Assessing Researcher Needs for a Virtual Biobank.

INTRODUCTION: Biosamples and associated clinical data accelerate translational and clinical research discoveries. A lack of high quality biosamples both stalls projects and limits research advances. In this study, we targeted a wide audience of University of California (UC) biobanking stakeholders who were either involved with the collection or the utilization of biosamples to assess the scope of their biobanking activities and their interest in virtual biobanking or cooperating in the formation of the UC-wide biorepository. MATERIALS AND METHODS: Each institutional review board from the five UC medical campuses' provided a dataset of potential researchers involved with biobanking. Once identified, a brief six item web-based questionnaire was administered electronically to these researchers. RESULTS: Most survey participants (80%) responded "yes" (n = 348) that they were actively collecting biosamples for research, and 68% of participants indicated they would either definitely (30%, n = 131) or maybe (38%, n = 166) request biosample materials now or within the next year. An equal proportion of participants responded yes (42% or n = 184) and maybe (42% or n = 182) when asked if they would voluntarily contribute specimens to a UC-wide virtual biobank. DISCUSSION: The results presented above show high levels of support among UC biobanking stakeholders for both requesting material from and contributing material to a UC-wide virtual biobank. In addition, a considerable number of individual researchers on our five UC medical campuses are conducting biospecimen research (i.e., well over n = 435 respondents).

T cells in the control of organ-specific autoimmunity.

Immune tolerance is critical to the avoidance of unwarranted immune responses against self antigens. Multiple, non-redundant checkpoints are in place to prevent such potentially deleterious autoimmune responses while preserving immunity integral to the fight against foreign pathogens. Nevertheless, a large and growing segment of the population is developing autoimmune diseases. Deciphering cellular and molecular pathways of immune tolerance is an important goal, with the expectation that understanding these pathways will lead to new clinical advances in the treatment of these devastating diseases. The vast majority of autoimmune diseases develop as a consequence of complex mechanisms that depend on genetic, epigenetic, molecular, cellular, and environmental elements and result in alterations in many different checkpoints of tolerance and ultimately in the breakdown of immune tolerance. The manifestations of this breakdown are harmful inflammatory responses in peripheral tissues driven by innate immunity and self antigen-specific pathogenic T and B cells. T cells play a central role in the regulation and initiation of these responses. In this Review we summarize our current understanding of the mechanisms involved in these fundamental checkpoints, the pathways that are defective in autoimmune diseases, and the therapeutic strategies being developed with the goal of restoring immune tolerance.

Tolerance induction and reversal of diabetes in mice transplanted with human embryonic stem cell-derived pancreatic endoderm.

Type 1 diabetes (T1D) is an autoimmune disease caused by T cell-mediated destruction of insulin-producing ß cells in the islets of Langerhans. In most cases, reversal of disease would require strategies combining islet cell replacement with immunotherapy that are currently available only for the most severely affected patients. Here, we demonstrate that immunotherapies that target T cell costimulatory pathways block the rejection of xenogeneic human embryonic-stem-cell-derived pancreatic endoderm (hESC-PE) in mice. The therapy allowed for long-term development of hESC-PE into islet-like structures capable of producing human insulin and maintaining normoglycemia. Moreover, short-term costimulation blockade led to robust immune tolerance that could be transferred independently of regulatory T cells. Importantly, costimulation blockade prevented the rejection of allogeneic hESC-PE by human PBMCs in a humanized model in vivo. These results support the clinical development of hESC-derived therapy, combined with tolerogenic treatments, as a sustainable alternative strategy for patients with T1D.

BPIFB1 is a lung-specific autoantigen associated with interstitial lung disease.

Interstitial lung disease (ILD) is a complex and heterogeneous disorder that is often associated with autoimmune syndromes. Despite the connection between ILD and autoimmunity, it remains unclear whether ILD can develop from an autoimmune response that specifically targets the lung parenchyma. We examined a severe form of autoimmune disease, autoimmune polyglandular syndrome type 1 (APS1), and established a strong link between an autoimmune response to the lung-specific protein BPIFB1 (bactericidal/permeability-increasing fold-containing B1) and clinical ILD. Screening of a large cohort of APS1 patients revealed autoantibodies to BPIFB1 in 9.6% of APS1 subjects overall and in 100% of APS1 subjects with ILD. Further investigation of ILD outside the APS1 disorder revealed BPIFB1 autoantibodies present in 14.6% of patients with connective tissue disease-associated ILD and in 12.0% of patients with idiopathic ILD. The animal model for APS1, Aire⁻/⁻ mice, harbors autoantibodies to a similar lung antigen (BPIFB9); these autoantibodies are a marker for ILD. We found that a defect in thymic tolerance was responsible for the production of BPIFB9 autoantibodies and the development of ILD. We also found that immunoreactivity targeting BPIFB1 independent of a defect in Aire also led to ILD, consistent with our discovery of BPIFB1 autoantibodies in non-APS1 patients. Overall, our results demonstrate that autoimmunity targeting the lung-specific antigen BPIFB1 may contribute to the pathogenesis of ILD in patients with APS1 and in subsets of patients with non-APS1 ILD, demonstrating the role of lung-specific autoimmunity in the genesis of ILD.

Distinct genetic control of autoimmune neuropathy and diabetes in the non-obese diabetic background.

The non-obese diabetic (NOD) mouse is susceptible to the development of autoimmune diabetes but also multiple other autoimmune diseases. Over twenty susceptibility loci linked to diabetes have been identified in NOD mice and progress has been made in the definition of candidate genes at many of these loci (termed Idd for insulin-dependent diabetes). The susceptibility to multiple autoimmune diseases in the NOD background is a unique opportunity to examine susceptibility genes that confer a general propensity for autoimmunity versus susceptibility genes that control individual autoimmune diseases. We previously showed that NOD mice deficient for the costimulatory molecule B7-2 (NOD-B7-2KO mice) were protected from diabetes but spontaneously developed an autoimmune peripheral neuropathy. Here, we took advantage of multiple NOD mouse strains congenic for Idd loci to test the role of these Idd loci the development of neuropathy and determine if B6 alleles at Idd loci that are protective for diabetes will also be for neuropathy. Thus, we generated NOD-B7-2KO strains congenic at Idd loci and examined the development of neuritis and clinical neuropathy. We found that the NOD-H-2(g7) MHC region is necessary for development of neuropathy in NOD-B7-2KO mice. In contrast, other Idd loci that significantly protect from diabetes did not affect neuropathy when considered individually. However, we found potent genetic interactions of some Idd loci that provided almost complete protection from neuritis and clinical neuropathy. In addition, defective immunoregulation by Tregs could supersede protection by some, but not other, Idd loci in a tissue-specific manner in a model where neuropathy and diabetes occurred concomitantly. Thus, our study helps identify Idd loci that control tissue-specific disease or confer general susceptibility to autoimmunity, and brings insight to the Treg-dependence of autoimmune processes influenced by given Idd region in the NOD background.

Current and future immunomodulation strategies to restore tolerance in autoimmune diseases.

Autoimmune diseases reflect a breakdown in self-tolerance that results from defects in thymic deletion of potentially autoreactive T cells (central tolerance) and in T-cell intrinsic and extrinsic mechanisms that normally control potentially autoreactive T cells in the periphery (peripheral tolerance). The mechanisms leading to autoimmune diseases are multifactorial and depend on a complex combination of genetic, epigenetic, molecular, and cellular elements that result in pathogenic inflammatory responses in peripheral tissues driven by self-antigen-specific T cells. In this article, we describe the different checkpoints of tolerance that are defective in autoimmune diseases as well as specific events in the autoimmune response which represent therapeutic opportunities to restore long-term tolerance in autoimmune diseases. We present evidence for the role of different pathways in animal models and the therapeutic strategies targeting these pathways in clinical trials in autoimmune diseases.

Defective autoimmune regulator-dependent central tolerance to myelin protein zero is linked to autoimmune peripheral neuropathy.

Chronic inflammatory demyelinating polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking Ags, and pathogenic effector mechanisms are not well defined. The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-Ags and deletion of self-reactive T cells. In this study, we used mice with hypomorphic Aire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets myelin protein zero, an Ag for which expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4(+) T cells are sufficient to transfer disease in mice and produce IFN-γ in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to myelin protein zero initiates an autoimmune Th1 effector response toward peripheral nerves.

Breakdown in peripheral tolerance in type 1 diabetes in mice and humans.

Type 1 Diabetes (T1D), also called juvenile diabetes because of its classically early onset, is considered an autoimmune disease targeting the insulin-producing ß cells in the pancreatic islets of Langerhans. T1D reflects a loss of tolerance to tissue self-antigens caused by defects in both central tolerance, which aims at eliminating potentially autoreactive lymphocytes developing in the thymus, and peripheral tolerance, which normally controls autoreactive T cells that escaped the thymus. Like in other autoimmune diseases, the mechanisms leading to T1D are multifactorial and depend on a complex combination of genetic, epigenetic, molecular, and cellular elements that result in the breakdown of peripheral tolerance. In this article, we discuss the contribution of these factors in the development of the autoimmune response targeting pancreatic islets in T1D and the therapeutic strategies currently being explored to correct these defects.

Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/ B7 family.

Positive and negative costimulation by members of the CD28 family is critical for the development of productive immune responses against foreign pathogens and their proper termination to prevent inflammation-induced tissue damage. In addition, costimulatory signals are critical for the establishment and maintenance of peripheral tolerance. This paradigm has been established in many animal models and has led to the development of immunotherapies targeting costimulation pathways for the treatment of cancer, autoimmune disease, and allograft rejection. During the last decade, the complexity of the biology of costimulatory pathways has greatly increased due to the realization that costimulation does not affect only effector T cells but also influences regulatory T cells and antigen-presenting cells. Thus, costimulation controls T-cell tolerance through both intrinsic and extrinsic pathways. In this review, we discuss the influence of costimulation on intrinsic and extrinsic pathways of peripheral tolerance, with emphasis on members of the CD28 family, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1), as well as the downstream cytokine interleukin-1 (IL-2).

Regulating the regulators: costimulatory signals control the homeostasis and function of regulatory T cells.

SUMMARY: Costimulation is a concept that goes back to the early 1980s when Lafferty and others hypothesized that cell surface and soluble molecules must exist that are essential for initiating immune responses subsequent to antigen exposure. The explosion in this field of research ensued as over a dozen molecules have been identified to function as second signals following T-cell receptor engagement. By 1994, it seemed clear that the most prominent costimulatory pathway CD28 and functionally related costimulatory molecules, such as CD154, were the major drivers of a positive immune response. Then the immunology world turned upside down. CD28 knockout mice, which were, in most cases, immunodeficient, led to increased autoimmunity when bred into the non-obese diabetic background. Another CD28 family member, cytotoxic T-lymphocyte-associated protein 4, which was presumed to be a costimulatory molecule on activated T cells, turned out to be critical in downregulating immunity. These results, coupled with the vast suppressor cell literature which had been largely rebuked, suggested that the immune system was not poised for response but controlled in such a way that regulation was dominant. Over the last decade, we have learned that these costimulatory molecules play a key role in the now classical CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) that provide critical control of unwanted autoimmune responses. In this review, we discuss the connections between costimulation and Tregs that have changed the costimulation paradigm.

A novel myelin P0-specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy.

Autoimmune-prone nonobese diabetic mice deficient for B7-2 spontaneously develop an autoimmune peripheral neuropathy mediated by inflammatory CD4(+) T cells that is reminiscent of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. To determine the etiology of this disease, CD4(+) T cell hybridomas were generated from inflamed tissue-derived CD4(+) T cells. A majority of T cell hybridomas were specific for myelin protein 0 (P0), which was the principal target of autoantibody responses targeting nerve proteins. To determine whether P0-specific T cell responses were sufficient to mediate disease, we generated a novel myelin P0-specific T cell receptor transgenic (POT) mouse. POT T cells were not tolerized or deleted during thymic development and proliferated in response to P0 in vitro. Importantly, when bred onto a recombination activating gene knockout background, POT mice developed a fulminant form of peripheral neuropathy that affected all mice by weaning age and led to their premature death by 3-5 wk of age. This abrupt disease was associated with the production of interferon gamma by P0-specific T cells and a lack of CD4(+) Foxp3(+) regulatory T cells. Collectively, our data suggest that myelin P0 is a major autoantigen in autoimmune peripheral neuropathy.

Spontaneous development of a pancreatic exocrine disease in CD28-deficient NOD mice.

Autoimmune pancreatitis (AIP) is a heterogeneous autoimmune disease in humans characterized by a progressive lymphocytic and plasmacytic infiltrate in the exocrine pancreas. In this study, we report that regulatory T cell-deficient NOD.CD28KO mice spontaneously develop AIP that closely resembles the human disease. NOD mouse AIP was associated with severe periductal and parenchymal inflammation of the exocrine pancreas by CD4(+) T cells, CD8(+) T cells, and B cells. Spleen CD4(+) T cells were found to be both necessary and sufficient for the development of AIP. Autoantibodies and autoreactive T cells from affected mice recognized a approximately 50-kDa protein identified as pancreatic amylase. Importantly, administration of tolerogenic amylase-coupled fixed spleen cells significantly ameliorated disease severity, suggesting that this protein functions as a key autoantigen. The establishment and characterization of this spontaneous pancreatic amylase-specific AIP in regulatory T cell-deficient NOD.CD28KO mice provides an excellent model for the study of disease pathogenesis and development of new therapies for human autoimmune pancreatitis.

B cell depletion: a novel therapy for autoimmune diabetes?

Autoimmune diabetes is believed to be mediated primarily by T cells. However, B cells have been implicated in the pathogenesis of the disease in NOD mice. Although preclinical studies have been limited by the absence of anti-CD20 reagents that can induce B cell depletion in mice, a clinical trial using the B cell-depleting anti-CD20 monoclonal antibody rituximab (Rituxan) is underway in type 1 diabetes patients. In this issue of the JCI, Hu et al. describe the generation of transgenic NOD mice that express human CD20 on B cells (see the related article beginning on page 3857). They show that anti-CD20 therapy induces B cell depletion in these mice and offers some level of protection against diabetes. Although many questions remain unanswered, this mouse model represents the first opportunity to evaluate the potential value of rituximab as a novel therapy for autoimmune diabetes.

Constitutive expression of B7-1 on B cells uncovers autoimmunity toward the B cell compartment in the nonobese diabetic mouse.

The NOD mouse is an invaluable model for the study of autoimmune diabetes. Furthermore, although less appreciated, NOD mice are susceptible to other autoimmune diseases that can be differentially manifested by altering the balance of T cell costimulatory pathways. In this study, we show that constitutively expressing B7-1 on B cells (NOD-B7-1B-transgenic mice) resulted in reduced insulitis and completely protected NOD mice from developing diabetes. Furthermore, B7-1 expression led to a dramatic reduction of the B cell compartment due to a selective deletion of follicular B cells in the spleen, whereas marginal zone B cells were largely unaffected. B cell depletion was dependent on B cell specificity, mediated by CD8(+) T cells, and occurred exclusively in the autoimmune-prone NOD background. Our results suggest that B cell deletion was a consequence of the specific activation of autoreactive T cells directed at peripheral self Ags presented by maturing B cells that expressed B7-1 costimulatory molecules. This study underscores the importance of B7 costimulatory molecules in controlling the amplitude and target of autoimmunity in genetically prone individuals and has important implications in the use of costimulatory pathway antagonists in the treatment of human autoimmune diseases.

Sensory neurons link the nervous system and autoimmune diabetes.

The initial factors that trigger the autoimmune response against pancreatic islets in the nonobese diabetic (NOD) mouse are still unknown. In this issue of Cell, propose that a defect in a subset of sensory neurons innervating the pancreas plays a major role in initiating the chain of events that will lead to local inflammation, islet destruction, and autoimmune diabetes.

Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway.

The past decade has seen a significant increase in the number of potentially tolerogenic therapies for treatment of new-onset diabetes. However, most treatments are antigen nonspecific, and the mechanism for the maintenance of long-term tolerance remains unclear. In this study, we developed an antigen-specific therapy, insulin-coupled antigen-presenting cells, to treat diabetes in nonobese diabetic mice after disease onset. Using this approach, we demonstrate disease remission, inhibition of pathogenic T cell proliferation, decreased cytokine production, and induction of anergy. Moreover, we show that robust long-term tolerance depends on the programmed death 1 (PD-1)-programmed death ligand (PD-L)1 pathway, not the distinct cytotoxic T lymphocyte-associated antigen 4 pathway. Anti-PD-1 and anti-PD-L1, but not anti-PD-L2, reversed tolerance weeks after tolerogenic therapy by promoting antigen-specific T cell proliferation and inflammatory cytokine production directly in infiltrated tissues. PD-1-PD-L1 blockade did not limit T regulatory cell activity, suggesting direct effects on pathogenic T cells. Finally, we describe a critical role for PD-1-PD-L1 in another powerful immunotherapy model using anti-CD3, suggesting that PD-1-PD-L1 interactions form part of a common pathway to selectively maintain tolerance within the target tissues.

Distinct effector mechanisms in the development of autoimmune neuropathy versus diabetes in nonobese diabetic mice.

NOD mice deficient for the costimulatory molecule B7-2 (NOD-B7-2KO mice) are protected from autoimmune diabetes but develop a spontaneous autoimmune peripheral neuropathy that resembles human diseases Guillain-Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy. Similar observations have now been made in conventional NOD mice. We have shown previously that this disease was mediated by autoreactive T cells inducing demyelination in the peripheral nervous system. In this study, we analyzed the molecular pathways involved in the disease. Our data showed that neuropathy developed in the absence of perforin or fas, suggesting that classic cytotoxicity pathways were dispensable for nerve damage in NOD-B7-2KO mice. In contrast, IFN-gamma played an obligatory role in the development of neuropathy as demonstrated by the complete protection from disease and infiltration in the nerves in NOD-B7-2KO mice deficient for IFN-gamma. This result was consistent with the inflammatory phenotype of T cells infiltrating the peripheral nerves. Importantly, the relative role of perforin, fas, and IFN-gamma appears completely different in autoimmune diabetes vs neuropathy. Thus, there are sharp contrasts in the pathogenesis of autoimmune diseases targeting different tissues in the same NOD background.

Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo.

Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as alpha-galactosylceramide (alpha GalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-gamma or IL-4 production by these cells. We hypothesized that alpha GalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in alpha GalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, alpha GalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells.

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function.

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.