Publications by authors named "Vanessa L Bryant"

28 Publications

  • Page 1 of 1

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Blood Adv 2021 Jun;5(11):2550-2562

The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.
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http://dx.doi.org/10.1182/bloodadvances.2020004139DOI Listing
June 2021

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

EMBO J 2021 Jun 5;40(11):e107333. Epub 2021 May 5.

ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER) , HER2 , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8 T cells characterized triple-negative and HER2 cancers but not ER tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.
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http://dx.doi.org/10.15252/embj.2020107333DOI Listing
June 2021

When B cells break bad: development of pathogenic B cells in Sjögren's syndrome.

Clin Exp Rheumatol 2020 Jul-Aug;38 Suppl 126(4):271-282. Epub 2020 Sep 22.

Immunology Division, Walter and Eliza Hall Institute for Medical Research, Parkville; Department of Medicine, The University of Melbourne and Department of Clinical Immunology and Allergy, The Royal Melbourne Hospital, Parkville, and Department of Clinical Immunology and Allergy, The Royal Melbourne Hospital, Parkville, Australia.

Primary Sjögren's syndrome (pSS) is often considered a B cell-mediated disease, yet the precise role of B cells in the pathogenesis is not fully understood. This is exemplified by the failure of multiple clinical trials directed at B cell depletion or inhibition. To date, most prognostic markers for severe disease outcomes are autoantibodies, but the underlying mechanisms by which B cells drive diverse disease presentations in pSS likely extend beyond autoantibody production. Here we outline an expanded role of B cells in disease pathogenesis drawing on examples from animal models of SS, and from other autoimmune diseases that share similar clinical or immunological abnormalities. We focus on recent findings from the detailed analysis of pathogenic B cells in patients with pSS to propose strategies for patient stratification to improve clinical trial outcomes. We conclude that an integrated cellular, molecular and genetic analysis of patients with pSS will reveal the underlying pathogenic mechanisms and guide precision medicine.
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October 2020

IL-17 production by tissue-resident MAIT cells is locally induced in children with pneumonia.

Mucosal Immunol 2020 09 28;13(5):824-835. Epub 2020 Feb 28.

Department of Respiratory Medicine, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Centre, State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou, Guangdong, China.

Community-acquired pneumonia (CAP) contributes substantially to morbidity and mortality in children under the age of 5 years. In examining bronchoalveolar lavages (BALs) of children with CAP, we found that interleukin-17 (IL-17) production was significantly increased in severe CAP. Immune profiling showed that mucosal-associated invariant T (MAIT) cells from the BALs, but not blood, of CAP patients actively produced IL-17 (MAIT17). Single-cell RNA-sequencing revealed that MAIT17 resided in a BAL-resident PLZFCD103 MAIT subset with high expression of hypoxia-inducible factor 1α (HIF-1α), reflecting the hypoxic state of the inflamed tissue. CAP BALs also contained a T-bet MAIT1 subset and a novel DDIT3 (DNA damage-inducible transcript 3-positive) MAIT subset with low expression of HIF1A. Furthermore, MAIT17 differed from T-helper type 17 (Th17) cells in the expression of genes related to tissue location, innateness, and cytotoxicity. Finally, we showed that BAL monocytes were hyper-inflammatory and elicited differentiation of MAIT17. Thus, tissue-resident MAIT17 cells are induced at the infected respiratory mucosa, likely influenced by inflammatory monocytes, and contribute to IL-17-mediated inflammation during CAP.
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http://dx.doi.org/10.1038/s41385-020-0273-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434594PMC
September 2020

TCF-1 limits the formation of Tc17 cells via repression of the MAF-RORγt axis.

J Exp Med 2019 07 29;216(7):1682-1699. Epub 2019 May 29.

Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

Interleukin (IL)-17-producing CD8 T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ-producing effector CD8 T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 () regulates CD8 T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1-driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8 T cell subsets. IL-17-producing CD8 T cells isolated from healthy humans were also distinct from CD8IL-17 T cells and enriched in pathways driven by and Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.
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http://dx.doi.org/10.1084/jem.20181778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605755PMC
July 2019

Fatal Enteroviral Encephalitis in a Patient with Common Variable Immunodeficiency Harbouring a Novel Mutation in NFKB2.

J Clin Immunol 2019 04 29;39(3):324-335. Epub 2019 Mar 29.

Department of Clinical Immunology and Allergy, Royal Melbourne Hospital, Parkville, VIC, 3052, Australia.

Common variable immunodeficiency is the most prevalent of the primary immunodeficiency diseases, yet its pathogenesis is largely poorly understood. Of the cases that are monogenic, many arise due to pathogenic variants in NFKB1 and NFKB2. Here, we report enteroviral encephalomyelitis as the cause of a fatal neurodegenerative condition in a patient with a novel heterozygous mutation in NFKB2 (c.2543insG, p.P850Sfs36*) that disrupts non-canonical NF-κB signaling. Investigations of primary and secondary lymphoid tissue demonstrated a complete absence of B cells and germinal centers. Despite multiple negative viral PCR testing of cerebrospinal fluid during her disease progression, post-mortem analysis of cerebral tissue revealed a chronic lymphocytic meningoencephalitis, in the presence of Cocksackie A16 virus, as the cause of death. The clinical features, and progression of disease reported here, demonstrate divergent clinical and immunological phenotypes of individuals within a single family. This is the first reported case of fatal enteroviral encephalomyelitis in a patient with NF-κB2 deficiency and mandates a low threshold for early brain biopsy and the administration of increased immunoglobulin replacement in any patient with a defect in this pathway and deterioration of neurological status.
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http://dx.doi.org/10.1007/s10875-019-00602-xDOI Listing
April 2019

Human IFN-γ immunity to mycobacteria is governed by both IL-12 and IL-23.

Sci Immunol 2018 12;3(30)

Department of Pediatric Immunology and Allergy, Ankara University School of Medicine, Ankara, Turkey.

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rβ1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rβ2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αβ T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4 T cells, including, in particular, mycobacterium-specific T1* cells (CD45RACCR6), is dependent on both IL-12 and IL-23. Last, we show that , , and have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rβ2 or IL-23R deficiency, relative to IL-12Rβ1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rβ2-deficient than IL-12Rβ1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.
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http://dx.doi.org/10.1126/sciimmunol.aau6759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380365PMC
December 2018

Pitfalls of immunotherapy: lessons from a patient with CTLA-4 haploinsufficiency.

Allergy Asthma Clin Immunol 2018 22;14:65. Epub 2018 Oct 22.

1Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC Australia.

Background: Daclizumab is a humanized monoclonal antibody that blocks CD25, the high affinity alpha subunit of the interleukin-2 receptor. Daclizumab therapy targets T regulatory cell and activated effector T cell proliferation to suppress autoimmune disease activity, in inflammatory conditions like relapsing and remitting multiple sclerosis. Here, we present the first report of agranulocytosis with daclizumab therapy in a patient with relapsing and remitting multiple sclerosis.

Case Presentation: Our patient was a 24-year-old Australian female with a clinical history of atopy, lymphocytic enteritis complicated by B12 deficiency, relapsing and remitting multiple sclerosis, recurrent lower respiratory tract infections, vulval/cervical intraepithelial neoplasia and melanoma. She was commenced on daclizumab therapy after failing several lines of treatment for relapsing and remitting multiple sclerosis. During a hospital admission for lymphocytic enteritis, she was incidentally diagnosed with combined immunodeficiency with hypogammaglobulinaemia and declined proposed regular intravenous immunoglobulin infusions. Following six months of daclizumab therapy, our patient presented to hospital with febrile neutropenia. No clear infective cause was found, despite numerous investigations. However, bone marrow biopsy revealed agranulocytosis with an apparent maturation block at the myeloblasts stage. Neustrophil recovery occurred following cessation of daclizumab and the initiation of T cell immunosuppressive agents including systemic corticosteroids and methotrexate. The patient was further investigated for combined immunodeficiency and whole exome sequencing revealed a novel heterozygous missense variant in (), leading to a diagnosis of CTLA-4 haploinsufficiency with autoimmune infiltration (CHAI).

Conclusion: This case demonstrates that autoimmune disease may be the presenting feature of primary immunodeficiency and should be appropriately investigated prior to the commencement of immunotherapy. Genetic clarification of underlying primary immunodeficiency may provide critical clinical information that alters the safety of the proposed treatment strategy.
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http://dx.doi.org/10.1186/s13223-018-0272-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196561PMC
October 2018

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1.

Front Immunol 2018 20;9:1809. Epub 2018 Aug 20.

Immune Therapies Group, Burnet Institute, Melbourne, VIC, Australia.

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.
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http://dx.doi.org/10.3389/fimmu.2018.01809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109644PMC
September 2019

Delayed Diagnosis and Complications of Predominantly Antibody Deficiencies in a Cohort of Australian Adults.

Front Immunol 2018 14;9:694. Epub 2018 May 14.

The Jeffrey Modell Diagnostic and Research Centre for Primary Immunodeficiencies, Melbourne, VIC, Australia.

Background: Predominantly antibody deficiencies (PADs) are the most common type of primary immunodeficiency in adults. PADs frequently pass undetected leading to delayed diagnosis, delayed treatment, and the potential for end-organ damage including bronchiectasis. In addition, PADs are frequently accompanied by comorbid autoimmune disease, and an increased risk of malignancy.

Objectives: To characterize the diagnostic and clinical features of adult PAD patients in Victoria, Australia.

Methods: We identified adult patients receiving, or having previously received immunoglobulin replacement therapy for a PAD at four hospitals in metropolitan Melbourne, and retrospectively characterized their clinical and diagnostic features.

Results: 179 patients from The Royal Melbourne, Alfred and Austin Hospitals, and Monash Medical Centre were included in the study with a median age of 49.7 years (range: 16-87 years), of whom 98 (54.7%) were female. The majority of patients (116; 64.8%) met diagnostic criteria for common variable immunodeficiency (CVID), and 21 (11.7%) were diagnosed with X-linked agammaglobulinemia (XLA). Unclassified hypogammaglobulinemia (HGG) was described in 22 patients (12.3%), IgG subclass deficiency (IGSCD) in 12 (6.7%), and specific antibody deficiency (SpAD) in 4 individuals (2.2%). The remaining four patients had a diagnosis of Good syndrome (thymoma with immunodeficiency). There was no significant difference between the age at diagnosis of the disorders, with the exception of XLA, with a median age at diagnosis of less than 1 year. The median age of reported symptom onset was 20 years for those with a diagnosis of CVID, with a median age at diagnosis of 35 years. CVID patients experienced significantly more non-infectious complications, such as autoimmune cytopenias and lymphoproliferative disease, than the other antibody deficiency disorders. The presence of non-infectious complications was associated with significantly reduced survival in the cohort.

Conclusion: Our data are largely consistent with the experience of other centers internationally, with clear areas for improvement, including reducing diagnostic delay for patients with PADs. It is likely that these challenges will be in part overcome by continued advances in implementation of genomic sequencing for diagnosis of PADs, and with that opportunities for targeted treatment of non-infectious complications.
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http://dx.doi.org/10.3389/fimmu.2018.00694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960671PMC
June 2019

Clinical Implications of Digenic Inheritance and Epistasis in Primary Immunodeficiency Disorders.

Front Immunol 2017 26;8:1965. Epub 2018 Jan 26.

School of Biological Sciences, University of Auckland, Auckland, New Zealand.

The existence of epistasis in humans was first predicted by Bateson in 1909. Epistasis describes the non-linear, synergistic interaction of two or more genetic loci, which can substantially modify disease severity or result in entirely new phenotypes. The concept has remained controversial in human genetics because of the lack of well-characterized examples. In humans, it is only possible to demonstrate epistasis if two or more genes are mutated. In most cases of epistasis, the mutated gene products are likely to be constituents of the same physiological pathway leading to severe disruption of a cellular function such as antibody production. We have recently described a digenic family, who carry mutations of /TACI as well as genes. Both genes lie in tandem along the immunoglobulin isotype switching and secretion pathway. We have shown they interact in an epistatic way causing severe immunodeficiency and autoimmunity in the digenic proband. With the advent of next generation sequencing, it is likely other families with digenic inheritance will be identified. Since digenic inheritance does not always cause epistasis, we propose an epistasis index which may help quantify the effects of the two mutations. We also discuss the clinical implications of digenic inheritance and epistasis in humans with primary immunodeficiency disorders.
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http://dx.doi.org/10.3389/fimmu.2017.01965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790765PMC
January 2018

Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes.

Immunol Cell Biol 2018 02 15;96(2):149-159. Epub 2017 Dec 15.

Division of Immunology, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

The discovery of cell division tracking properties of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) by Lyons and Parish in 1994 led to a broad range of new methods and numerous important biological discoveries. After labeling, CFSE is attached to free amine groups and intracellular proteins in the cytoplasm and nucleus of a cell, and halves in fluorescence intensity with each round of cell division, enabling enumeration of the number of divisions a cell has undergone. A range of popular division tracking dyes were subsequently developed, including CellTrace Violet (CTV), making available the green fluorescent channel previously occupied by CFSE. More recently, CellTrace Yellow (CTY) and CellTrace Far Red (CTFR), each with unique fluorescence properties, were introduced. In a comparison, we found that the fluorescence values of both dyes were well separated from autofluorescence, and enabled a greater number of divisions to be identified than CTV, before this limit was reached. These new dyes provided clear and well-separated peaks for both murine and human B lymphocytes, and should find wide application. The range of excitation/emission spectra available for division tracking dyes now also facilitates multiplexing, that is, the labeling of cells with different combinations of dyes to give a unique fluorescence signature, allowing single cell in vitro and in vivo tracking. The combinatorial possibilities are significantly increased with these additional dyes.
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http://dx.doi.org/10.1111/imcb.1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446909PMC
February 2018

Epistatic interactions between mutations of TACI () and result in a severe primary immunodeficiency disorder and systemic lupus erythematosus.

Clin Transl Immunology 2017 Oct 20;6(10):e159. Epub 2017 Oct 20.

Department of Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Common variable immunodeficiency disorders (CVID) are a group of primary immunodeficiencies where monogenetic causes account for only a fraction of cases. On this evidence, CVID is potentially polygenic and epistatic although there are, as yet, no examples to support this hypothesis. We have identified a non-consanguineous family, who carry the C104R (c.310T>C) mutation of the Transmembrane Activator Calcium-modulator and cyclophilin ligand Interactor (TACI, ) gene. Variants in /TACI are identified in up to 10% of CVID patients, and are associated with, but not solely causative of CVID. The proband is heterozygous for the /TACI C104R mutation and meets the Ameratunga diagnostic criteria for CVID and the American College of Rheumatology criteria for systemic lupus erythematosus (SLE). Her son has type 1 diabetes, arthritis, reduced IgG levels and IgA deficiency, but has not inherited the /TACI mutation. Her brother, homozygous for the /TACI mutation, is in good health despite profound hypogammaglobulinemia and mild cytopenias. We hypothesised that a second unidentified mutation contributed to the symptomatic phenotype of the proband and her son. Whole-exome sequencing of the family revealed a nonsense mutation (T168fsX191) in the Transcription Factor 3 () gene encoding the E2A transcription factors, present only in the proband and her son. We demonstrate mutations of /TACI impair immunoglobulin isotype switching and antibody production predominantly via T-cell-independent signalling, while mutations of impair both T-cell-dependent and -independent pathways of B-cell activation and differentiation. We conclude that epistatic interactions between mutations of the /TACI and signalling networks lead to the severe CVID-like disorder and SLE in the proband.
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http://dx.doi.org/10.1038/cti.2017.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671988PMC
October 2017

Review: Diagnosing Common Variable Immunodeficiency Disorder in the Era of Genome Sequencing.

Clin Rev Allergy Immunol 2018 Apr;54(2):261-268

Auckland Hospital, Park Rd, Grafton, Auckland, 1010, New Zealand.

Common variable immunodeficiency disorders (CVID) are an enigmatic group of often heritable conditions, which may manifest for the first time in early childhood or as late as the eighth decade of life. In the last 5 years, next generation sequencing (NGS) has revolutionised identification of genetic disorders. However, despite the best efforts of researchers around the globe, CVID conditions have been slow to yield their molecular secrets. We have previously described the many clinical advantages of identifying the genetic basis of primary immunodeficiency disorders (PIDs). In a minority of CVID patients, monogenic defects have now been identified. If a causative mutation is identified, these conditions are reclassified as CVID-like disorders. Here we discuss recent advances in the genetics of CVID and discuss how NGS can be optimally deployed to identify the causal mutations responsible for the protean clinical manifestations of these conditions. Diagnostic criteria such as the Ameratunga et al. criteria will continue to play an important role in patient management as well as case selection and sequencing strategy design until the genetic conundrum of CVID is solved.
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http://dx.doi.org/10.1007/s12016-017-8645-0DOI Listing
April 2018

Life, death, and antibodies.

Science 2017 10;358(6360):171-172

Division of Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.

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http://dx.doi.org/10.1126/science.aap8728DOI Listing
October 2017

The TNF Receptor Superfamily-NF-κB Axis Is Critical to Maintain Effector Regulatory T Cells in Lymphoid and Non-lymphoid Tissues.

Cell Rep 2017 Sep 7;20(12):2906-2920. Epub 2017 Sep 7.

Molecular Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia. Electronic address:

After exiting the thymus, Foxp3 regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans.
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http://dx.doi.org/10.1016/j.celrep.2017.08.068DOI Listing
September 2017

The Expanding Spectrum of NFkB1 Deficiency.

J Clin Immunol 2016 08 23;36(6):531-2. Epub 2016 Jun 23.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

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http://dx.doi.org/10.1007/s10875-016-0310-5DOI Listing
August 2016

Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency.

Am J Hum Genet 2015 Sep 13;97(3):389-403. Epub 2015 Aug 13.

Center for Chronic Immunodeficiency, University Medical Center Freiburg and University of Freiburg, Freiburg 79108, Germany; Institute of Immunity and Transplantation, University College London, London WC1E 6BT, UK. Electronic address:

Common variable immunodeficiency (CVID), characterized by recurrent infections, is the most prevalent symptomatic antibody deficiency. In ∼90% of CVID-affected individuals, no genetic cause of the disease has been identified. In a Dutch-Australian CVID-affected family, we identified a NFKB1 heterozygous splice-donor-site mutation (c.730+4A>G), causing in-frame skipping of exon 8. NFKB1 encodes the transcription-factor precursor p105, which is processed to p50 (canonical NF-κB pathway). The altered protein bearing an internal deletion (p.Asp191_Lys244delinsGlu; p105ΔEx8) is degraded, but is not processed to p50ΔEx8. Altered NF-κB1 proteins were also undetectable in a German CVID-affected family with a heterozygous in-frame exon 9 skipping mutation (c.835+2T>G) and in a CVID-affected family from New Zealand with a heterozygous frameshift mutation (c.465dupA) in exon 7. Given that residual p105 and p50—translated from the non-mutated alleles—were normal, and altered p50 proteins were absent, we conclude that the CVID phenotype in these families is caused by NF-κB1 p50 haploinsufficiency.
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http://dx.doi.org/10.1016/j.ajhg.2015.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564940PMC
September 2015

Chemokines, their receptors and human disease: the good, the bad and the itchy.

Immunol Cell Biol 2015 Apr;93(4):364-71

Walter and Eliza Hall Institute, Melbourne, Parkville, Australia.

Chemokines are a highly specialized group of cytokines that coordinate trafficking and homing of leucocytes between bone marrow, lymphoid organs and sites of infection or inflammation. They are also responsible for structural organization within lymphoid organs. Aberrant expression or function of these molecules, or their receptors, has been linked to protection or susceptibility to specific infectious diseases, as well as the risk of autoimmune disease and malignancy, revealing critical roles of chemokines and their receptors in human health, disease and therapeutics. In this review, we focus on human diseases that provide lessons regarding the critical role of these specialized and complex cytokines.
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http://dx.doi.org/10.1038/icb.2015.23DOI Listing
April 2015

Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.

J Leukoc Biol 2014 Apr 16;95(4):667-76. Epub 2013 Dec 16.

2.Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14(+) cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.
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http://dx.doi.org/10.1189/jlb.0513250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958742PMC
April 2014

Partial IFN-γR2 deficiency is due to protein misfolding and can be rescued by inhibitors of glycosylation.

Blood 2013 Oct 20;122(14):2390-401. Epub 2013 Aug 20.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY;

We report a molecular study of the two known patients with autosomal recessive, partial interferon-γ receptor (IFN-γR)2 deficiency (homozygous for mutations R114C and G227R), and three novel, unrelated children, homozygous for S124F (P1) and G141R (P2 and P3). IFN-γR2 levels on the surface of the three latter patients' cells are slightly lower than those on control cells. The patients' cells also display impaired, but not abolished, response to IFN-γ. Moreover, the R114C, S124F, G141R and G227R IFNGR2 hypomorphic alleles all encode misfolded proteins with abnormal N-glycosylation. The mutants are largely retained in the endoplasmic reticulum, although a small proportion reach and function at the cell surface. Strikingly, the IFN-γ response of the patients' cells is enhanced by chemical modifiers of N-glycosylation, as previously shown for patients with gain-of-glysosylation T168N and misfolding 382-387dup null mutations. All four in-frame IFNGR2 hypomorphic mutant alleles encoding surface-expressed receptors are thus deleterious by a mechanism involving abnormal N-glycosylation and misfolding of the IFN-γR2 protein. The diagnosis of partial IFN-γR2 deficiency is clinically useful, as affected patients should be treated with IFN-γ, [corrected] unlike patients with complete IFN-γR2 deficiency. Moreover, inhibitors of glycosylation might be beneficial in patients with complete or partial IFN-γR2 deficiency due to misfolding or gain-of-glycosylation receptors.
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http://dx.doi.org/10.1182/blood-2013-01-480814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790508PMC
October 2013

Haploinsufficiency at the human IFNGR2 locus contributes to mycobacterial disease.

Hum Mol Genet 2013 Feb 16;22(4):769-81. Epub 2012 Nov 16.

St Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065, USA.

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome, the known genetic etiologies of which impair the production of, or the response to interferon-gamma (IFN-γ). We report here a patient (P1) with MSMD whose cells display mildly impaired responses to IFN-γ, at levels, however, similar to those from MSMD patients with autosomal recessive (AR) partial IFN-γR2 or STAT1 deficiency. Whole-exome sequencing (WES) and Sanger sequencing revealed only one candidate variation for both MSMD-causing and IFN-γ-related genes. P1 carried a heterozygous frame-shift IFNGR2 mutation inherited from her father. We show that the mutant allele is intrinsically loss-of-function and not dominant-negative, suggesting haploinsufficiency at the IFNGR2 locus. We also show that Epstein-Barr virus transformed B lymphocyte cells from 10 heterozygous relatives of patients with AR complete IFN-γR2 deficiency respond poorly to IFN-γ, in some cases as poorly as the cells of P1. Naive CD4(+) T cells and memory IL-4-producing T cells from these individuals also responded poorly to IFN-γ, whereas monocytes and monocyte-derived macrophages (MDMs) did not. This is consistent with the lower levels of expression of IFN-γR2 in lymphoid than in myeloid cells. Overall, MSMD in this patient is probably due to autosomal dominant (AD) IFN-γR2 deficiency, resulting from haploinsufficiency, at least in lymphoid cells. The clinical penetrance of AD IFN-γR2 deficiency is incomplete, possibly due, at least partly, to the variability of cellular responses to IFN-γ in these individuals.
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http://dx.doi.org/10.1093/hmg/dds484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554203PMC
February 2013

Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

Science 2012 Sep 2;337(6102):1684-8. Epub 2012 Aug 2.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA.

ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes-granulocyte, in particular-reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.
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http://dx.doi.org/10.1126/science.1224026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507439PMC
September 2012

IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4.

J Immunol 2008 Aug;181(3):1767-79

Immunology and Inflammation Group, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-beta. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1(+) cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA(+) cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1(+) cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.
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http://dx.doi.org/10.4049/jimmunol.181.3.1767DOI Listing
August 2008

STAT3 is required for IL-21-induced secretion of IgE from human naive B cells.

Blood 2008 Sep 25;112(5):1784-93. Epub 2008 Jun 25.

Immunology and Inflammation Group, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

The production of immunoglobulin E (IgE) is tightly regulated. This is evidenced by the fact that it comprises less than 0.0001% of serum Ig, and aberrant production causes atopic conditions, including allergy, rhinitis, and anaphylaxis. Interleukin-4 (IL-4) is a well-characterized inducer of IgE by human and murine B cells, whereas interferon-gamma can antagonize this effect. IL-21 has also been recognized for its ability to suppress IL-4-induced IgE production by murine B cells. Here, we identified IL-21 as an inducer of IgE production by CD40L-stimulated human naive B cells. Furthermore, there was a striking synergy between IL-4 and IL-21 on inducing IgE secretion by CD40L-stimulated human B cells, such that the levels detected under these conditions exceeded those induced by IL-4 or IL-21 alone by more than 10-fold. IL-21 induced activation of STAT3 and analysis of B cells from patients with loss-of-function STAT3 mutations revealed that the ability of IL-21 to induce IgE secretion, and augment that driven by IL-4, was STAT3-dependent. These findings highlight a fundamental difference between the regulation of IgE production by human and murine B cells and have implications for the dysregulated production of IgE in conditions characterized by extremely high levels of serum IgE.
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http://dx.doi.org/10.1182/blood-2008-02-142745DOI Listing
September 2008

Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells.

J Immunol 2007 Dec;179(12):8180-90

Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets--neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells--into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7- T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.
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http://dx.doi.org/10.4049/jimmunol.179.12.8180DOI Listing
December 2007

Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21.

J Immunol 2006 Oct;177(8):5236-47

Centenary Institute of Cancer Medicine and Cell Biology, New South Wales, Australia.

Although recent studies indicated that IL-21 is an important regulator of human B cell activation, detailed comparison of the effects of IL-21 on distinct B cell subsets have not been performed. Our studies revealed that IL-21R is expressed by naive and germinal center B cells, but not memory or plasma cells. IL-21R was increased on naive and memory B cells following in vitro activation. Investigation into the kinetics and magnitude of responses of human B cells to IL-21 revealed that IL-21 potently augmented proliferation of CD40L-stimulated neonatal, splenic naive, and memory and tonsil germinal center B cells. This response exceeded that induced by IL-4, IL-10, and IL-13, cytokines that also induce B cell proliferation. Remarkably, CD40L/IL-21-stimulated naive B cells underwent the same number of divisions as memory cells and exhibited a greater enhancement in their response compared with CD40L alone than memory B cells. Therefore, IL-21 is a powerful growth factor for naive B cells. This may result from the higher expression of IL-21R on naive, compared with memory, B cells. Stimulation of human B cells with CD40L/IL-21 also induced IL-10 production and activation of STAT3. We propose that IL-21 may have therapeutic application in conditions of immunodeficiency where it could expand naive B cells, the predominant B cell subset in such patients. Conversely, because IL-21 is increased in murine models of lupus, dysregulated IL-21 production may contribute to perturbed B cell homeostasis observed in systemic lupus erythematosus. Thus, antagonizing IL-21 may be a novel strategy for treating Ab-mediated autoimmune diseases.
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http://dx.doi.org/10.4049/jimmunol.177.8.5236DOI Listing
October 2006

BAFF, APRIL and human B cell disorders.

Semin Immunol 2006 Oct 17;18(5):305-17. Epub 2006 Aug 17.

Centenary Institute of Cancer Medicine and Cell Biology, Newtown, NSW, Australia.

B cells require signals from multiple sources for their development from precursor cells, and differentiation into effector cells. BAFF has been identified as a critical regulator of B cell development and differentiation. Defects in the production of BAFF and/or expression of its receptors have been associated with a diverse array of human immunopathologies characterised by perturbed B cell function and behaviour, including autoimmunity, malignancy, and immunodeficiency. This review will discuss the role of BAFF in the pathogenesis of these human immune disorders. It will also highlight relevant differences between the function of BAFF in humans and mice and the impact of this on the therapeutic utility of BAFF antagonists in the treatment of different human diseases.
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http://dx.doi.org/10.1016/j.smim.2006.04.004DOI Listing
October 2006