Response to comment on 'AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies'.

In 2016, we reported four substantial observations of APECED/APS1 patients, who are deficient in AIRE, a major regulator of central T cell tolerance (Meyer et al., 2016). Two of those observations have been challenged. Specifically, 'private' autoantibody reactivities shared by only a few patients but collectively targeting >1000 autoantigens have been attributed to false positives (Landegren, 2019). While acknowledging this risk, our study-design included follow-up validation, permitting us to adopt statistical approaches to also limit false negatives. Importantly, many such private specificities have now been validated by multiple, independent means including the autoantibodies' molecular cloning and expression. Second, a significant correlation of antibody-mediated IFNα neutralization with an absence of disease in patients highly disposed to Type I diabetes has been challenged because of a claimed failure to replicate our findings (Landegren, 2019). However, flaws in design and implementation invalidate this challenge. Thus, our results present robust, insightful, independently validated depictions of APECED/APS1, that have spawned productive follow-up studies.

Autoantibody Repertoire in APECED Patients Targets Two Distinct Subgroups of Proteins.

High titer autoantibodies produced by B lymphocytes are clinically important features of many common autoimmune diseases. APECED patients with deficient autoimmune regulator (AIRE) gene collectively display a broad repertoire of high titer autoantibodies, including some which are pathognomonic for major autoimmune diseases. AIRE deficiency severely reduces thymic expression of gene-products ordinarily restricted to discrete peripheral tissues, and developing T cells reactive to those gene-products are not inactivated during their development. However, the extent of the autoantibody repertoire in APECED and its relation to thymic expression of self-antigens are unclear. We here undertook a broad protein array approach to assess autoantibody repertoire in APECED patients. Our results show that in addition to shared autoantigen reactivities, APECED patients display high inter-individual variation in their autoantigen profiles, which collectively are enriched in evolutionarily conserved, cytosolic and nuclear phosphoproteins. The APECED autoantigens have two major origins; proteins expressed in thymic medullary epithelial cells and proteins expressed in lymphoid cells. These findings support the hypothesis that specific protein properties strongly contribute to the etiology of B cell autoimmunity.

Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

OBJECTIVE: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. METHODS: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNß reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. RESULTS: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNß promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. CONCLUSION: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies.

Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.

AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

In vitro reconstitution of PHO5 promoter chromatin remodeling points to a role for activator-nucleosome competition in vivo.

The yeast PHO5 promoter is a classical model for studying the role of chromatin in gene regulation. To enable biochemical dissection of the mechanism leading to PHO5 activation, we reconstituted the process in vitro. Positioned nucleosomes corresponding to the repressed PHO5 promoter state were assembled using a yeast extract-based in vitro system. Addition of the transactivator Pho4 yielded an extensive DNase I-hypersensitive site resembling induced PHO5 promoter chromatin. Importantly, this remodeling was energy dependent. In contrast, little or no chromatin remodeling was detected at the PHO8 or PHO84 promoter in this in vitro system. Only the PHO5 promoter harbors a high-affinity intranucleosomal Pho4 binding site (UASp) where Pho4 binding can compete with nucleosome formation, prompting us to test the importance of such competition for chromatin remodeling by analysis of UASp mutants in vivo. Indeed, the intranucleosomal location of the UASp element was critical, but not essential, for complete remodeling at the PHO5 promoter in vivo. Further, binding of just the Gal4 DNA binding domain to an intranucleosomal site could increase PHO5 promoter opening. These data establish an auxiliary role for DNA binding competition between Pho4 and histones in PHO5 promoter chromatin remodeling in vivo.

A role for noncoding transcription in activation of the yeast PHO5 gene.

Noncoding, or intergenic, transcription by RNA polymerase II (RNAPII) is remarkably widespread in eukaryotic organisms, but the effects of such transcription remain poorly understood. Here we show that noncoding transcription plays a role in activation, but not repression, of the Saccharomyces cerevisiae PHO5 gene. Histone eviction from the PHO5 promoter during activation occurs with normal kinetics even in the absence of the PHO5 TATA box, showing that transcription of the gene itself is not required for promoter remodeling. Nevertheless, we find that mutations that impair transcript elongation by RNAPII affect the kinetics of histone eviction from the PHO5 promoter. Most dramatically, inactivation of RNAPII itself abolishes eviction completely. Under repressing conditions, an approximately 2.4-kb noncoding exosome-degraded transcript is detected that originates near the PHO5 termination site and is transcribed in the antisense direction. Abrogation of this transcript delays chromatin remodeling and subsequent RNAPII recruitment to PHO5 upon activation. We propose that noncoding transcription through positioned nucleosomes can enhance chromatin plasticity so that chromatin remodeling and activation of traversed genes occur in a timely manner.

Nucleosome stability at the yeast PHO5 and PHO8 promoters correlates with differential cofactor requirements for chromatin opening.

The coregulated PHO5 and PHO8 genes in Saccharomyces cerevisiae provide typical examples for the role of chromatin in promoter regulation. It has been a long-standing question why the cofactors Snf2 and Gcn5 are essential for full induction of PHO8 but dispensable for opening of the PHO5 promoter. We show that this discrepancy may result from different stabilities of the two promoter chromatin structures. To test this hypothesis, we used our recently established yeast extract in vitro chromatin assembly system, which generates the characteristic PHO5 promoter chromatin. Here we show that this system also assembles the native PHO8 promoter nucleosome pattern. Remarkably, the positioning information for both native patterns is specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract does not support proper nucleosome positioning unless supplemented with yeast extract. By competitive assemblies in the yeast extract system we show that the PHO8 promoter has greater nucleosome positioning power and that the properly positioned nucleosomes are more stable than those at the PHO5 promoter. Thus we provide evidence for the correlation of inherently more stable chromatin with stricter cofactor requirements.

Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma.

In classical Hodgkin lymphoma the malignant Hodgkin/Reed-Sternberg (HRS) cells characteristically constitute only a small minority of the tumour load. Their origin has been debated for decades, but on the basis of rearrangement and somatic hypermutations of their immunoglubulin (Ig) genes, HRS cells are now ascribed to the B-cell lineage. Nevertheless, phenotypically HRS cells have lost their B cell identity: they usually lack common B cell-specific surface markers such as CD19 and CD79a as well as Ig gene transcripts. Here we demonstrate that Ig promoters as well as both intronic and 3' enhancer sequences are transcriptionally inactive in HRS cell lines. This inactivity correlates with either reduced levels or even a complete lack of several B cell-specific transcription factors required for their expression: Oct-2, OBF-1, PU.1, E47/E12, PAX-5 and EBF. Moreover, we demonstrate that PU.1 and PAX-5 are significantly down-regulated in HRS cells in pathological specimens from primary tumour tissues. However, forced expression of these transcription factors can activate regulatory sequences of silenced B cell marker genes, and in one instance also transcription from a silenced endogenous locus. Thus, HRS cells are dedifferentiated B cells with global down-regulation of B cell-specific genes.