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

Ikbkb Gain-of-Function in human disease

The NFkB signaling pathway is important in regulating numerous immune and inflammatory responses. NFkB family members can modulate the transcription of cytokines, as well as regulate genes involved in cellular differentiation, survival, proliferation, and immune cell function. Multiple groups have identified hypomorphic mutations in IKBKB (encodes IKKB) in patients suffering from immunodeficiency, where impaired NFkB activity was observed. My PhD has focused on the effects of an IKKB gain-of-function mutation and its role in human disease. In this thesis I describe experiments conducted to investigate the effects of a novel heterozygous de novo missense mutation that we identified in a proband with immunodeficiency. The mutation was found to result in a valine to isoleucine amino acid substitution within the kinase domain of the IKKB domain sequence, and resulted in a gain-of-function effect on IKKB. This enabled overactivation of the NFkB signaling pathway. To prove causation of this mutation, we generated a CRISPR-cas9 mouse model carrying the orthologous mutation. Biochemical and cellular analysis revealed similarities between the human and the mouse, therefore conferring a causative role of the mutation in the proband's immunodeficiency (Chapter 3). Through aging and observation of the mouse model, we identified the development of an inflammatory condition that involved the skin and bone/joints, and closely resembled the IL-17-mediated human disease, psoriatic arthritis (PsA) (Chapter 4). A gene dosage effect was evident where a skin-only disease was present in mice that were heterozygous for the IkbkbV203I variant, whilst a skin and systemic inflammatory illness developed when mice carry a double dose of the IkbkbV203I variant. The Ikbkb gain-of-function mutation generated a remarkable Treg population that abnormally produces increased IL-17 both in lymphoid tissues and at the sites of inflammation (Chapter 5). Single-cell RNA sequencing enabled the identification of an abnormally abundant Treg cluster within the spleen and bone marrow of mice homozygous for the IkbkbV203I. This cluster resembled a gene signature similar to an established non-lymphoid tissue Treg population, as well as a strong NFkB signature mediated by the gain-of-function mutation (Chapter 6). In this thesis I have investigated and identified the effects of an overactive IKKB protein within the immune system to result in primary immunodeficiency disease (PID) and the IL-17-mediated inflammatory condition, psoriatic arthritis in a mouse model. Our findings provide evidence that a fine balance of the NFkB is required to maintain immune system homeostasis. Furthermore, we have identified a biomarker for progression from skin-only inflammation to psoriatic arthritis through the presence of IL-17+ Tregs, mediated by GoF IKKB. We expect that these findings will be important in better defining diagnosis methods, as well as novel therapeutic targets for PIDs and PsA

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

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.

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

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

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