Publications by authors named "Robert Brink"

113 Publications

Loss-of-function of Fbxo10, encoding a post-translational regulator of BCL2 in lymphomas, has no discernible effect on BCL2 or B lymphocyte accumulation in mice.

PLoS One 2021 29;16(4):e0237830. Epub 2021 Apr 29.

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

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237830PLOS
April 2021

Positive selection of IgG over IgM B cells in the germinal center reaction.

Immunity 2021 Apr 7. Epub 2021 Apr 7.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, UNSW Sydney, Sydney, NSW 2010, Australia. Electronic address:

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM B cells; IgG B cells subsequently increased in frequency, dominating GC responses 14-21 days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM and IgG GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM GC B cells. Instead, high-affinity IgG GC B cells outcompeted high-affinity IgM GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.
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http://dx.doi.org/10.1016/j.immuni.2021.03.013DOI Listing
April 2021

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

BAFFR controls early memory B cell responses but is dispensable for germinal center function.

J Exp Med 2021 Feb;218(2)

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

The TNF superfamily ligand BAFF maintains the survival of naive B cells by signaling through its surface receptor, BAFFR. Activated B cells maintain expression of BAFFR after they differentiate into germinal center (GC) or memory B cells (MBCs). However, the functions of BAFFR in these antigen-experienced B cell populations remain unclear. Here, we show that B cell-intrinsic BAFFR does not play a significant role in the survival or function of GC B cells or in the generation of the somatically mutated MBCs derived from them. Instead, BAFF/BAFFR signaling was required to generate the unmutated, GC-independent MBCs that differentiate directly from activated B cell blasts early in the response. Furthermore, amplification of BAFFR signaling in responding B cells did not affect GCs or the generation of GC-derived MBCs but greatly expanded the GC-independent MBC response. Although BAFF/BAFFR signaling specifically controlled the formation of the GC-independent MBC response, both types of MBCs required input from this pathway for optimal long-term survival.
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http://dx.doi.org/10.1084/jem.20191167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604765PMC
February 2021

Conformational diversity facilitates antibody mutation trajectories and discrimination between foreign and self-antigens.

Proc Natl Acad Sci U S A 2020 09 27;117(36):22341-22350. Epub 2020 Aug 27.

Garvan Institute of Medical Research, University of New South Wales (UNSW) Sydney, Darlinghurst, NSW 2010, Australia;

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.
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http://dx.doi.org/10.1073/pnas.2005102117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486785PMC
September 2020

Genetic loss of AMPK-glycogen binding destabilises AMPK and disrupts metabolism.

Mol Metab 2020 11 29;41:101048. Epub 2020 Jun 29.

Exercise and Nutrition Research Program, Mary MacKillop Institute for Health Research, Australian Catholic University, Level 5, 215 Spring Street, Melbourne, Victoria 3000, Australia. Electronic address:

Objective: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions.

Methods: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice.

Results: While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT.

Conclusions: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2020.101048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393401PMC
November 2020

A Future Outlook on Molecular Mechanisms of Immunity.

Trends Immunol 2020 07 2;41(7):549-555. Epub 2020 Jun 2.

The Scripps Research Institute, Department of Immunology and Microbiology, La Jolla, CA, USA. Electronic address:

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http://dx.doi.org/10.1016/j.it.2020.05.005DOI Listing
July 2020

Restriction of memory B cell differentiation at the germinal center B cell positive selection stage.

J Exp Med 2020 07;217(7)

Immunity and Cancer, Francis Crick Institute, London, UK.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC-MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC-MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC-MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.
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http://dx.doi.org/10.1084/jem.20191933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336312PMC
July 2020

Germinal centers and autoantibodies.

Immunol Cell Biol 2020 07 18;98(6):480-489. Epub 2020 Mar 18.

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

Preventing self-reactive lymphocytes from participating in effector responses is fundamental to maintaining immunological self-tolerance and circumventing autoimmunity. A range of complementary mechanisms are known to act upon the primary B- and T-cell repertoires to this effect, eliminating or silencing lymphocytes expressing self-reactive antigen receptors generated through V(D)J recombination in early lymphoid precursors. In the case of B cells, secondary diversification of antigen receptor repertoire by somatic hypermutation (SHM) provides an additional challenge, especially because this occurs in germinal center (GC) B cells that are actively responding to antigen and primed for differentiation into antibody-producing plasma cells. While it is clear that self-tolerance mechanisms do act to prevent antibody production by self-reactive GC B cells, it is also apparent that most pathogenic autoantibodies carry somatic mutations and so have derived from a GC response. Recent advances in the analysis of autoantibody-producing cells associated with human autoimmune diseases together with insights gained from animal models have increased our understanding of the relationships between GCs, SHM and autoantibody production. Here we discuss these developments and focus in particular on how they have illuminated the genesis and pathogenesis of one archetypal autoantibody, rheumatoid factor.
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http://dx.doi.org/10.1111/imcb.12321DOI Listing
July 2020

Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Cell 2020 03 13;180(5):878-894.e19. Epub 2020 Feb 13.

Kirby Institute for Infection and Immunity, UNSW Sydney, Sydney, NSW 2052, Australia; School of Medical Sciences and Cellular Genomics Futures Institute, UNSW Sydney, Sydney, NSW 2052, Australia.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.
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http://dx.doi.org/10.1016/j.cell.2020.01.029DOI Listing
March 2020

Activated PI3Kδ breaches multiple B cell tolerance checkpoints and causes autoantibody production.

J Exp Med 2020 02;217(2)

Immunity and Inflammatory Diseases, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.
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http://dx.doi.org/10.1084/jem.20191336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041712PMC
February 2020

Selection in the germinal center.

Curr Opin Immunol 2020 04 11;63:29-34. Epub 2019 Dec 11.

Immunology Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia; St Vincent's Clinical School, UNSW Australia, 390 Victoria St, Darlinghurst, NSW 2010, Australia. Electronic address:

Germinal centers (GCs) are well known for their important role in shaping the secondary B cell repertoire to generate antibodies capable of binding with high-affinity and specificity to foreign antigens. Somatic hypermutation of the Ig variable region genes in GC B cells represents a highly efficient mechanism for generating new antibody variants with increased antigen affinity. To be effective, however, this process needs to be intimately linked with equally efficient processes that positively select high-affinity clones for perpetuation in the GC and, ultimately, for differentiation into plasma cell and memory B cell effector populations. Just as important is the need for mechanisms of negative selection that remove GC B cell clones with unwanted specificities, particularly those that have gained reactivity with self-components. Here, we discuss recent advances in our understanding of the various selective processes that occur within the GC and identify the major questions in this field that remain to be answered.
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http://dx.doi.org/10.1016/j.coi.2019.11.001DOI Listing
April 2020

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Nat Immunol 2019 10 18;20(10):1299-1310. Epub 2019 Sep 18.

Research School of Chemistry, The Australian National University, Canberra, Australian Capital Territory, Australia.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.
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http://dx.doi.org/10.1038/s41590-019-0492-0DOI Listing
October 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

Activating mutations in PIK3CD disrupt the differentiation and function of human and murine CD4 T cells.

J Allergy Clin Immunol 2019 07 6;144(1):236-253. Epub 2019 Feb 6.

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

Background: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8 T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4 T cells in disease pathogenesis.

Objective: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4 T-cell function.

Methods: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4 T cells and a novel murine disease model caused by overactive PI3K signaling.

Results: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (T) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human T cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a T cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4 T cells also acquired an aberrant T phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and T cells was largely CD4 T-cell extrinsic, whereas changes in cytokine production and T cell function were cell intrinsic.

Conclusion: Our studies reveal that CD4 T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations.
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http://dx.doi.org/10.1016/j.jaci.2019.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612302PMC
July 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

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

SAMHD1 enhances immunoglobulin hypermutation by promoting transversion mutation.

Proc Natl Acad Sci U S A 2018 05 18;115(19):4921-4926. Epub 2018 Apr 18.

Centenary Institute, Camperdown NSW 2050, Australia;

Activation-induced deaminase (AID) initiates hypermutation of genes in activated B cells by converting C:G into U:G base pairs. G-phase variants of uracil base excision repair (BER) and mismatch repair (MMR) then deploy translesion polymerases including REV1 and Pol η, which exacerbates mutation. dNTP paucity may contribute to hypermutation, because dNTP levels are reduced in G phase to inhibit viral replication. To derestrict G-phase dNTP supply, we CRISPR-inactivated SAMHD1 (which degrades dNTPs) in germinal center B cells. inactivation increased B cell virus susceptibility, increased transition mutations at C:G base pairs, and substantially decreased transversion mutations at A:T and C:G base pairs in both strands. We conclude that SAMHD1's restriction of dNTP supply enhances AID's mutagenicity and that the evolution of hypermutation included the repurposing of antiviral mechanisms based on dNTP starvation.
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http://dx.doi.org/10.1073/pnas.1719771115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948975PMC
May 2018

Knockout of glucose transporter GLUT6 has minimal effects on whole body metabolic physiology in mice.

Am J Physiol Endocrinol Metab 2018 08 17;315(2):E286-E293. Epub 2018 Apr 17.

School of Biotechnology and Biomolecular Sciences, University of New South Wales , Sydney, New South Wales , Australia.

Glucose transporter 6 (GLUT6) is a member of the facilitative glucose transporter family. GLUT6 is upregulated in several cancers but is not widely expressed in normal tissues. Previous studies have shown that GLUT6 knockdown kills endometrial cancer cells that express elevated levels of the protein. However, whether GLUT6 represents a viable anticancer drug target is unclear because the role of GLUT6 in normal metabolic physiology is unknown. Herein we generated GLUT6 knockout mice to determine how loss of GLUT6 affected whole body glucose homeostasis and metabolic physiology. We found that the mouse GLUT6 ( Slc2a6) gene expression pattern was similar to humans with mRNA found primarily in brain and spleen. CRISPR-Cas9-mediated deletion of Slc2a6 did not alter mouse development, growth, or whole body glucose metabolism in male or female mice fed either a chow diet or Western diet. GLUT6 deletion did not impact glucose tolerance or blood glucose and insulin levels in male or female mice fed either diet. However, compared with wild-type littermate controls, GLUT6 null female mice had a relatively minor decrease in fat accumulation when fed Western diet and had a lower respiratory exchange ratio when fed chow diet. Collectively, these data show that GLUT6 is not a major regulator of whole body metabolic physiology; therefore, GLUT6 inhibition may have minimal adverse effects if targeted for cancer therapy.
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http://dx.doi.org/10.1152/ajpendo.00082.2018DOI Listing
August 2018

Germinal center antibody mutation trajectories are determined by rapid self/foreign discrimination.

Science 2018 04;360(6385):223-226

Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia.

Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.
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http://dx.doi.org/10.1126/science.aao3859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922412PMC
April 2018

Atypical chemokine receptor 4 shapes activated B cell fate.

J Exp Med 2018 03 31;215(3):801-813. Epub 2018 Jan 31.

Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia

Activated B cells can initially differentiate into three functionally distinct fates-early plasmablasts (PBs), germinal center (GC) B cells, or early memory B cells-by mechanisms that remain poorly understood. Here, we identify atypical chemokine receptor 4 (ACKR4), a decoy receptor that binds and degrades CCR7 ligands CCL19/CCL21, as a regulator of early activated B cell differentiation. By restricting initial access to splenic interfollicular zones (IFZs), ACKR4 limits the early proliferation of activated B cells, reducing the numbers available for subsequent differentiation. Consequently, ACKR4 deficiency enhanced early PB and GC B cell responses in a CCL19/CCL21-dependent and B cell-intrinsic manner. Conversely, aberrant localization of ACKR4-deficient activated B cells to the IFZ was associated with their preferential commitment to the early PB linage. Our results reveal a regulatory mechanism of B cell trafficking via an atypical chemokine receptor that shapes activated B cell fate.
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http://dx.doi.org/10.1084/jem.20171067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839757PMC
March 2018

GPR65 inhibits experimental autoimmune encephalomyelitis through CD4 T cell independent mechanisms that include effects on iNKT cells.

Immunol Cell Biol 2018 02 19;96(2):128-136. Epub 2017 Dec 19.

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

The G protein-coupled receptor 65 (GPR65) gene has been genetically associated with several autoimmune diseases, including multiple sclerosis (MS). GPR65 is predominantly expressed in lymphoid organs and is activated by extracellular protons. In this study, we tested whether GPR65 plays a functional role in demyelinating autoimmune disease. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we found that Gpr65-deficient mice develop exacerbated disease. CD4 helper T cells are key drivers of EAE pathogenesis, however, Gpr65 deficiency in these cells did not contribute to the observed exacerbated disease. Instead, Gpr65 expression levels were found to be highest on invariant natural killer T (iNKT) cells. EAE severity in Gpr65-deficient mice was normalized in the absence of iNKT cells (CD1d-deficient mice), suggesting that GPR65 signals in iNKT cells are important for suppressing autoimmune disease. These findings provide functional support for the genetic association of GPR65 with MS and demonstrate GPR65 signals suppress autoimmune activity in EAE.
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http://dx.doi.org/10.1111/imcb.1031DOI Listing
February 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

CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

Immunity 2017 12;47(6):1142-1153.e4

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, UNSW Sydney, Darlinghurst, NSW 2010, Australia. Electronic address:

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses.
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http://dx.doi.org/10.1016/j.immuni.2017.11.022DOI Listing
December 2017

Structural basis of antigen recognition: crystal structure of duck egg lysozyme.

Acta Crystallogr D Struct Biol 2017 Nov 25;73(Pt 11):910-920. Epub 2017 Oct 25.

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

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported. Using structural analyses in combination with mass spectrometry, non-ambiguous DEL primary sequences are reported. Furthermore, the structures and sequences determined here enable rationalization of the binding affinity of DEL for well documented landmark anti-lysozyme antibodies.
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http://dx.doi.org/10.1107/S2059798317013730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683014PMC
November 2017

IL-2 Shapes the Survival and Plasticity of IL-17-Producing γδ T Cells.

J Immunol 2017 10 23;199(7):2366-2376. Epub 2017 Aug 23.

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

IL-17-producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine. We found γδT-17 cells to be enriched, not depleted, in IL-2-deficient mice. The absence of IL-2 also resulted in higher IL-17 production and the emergence of IL-17IFN-γ double producers. Furthermore, the addition of IL-2 to in vitro cultures of sorted γδT-17 cells was able to moderate IL-17 and affect differentiation into polyfunctional cytokine-producing cells. Interestingly, the Vγ6 subset was more susceptible to the effects of IL-2 than Vγ4 γδT-17 cells. We also found that unlike other γδ T cells, γδT-17 cells do not produce IL-2, but express Blimp-1, a known transcriptional repressor of IL-2. Although IL-2 was able to induce robust proliferation of γδT-17 cells, it did not sustain viability, negatively impacting their survival via downregulation of the IL-7R. Taken together, these data indicate that IL-2 can augment the γδT-17 response in favor of short-lived effectors with limited plasticity, particularly in the presence of IL-1β and IL-23. In this way, IL-2 may act to curtail the innate-like response of γδT-17 cells upon arrival of IL-2-producing adaptive immune cells at the site of inflammation.
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http://dx.doi.org/10.4049/jimmunol.1700335DOI Listing
October 2017

Murine LRBA deficiency causes CTLA-4 deficiency in Tregs without progression to immune dysregulation.

Immunol Cell Biol 2017 10 14;95(9):775-788. Epub 2017 Jun 14.

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

Inherited mutations in lipopolysaccharide-responsive beige-like anchor (LRBA) cause a recessive human immune dysregulation syndrome with memory B-cell and antibody deficiency (common variable immunodeficiency), inflammatory bowel disease, enlarged spleen and lymph nodes, accumulation of activated T cells and multiple autoimmune diseases. To understand the pathogenesis of the syndrome, C57BL/6 mice carrying a homozygous truncating mutation in Lrba were produced using CRISPR/Cas9-mediated gene targeting. These mice revealed that LRBA has a critical, cell-autonomous role in promoting cytotoxic T-lymphocyte antigen-4 (CTLA-4) accumulation within CD4 effector T cells and FOXP3 T-regulatory cells (Tregs). In young mice, or in chimeric mice where only half of the T cells are LRBA deficient, low CTLA-4 was the only detectable abnormality in Tregs, whereas in old mice FOXP3 was also decreased. Low CTLA-4 did not translate into increased CD86 on B cells unless the LRBA-deficient mice were immunised, and neither immunisation nor chronic lymphocytic choriomeningitis virus infection precipitated immune dysregulation. LRBA deficiency did not alter antigen-specific B-cell activation, germinal centre (GC) formation, isotype switching or affinity maturation. Paradoxically, CD86 was decreased on GC B cells in LRBA-deficient mice, pointing to compensatory mechanisms for controlling CD86 in the face of low CTLA-4. These results add to the experimental rationale for treating LRBA deficiency with the CTLA4-Ig fusion protein, Abatacept, and pose questions about the limitations of laboratory experiments in mice to reproduce human disease in natura.
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http://dx.doi.org/10.1038/icb.2017.50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636941PMC
October 2017

In vivo photolabeling of tumor-infiltrating cells reveals highly regulated egress of T-cell subsets from tumors.

Proc Natl Acad Sci U S A 2017 05 15;114(22):5677-5682. Epub 2017 May 15.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia;

Immune therapy is rapidly gaining prominence in the clinic as a major weapon against cancer. Whereas much attention has been focused on the infiltration of tumors by immune cells, the subsequent fate of these infiltrates remains largely unexplored. We therefore established a photoconversion-based model that allowed us to label tumor-infiltrating immune cells and follow their migration. Using this system, we identified a population of tumor-experienced cells that emigrate from primary tumors to draining lymph nodes via afferent lymphatic vessels. Although the majority of tumor-infiltrating cells were myeloid, T cells made up the largest population of tumor-egressing leukocytes. Strikingly, the subset composition of tumor-egressing T cells was greatly skewed compared with those that had infiltrated the tumor and those resident in the draining lymph node. Some T-cell subsets such as CD8 T cells emigrated more readily; others including CD4CD8 T cells were preferentially retained, suggesting that specific mechanisms guide immune cell egress from tumors. Furthermore, tumor-egressing T cells were more activated and displayed enhanced effector function in comparison with their lymph node counterparts. Finally, we demonstrated that tumor-infiltrating T cells migrate to distant secondary tumors and draining lymph nodes, highlighting a mechanism whereby tumor-experienced effector T cells may mediate antitumor immunity at metastatic sites. Thus, our results provide insights into migration and function of tumor-infiltrating immune cells and the role of these cells in tumor immunity outside of primary tumor deposits.
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http://dx.doi.org/10.1073/pnas.1618446114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465915PMC
May 2017

Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells.

J Exp Med 2017 05 31;214(5):1259-1267. Epub 2017 Mar 31.

Immunology Division, Garvan Institute of Medical Research, Darlinghurst NSW 2010, Australia

Plasma cells (PCs) derived from germinal centers (GCs) secrete the high-affinity antibodies required for long-term serological immunity. Nevertheless, the process whereby GC B cells differentiate into PCs is uncharacterized, and the mechanism underlying the selective PC differentiation of only high-affinity GC B cells remains unknown. In this study, we show that differentiation into PCs is induced among a discrete subset of high-affinity B cells residing within the light zone of the GC. Initiation of differentiation required signals delivered upon engagement with intact antigen. Signals delivered by T follicular helper cells were not required to initiate differentiation but were essential to complete the differentiation process and drive migration of maturing PCs through the dark zone and out of the GC. This bipartite or two-signal mechanism has likely evolved to both sustain protective immunity and avoid autoantibody production.
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http://dx.doi.org/10.1084/jem.20161533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413338PMC
May 2017