High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.

Targeted plasma proteomics reveals signatures discriminating COVID-19 from sepsis with pneumonia.

BACKGROUND: COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. METHODS: We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. RESULTS: We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. CONCLUSIONS: This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

Defining the proteolytic landscape during enterovirus infection.

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

The Karolinska KI/K COVID-19 Immune Atlas: An open resource for immunological research and educational purposes.

The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allow scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.

Human Enterovirus Group B Viruses Rely on Vimentin Dynamics for Efficient Processing of Viral Nonstructural Proteins.

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with ß, ß'-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, smaller amounts of nonstructural proteins did not result from proteasomal degradation but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1 but had less effect on 2A. The results suggest that the vimentin dynamics regulate viral nonstructural protein synthesis while having less effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and nonstructural proteins of enteroviruses, having consequences on host cell survival.IMPORTANCE A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and nonstructural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the nonstructural proteins orchestrate the takeover of the host cell and its functions. Here, we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

Rationale for enteroviral vaccination and antiviral therapies in human type 1 diabetes.

In type 1 diabetes, pancreatic beta cells are destroyed by chronic autoimmune responses. The disease develops in genetically susceptible individuals, but a role for environmental factors has been postulated. Viral infections have long been considered as candidates for environmental triggers but, given the lack of evidence for an acute, widespread, cytopathic effect in the pancreas in type 1 diabetes or for a closely related temporal association of diabetes onset with such infections, a role for viruses in type 1 diabetes remains unproven. Moreover, viruses have rarely been isolated from the pancreas of individuals with type 1 diabetes, mainly (but not solely) due to the inaccessibility of the organ. Here, we review past and recent literature to evaluate the proposals that chronic, recurrent and, possibly, persistent enteroviral infections occur in pancreatic beta cells in type 1 diabetes. We also explore whether these infections may be sustained by different virus strains over time and whether multiple viral hits can occur during the natural history of type 1 diabetes. We emphasise that only a minority of beta cells appear to be infected at any given time and that enteroviruses may become replication defective, which could explain why they have been isolated from the pancreas only rarely. We argue that enteroviral infection of beta cells largely depends on the host innate and adaptive immune responses, including innate responses mounted by beta cells. Thus, we propose that viruses could play a role in type 1 diabetes on multiple levels, including in the triggering and chronic stimulation of autoimmunity and in the generation of inflammation and the promotion of beta cell dysfunction and stress, each of which might then contribute to autoimmunity, as part of a vicious circle. We conclude that studies into the effects of vaccinations and/or antiviral drugs (some of which are currently on-going) is the only means by which the role of viruses in type 1 diabetes can be finally proven or disproven.

Genetic and Environmental Interaction in Type 1 Diabetes: a Relationship Between Genetic Risk Alleles and Molecular Traits of Enterovirus Infection?

PURPOSE OF REVIEW: We provide an overview of the current knowledge regarding the natural history of human type 1 diabetes (T1D) and the documented associations between virus infections (in particular the enteroviruses) and disease development. We review studies that examine whether T1D-specific risk alleles in genes involved in the function of the immune system can alter susceptibility to virus infections or affect the magnitude of the host antiviral response. We also highlight where the major gaps in our knowledge exist and consider possible implications that new insights gained from the discussed gene-environment interaction studies may bring. RECENT FINDINGS: A commonality between several of the studied T1D risk variants studied is their role in modulating the host immune response to viral infection. Generally, little support exists indicating that the risk variants increase susceptibility to infection and moreover, they usually appear to predispose the immune system towards a hyper-reactive state, decrease the risk of infection, and/or favor the establishment of viral persistence. In conclusion, although the current number of studies is limited, this type of research can provide important insights into the mechanisms that are central to disease pathogenesis and further describe how genetic and environmental factors jointly influence the risk of T1D development. The latter may provide genetic markers that could be used for patient stratification and for the selection of method(s) for disease prevention.

A Coxsackievirus B vaccine protects against virus-induced diabetes in an experimental mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Epidemiological studies suggest a role for Coxsackievirus B (CVB) serotypes in the pathogenesis of type 1 diabetes, but their actual contribution remains elusive. In the present study, we have produced a CVB1 vaccine to test whether vaccination against CVBs can prevent virus-induced diabetes in an experimental model. METHODS: NOD and SOCS1-tg mice were vaccinated three times with either a formalin-fixed non-adjuvanted CVB1 vaccine or a buffer control. Serum was collected for measurement of neutralising antibodies using a virus neutralisation assay. Vaccinated and buffer-treated mice were infected with CVB1. Viraemia and viral replication in the pancreas were measured using standard plaque assay and PCR. The development of diabetes was monitored by blood glucose measurements. Histological analysis and immunostaining for viral capsid protein 1 (VP1), insulin and glucagon in formalin-fixed paraffin embedded pancreas was performed. RESULTS: The CVB1 vaccine induced strong neutralising antibody responses and protected against viraemia and the dissemination of virus to the pancreas in both NOD mice (n = 8) and SOCS1-tg mice (n = 7). Conversely, 100% of the buffer-treated NOD and SOCS1-tg mice were viraemic on day 3 post infection. Furthermore, half (3/6) of the buffer-treated SOCS1-tg mice developed diabetes upon infection with CVB1, with a loss of the insulin-positive beta cells and damage to the exocrine pancreas. In contrast, all (7/7) vaccinated SOCS1-tg mice were protected from virus-induced diabetes and showed no signs of beta cell loss or pancreas destruction (p < 0.05). CONCLUSIONS/INTERPRETATION: CVB1 vaccine can efficiently protect against both CVB1 infection and CVB1-induced diabetes. This preclinical proof of concept study provides a base for further studies aimed at developing a vaccine for use in elucidating the role of enteroviruses in human type 1 diabetes.

Composition and functionality of the intrahepatic innate lymphoid cell-compartment in human nonfibrotic and fibrotic livers.

Human innate lymphoid cells have been described to exist in different organs, with functional deregulation of these cells contributing to several disease states. Here, we performed the first detailed characterization of the phenotype, tissue-residency properties, and functionality of ILC1s, ILC2s, and ILC3s in the human adult and fetal liver. In addition, we investigated changes in the ILC compartment in liver fibrosis. A unique composition of tissue-resident ILCs was observed in nonfibrotic livers as compared with that in mucosal tissues, with NKp44- ILC3s accounting for the majority of total intrahepatic ILCs. The frequency of ILC2s, representing a small fraction of ILCs in nonfibrotic livers, increased in liver fibrosis and correlated directly with the severity of the disease. Notably, intrahepatic ILC2s secreted the profibrotic cytokine IL-13 when exposed to IL-33 and thymic stromal lymphopoetin (TSLP); these cytokines were produced by hepatocytes, hepatic stellate cells (HSCs), and Kupffer cells in response to TLR-3 stimulation. In summary, the present results provide the first detailed characterization of intrahepatic ILCs in human adult and fetal liver. The results indicate a role for ILC2s in human liver fibrosis, implying that targeting ILC2s might be a novel therapeutic strategy for its treatment.

A Link Between a Common Mutation in CFTR and Impaired Innate and Adaptive Viral Defense.

Acute respiratory virus infections predispose the cystic fibrosis (CF) lung to chronic bacterial colonization, which contributes to high mortality. For reasons unknown, respiratory virus infections have a prolonged duration in CF. Here, we demonstrate that mice carrying the most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutation in humans, ΔF508, show increased morbidity and mortality following infection with a common human enterovirus. ΔF508 mice demonstrated impaired viral clearance, a slower type I interferon response and delayed production of virus-neutralizing antibodies. While the ΔF508 mice had a normal immune cell repertoire, unchanged serum immunoglobulin concentrations and an intact immune response to a T-cell-independent antigen, their response to a T-cell-dependent antigen was significantly delayed. Our studies reveal a novel function for CFTR in antiviral immunity and demonstrate that the ΔF508 mutation in cftr is coupled to an impaired adaptive immune response. This important insight could open up new approaches for patient care and treatment.

Enteroviral proteases: structure, host interactions and pathogenicity.

Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd.

Coxsackievirus counters the host innate immune response by blocking type III interferon expression.

Type I IFNs play an important role in the immune response to enterovirus infections. Their importance is underscored by observations showing that many enteroviruses including coxsackie B viruses (CVBs) have developed strategies to block type I IFN production. Recent studies have highlighted a role for the type III IFNs (also called IFNλs) in reducing permissiveness to infections with enteric viruses including coxsackievirus. However, whether or not CVBs have measures to evade the effects of type III IFNs remains unknown. By combining virus infection studies and different modes of administrating the dsRNA mimic poly I : C, we discovered that CVBs target both TLR3- and MDA5/RIG-I-mediated type III IFN expression. Consistent with this, the cellular protein expression levels of the signal transduction proteins TRIF and IPS1 were reduced and no hyperphosphorylation of IRF-3 was observed following infection with the virus. Notably, decreased expression of full-length TRIF and IPS1 and the appearance of cleavage products was observed upon both CVB3 infection and in cellular protein extracts incubated with recombinant 2Apro, indicating an important role for the viral protease in subverting the cellular immune system. Collectively, our study reveals that CVBs block the expression of type III IFNs, and that this is achieved by a similar mechanism as the virus uses to block type I IFN production. We also demonstrate that the virus blocks several intracellular viral recognition pathways of importance for both type I and III IFN production. The simultaneous targeting of numerous arms of the host immune response may be required for successful viral replication and dissemination.

Depletion of IL-2 receptor ß-positive cells protects from diabetes in non-obese diabetic mice.

The destruction of ß-cells in type 1 diabetes (T1D) progresses silently until only a minor fraction of the ß-cells remain. A late acting therapy leading to the prevention of further ß-cell killing would therefore be desirable. CD122, the ß chain of the interleukin-2 receptor, is highly expressed on natural killer (NK) cells and on a subpopulation of CD8 T cells. In this study, we have treated non-obese diabetic (NOD) mice with a depleting antibody against CD122. The treatment protected from diabetes, even when initiated just before disease onset. The degree of leukocyte infiltration into islets was unaffected by the treatment, further supporting effectiveness late in the disease process. It effectively removed all NK cells from the spleen, pancreas and pancreatic lymph nodes and abolished NK cell activity. Interestingly, despite the lack of CD122 expression on CD8 T cells in the pancreas, the overall frequency of CD8 cells decreased in this organ, whereas it was unaffected in the spleen. T cells were also still capable to respond against a foreign antigen. Conclusively, targeting of CD122(+) cells could represent a novel treatment strategy against T1D.

A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models.

AIMS/HYPOTHESIS: Enterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus. METHODS: A formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development. RESULTS: Vaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice. CONCLUSIONS/INTERPRETATION: We conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.

Novel role for matricellular proteins in the regulation of islet ß cell survival: the effect of SPARC on survival, proliferation, and signaling.

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining ß cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of ß cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in ß cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 ß cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 ß cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and ß cell growth.

Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.

AIMS/HYPOTHESIS: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. METHODS: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. RESULTS: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. CONCLUSIONS/INTERPRETATION: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression.

CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells.

BACKGROUND: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). Although patients with CF often have disturbances in glucose metabolism including impaired insulin release, no previous studies have tested the hypothesis that CFTR has a biological function in pancreatic beta-cells. METHODS: Experiments were performed on islets and single beta-cells from human donors and NMRI-mice. Detection of CFTR was investigated using PCR and confocal microscopy. Effects on insulin secretion were measured with radioimmunoassay (RIA). The patch-clamp technique was used to measure ion channel currents and calcium-dependent exocytosis (as changes in membrane capacitance) on single cells with high temporal resolution. Analysis of ultrastructure was done on transmission electron microscopy (TEM) images. RESULTS: We detected the presence of CFTR and measured a small CFTR conductance in both human and mouse beta-cells. The augmentation of insulin secretion at 16.7 mM glucose by activation of CFTR by cAMP (forskolin (FSK) or GLP-1) was significantly inhibited when CFTR antagonists (GlyH-101 and/or CFTRinh-172) were added. Likewise, capacitance measurements demonstrated reduced cAMP-dependent exocytosis upon CFTR-inhibition, concomitant with a decreased number of docked insulin granules. Finally, our studies demonstrate that CFTR act upstream of the chloride channel Anoctamin 1 (ANO1; TMEM16A) in the regulation of cAMP- and glucose-stimulated insulin secretion. CONCLUSION: Our work demonstrates a novel function for CFTR as a regulator of pancreatic beta-cell insulin secretion and exocytosis, and put forward a role for CFTR as regulator of ANO1 and downstream priming of insulin granules prior to fusion and release of insulin. The pronounced regulatory effect of CFTR on insulin secretion is consistent with impaired insulin secretion in patients with CF.

Induction of an antiviral state and attenuated coxsackievirus replication in type III interferon-treated primary human pancreatic islets.

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.

Coxsackievirus infection induces direct pancreatic ß cell killing but poor antiviral CD8+ T cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.