Nuclear Factor-kappaB in Autoimmunity: Man and Mouse

NF-κB (nuclear factor-kappa B) is a transcription complex crucial for host defense mediated by innate and adaptive immunity, where canonical NF-κB signaling, mediated by nuclear translocation of RelA, c-Rel, and p50, is important for immune cell activation, differentiation, and survival. Non-canonical signaling mediated by nuclear translocation of p52 and RelB contributes to lymphocyte maturation and survival and is also crucial for lymphoid organogenesis. We outline NF-κB signaling and regulation, then summarize important molecular contributions of NF-κB to mechanisms of self-tolerance. We relate these mechanisms to autoimmune phenotypes described in what is now a substantial catalog of immune defects conferred by mutations in NF-κB pathways in mouse models. Finally, we describe Mendelian autoimmune syndromes arising from human NF-κB mutations, and speculate on implications for understanding sporadic autoimmune disease.This work was supported by NHMRC program grant 1113577, NHMRC project grant 1079648, and NHMRC Centre of Research Excellence grant 1107464

Nuclear Factor-kappaB in Autoimmunity: Man and Mouse

NF-κB (nuclear factor-kappa B) is a transcription complex crucial for host defense mediated by innate and adaptive immunity, where canonical NF-κB signaling, mediated by nuclear translocation of RelA, c-Rel, and p50, is important for immune cell activation, differentiation, and survival. Non-canonical signaling mediated by nuclear translocation of p52 and RelB contributes to lymphocyte maturation and survival and is also crucial for lymphoid organogenesis. We outline NF-κB signaling and regulation, then summarize important molecular contributions of NF-κB to mechanisms of self-tolerance. We relate these mechanisms to autoimmune phenotypes described in what is now a substantial catalog of immune defects conferred by mutations in NF-κB pathways in mouse models. Finally, we describe Mendelian autoimmune syndromes arising from human NF-κB mutations, and speculate on implications for understanding sporadic autoimmune disease

Interactions Between Gibberellins and Other Hormones

In this chapter we discuss interactions between the gibberellins (GAs) and other plant hormones. Auxin and ethylene exert strong effects on GA levels, and at least some of the physiological responses to auxin are mediated by increased GA content. Abscisic acid (ABA) and GA appear to negatively regulate each other's levels in seeds, consistent with their antagonistic effects on germination. It has been suggested that several non-GA hormones affect the stability of DELLAs, the key signalling proteins that are destabilised by GAs. However, it appears that the reported effects of these additional hormones on DELLA stability are either mediated by changes in GA content, or have not yet been confirmed. Recent papers provide evidence that DELLAs physically interact with transcriptional regulators from the signalling pathways of the brassinosteroids, ethylene, jasmonic acid and ABA. This may represent a physiologically significant way in which GAs interact with other signalling pathways, and helps to explain how DELLAs, which lack a conserved DNA binding domain, can regulate gene transcriptio

Modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by CRISPR/Cas9

Introduction/Background: Next generation sequencing has resulted in substantial progress in identification of Mendelian immune deficiency syndromes. In some cases, however, putative causal mutations occur in single kindreds, or even individual patients. Under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. Objectives: Understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. One method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency Methods: Mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. We have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the NF-kB pathway. Results: So far, defects in both canonical and non-canonical pathways of NF-kB activation have been shown to cause immune deficiency, often associated with immune dysregulation. We describe a known defects and novel putative defect identified in the canonical NF-kB pathway Conclusions: CRISPR-cas9 mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative

Hypomorphic caspase activation and recruitment domain 11 (CARD11) mutations associated with diverse immunologic phenotypes with or without atopic disease

Background Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. Objectives We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. Methods Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. Results Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked–like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. Conclusion These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activityWe thank Drs James R. Lupski, Richard A. Gibbs, and Zeynep H. Coban Akdemir for providing WES and bioinformatics support in the BaylorHopkins Center for Mendelian Genomics (National Institutes of Health grant UM1HG006542) and T. D. Andrews and M. Field for bioinformatics support at the John Curtin School of Medical Research

IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Acknowledgements: The authors thank the Australian Phenomics Facility staff for husbandry and genotyping, and the Flow Cytometry facility, Biomolecular Resource Facility, the Phenomics Translation Initiative team, and the ANU Bioinformatics Consultancy at the John Curtin School of Medical Research for their services. This work was funded by the National Health and Medical Research Council Program Grant APP1113577 (CGV, MCC), CRE APP1079648 (CGV, MCC) and Project Grant APP1107464 (MCC); the Alan Harvey CVID Research Endowment (CC); Royal Society Wolfson Fellowship RSWF\R2\222004 (MCC). This study utilized the Australian Phenomics Network Histopathology and Organ Pathology Service of the University of Melbourne. The Phenomics Translation Initiative is supported by the Medical Research Future Fund (EPCD000035).Funder: Alan Harvey CVID Research EndowmentLoss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation

Gain-of-function IKBKB mutation causes human combined immune deficiency

Genetic mutations account for many devastating early onset immune deficiencies. In contrast, less severe and later onset immune diseases, including in patients with no prior family history, remain poorly understood. Whole exome sequencing in two cohorts of such patients identified a novel heterozygous de novo IKBKB missense mutation (c.607G>A) in two separate kindreds in whom probands presented with immune dysregulation, combined T and B cell deficiency, inflammation, and epithelial defects. IKBKB encodes IKK2, which activates NF-κB signaling. IKK2V203I results in enhanced NF-κB signaling, as well as T and B cell functional defects. IKK2V203 is a highly conserved residue, and to prove causation, we generated an accurate mouse model by introducing the precise orthologous codon change in Ikbkb using CRISPR/Cas9. Mice and humans carrying this missense mutation exhibit remarkably similar cellular and biochemical phenotypes. Accurate mouse models engineered by CRISPR/Cas9 can help characterize novel syndromes arising from de novo germline mutations and yield insight into pathogenesis.The study was funded by National Health and Medical Research Council grants 1107464 (to M.C. Cook), 1079648 (to C.G. Vinuesa and M.C. Cook), and 1113577 (to C.G. Vinuesa and M.C. Cook); The Bev and Alan Harvey Bequest; Japan Society for the Promotion of Science grants KAKENHI JP16H05355 (to S. Okada), 16K15528 (to S. Okada), JP26461570 (to H. Kanegane), and JP17K10099 (to H. Kanegane); and the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4 + T cells

Acknowledgements: We thank Harpreet Vohra and Michael Devoy at the Flow Cytometry Facility and Maxim Nekrasov at the Australian Cancer Research Foundation (ACRF) Biomolecular Resource Facility of the John Curtin School of Medical Research for technical support and Ann-Maree Hatch and Anastasia Wilson for assistance with obtaining blood and tonsil samples. We thank Dominik Spensberger and Gaetan Burgio at John Curtin School of Medical Research for their help with mouse model construction. The study was supported by NHMRC grants APP1113577 (MCC, CGV) and APP1079648 (MCC, CGV), and grant APP1130330 awarded through the Priority-drive Collaborative Cancer Research Scheme and funded by Cancer Australia (MCC, DY, SY).As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection