Publications by authors named "Tri Giang Phan"

64 Publications

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption.

Cell 2021 Mar 25;184(5):1330-1347.e13. Epub 2021 Feb 25.

Healthy Ageing Theme, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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http://dx.doi.org/10.1016/j.cell.2021.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938889PMC
March 2021

The dormant cancer cell life cycle.

Nat Rev Cancer 2020 07 2;20(7):398-411. Epub 2020 Jun 2.

St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia.

The success of targeted therapies and immunotherapies has created optimism that cancers may be curable. However, not all patients respond, drug resistance is common and many patients relapse owing to dormant cancer cells. These rare and elusive cells can disseminate early and hide in specialized niches in distant organs before being reactivated to cause disease relapse after successful treatment of the primary tumour. Despite their importance, we are yet to leverage knowledge generated from experimental models and translate the potential of targeting dormant cancer cells to prevent disease relapse in the clinic. This is due, at least in part, to the lack of adherence to consensus definitions by researchers, limited models that faithfully recapitulate this stage of metastatic spread and an absence of interdisciplinary approaches. However, the application of new high-resolution, single-cell technologies is starting to revolutionize the field and transcend classical reductionist models of studying individual cell types or genes in isolation to provide a global view of the complex underlying cellular ecosystem and transcriptional landscape that controls dormancy. In this Perspective, we synthesize some of these recent advances to describe the hallmarks of cancer cell dormancy and how the dormant cancer cell life cycle offers opportunities to target not only the cancer but also its environment to achieve a durable cure for seemingly incurable cancers.
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http://dx.doi.org/10.1038/s41568-020-0263-0DOI Listing
July 2020

The Clinical Immunogenomics Research Consortium Australasia (CIRCA): a Distributed Network Model for Genomic Healthcare Delivery.

J Clin Immunol 2020 07 1;40(5):763-766. Epub 2020 Jun 1.

Immunity and Inflammation Theme, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, New South Wales, 2010, Australia.

The Clinical Immunogenomics Research Consortium Australasia (CIRCA) crowdsources expertise in medicine, genomics, data science, and fundamental biology to diagnose and treat patients with rare inborn errors of immunity. This distributed network model operates free of geographic borders and allows rapid progression through the full research/translation/clinical management pipeline, from initial gene variant discovery, through functional validation, and on to precision mechanism-based treatment of patients throughout Australia and New Zealand. The model is scalable and applicable to other rare diseases where clinical experience and scientific know-how are limited, and enables efficient delivery of genomics for all.
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http://dx.doi.org/10.1007/s10875-020-00787-6DOI Listing
July 2020

The geography of memory B cell reactivation in vaccine-induced immunity and in autoimmune disease relapses.

Immunol Rev 2020 07 30;296(1):62-86. Epub 2020 May 30.

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

Memory B cells (Bmem) provide an active second layer of defense against re-infection by pathogens that have bypassed the passive first layer provided by neutralizing antibodies. Here, we review recent progress in our understanding of Bmem heterogeneity in terms of their origin (germinal center-dependent vs center-independent), phenotype (canonical vs atypical vs age-associated B cells), trafficking (recirculating vs tissue-resident), and fate (plasma cell vs germinal center differentiation). The development of transgenic models and intravital imaging technologies has made it possible to track the cellular dynamics of Bmem reactivation by antigen, their interactions with follicular memory T cells, and differentiation into plasma cells in subcapsular proliferative foci in the lymph nodes of immune animals. Such in situ studies have reinforced the importance of geography in shaping the outcome of the secondary antibody response. We also review the evidence for Bmem reactivation and differentiation into short-lived plasma cells in the pathogenesis of disease flares in relapsing-remitting autoimmune diseases. Elucidating the mechanisms that control the Bmem fate decision to differentiate into plasma cells or germinal center B cells will aid future efforts to more precisely engineer fit-for-purpose vaccines as well as to treat antibody-mediated autoimmune diseases.
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http://dx.doi.org/10.1111/imr.12862DOI Listing
July 2020

Prostate cancer cell-intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone.

EMBO Rep 2020 06 21;21(6):e50162. Epub 2020 Apr 21.

La Trobe Institute for Molecular Science, Department of Biochemistry and Genetics, La Trobe University, Melbourne, Vic., Australia.

The latency associated with bone metastasis emergence in castrate-resistant prostate cancer is attributed to dormancy, a state in which cancer cells persist prior to overt lesion formation. Using single-cell transcriptomics and ex vivo profiling, we have uncovered the critical role of tumor-intrinsic immune signaling in the retention of cancer cell dormancy. We demonstrate that loss of tumor-intrinsic type I IFN occurs in proliferating prostate cancer cells in bone. This loss suppresses tumor immunogenicity and therapeutic response and promotes bone cell activation to drive cancer progression. Restoration of tumor-intrinsic IFN signaling by HDAC inhibition increased tumor cell visibility, promoted long-term antitumor immunity, and blocked cancer growth in bone. Key findings were validated in patients, including loss of tumor-intrinsic IFN signaling and immunogenicity in bone metastases compared to primary tumors. Data herein provide a rationale as to why current immunotherapeutics fail in bone-metastatic prostate cancer, and provide a new therapeutic strategy to overcome the inefficacy of immune-based therapies in solid cancers.
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http://dx.doi.org/10.15252/embr.202050162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271653PMC
June 2020

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes.

Nat Commun 2019 07 16;10(1):3120. Epub 2019 Jul 16.

Garvan Institute of Medical Research, Sydney, NSW, 2010, Australia.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.
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http://dx.doi.org/10.1038/s41467-019-11049-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635368PMC
July 2019

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells.

Blood 2019 07 25;134(1):30-43. Epub 2019 Apr 25.

Department of Hematology and Immunology, Vrije Universiteit Brussel, Brussels, Belgium.

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
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http://dx.doi.org/10.1182/blood.2018880930DOI Listing
July 2019

Hematopoietic stem cell transplant effectively rescues lymphocyte differentiation and function in DOCK8-deficient patients.

JCI Insight 2019 04 25;5. Epub 2019 Apr 25.

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

Bi-allelic inactivating mutations in DOCK8 cause a combined immunodeficiency characterised by severe pathogen infections, eczema, allergies, malignancy and impaired humoral responses. These clinical features result from functional defects in most lymphocyte lineages. Thus, DOCK8 plays a key role in immune cell function. Hematopoietic stem cell transplantation (HSCT) is curative for DOCK8 deficiency. While previous reports have described clinical outcomes for DOCK8 deficiency following HSCT, the effect on lymphocyte reconstitution and function has not been investigated. Our study determined whether defects in lymphocyte differentiation and function in DOCK8-deficient patients were restored following HSCT. DOCK8-deficient T and B lymphocytes exhibited aberrant activation and effector function in vivo and in vitro. Frequencies of αβ T and MAIT cells were reduced while γδT cells were increased in DOCK8-deficient patients. HSCT improved, abnormal lymphocyte function in DOCK8-deficient patients. Elevated total and allergen-specific IgE in DOCK8-deficient patients decreased over time following HSCT. Our results document the extensive catalogue of cellular defects in DOCK8-deficient patients, and the efficacy of HSCT to correct these defects, concurrent with improvements in clinical phenotypes. Overall, our findings provide mechanisms at a functional cellular level for improvements in clinical features of DOCK8 deficiency post-HSCT, identify biomarkers that correlate with improved clinical outcomes, and inform the general dynamics of immune reconstitution in patients with monogenic immune disorders following HSCT.
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http://dx.doi.org/10.1172/jci.insight.127527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629099PMC
April 2019

B cell-intrinsic requirement for STK4 in humoral immunity in mice and human subjects.

J Allergy Clin Immunol 2019 06 20;143(6):2302-2305. Epub 2019 Feb 20.

Immunology Division, Garvan Institute of Medical Research, Sydney, Australia; St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.02.010DOI Listing
June 2019

Subcapsular Sinus Macrophages: The Seat of Innate and Adaptive Memory in Murine Lymph Nodes.

Trends Immunol 2019 01 27;40(1):35-48. Epub 2018 Nov 27.

Immunology Division, Garvan Institute of Medical Research, Sydney, Australia; St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia. Electronic address:

Subcapsular sinus (SCS) macrophages are strategically positioned at the lymph-tissue interface in the lymph node to trap and present antigen to B cells. Recent murine data has shown that SCS macrophages also prevent the systemic spread of lymph-borne pathogens and are capable of activating a diverse range of innate effector and adaptive memory cells, including follicular memory T cells and memory B cells (B), that are either pre-positioned or rapidly recruited to the subcapsular niche following infection and inflammation. Furthermore, B are rapidly reactivated to differentiate into plasma cells in subcapsular proliferative foci (SPF). Thus, understanding how SCS macrophages coordinate both innate and adaptive memory responses in the subcapsular niche can provide new opportunities to bolster immunity against pathogens and cancer.
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http://dx.doi.org/10.1016/j.it.2018.11.004DOI Listing
January 2019

Memory B cells are reactivated in subcapsular proliferative foci of lymph nodes.

Nat Commun 2018 08 22;9(1):3372. Epub 2018 Aug 22.

Immunology Division, Garvan Institute of Medical Research, Sydney, NSW, 2010, Australia.

Vaccine-induced immunity depends on the generation of memory B cells (MBC). However, where and how MBCs are reactivated to make neutralising antibodies remain unknown. Here we show that MBCs are prepositioned in a subcapsular niche in lymph nodes where, upon reactivation by antigen, they rapidly proliferate and differentiate into antibody-secreting plasma cells in the subcapsular proliferative foci (SPF). This novel structure is enriched for signals provided by T follicular helper cells and antigen-presenting subcapsular sinus macrophages. Compared with contemporaneous secondary germinal centres, SPF have distinct single-cell molecular signature, cell migration pattern and plasma cell output. Moreover, SPF are found both in human and mouse lymph nodes, suggesting that they are conserved throughout mammalian evolution. Our data thus reveal that SPF is a seat of immunological memory that may be exploited to rapidly mobilise secondary antibody responses and improve vaccine efficacy.
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http://dx.doi.org/10.1038/s41467-018-05772-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105623PMC
August 2018

Single Cell RNA Sequencing of Rare Immune Cell Populations.

Front Immunol 2018 4;9:1553. Epub 2018 Jul 4.

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

Single-cell RNA sequencing (scRNA-Seq) is transforming our ability to characterize cells, particularly rare cells that are often overlooked in bulk population analytical approaches. This has lead to the discovery of new cell types and cellular states that echo the underlying heterogeneity and plasticity in the immune system. Technologies for the capture, sequencing, and bioinformatic analysis of single cells are rapidly improving, and scRNA-Seq is now becoming much more accessible to non-specialized laboratories. Here, we describe our experiences in adopting scRNA-Seq to the study of rare immune cells in their microanatomical niches.
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http://dx.doi.org/10.3389/fimmu.2018.01553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039576PMC
July 2018

Germline-activating mutations in compromise B cell development and function.

J Exp Med 2018 08 17;215(8):2073-2095. Epub 2018 Jul 17.

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

Gain-of-function (GOF) mutations in , encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.
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http://dx.doi.org/10.1084/jem.20180010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080914PMC
August 2018

B cells race the clock to get a second wind.

Nat Immunol 2018 08;19(8):791-793

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

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http://dx.doi.org/10.1038/s41590-018-0166-3DOI Listing
August 2018

Removing physiological motion from intravital and clinical functional imaging data.

Elife 2018 07 9;7. Epub 2018 Jul 9.

Kinghorn Cancer Centre, Garvan Institute of Medical Research, University of New South Wales, Sydney, Australia.

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark , a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
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http://dx.doi.org/10.7554/eLife.35800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037484PMC
July 2018

Generation of memory B cells and their reactivation.

Immunol Rev 2018 05;283(1):138-149

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

The successful establishment of humoral memory response depends on at least two layers of defense. Pre-existing protective antibodies secreted by long-lived plasma cells act as a first line of defense against reinfection ("constitutive humoral memory"). Previously, a second line of defense in which pathogen-experienced memory B cells are rapidly reactivated to produce antibodies ("reactive humoral memory"), was considered as simply a back-up system for the first line (particularly for re-infection with homologous viruses). However, in the case of re-infection with similar but different strains of viruses, or in response to viral escape mutants, the reactive humoral memory plays a crucial role. Here, we review recent progress in our understanding of how memory B cells are generated in the pre-GC stage and during the GC reaction, and how these memory B cells are robustly reactivated with the help of memory Tfh cells to generate the secondary antibody response. In addition, we discuss how these advances may be relevant to the quest for a vaccine that can induce broadly reactive antibodies against influenza and HIV.
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http://dx.doi.org/10.1111/imr.12640DOI Listing
May 2018

Self-Reactive B Cells in the Germinal Center Reaction.

Annu Rev Immunol 2018 04 22;36:339-357. Epub 2018 Jan 22.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia; email: ,

Maintenance of immunological self-tolerance requires lymphocytes carrying self-reactive antigen receptors to be selectively prevented from mounting destructive or inflammatory effector responses. Classically, self-tolerance is viewed in terms of the removal, editing, or silencing of B and T cells that have formed self-reactive antigen receptors during their early development. However, B cells activated by foreign antigen can enter germinal centers (GCs), where they further modify their antigen receptor by somatic hypermutation (SHM) of their immunoglobulin genes. The inevitable emergence of activated, self-reactive GC B cells presents a unique challenge to the maintenance of self-tolerance that must be rapidly countered to avoid autoantibody production. Here we discuss current knowledge of the mechanisms that enforce B cell self-tolerance, with particular focus on the control of self-reactive GC B cells. We also consider how self-reactive GC B cells can escape self-tolerance to initiate autoantibody production or instead be redeemed via SHM and used in productive antibody responses.
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http://dx.doi.org/10.1146/annurev-immunol-051116-052510DOI Listing
April 2018

Here, there and everywhere: T follicular helper cells on the move.

Immunology 2017 11 7;152(3):382-387. Epub 2017 Aug 7.

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

T follicular helper (Tfh) cells have the important function of providing B-cell help for the induction of antigen-specific antibody production. As such, it is important to determine the factors that regulate the development, differentiation and function of Tfh cells. This review highlights some of the recent advances in our understanding of Tfh cell migration, Tfh cell memory and the origins and fate of circulating Tfh cells in the blood, that have been revealed from studies in humans and mice.
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http://dx.doi.org/10.1111/imm.12793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629422PMC
November 2017

Impaired Intestinal Permeability Contributes to Ongoing Bowel Symptoms in Patients With Inflammatory Bowel Disease and Mucosal Healing.

Gastroenterology 2017 09 8;153(3):723-731.e1. Epub 2017 Jun 8.

Faculty of Medicine, UNSW Australia, Sydney, Australia; Immunology Division, The Garvan Institute of Medical Research, Sydney, Australia.

Background & Aims: Many patients with inflammatory bowel diseases (IBD) have ongoing bowel symptoms of diarrhea or abdominal pain despite mucosal healing. We investigated whether impaired intestinal permeability contributes to these symptoms.

Methods: We performed a prospective study of intestinal permeability, measured by endoscopic confocal laser endomicroscopy in 110 consecutive subjects (31 with ulcerative colitis [UC], 57 with Crohn's disease [CD], and 22 healthy individuals [controls]) in Sydney, Australia from May 2009 and September 2015. Symptomatic CD was defined by a CD Activity Index score of 150 or more and symptomatic UC by a partial Mayo score of 2 or more. Mucosal healing was defined as CD Endoscopic Index of Severity of 0 in CD or Mayo endoscopic sub-score of 0-1 for patients with UC. Intestinal permeability was quantified by the Confocal Leak Score (CLS; range: 0=no impaired permeability to 100=complete loss of barrier function). The primary endpoint was intestinal permeability in patients with symptomatic IBD in mucosal healing vs patients with asymptomatic IBD in mucosal healing. We determined the sensitivity and specificity of CLS in determining symptoms based on receiver operating characteristic analysis.

Results: Ongoing bowel symptoms were present in 16.3% of patients with IBD and mucosal healing (15.4% of patients with CD, 17.4% with UC). Patients with symptomatic IBD had a significantly higher median CLS (19.0) than patients with asymptomatic IBD (7.3; P < .001) or controls (5.9, P < .001). There were no significant differences between patients with IBD in remission vs controls (P = .261). Median CLS was significantly higher in patients with symptomatic than asymptomatic CD (17.7 vs 8.1; P = .009) and patients with symptomatic than asymptomatic UC (22.2 vs 6.9; P = .021). A CLS of 13.1 or more identified ongoing bowel symptoms in patients with IBD and mucosal healing with 95.2% sensitivity and 97.6% specificity; the receiver operating characteristic area under curve value was 0.88. Based on this cutoff, 36.2% of patients with IBD in mucosal healing have increased intestinal permeability. On regression analysis, every increase in CLS of 1.9 correlated with an additional diarrheal motion per day (P = .008).

Conclusions: In a prospective study of intestinal permeability in patients with IBD and mucosal healing, we associated impaired intestinal permeability with ongoing bowel symptoms; increases in permeability correlated with increased severity of diarrhea. Resolution of mucosal permeability beyond mucosal healing might improve outcomes of patients with IDB (ANZCTR.org.au: ACTRN12613001248752).
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http://dx.doi.org/10.1053/j.gastro.2017.05.056DOI Listing
September 2017

Fate Mapping and Transcript Profiling of Germinal Center Cells by Two-Photon Photoconversion.

Methods Mol Biol 2017 ;1623:59-72

Immunology Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW, 2010, Australia.

The germinal center (GC) reaction is the key process for the generation of high affinity antibodies to foreign antigen. Standard experimental techniques such as fluorescence-activated cell sorting and histology have provided numerous insights into the composition and function of the GC. However, these approaches are limited to a "snapshot" in time and are unable to fully capture the dynamic nature of the GC. Intravital two-photon microscopy overcomes these disadvantages and has led to several major advances in the field but is restricted by practical and technical limits that prevent long-range mapping and molecular studies. Here we describe procedures for optical marking or "tagging" of cells in precise microanatomical compartments by two-photon photoconversion that can be used for long-term fate mapping and transcript profiling of GC T and B cells.
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http://dx.doi.org/10.1007/978-1-4939-7095-7_5DOI Listing
March 2018

Defective protein prenylation is a diagnostic biomarker of mevalonate kinase deficiency.

J Allergy Clin Immunol 2017 09 10;140(3):873-875.e6. Epub 2017 May 10.

Bone Biology Division, Garvan Institute of Medical Research, and St Vincent's Clinical School, UNSW Sydney, Sydney, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.02.033DOI Listing
September 2017

Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells.

Nat Commun 2017 05 12;8:15373. Epub 2017 May 12.

Immunology Division, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.

Interleukin-2 (IL-2) is an established therapeutic agent used for cancer immunotherapy. Since treatment efficacy is mediated by CD8 and NK cell activity at the tumour site, considerable efforts have focused on generating variants that expand these subsets systemically, as exemplified by IL-2/antibody complexes and 'superkines'. Here we describe a novel determinant of antitumour activity using fusion proteins consisting of IL-2 and the antibody fragment crystallizable (Fc) region. Generation of long-lived IL-2-Fc variants in which CD25 binding is abolished through mutation effectively prevents unwanted activation of CD25 regulatory T-cells (Tregs) and results in strong expansion of CD25 cytotoxic subsets. Surprisingly, however, such variants are less effective than wild-type IL-2-Fc in mediating tumour rejection. Instead, we report that efficacy is crucially dependent on depletion of Tregs through Fc-mediated immune effector functions. Our results underpin an unexpected mechanism of action and provide important guidance for the development of next generation IL-2 therapeutics.
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http://dx.doi.org/10.1038/ncomms15373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437307PMC
May 2017

Memory B cells: total recall.

Curr Opin Immunol 2017 Apr 28;45:132-140. Epub 2017 Mar 28.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; St Vincent's Clinical School, University of NSW, Australia. Electronic address:

Immunological memory is a cornerstone of adaptive immune responses in higher vertebrates. The remarkable ability to generate memory cells following Ag exposure, in the context of natural infection or immunization, provides long-lived protection against infectious diseases, often for the hosts' lifetime. Indeed, the generation of memory B cells and long-lived plasma cells underpins the success of most vaccines. The concept of immunological memory is not new-it was first proposed nearly 2500 years ago. While our understanding of the complexities of humoral and cell-mediated memory continues to evolve, important aspects of this process remain unresolved. Here, we will provide an overview of recent advances in B-cell memory in mice and humans, and in health and disease.
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http://dx.doi.org/10.1016/j.coi.2017.03.005DOI Listing
April 2017

Dedicator of cytokinesis 8-deficient CD4 T cells are biased to a T2 effector fate at the expense of T1 and T17 cells.

J Allergy Clin Immunol 2017 Mar 20;139(3):933-949. Epub 2016 Aug 20.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, Australia; St Vincent's Clinical School, University of New South Wales, Darlinghurst, Australia. Electronic address:

Background: Dedicator of cytokinesis 8 (DOCK8) deficiency is a combined immunodeficiency caused by autosomal recessive loss-of-function mutations in DOCK8. This disorder is characterized by recurrent cutaneous infections, increased serum IgE levels, and severe atopic disease, including food-induced anaphylaxis. However, the contribution of defects in CD4 T cells to disease pathogenesis in these patients has not been thoroughly investigated.

Objective: We sought to investigate the phenotype and function of DOCK8-deficient CD4 T cells to determine (1) intrinsic and extrinsic CD4 T-cell defects and (2) how defects account for the clinical features of DOCK8 deficiency.

Methods: We performed in-depth analysis of the CD4 T-cell compartment of DOCK8-deficient patients. We enumerated subsets of CD4 T helper cells and assessed cytokine production and transcription factor expression. Finally, we determined the levels of IgE specific for staple foods and house dust mite allergens in DOCK8-deficient patients and healthy control subjects.

Results: DOCK8-deficient memory CD4 T cells were biased toward a T2 type, and this was at the expense of T1 and T17 cells. In vitro polarization of DOCK8-deficient naive CD4 T cells revealed the T2 bias and T17 defect to be T-cell intrinsic. Examination of allergen-specific IgE revealed plasma IgE from DOCK8-deficient patients is directed against staple food antigens but not house dust mites.

Conclusion: Investigations into the DOCK8-deficient CD4 T cells provided an explanation for some of the clinical features of this disorder: the T2 bias is likely to contribute to atopic disease, whereas defects in T1 and T17 cells compromise antiviral and antifungal immunity, respectively, explaining the infectious susceptibility of DOCK8-deficient patients.
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http://dx.doi.org/10.1016/j.jaci.2016.07.016DOI Listing
March 2017

Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

J Exp Med 2016 07 11;213(8):1589-608. Epub 2016 Jul 11.

Department of Immunology, Westmead Hospital, University of Sydney, Westmead 2145, Australia.

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.
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http://dx.doi.org/10.1084/jem.20151467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986526PMC
July 2016

Disentangling Tfr cells from Treg cells and Tfh cells: How to untie the Gordian knot.

Eur J Immunol 2016 05 24;46(5):1101-4. Epub 2016 Apr 24.

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

T follicular regulatory (Tfr) cells are a subpopulation of Treg cells that have adopted the T follicular helper cell program to localize to the B-cell follicle. Because of the difficulties in generating mouse models in which Tfr cells are selectively affected, determining where and how Tfr cells regulate the germinal center response remains to be resolved. In this issue of the European Journal of Immunology, Dent and colleagues [Eur. J. Immunol. 2016. 46: 1152-1161] describe a simple, elegant mouse model to conditionally delete Tfr cells without impacting on the Treg- and Tfh-cell populations. Their initial studies suggest that Tfr cells have a more complex role than previously thought, particularly with respect to the regulation of immunoglobulin isotype switching to IgA.
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http://dx.doi.org/10.1002/eji.201646389DOI Listing
May 2016

MicroRNA-155 controls affinity-based selection by protecting c-MYC+ B cells from apoptosis.

J Clin Invest 2016 Jan 14;126(1):377-88. Epub 2015 Dec 14.

The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes.
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http://dx.doi.org/10.1172/JCI82914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701536PMC
January 2016