Revisiting B cell tolerance and autoantibodies in seropositive and seronegative autoimmune rheumatic disease (AIRD).

Autoimmune rheumatic diseases (AIRD) are categorized seropositive or seronegative, dependent upon the presence or absence of specific autoreactive antibodies, including rheumatoid factor and anti-citrullinated protein antibodies. Autoantibody-based diagnostics have proved helpful in patient care, not only for diagnosis but also for monitoring of disease activity and prediction of therapy responsiveness. Recent work demonstrates that AIRD patients develop autoantibodies beyond those contained in the original categorization. In this study we discuss key mechanisms that underlie autoantibody development in AIRD: defects in early B cell development, genetic variants involved in regulating B cell and T cell tolerance, environmental triggers and antigen modification. We describe how autoantibodies can directly contribute to AIRD pathogenesis through innate and adaptive immune mechanisms, eventually culminating in systemic inflammation and localized tissue damage. We conclude by discussing recent insights that suggest distinct AIRD have incorrectly been denominated seronegative.

F-actin remodeling defects as revealed in primary immunodeficiency disorders.

Primary immunodeficiencies (PIDs) are a heterogeneous group of immune-related diseases. PIDs develop due to defects in gene-products that have consequences to immune cell function. A number of PID-proteins is involved in the remodeling of filamentous actin (f-actin) to support the generation of a contact zone between the antigen-specific T cell and antigen presenting cell (APC): the immunological synapse (IS). IS formation is the first step towards T-cell activation and essential for clonal expansion and acquisition of effector function. We here evaluated PIDs in which aberrant f-actin-driven IS formation may contribute to the PID disease phenotypes as seen in patients. We review examples of such contributions to PID phenotypes from literature, and highlight cases in which PID-proteins were evaluated for a role in f-actin polymerization and IS formation. We conclude with the proposition that patient groups might benefit from stratifying them in distinct functional groups in regard to their f-actin remodeling phenotypes in lymphocytes.

Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy.

Replicon particles of Rift Valley fever virus, referred to as nonspreading Rift Valley fever virus (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8(+) T cells in vitro and that prophylactic and therapeutic vaccinations of mice with NSR encoding a tumor-associated CD8 peptide can control the outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.

Cognate CD4 T-cell licensing of dendritic cells heralds anti-cytomegalovirus CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation.

UNLABELLED: Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells (DCs) is required to generate effective CD8+ T-cell responses. For humans, this was not fully understood. We here show that CD4+ T cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65-specific CD4+ T cells precedes the rise in CMV-pp65-specific CD8+ T cells. Second, the elicitation of CMV-pp65-specific CD8+ T cells from rare naive precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of gamma interferon (IFN-γ) by pp65-specific CD4+ T cells. Together, these data show that human DCs require licensing by cognate antigen-specific CD4+ T cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients. IMPORTANCE: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) that they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T cells, called helper T cells, is required.

Increased prevalence of gastrointestinal viruses and diminished secretory immunoglobulin a levels in antibody deficiencies.

PURPOSE: Gastrointestinal disease occurs frequently in antibody deficiencies. This study aims to explore the relation between gastrointestinal infections and mucosal homeostasis in patients with antibody deficiencies. METHODS: We performed an observational study including 54 pediatric antibody deficient patients (48 % CVID, 41 % CVID-like, 11 % XLA) and 66 healthy controls. Clinical symptom scores and stool samples were collected prospectively. Stool samples were evaluated for bacteria, parasites, viruses, secretory IgA- and for calprotectin levels. Results were compared between patients and controls. RESULTS: 24 % of antibody deficient patients versus 9 % of healthy controls tested positive for gastrointestinal viruses (p = 0.028). Fecal calprotectin levels were significantly higher in virus positive patients compared to virus negative patients (p = 0.002). However, in controls, fecal calprotectin levels were similar between virus positive and virus negative controls. Moreover, gastrointestinal virus positive patients had low serum IgA levels in 13/14 cases (94 %) versus 40/62 (62 %) patients in the virus negative patient group (p = 0.04). The virus positive patient group also displayed significantly lower secretory IgA levels in stool (median 13 ug/ml) than patients without gastrointestinal viruses detected or healthy controls (median 155 ug/ml) (p = 0.046). CONCLUSION: We here report an increased prevalence of gastrointestinal viruses and gastrointestinal complaints in antibody deficient patients. Patients that tested positive for gastrointestinal viruses showed diminished serum- and secretory IgA levels, and only in patients, virus positivity was associated with signs of mucosal inflammation. These findings suggest that particularly patients with low IgA are at risk for longstanding replication of gastrointestinal viruses, which may eventually result in CVID-related enteropathy.

Invariant natural killer T cells in adipose tissue: novel regulators of immune-mediated metabolic disease.

Adipose tissue (AT) represents a microenvironment where intersection takes place between immune processes and metabolic pathways. A variety of immune cells have been characterized in AT over the past decades, with the most recent addition of invariant natural killer T (iNKT) cells. As members of the T cell family, iNKT cells represent a subset that exhibits both innate and adaptive characteristics and directs ensuing immune responses. In disease conditions, iNKT cells have established roles that include disorders in the autoimmune spectrum in malignancies and infectious diseases. Recent work supports a role for iNKT cells in the maintenance of AT homeostasis through both immune and metabolic pathways. The deficiency of iNKT cells can result in AT metabolic disruptions and insulin resistance. In this review, we summarize recent work on iNKT cells in immune regulation, with an emphasis on AT-resident iNKT cells, and identify the potential mechanisms by which adipocytes can mediate iNKT cell activity.

Strategies to overcome low MHC-I expression in paediatric and adult tumours.

Immunotherapy has made significant advancements in cancer treatments, improving patients' survival rates and quality of life. Several challenges still need to be addressed, which include the considerable fraction of incomplete curative responses in cancer patients, the development of therapy resistance by tumours, and the occurrence of adverse effects, such as inflammatory and autoimmune complications. Paediatric tumours usually exhibit lower responsiveness to immunotherapies compared to adult tumours. Although the underlying reasons are not yet fully understood, one known mechanism by which tumours avoid immune recognition is through reduced cell surface expression of major histocompatibility complex class I (MHC-I) complexes. Accordingly, the reduced presentation of neoantigens by MHC-I hinders the recognition and targeting of tumour cells by CD8+ T cells, impeding T-cell-mediated cytotoxic anti-tumour responses. MHC-I downregulation indeed often correlates with a poorer prognosis and diminished response to immunotherapy. Understanding the mechanisms underlying MHC-I downregulation in different types of paediatric and adult tumours is crucial for developing strategies to restore MHC-I expression and enhance anti-tumour immune responses. We here discuss progress in MHC-I-based immunotherapies against cancers.

Antigen cross-presentation: extending recent laboratory findings to therapeutic intervention.

The initiation of adaptive immune responses requires antigen presentation to lymphocytes. In particular, dendritic cells (DCs) are equipped with specialized machinery that promote effective display of peptide/major histocompatibility complexes (MHC), rendering them the most potent stimulators of naive T lymphocytes. Antigen cross-presentation to CD8(+) T cells is an important mechanism for the development of specific cytotoxic T lymphocyte (CTL) responses against tumours and viruses that do not infect antigen-presenting cells. Here, we review recent findings concerning antigen cross-presentation to CD8(+) T lymphocytes. Specific subtypes of DCs in the mouse have been defined as being especially endowed for antigen cross-presentation, and a human homologue of these DCs has recently been described. DC vaccination strategies for the prevention and treatment of human diseases have been under investigation in recent years, but have not generally reached satisfying results. We here provide an overview of new findings in antigen cross-presentation research and how they can be used for development of the next generation of human DC vaccines.

Airway and interstitial lung disease are distinct entities in paediatric common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a common primary immune deficiency, caused by undefined defects in lymphocyte function, and is treated routinely by immunoglobulin substitution. CVID complications include airway disease (AD) and interstitial lung disease (ILD). It was not known if AD and ILD in CVID have a common immunological aetiology and should be considered separate features of the same disease, or as distinct syndromes that require specialized monitoring and treatment. We used high-resolution computed tomography (CT) to diagnose AD or ILD in paediatric CVID patients. Spirometry and body plethysmography did not differentiate between ILD and AD. Patients with AD (n = 11, 20%) developed more pneumonias while children with ILD (n = 8, 15%) showed immune dysregulation characterized by autoimmune complications, more severe memory B cell reduction and expansion of non-naive cytotoxic T cells. In conclusion, ILD and AD in CVID have dissimilar clinical and immunological characteristics, suggesting distinct aetiology requiring tailored monitoring and treatment of these patient subgroups.

Deletion of calcineurin and myocyte enhancer factor 2 (MEF2) binding domain of Cabin1 results in enhanced cytokine gene expression in T cells.

Cabin1 binds calcineurin and myocyte enhancer factor 2 (MEF2) through its COOH-terminal region. In cell lines, these interactions were shown to inhibit calcineurin activity after T cell receptor (TCR) signaling and transcriptional activation of Nur77 by MEF2. The role of these interactions under physiological conditions was investigated using a mutant mouse strain that expresses a truncated Cabin1 lacking the COOH-terminal calcineurin and MEF2 binding domains. T and B cell development and thymocyte apoptosis were normal in mutant mice. In response to anti-CD3 stimulation, however, mutant T cells expressed significantly higher levels of interleukin (IL)-2, IL-4, IL-9, IL-13, and interferon gamma than wild-type T cells. The enhanced cytokine gene expression was not associated with change in nuclear factor of activated T cells (NF-AT)c or NF-ATp nuclear translocation but was preceded by the induction of a phosphorylated form of MEF2D in mutant T cells. Consistent with the enhanced cytokine expression, mutant mice had elevated levels of serum immunoglobulin (Ig)G1, IgG2b, and IgE and produced more IgG1 in response to a T cell-dependent antigen. These findings suggest that the calcineurin and MEF2 binding domain of Cabin1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression probably through modulation of MEF2 activity.

A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.

To evaluate the role of natural immunoglobulin (Ig)M in the immediate response against microbial infection, we tested mutant mice that are deficient in secreted (s)IgM in an acute peritonitis model induced by cecal ligation and puncture (CLP). 20% of wild-type mice died within 32 h of CLP, whereas 70% of sIgM-deficient mice died within the same time period. The increased susceptibility was associated with a reduced level of tumor necrosis factor (TNF)-alpha, a decreased neutrophil recruitment and an increased bacterial load in the peritoneum, and elevated levels of endotoxin and proinflammatory cytokines in the circulation. Resistance to CLP by sIgM-deficient mice was restored by reconstitution with polyclonal IgM from normal mouse serum. Reconstitution with a monoclonal IgM specific to phosphatidylcholine, a conserved cell membrane component, has a modest effect but a monoclonal IgM specific to phosphocholine is not protective. These findings demonstrate a critical role of natural IgM in the immediate defense against severe bacterial infection.

Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor.

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion.

Proof of progression over time: finally fulminant brain, muscle, and liver affection in Alpers syndrome associated with the A467T POLG1 mutation.

This case concerns a 17-year-old boy, who was given the diagnosis of Alpers syndrome only postmortem when a homozygous 1399G-->A (A467T) mutation was found in the linker-region of POLG1. Serial muscle and liver biopsies as well as brain MRI scans in our patient ranging from early childhood to postmortem analyses showed that (i) routine diagnostic procedures can be normal in the early stage of the disorder and that (ii) central nervous system and further organ affection may only develop in the time course of the disease. Consecutive diagnostic examinations clearly reflected the devastating clinical course and cerebral deterioration evolving over time in Alpers syndrome.

Review of immune tolerance induction in hemophilia A.

At first sight the bleeding disorder hemophilia A seems to have little in common with immune disorders, but immunology research intersects with other disciplines including hematology. Nowadays, the most important complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against exogenous administered factor VIII (FVIII), which occurs in approximately 30% of all patients with severe hemophilia A. This antibody response renders FVIII replacement therapy ineffective, thereby increasing the risk for uncontrollable bleeding and morbidity, decreasing quality of life and increasing healthcare costs. The only proven effective therapy to eradicate these inhibitors is immune-based. Using a protocol called "immune tolerance induction" (ITI), the repeated and frequent administration of FVIII under non-inflammatory conditions downregulates the established antibody response and induces immune tolerance. There has been progress in research clarifying the mechanisms that mediate tolerance induction using ITI, both from patient studies and from research in cell culture and animal-based models. Peripheral tolerance induction to FVIII involves the apoptosis of antigen-specific B-memory cells, anergy induction in antigen-specific effector T-cells (Teff), induction of regulatory T-cells (Treg) and the formation of anti-idiotypic antibodies. In this review hemophilia A will be used as an example to discuss current concepts of tolerance induction as they are applied in patient care. Where possible, we will extrapolate tolerance findings in hemophilia A to related pathways known to affect auto-immune disorders or allergy.

Vascular transport of insulin to rat cardiac muscle. Central role of the capillary endothelium.

Using intact, beating hearts, we have assessed the interaction of insulin with capillary endothelium and the subsequent appearance of insulin in cardiac muscle. Rat hearts were perfused with 125I-insulin (10(-10) M) alone or in combination with unlabeled insulin (10(-9)-10(-5) M). 125I grains (shown to represent greater than 90% intact insulin) over both capillary endothelium and cardiac muscle decreased in a dose-dependent manner when hearts were co-perfused with labeled insulin and increasing concentrations of unlabeled insulin. Perfusion of 125I-desoctapeptide (DOP) insulin, a low affinity insulin analogue, with unlabeled insulin (10(-9)-10(-5) M) had no effect on the appearance of 125I-DOP insulin over microvessel endothelium and muscle. When capillary receptors were first destroyed by trypsin treatment or blocked by anti-receptor antibodies, the appearance of 125I-insulin in cardiac muscle decreased proportional to the inhibition of insulin binding to the capillary receptors. We conclude that insulin binding to capillary endothelial receptors is a central step in the transport of intravascular insulin to rat cardiac muscle.

Transcriptional and posttranscriptional regulation of insulin-like growth factor binding protein-3 by cyclic adenosine 3',5'-monophosphate: messenger RNA stabilization is accompanied by decreased binding of a 42-kDa protein to a uridine-rich domain in the 3'-untranslated region.

The Madin Darby bovine kidney (MDBK) cell line was used to investigate the mechanisms underlying the cAMP regulation of insulin-like growth factor binding protein-3 (IGFBP-3) gene expression. Treatment of confluent monolayers either with forskolin or cAMP produced a 60- to 75-fold induction of IGFBP-3 mRNA and protein levels. This effect did not require new protein synthesis as inhibition of translation by cycloheximide actually caused a 2-fold increase in the cAMP induction. The rates of IGFBP-3 gene transcription, assessed by nuclear run-on assays, increased approximately 15-fold in cells exposed to cAMP. In addition, the half-life of the IGFBP-3 mRNA transcript was increased approximately 3-fold in the presence of cAMP. Gel mobility shift and competition experiments revealed the specific binding of an approximately 42-kDa cytoplasmic protein factor to the 3'-untranslated region (3'-UTR) of the IGFBP-3 mRNA. A 21-nucleotide uridine-rich segment that contained no AUUUA motif was sufficient for the specific binding. The binding activity of this protein was reduced after cAMP treatment but was increased by phosphatase treatment. In conclusion, the cAMP induction of IGFBP-3 mRNA in MDBK cells occurred at both the transcriptional and posttranscriptional levels. The IGFBP-3 mRNA stabilization in MDBK cells probably involved the phosphorylation of a member of the family of U-rich region mRNA-binding proteins and is the first reported member whose RNA-binding activity is reduced by cAMP.

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

Role of natural and immune IgM antibodies in immune responses.

IgM antibodies constitute the major component of the natural antibodies and is also the first class of antibodies produced during a primary antibody response. The IgM-type antibodies differ from other classes of antibodies in that they are predominantly produced by B1 cells, in the absence of apparent stimulation by specific antigens. In addition, IgM antibodies are mostly encoded by germline V gene segments and have low affinities but broad specificites to both foreign and self structures. New developments regarding the function of both immune IgM antibodies and natural IgM antibodies will be examined here.