 2 diabetes that show a potential overlap with type 1 diabetes.

Islet stress, degradation and autoimmunity.

ß-cell destruction in type 1 diabetes (T1D) results from the effect of inflammation and autoimmunity. In response to inflammatory signals, islet cells engage adaptive mechanisms to restore and maintain cellular homeostasis. Among these mechanisms, the unfolded protein response (UPR) leads to a reduction of the general protein translation rate, increased production of endoplasmic reticulum chaperones and the initiation of degradation by activation of the ER associated degradation pathway (ERAD) in which newly synthetized proteins are ubiquitinylated and processed through the proteasome. This adaptive phase is also believed to play a critical role in the development of autoimmunity by the generation of neoantigens. While we have previously investigated the effect of stress on transcription, translation and post-translational events as possible source for neoantigens, the participation of the degradation machinery, yet crucial in the generation of antigenic peptides, remains to be investigated in the context of T1D pathology. In this review, we will describe the relation between the unfolded protein response and the Ubiquitin Proteasome System (UPS) and address the role of the cellular degradation machinery in the generation of antigens. Learning from tumour immunology, we propose how these processes may unmask ß-cells by triggering the generation of aberrant peptides recognized by the immune cells.

Immunogenicity of human embryonic stem cell-derived beta cells.

AIMS/HYPOTHESIS: To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells. METHODS: We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice. RESULTS: We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells. CONCLUSIONS/INTERPRETATION: hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.

Innate and adaptive immunity to human beta cell lines: implications for beta cell therapy.

AIMS/HYPOTHESIS: Genetically engineered human beta cell lines provide a novel source of human beta cells to study metabolism, pharmacology and beta cell replacement therapy. Since the immune system is essentially involved in beta cell destruction in type 1 diabetes and after beta cell transplantation, we investigated the interaction of human beta cell lineswith the immune system to resolve their potential for immune intervention protocol studies. METHODS: Human pancreatic beta cell lines (EndoC-ßH1 and ECi50) generated by targeted oncogenesis in fetal pancreas were assessed for viability after innate and adaptive immune challenges. Beta cell lines were pre-conditioned with T helper type 1 (Th1) cytokines or high glucose to mimic inflammatory and hyperglycaemia-stressed conditions. Beta cells were then co-cultured with auto- and alloreactive cytotoxic T cells (CTL), natural killer (NK) cells, supernatant fraction from activated autoreactive Th1 cells, or alloantibodies in the presence of complement or effector cells. RESULTS: Low HLA expression protected human beta cell lines from adaptive immune destruction, but it was associated with direct killing by activated NK cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta cell apoptosis and upregulation of HLA, increasing beta cell vulnerability to killing by auto- and alloreactive CTL and alloreactive antibodies. CONCLUSIONS/INTERPRETATION: We demonstrate that genetically engineered human beta cell lines can be used in vitro to assess diverse immune responses that may be involved in the pathogenesis of type 1 diabetes in humans and beta cell transplantation, enabling preclinical evaluation of novel immune intervention strategies protecting beta cells from immune destruction.

Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis.

OBJECTIVES: Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA. METHODS: Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry. RESULTS: By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture. CONCLUSIONS: We developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets.