Biology subjects

Sims, E. K.

Publications and source records attributed to Sims, E. K..

9 recordsLinked to original sources

bioRxiv2026

A Novel PD-L1 Splice Isoform Modulates β Cell Communication in Response to Interferon Signaling

{beta} cell expression of the immune checkpoint ligand PD-L1 (encoded by CD274) limits autoimmune {beta} cell destruction in type 1 diabetes (T1D). {beta} cell display PD-L1 not only at the cell surface but also secreted on extracellular vesicles (EVs), which bind PD-1 and restrain CD8+ T cell activation. {beta} cell IFN signaling is an early driver of T1D pathogenesis, yet the mechanisms linking IFN signaling to the fate of PD-L1 protein remain undefined. Here, we identify an IFN- and coxsackievirus-inducible alternatively spliced isoform of PD-L1, PD-L1{Delta}3, lacking cassette exon 3 and generated in human {beta} cells and islets in response to IFN signaling or viral infection. PD-L1{Delta}3 transcripts are elevated in islets from donors with single autoantibody positivity (AAB+) and T1D. Unlike full-length PD-L1, which localizes to the plasma membrane, PD-L1{Delta}3 is retained intracellularly and loses the capacity to bind PD-1. Functionally, PD-L1{Delta}3 fails to suppress CD8+ and CD4+ T cell proliferation, activation, and cytotoxic cytokine release, and is not efficiently sorted into EVs. Convergently, a germline CD274 splice-site variant (c.682+1G>A) found in siblings with neonatal T1D yields a protein with the same intracellular retention, reduced {beta} cell PD-1 binding, and reduced {beta} cell and circulating EV PD-L1. Together these findings define PD-L1{Delta}3 as an IFN-induced splice variant that diverts PD-L1 away from its immunoregulatory, EV-competent form, revealing a post-transcriptional axis that shapes {beta} cell immune communication.

cell biology↗

bioRxiv2026

Carbamylome analysis reveals regulatory roles for lysine carbamylation in β-cell glycolytic and insulin-processing enzymes

In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFN{gamma} + IL-1{beta} + TNF identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced {beta}-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for {beta}-cell metabolism and insulin production in the context of islet inflammation.

biochemistry↗

bioRxiv2026

Assessing extracellular vesicle proteins as predictive biomarkers for developing type 1 diabetes

Plasma extracellular vesicles (EVs) are considered excellent sources for biomarker discovery since they carry signatures of their cellular origin and disease processes. In this paper, we evaluate the potential of plasma EV proteomics analysis for identifying predictive biomarkers of developing type 1 diabetes (T1D), which results from autoimmune destruction of insulin-producing {beta} cells in the islet. We used strong anion exchange beads (Mag-Net) to capture plasma EVs from 19 donors with islet autoimmunity (diagnosed by circulating autoantibodies against islet proteins - AAB+) vs. 17 control individuals and analyzed their protein cargo by mass spectrometry. The analysis identified and quantified 5,480 proteins, a 3.2-fold increase in proteome coverage compared to our previous T1D biomarker proteomics study that used whole plasma depleted of the 14 most abundant proteins. The Mag-Net approach also detected 1,306 out of the 1,717 proteins (76%) that we previously verified as EV proteins. Statistical tests revealed 448 proteins to be differentially abundant in AAB+ vs control volunteers, including 69 previously verified EV proteins. A functional-enrichment analysis resulted in overrepresentation of 25 pathways among the differentially abundant proteins, including pathways related to autoimmune response and lipid metabolism. The capacity of this data to predict AAB+ was tested with a machine learning analysis using a random forest model, resulting in a receiver operating characteristic-area under the curve of 0.81. Overall, our study indicates that plasma EV proteomics analysis can be an exciting approach for studying biomarkers for developing T1D. Significance of the studyType 1 diabetes (T1D) is a disease characterized by the bodys inability to produce insulin and consequently, to control blood glucose levels. Despite the initial trigger being unclear, the disease development process involves an autoimmune response to the islets of Langerhans, resulting in the death of insulin-producing {beta} cells. There is no cure for the disease, and treatment relies on exogenous administration of insulin. Therefore, preventive therapies that block the autoimmune process are attractive for treating T1D. In fact, anti-CD3 antibody (Teplizumab) delays the onset of T1D by 2 years by targeting T cells. Predictive biomarkers for developing T1D are needed to aid the development and implementation of new therapies and to identify the initial trigger and mechanisms of the islet autoimmune process. In this paper, we assess the potential of plasma extracellular vesicle (EV) proteomics analysis for identifying predictive biomarkers of T1D. Our results show excellent potential of the approach, opening opportunities to perform broader studies to identify biomarkers for developing T1D.

systems biology↗

bioRxiv2025

An Antibody Cocktail-Based Immunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification of Circulating Proinsulin Proteoforms and C-peptide

Accurately measuring circulating proinsulin proteoforms is crucial for clinical investigation of diabetes, but was previously not feasible owing to limited assay specificity/sensitivity. Here we devised a highly sensitive LC-MS-based strategy to quantify intact proinsulin, des-31,32 and des-64,65 proinsulin, and C-peptide in circulation. The method involves: i)quantitative, robust affinity capture using an optimized antibody cocktail, eliminating the severe quantitative bias across multiple proteomes typically introduced when using a single antibody; ii)Lys-C digestion producing unique signature peptides for each proteoform, and iii)trapping-nano-LC coupled with FAIMS/dCV-MS for an ultra-sensitive analysis. The selective trapping/delivery ensured sensitive/selective analysis of the targets while achieving excellent analytical robustness that is critical for clinical assays, and the FAIMS/dCV substantially reduces baseline noise/interferences, further enhancing S/N. The assay achieved exceptional sensitivity, with serum LOQs of 1.7, 2.3, and 3.6 pg/mL respectively for intact-proinsulin, des-31,32 and des-64,65, representing the first assay capable of sensitively quantifying these major circulating proinsulin proteoforms. We applied this assay to 78 subjects, including autoantibody positive(n=20) and new-onset type-1-diabetes(T1D, n=19) with respective age/sex/BMI-matched controls, enabling the first accurate profiling of proinsulin proteoforms in clinical groups. The assay results demonstrated a clear separation of control and new-onset T1D groups that a parallel total-proinsulin ELISA assay fails to capture. Furthermore, distinct expression patterns in relative abundance ratios among proteoforms were observed across clinical groups. This assay may provide valuable insights into the {beta}-cell functions and the onset/progression of diabetes and other associated conditions. Moreover, the strategy is broadly applicable to targeted measurement of other biomarker proteoforms.

biochemistry↗

bioRxiv2025

Engineering sialylation in pigs: A promising strategy for overcoming xenograft rejection and revolutionizing organ transplantation.

The organ shortage crisis leaves over 100,000 people waiting for transplants, causing 6,000 deaths annually. To address this, pigs are being explored as potential donors. Despite advances like the FDA-approved GalSafe pig, immunological challenges remain. Key issues include strong antibody, innate, and cellular immune responses, along with coagulation problems due to differences in glycosides and sialic acid linkages, which prevent long-term xenograft survival. Hyperacute rejection, caused by instant blood-mediated immune reaction (IBMIR), is a persistent problem, characterized by inflammatory and thrombotic responses when, for example, xenogeneic islets contact blood or reperfusion post anastomosis of transplanted organ. To overcome IBMIR, and other innate immune mediated rejection, we expressed human sialyltransferase (ST8Sia6) in otherwise wild type, porcine endothelium, creating human-like sialic linkages on porcine glycoproteins and lipids. This increased expression of human sialic acid 2,8- linkage on porcine endothelial cells, enhanced sialic-acid-binding immunoglobulin-like lectin (Siglec) binding reducing immune effector mechanisms such as complement activation and cell cytotoxicity. It also correlated with reduced CTL-targeted killing, lower levels of CD107a, perforin, and IFN-{gamma} production. This coincided with higher immunoreceptor tyrosine-based inhibition motif (ITIM) induction, mirroring immune tolerance seen in fetal development and tumor immune evasion. Moreover, the landscape of the expression induced transcriptome of ST8Sia6, overexpression in porcine kidney cells revealed differential expression of genes involved in immune downregulation, cell signaling, and metabolic alteration. Expression of 2,8-linked disialic acids on porcine cells protected against immune effector mechanisms, reducing complement activation, immune cell activation, and CTL killing. These findings suggest that enhanced 2,8- linked disialic acid expression can modulate the innate and adaptive immune response, reducing xenograft rejection. This approach may improve xenotransplantation success, mitigate primary non-function in xenografts, and be applied to human iPSC-derived islets and other cell products. Further research into the specific mechanisms of these immunomodulatory effects could guide the development of effective strategies for xenotransplantation.

immunology↗

bioRxiv2024

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes

Aims/hypothesisSurviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. MethodsBeta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. ResultsPD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN- or IFN-{square} treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretationIFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death. Research in contextO_ST_ABSWhat is already known about this subject? (maximum of 3 bullet points)C_ST_ABSO_LIExtracellular vesicles (EVs) serve as paracrine effectors in the islet microenvironment in health and disease. C_LIO_LIInterferon-alpha (IFN-) and IFN-gamma (IFN-{square}) are key cytokines contributing to type 1 diabetes pathophysiology and islet IFN signalling increases beta cell programmed death-ligand 1 (PD-L1) expression. C_LIO_LIUp-regulation of beta cell PD-L1 in the non-obese diabetic (NOD) mouse model delays progression of type 1 diabetes. C_LI What is the key question? (one bullet point only; formatted as a question)O_LIDo beta cells exposed to IFNs upregulate EV PD-L1 and can these changes be detected in circulation? C_LI What are the new findings? (maximum of 3 bullet points)O_LIIFN- or IFN-{square} exposure increases beta cell EV PD-L1 cargo in beta cell lines and human islets. C_LIO_LIPD-L1 is present on the surface of beta cell EVs, binds PD-1 and EV PD-L1 inhibits proliferation, activation and cytotoxicity of murine CD8 T cells. C_LIO_LIEV PD-L1 levels are higher in islet autoantibody positive individuals compared to nondiabetic controls and levels of circulating EV PD-L1 positively correlate with residual beta cell function in islet autoantibody positive individuals as well as in individuals with recent-onset type 1 diabetes. C_LI How might this impact on clinical practice in the foreseeable future? (one bullet point only)O_LIA beneficial effect of PD-L1+ EVs could ultimately be harnessed as an intervention to prevent autoimmune beta cell destruction. Circulating EV PD-L1 cargo has potential as a minimally invasive and informative biomarker to offer insights into the pathogenesis and progression of type 1 diabetes. C_LI

cell biology↗

bioRxiv2024

Proinflammatory stress activates neutral sphingomyelinase 2 based generation of a ceramide-enriched β cell EV subpopulation

{beta} cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting {beta} cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that {beta} cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched EVs that could impact surrounding {beta} cells. Consistent with this, proinflammatory cytokine treatment of INS-1 {beta} cells and human islets concurrently increased {beta} cell nSMase2 and ceramide expression, as well as EV ceramide staining. Direct chemical activation or genetic knockdown of nSMase2, or treatment with a GLP-1 receptor agonist also modulated cellular and EV ceramide. Small RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to {beta} cell function and identity. Coculture experiments using CD9-GFP tagged INS-1 cell EVs demonstrated that either cytokine treatment or chemical nSMase2 activation increased EV transfer to recipient cells. Children with recent-onset T1D showed no abnormalities in circulating ceramide-enriched EVs, suggesting a localized, rather than systemic phenomenon. These findings highlight nSMase2 as a regulator of {beta} cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of {beta} cell inflammatory stress. Article HighlightsO_ST_ABSa. Why did we undertake this study?C_ST_ABSMechanisms connecting {beta} cell stress to changes in extracellular vesicle (EV) cargo and potential impacts on diabetes are poorly defined. b. What is the specific question we wanted to answer?Does {beta} cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways to generate ceramide-enriched EVs. c. What did we find?Proinflammatory cytokine treatment of {beta} cells increased {beta} cell ceramide expression, along with EV ceramide in part via increases in nSMase2. Ceramide-enriched EVs housed a distinct set of miRNAs linked to insulin signaling. Both cytokine treatment and nSMase2 activation increase EV transfer to other {beta} cells. d. What are the implications of our findings?Our findings highlight nSMase2 as a regulator of {beta} cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of {beta} cell inflammatory stress.

molecular biology↗

bioRxiv2022

Meta-analysis refinement of plasma extracellular vesicle composition identifies proplatelet basic protein as a signaling messenger in type 1 diabetes

Extracellular vesicles (EVs) play important roles in cell-to-cell communication and are potential biomarkers as they carry markers of their derived tissues and disease signatures. However, obtaining pure EV preparations from biofluids is challenging due to contaminants with similar physicochemical properties. Here, we performed a meta-analysis of plasma EV proteomics data deposited in public repositories to refine the protein composition of EVs and investigate potential roles in type 1 diabetes development. With the concept that each purification method yields different proportions of distinct contaminants, we grouped proteins into clusters based on their abundance profiles. This allowed us to separate clusters with classical EV markers, such as CD9, CD40, C63 and CD81, from clusters of well-known contaminants, such as serum albumin, apolipoproteins and components of the complement and coagulation pathways. Two clusters containing a total of 1720 proteins combined were enriched with EV markers and depleted in common contaminants; therefore, they were considered to contain bona fide EV components. As possible origins of plasma EVs, these clusters had markers of tissues such as spleen, liver, brain, lungs, pancreas, and blood/immune cells. These clusters were also enriched in cell surface markers CD antigens, and proteins from cell-to-cell communication and signaling pathways, such as chemokine signaling and antigen presentation. We also show that the EV component and type 1 diabetes biomarker, platelet basic protein (PPBP/CXCL7) regulates apoptosis in both beta and macrophage cell lines. Overall, our meta-analysis refined the composition of plasma EVs, reinforcing a primary function as messengers for cell-to-cell communication and signaling. Furthermore, this analysis identifies optimal avenues to target EVs for development of disease biomarkers.

cell biology↗

bioRxiv2019

Abnormalities in Proinsulin Processing in Islets from Individuals with Longstanding T1D

Work by our group and others has suggested that elevations in circulating proinsulin relative to C-peptide is associated with development of Type 1 diabetes (T1D). We recently described the persistence of detectable serum proinsulin in a large majority (95.9%) of individuals with longstanding T1D, including individuals with undetectable serum C-peptide. Here we describe analyses performed on human pancreatic sections from the nPOD collection (n=30) and isolated human islets (n=10) to further explore mechanistic etiologies of persistent proinsulin secretion in T1D. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with an inflammatory cytokine cocktail reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced {beta} cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.

pathology↗