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Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell–dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR–CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection. Heeger and colleagues report that activated B cells dynamically regulate the expression of complement regulatory proteins via the transcription factor BCL6. C3 convertase activity and C3aR1–C5aR1 signaling were both necessary for optimal B cell activation and germinal center formation.

Type 1 diabetes (T1D) is a progressive autoimmune condition that culminates in loss of insulin-producing beta cells. Pancreatic histopathology provides essential insights into disease initiation/progression yet an integrated perspective onto in situ pathogenic processes is lacking. Here, we combined multiplexed immunostaining, high-magnification whole-slide imaging, digital pathology, and semi-automated image analyses to interrogate pancreatic tail and head sections across T1D stages, including at-risk and at-onset cases. Deconvolution of architectural features, endocrine cell composition, immune cell burden, and spatial relations of ~25,000 islets effectively contextualizes previously established and additional pancreatic hallmarks in health and T1D. Our results reveal a spatially homogenous and islet size-contingent architectural organization of the endocrine pancreas, a notable coordination of organ-wide pathogenic processes, and multiple histopathological correlates that foreshadow distinctive T1D histopathology already at the preclinical stage. Altogether, we propose a revised natural history of T1D with implications for further histopathological investigations and considerations of pathogenetic modalities. Histological analysis of the human pancreas provides insight into initiation and progression of type 1 diabetes (T1D). Here the authors utilize pancreatic tissue sections across different disease stages and apply whole slide imaging and digital pathology to identify endocrine cell composition, immune cell burden and spatial islet relationships in health and over the course of T1D.

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase-dependent but not nuclear factor-κB-dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

Preservation and expansion of β-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPβ) is a nuclear effector of hyperglycemic stress occurring in β-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPβ is necessary for adaptive β-cell expansion in response to metabolic challenges. Conversely, chronic excessive β-cell-specific overexpression of ChREBPβ results in loss of β-cell identity, apoptosis, loss of β-cell mass, and diabetes. Furthermore, β-cell “glucolipotoxicity” can be prevented by deletion of ChREBPβ. Moreover, ChREBPβ-mediated cell death is mitigated by overexpression of the alternate CHREBP gene product, ChREBPα, or by activation of the antioxidant Nrf2 pathway in rodent and human β-cells. We conclude that ChREBPβ, whether adaptive or maladaptive, is an important determinant of β-cell fate and a potential target for the preservation of β-cell mass in diabetes. ChREBP is a glucose-responsive transcription factor, which regulates glucose-mediated proliferation and cell death in pancreatic β-cells. Here the authors show that the acute feed forward induction of ChREBPβ is required for adaptive β-cell expansion, that chronic overexpression of ChREBPβ is toxic to β-cells, and offer mitigation strategies

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms.

The determinants of protective CD8+ memory T cell (CD8+TM) immunity remain incompletely defined and may in fact constitute an evolving agency as aging CD8+TM progressively acquire enhanced rather than impaired recall capacities. Here, we show that old as compared to young antiviral CD8+TM more effectively harness disparate molecular processes (cytokine signaling, trafficking, effector functions, and co-stimulation/inhibition) that in concert confer greater secondary reactivity. The relative reliance on these pathways is contingent on the nature of the secondary challenge (greater for chronic than acute viral infections) and over time, aging CD8+TM re-establish a dependence on the same accessory signals required for effective priming of naïve CD8+T cells in the first place. Thus, our findings reveal a temporal regulation of complementary recall response determinants that is consistent with the recently proposed \"rebound model\" according to which aging CD8+TM properties are gradually aligned with those of naïve CD8+T cells; our identification of a broadly diversified collection of immunomodulatory targets may further provide a foundation for the potential therapeutic \"tuning\" of CD8+TM immunity.

Background An association between hepatitis C virus (HCV) and type 2 diabetes (T2D) is supported by numerous epidemiologic studies. We hypothesized that HCV could infect human pancreatic islet cells in vitro. Methods Measures of HCV RNA synthesis and protein production were used to evaluate HCV infection of pancreatic islets recovered from human donors. Results Significant co-staining of insulin and the HCV entry factor CD81 was observed in pancreatic islets. Positive- and negative-sense HCV RNA were detected in HCV-exposed islets at days 1, 3, 7, and 14 post-infection. The HCV core and NS3 proteins were expressed and increased with time providing further evidence of viral replication. Interferon and an HCV polymerase inhibitor reduced viral replication in islet cells. In HCV-infected islets, TNFα levels were elevated at days 1, 3, and 7 post-infection, while IL-6 levels were elevated at day 1 but not days 3 or 7. Overall, the expression of miR-122 was low in islets compared to the Huh7.5 hepatocyte-derived cell line, although the relative expression of miR-122 increased in islet cells after viral infection (1, 6.63, and 5.83 at days 1, 3, and 7, respectively). Conclusions In this pilot study, viral infection was demonstrated in pancreatic islet cells from multiple donors using complementary measures of viral replication, thus providing evidence of in vitro infection. Altered cytokine expression may contribute to the development of insulin deficiency, and understanding the etiology of diabetes in individuals with HCV infection may facilitate the development of novel treatment modalities and prevention strategies. This in vitro system provides an important model for mechanistic studies of HCV-pancreas interactions and facilitates future studies of the potential impact of viral infection on islet cell function.

The destruction of infected cells by cytotxic T lymphocytes (CTL) is integral to the effective control of viral and bacterial diseases, and CTL function at large has long been regarded as a distinctive property of the CD8(+)T cell subset. In contrast, and despite their first description more than three decades ago, the precise contribution of cytotoxic CD4(+)T cells to the resolution of infectious diseases has remained a matter of debate. In particular, the CTL activity of pathogen-specific CD4(+) \"helper\" T cells constitutes a single trait among a diverse array of other T cell functionalities, and overall appears considerably weaker than the cytolytic capacity of CD8(+) effector T cells. Here, using an in vivo CTL assay, we report that cytotoxic CD4(+)T cells are readily generated against both viral and bacterial pathogens, and that the efficiency of MHC-II-restricted CD4(+)T cell killing adjusted for effector:target cell ratios, precise specificities and functional avidities is comparable in magnitude to that of CD8(+)T cells. In fact, the only difference between specific CD4(+) and CD8(+)T cells pertains to the slightly delayed killing kinetics of the former demonstrating that potent CTL function is a cardinal property of both antiviral CD8(+) and CD4(+)T cells.

Heightened protection from infectious disease as conferred by vaccination or pathogen exposure relies on the effective generation and preservation of specific immunological memory. T cells are irreducibly required for the control of most viral infections, and maintenance of CD8+T cell memory is regulated by at least two cytokines, IL-7 and IL-15, which support survival (IL-7, IL-15) and basal homeostatic proliferation (IL-15) of specific CD8+memory T cells ( TM). In contrast, the factors governing the homeostasis of pathogen-specific CD4+TMremain at present unknown. Here, we used a physiologic in vivo model system for viral infection to delineate homeostatic features and mechanisms of antiviral CD4+TMpreservation in direct juxtaposition to CD8+T cell memory. Basal homeostatic proliferation is comparable between specific CD4+and CD8+TMand independent of immunodominant determinants and functional avidities but regulated in a tissue-specific fashion. IL-7, identified as the dominant cytokine, and IL-15, an accessory cytokine, regulate basal homeostatic proliferation and survival of antiviral CD4+TM. Interestingly, a role for these cytokines in regulation of CD4+T cell memory is not readily discernible in the generic \"memory-phenotype\" population, apparently a consequence of its heterogeneous composition. We also describe a prominent, nonredundant role for IL-7 in supporting basal homeostatic proliferation of CD8+TM. We propose that homeostatic control of antiviral CD4+and CD8+T cell memory is fundamentally similar and characterized by quantitative, rather than qualitative, differences.