Publications by authors named "Christopher C Goodnow"

150 Publications

A Point Mutation in IKAROS ZF1 Causes a B Cell Deficiency in Mice.

J Immunol 2021 Apr 3;206(7):1505-1514. Epub 2021 Mar 3.

Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2601, Australia;

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through -ethyl--nitrosourea mutagenesis, we have discovered a novel mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine mutations, this L132P point mutation ( ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1 overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1 Furthermore, mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in mice.
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http://dx.doi.org/10.4049/jimmunol.1901464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987828PMC
April 2021

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 Mar 20;591(7848):131-136. Epub 2021 Jan 20.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
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http://dx.doi.org/10.1038/s41586-021-03218-7DOI Listing
March 2021

DOCK8 deficiency diminishes thymic T-regulatory cell development but not thymic deletion.

Clin Transl Immunology 2021 7;10(1):e1236. Epub 2021 Jan 7.

Infection and Immunity Program Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology Monash University Melbourne VIC Australia.

Objective: To define the effect of DOCK8 deficiency on thymic tolerance in mice.

Methods: Thymocytes from wild-type ( ) and DOCK8-deficient ( ) mice were examined by flow cytometry. Some mice had transgenic expression of the BCL2 anti-apoptotic protein in haemopoietic cells. Some mice expressed the transgenic 3A9 T-cell receptor (TCR), which triggers thymocyte deletion in mice also expressing hen egg lysozyme under the insulin promoter.

Results: In mice, the proportion of thymocytes induced to acquire tolerance at the immature CCR7 stage was normal. Deletion of strongly self-reactive CD4 thymocytes occurred efficiently in mice in a TCR-transgenic model that requires self-antigen transfer from epithelial cells to bone marrow (BM)-derived antigen-presenting cells. Thymic Foxp3 T-regulatory cells (T) and Helios Foxp3 T precursors were decreased in mice, including when apoptosis was inhibited by BCL2 transgene expression. thymic T expressed CD25 and CTLA-4 at normal levels. The results suggest that DOCK8 deficiency does not affect the function of BM-derived antigen-presenting cells in the thymus, the TCR self-reactivity threshold that activates tolerance mechanisms in thymocytes or the apoptotic deletion of these thymocytes. However, DOCK8 is required to prevent a subset of developing T cells from undergoing cell death via a mechanism that is distinct from apoptosis.

Conclusion: DOCK8 deficiency diminishes T development in the thymus without compromising thymocyte deletion.
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http://dx.doi.org/10.1002/cti2.1236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790591PMC
January 2021

Loss of hnRNPLL-dependent splicing of Ptprc has no impact on B-cell development, activation and terminal differentiation into antibody-secreting cells.

Immunol Cell Biol 2020 Dec 17. Epub 2020 Dec 17.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.

The RNA-binding protein heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) controls alternative splicing of protein tyrosine phosphatase receptor type C (Ptprc) which encodes CD45. hnRNPLL deficiency leads to a failure in silencing Ptprc exons 4-6 causing aberrant expression of the corresponding CD45 isoforms, namely, CD45RA, RB and RC. While an N-ethyl-N-nitrosourea-induced point mutation in murine Hnrnpll results in loss of peripheral naïve T cells, its role in B-cell biology remains unclear. Here, we demonstrate that B-cell development in the bone marrow of Hnrnpll mice is normal and the number of mature B-cell subsets in the spleen and peritoneal cavity is comparable to control littermates. In response to in vivo immunization, Hnrnpll mice were deficient in generating germinal center (GC) B cells, and analysis of mixed bone marrow chimeras revealed that the GC B-cell deficiency was a B-cell extrinsic effect of the hnRNPLL mutation. Mature Hnrnpll B cells proliferated normally in response to various B-cell receptor- and Toll-like receptor-mediated stimuli. Similarly, in vitro stimulation of mutant B cells led to normal generation of plasmablasts, but mutant plasmablasts failed to downregulate B220 expression because of the inability of cells to undergo proper CD45 pre-messenger RNA alternative splicing. These findings collectively suggest that, like in T and natural killer T cells, the mutation disrupts hnRNPLL-mediated alternative splicing of the Ptprc gene in plasmablasts, but this dysregulation of Ptprc alternative splicing does not affect the development and function of B cells.
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http://dx.doi.org/10.1111/imcb.12433DOI Listing
December 2020

COVID-19, varying genetic resistance to viral disease and immune tolerance checkpoints.

Immunol Cell Biol 2021 02 23;99(2):177-191. Epub 2020 Nov 23.

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

Coronavirus disease 2019 (COVID-19) is a zoonosis like most of the great plagues sculpting human history, from smallpox to pandemic influenza and human immunodeficiency virus. When viruses jump into a new species the outcome of infection ranges from asymptomatic to lethal, historically ascribed to "genetic resistance to viral disease." People have exploited these differences for good and bad, for developing vaccines from cowpox and horsepox virus, controlling rabbit plagues with myxoma virus and introducing smallpox during colonization of America and Australia. Differences in resistance to viral disease are at the core of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) crisis, yet our understanding of the mechanisms in any interspecies leap falls short of the mark. Here I review how the two key parameters of viral disease are countered by fundamentally different genetic mechanisms for resistance: (1) virus transmission, countered primarily by activation of innate and adaptive immune responses; and (2) pathology, countered primarily by tolerance checkpoints to limit innate and adaptive immune responses. I discuss tolerance thresholds and the role of CD8 T cells to limit pathological immune responses, the problems posed by tolerant superspreaders and the signature coronavirus evasion strategy of eliciting only short-lived neutralizing antibody responses. Pinpointing and targeting the mechanisms responsible for varying pathology and short-lived antibody were beyond reach in previous zoonoses, but this time we are armed with genomic technologies and more knowledge of immune checkpoint genes. These known unknowns must now be tackled to solve the current COVID-19 crisis and the inevitable zoonoses to follow.
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http://dx.doi.org/10.1111/imcb.12419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894315PMC
February 2021

Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility.

J Exp Med 2021 Feb;218(2)

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.
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http://dx.doi.org/10.1084/jem.20200476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595743PMC
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

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.

The Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2052, Australia. Electronic address:

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

Clonal redemption and clonal anergy as mechanisms to balance B cell tolerance and immunity.

Immunol Rev 2019 11 26;292(1):61-75. Epub 2019 Sep 26.

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

The adaptive immune system is tasked with producing antibodies that recognize a wide scope of potential pathogens, including those never before encountered, and concurrently avoiding formation of antibodies binding host tissues. The diverse repertoire of antibodies produced by V(D)J recombination inevitably includes autoantibodies that bind to self-antigens, estimated to be as much as 70% of nascent antibodies on immature B cells. Early theoretical models of tolerance hypothesized that such self-reactive clones could not possibly be allowed to survive and mature. However from the first direct view of the fate of nascent B cells carrying a self-binding antibody it was clear that many "forbidden clones" circulate to secondary lymphoid tissues, where they adopt an IgM IgD+ cell surface phenotype and are prevented from secreting autoantibodies by a series of tolerance checkpoints referred to as "clonal anergy." Since anergic B cells can be reactivated to secrete pathogenic autoantibodies in certain settings, the advantage of controlling self-reactive antibodies by clonal anergy has until recently remained enigmatic. Here we review this topic and recent advances showing that anergic B cells are recruited into the germinal center to mutate away from self-reactivity, undergoing "clonal redemption" into cells making antibodies with exquisite specificity for foreign immunogens.
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http://dx.doi.org/10.1111/imr.12808DOI Listing
November 2019

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.

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

Preponderance of Variation Associated With Autosomal Dominant Immune Dysregulation in the MYPPPY Motif.

Front Immunol 2019 23;10:1544. Epub 2019 Jul 23.

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

One of the primary targets of immune checkpoint inhibition is the negative immune regulatory molecule CTLA-4. Immune-related adverse events are commonly observed following CTLA-4 inhibition in melanoma treatment, and a spectrum of these conditions are also observed in individuals with germline haploinsufficiency of . Here we describe a heterozygous missense variant of in a young girl with childhood-onset autoimmune hepatitis and polyarthritis, the latter responding to treatment with CTLA-4-Ig fusion protein. This variant lay within the highly conserved MYPPPY motif of CTLA-4: a critical structural determinant of ligand binding, which is also bound by the anti-CTLA-4 monoclonal antibody ipilimumab. Within the spectrum of variants reported, missense variants in the MYPPPY motif were overrepresented when compared to variants within a control population, highlighting the physiological importance of this motif in both the genetic and pharmacological regulation of autoimmunity and anti-tumor immunity.
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http://dx.doi.org/10.3389/fimmu.2019.01544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664875PMC
November 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

IRF2 transcriptionally induces expression for pyroptosis.

Sci Signal 2019 05 21;12(582). Epub 2019 May 21.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of A forward genetic screen with -ethyl--nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. expression was substantially attenuated in deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the promoter to directly drive transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.
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http://dx.doi.org/10.1126/scisignal.aax4917DOI Listing
May 2019

CARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3 regulatory T cells.

Immunol Cell Biol 2019 09 26;97(8):740-752. Epub 2019 Jun 26.

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

FOXP3 regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1 (NRP1 ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1 Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4 T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.
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http://dx.doi.org/10.1111/imcb.12268DOI Listing
September 2019

Deletion of self-reactive CCR7- thymocytes in the absence of MHC expression on thymic epithelial cells.

Cell Death Differ 2019 12 24;26(12):2727-2739. Epub 2019 Apr 24.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia.

The selection of αβ T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αβ T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRβ + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRβ chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.
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http://dx.doi.org/10.1038/s41418-019-0331-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224288PMC
December 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

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns.

Cancer Cell 2019 02;35(2):297-314.e8

Epigenetics Research Laboratory, Genomics and Epigenetics Division, Garvan Institute of Medical Research, 384 Victoria St, Sydney, NSW 2010, Australia; St Vincent's Clinical School, UNSW, Sydney, NSW 2010, Australia. Electronic address:

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.
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http://dx.doi.org/10.1016/j.ccell.2019.01.004DOI Listing
February 2019

Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood.

Methods Mol Biol 2018 ;1827:287-309

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

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).
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http://dx.doi.org/10.1007/978-1-4939-8648-4_15DOI Listing
April 2019

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8 T Cell Tolerance Checkpoint to High-Dose Antigen.

Cell Rep 2018 07;24(3):577-584

John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia; Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. Electronic address:

Escape from peripheral tolerance checkpoints that control cytotoxic CD8 T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8 T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8 T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVA mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8 T cell tolerance checkpoint, with a different mechanism to CD4 T cells, and indicates that CD8 T cell deletion and anergy are molecularly separable checkpoints.
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http://dx.doi.org/10.1016/j.celrep.2018.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112980PMC
July 2018

Understanding Immune Tolerance of Cancer: Re-Purposing Insights from Fetal Allografts and Microbes.

Bioessays 2018 08 4;40(8):e1800050. Epub 2018 Jun 4.

The Garvan Institute of Medical Research, Darlinghurst, 2010, NSW, Australia.

Cancer cells seem to exploit mechanisms that evolve as part of physiological tolerance, which is a complementary and often beneficial form of defense. The study of physiological systems of tolerance can therefore provide insights into the development of a state of host tolerance of cancer, and how to break it. Analysis of these models has the potential to improve our understanding of existing immunological therapeutic targets, and help to identify future targets and rational therapeutic combinations. The treatment of cancer with immune checkpoint inhibitors aims to reverse the progression to tolerance of cancer, and achieve an immunogenic, rather than tolerogenic, homeostasis. Broadening the efficacy and durability of checkpoint inhibitors focuses on reversing tolerance and stimulating immunogenicity in the cancer, host, and environment. Two examples of important physiological states of tolerance that may inform tolerance of cancer are microbial infection and placental reproduction. These states of tolerance result from bilateral shaping of host and non-self, akin to immunoediting in cancer, and offer reliable models to study the immune tolerance paradigm.
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http://dx.doi.org/10.1002/bies.201800050DOI Listing
August 2018

αβ T-cell receptors with a central CDR3 cysteine are enriched in CD8αα intraepithelial lymphocytes and their thymic precursors.

Immunol Cell Biol 2018 07 3;96(6):553-561. Epub 2018 May 3.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia.

The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαβ CD8αα intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRβ chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3 regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.
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http://dx.doi.org/10.1111/imcb.12047DOI Listing
July 2018

Molecular Profiling and Clonal Tracking of Secreted Rheumatoid Factors in Primary Sjögren's Syndrome.

Arthritis Rheumatol 2018 10 3;70(10):1617-1625. Epub 2018 Aug 3.

Flinders University and SA Pathology, Bedford Park, South Australia, Australia.

Objective: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking.

Methods: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring.

Results: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig V ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig V 4-34 IgM-RF decreased following immunosuppression and remission of MC.

Conclusion: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.
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http://dx.doi.org/10.1002/art.40539DOI Listing
October 2018

B-cell receptor reconstruction from single-cell RNA-seq with VDJPuzzle.

Bioinformatics 2018 08;34(16):2846-2847

Kirby Institute for Infection and Immunity, Sydney, Australia.

Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature.

Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing.

Availability And Implementation: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty203DOI 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

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Immunology 2018 07 27;154(3):522-532. Epub 2018 Feb 27.

Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.
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http://dx.doi.org/10.1111/imm.12904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002238PMC
July 2018

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

PLoS Genet 2017 Nov 8;13(11):e1007072. Epub 2017 Nov 8.

Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Darlinghurst, NSW, Australia.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.
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http://dx.doi.org/10.1371/journal.pgen.1007072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695588PMC
November 2017

Synergistic cooperation and crosstalk between and mutations that dysregulate CD79B and surface IgM.

J Exp Med 2017 Sep 12;214(9):2759-2776. Epub 2017 Jul 12.

Australian Cancer Research Foundation Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, Australia

and mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of and mutations individually and combined in normal activated mouse B lymphocytes. mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing decreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of In B cells chronically stimulated by self-antigen, and mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by and provide an explanation for the co-occurrence of and mutations in lymphomas.
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http://dx.doi.org/10.1084/jem.20161454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584117PMC
September 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

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion.

Proc Natl Acad Sci U S A 2017 06 12;114(26):E5216-E5225. Epub 2017 Jun 12.

John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia;

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8 and CD4 T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.
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http://dx.doi.org/10.1073/pnas.1705795114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495275PMC
June 2017