Publications by authors named "Anselm Enders"

44 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

Structural determinants of the IRF4/DNA homodimeric complex.

Nucleic Acids Res 2021 02;49(4):2255-2265

Eccles Institute of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra 2600, Australia.

Interferon regulatory factor 4 (IRF4) is a key transcription factor (TF) in the regulation of immune cells, including B and T cells. It acts by binding DNA as both a homodimer and, in conjunction with other TFs, as a heterodimer. The choice of homo and heterodimeric/ DNA interactions is a critical aspect in the control of the transcriptional program and cell fate outcome. To characterize the nature of this interaction in the homodimeric complex, we have determined the crystal structure of the IRF4/ISRE homodimeric complex. We show that the complex formation is aided by a substantial DNA deformation with co-operative binding achieved exclusively through protein-DNA contact. This markedly contrasts with the heterodimeric form where DNA bound IRF4 is shown to physically interact with PU.1 TF to engage EICE1. We also show that the hotspot residues (Arg98, Cys99 and Asn102) contact both consensus and non-consensus sequences with the L1 loop exhibiting marked flexibility. Additionally, we identified that IRF4L116R, a mutant associated with chronic lymphocytic leukemia, binds more robustly to DNA thereby providing a rationale for the observed gain of function. Together, we demonstrate key structural differences between IRF4 homo and heterodimeric complexes, thereby providing molecular insights into IRF4-mediated transcriptional regulation.
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http://dx.doi.org/10.1093/nar/gkaa1287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913761PMC
February 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 2021 May 31;99(5):532-541. Epub 2021 Jan 31.

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
May 2021

Mutations of the gene FNIP1 associated with a syndromic autosomal recessive immunodeficiency with cardiomyopathy and pre-excitation syndrome.

Eur J Immunol 2020 07 20;50(7):1078-1080. Epub 2020 Apr 20.

Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany.

AMPK (adenosine monophosphate-activated protein kinase) is phosphorylated (AMPK-P) in response to low energy through allosteric activation by Adenosine mono- or diphosphate (AMP/ADP). Folliculin (FLCN) and the FLCN-interacting proteins 1 and 2 (FNIP1, 2) modulate AMPK. FNIP1 deficiency patients have a AMPK-P gain of function phenotype with hypertrophic cardiomyopathy, Wolff-Parkinson-White pre-excitation syndrome, myopathy of skeletal muscles and combined immunodeficiency.
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http://dx.doi.org/10.1002/eji.201948504DOI Listing
July 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

Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus.

Nat Commun 2019 05 17;10(1):2201. Epub 2019 May 17.

Department of Immunology, The Canberra Hospital, Garran, 2601, ACT, Australia.

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.
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http://dx.doi.org/10.1038/s41467-019-10242-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525203PMC
May 2019

Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets.

Blood Adv 2019 02;3(3):219-229

Division of Biomedical Science and Biochemistry, Research School of Biology.

The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.
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http://dx.doi.org/10.1182/bloodadvances.2018023473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373741PMC
February 2019

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

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

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

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

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

ASCT2 (SLC1A5)-Deficient Mice Have Normal B-Cell Development, Proliferation, and Antibody Production.

Front Immunol 2017 12;8:549. Epub 2017 May 12.

Research School of Biology, The Australian National University, Canberra, ACT, Australia.

SLC1A5 (solute carrier family 1, member 5) is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival . One of SLC1A5's primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients.
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http://dx.doi.org/10.3389/fimmu.2017.00549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427077PMC
May 2017

IgD attenuates the IgM-induced anergy response in transitional and mature B cells.

Nat Commun 2016 11 10;7:13381. Epub 2016 Nov 10.

Department of Immunology, John Curtin School of Medical Research, The Australian National University, 131 Garran Rd, Acton, Australian Capital Territory 2601, Australia.

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.
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http://dx.doi.org/10.1038/ncomms13381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5109548PMC
November 2016

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Nat Immunol 2016 Nov 26;17(11):1300-1311. Epub 2016 Sep 26.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Australia.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.
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http://dx.doi.org/10.1038/ni.3565DOI Listing
November 2016

German Society for Immunology and Australasian Society for Immunology joint Workshop 3(rd) -4(th) December 2015 - Meeting report.

Eur J Immunol 2016 Feb;46(2):265-8

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

The German Society for Immunology (DGfI) and the Australasian Society for Immunology (ASI) hosted the first DGfI-ASI joint workshop from December 3-4, 2015 in Canberra, Australia. A delegation of 15 distinguished German immunologists discussed the workshop topic "immune regulation in infections and immune mediated diseases" with the aim to establish new German-Australasian collaborations, discuss new concepts in the field of immune regulation and build a scientific network to create more utilizable resources for excellent (trans-border) immunological research. The workshop was associated with the 45(th) Annual Scientific Meeting of the ASI held from Nov 29-Dec 3, 2015, opening up even more opportunities for finding new collaboration partners. A return meeting will be linked to the annual DGfI meeting that will take place in 2017 in Erlangen.
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http://dx.doi.org/10.1002/eji.201670024DOI Listing
February 2016

ATP11C Facilitates Phospholipid Translocation across the Plasma Membrane of All Leukocytes.

PLoS One 2016 22;11(1):e0146774. Epub 2016 Jan 22.

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

Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D- (7-AAD-) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD- developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146774PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723305PMC
July 2016

Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency.

Blood 2015 Oct 19;126(14):1658-69. Epub 2015 Aug 19.

Center for Chronic Immunodeficiency (CCI), University Medical Center and University of Freiburg, Freiburg, Germany; Center of Pediatric and Adolescent Medicine, University Medical Center and University of Freiburg, Freiburg, Germany;

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.
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http://dx.doi.org/10.1182/blood-2015-03-631374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654427PMC
October 2015

Comparison of predicted and actual consequences of missense mutations.

Proc Natl Acad Sci U S A 2015 Sep 12;112(37):E5189-98. Epub 2015 Aug 12.

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

Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.
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http://dx.doi.org/10.1073/pnas.1511585112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577149PMC
September 2015

Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers.

J Exp Med 2015 Jun 22;212(7):1095-108. Epub 2015 Jun 22.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity and Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia

Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid-related orphan receptor γt (RORγt), whereas RORγt(lo) MAIT cells predominantly express T-bet and produce IFN-γ. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.
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http://dx.doi.org/10.1084/jem.20142110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493408PMC
June 2015

Reducing the search space for causal genetic variants with VASP.

Bioinformatics 2015 Jul 8;31(14):2377-9. Epub 2015 Mar 8.

Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra City, ACT 2601, Australia, Immunogenomics Group, Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia.

Motivation: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria.

Availability And Implementation: Source code available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/VASP.
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http://dx.doi.org/10.1093/bioinformatics/btv135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495293PMC
July 2015

T cell expansion is the limiting factor of virus control in mice with attenuated TCR signaling: implications for human immunodeficiency.

J Immunol 2015 Mar 11;194(6):2725-34. Epub 2015 Feb 11.

Center for Chronic Immunodeficiency, University Medical Center Freiburg and University of Freiburg, 79106 Freiburg, Germany;

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.
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http://dx.doi.org/10.4049/jimmunol.1400328DOI Listing
March 2015

Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span.

J Biol Chem 2014 Jul 4;289(28):19531-7. Epub 2014 Jun 4.

From the Ramaciotti Immunization Genomics Laboratory and

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.
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http://dx.doi.org/10.1074/jbc.C114.570267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094063PMC
July 2014

Zinc-finger protein ZFP318 is essential for expression of IgD, the alternatively spliced Igh product made by mature B lymphocytes.

Proc Natl Acad Sci U S A 2014 Mar 10;111(12):4513-8. Epub 2014 Mar 10.

Department of Immunology and Australian Phenomics Facility, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia.

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.
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http://dx.doi.org/10.1073/pnas.1402739111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970522PMC
March 2014

The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA.

Genome Biol 2014 Jan 29;15(1):R26. Epub 2014 Jan 29.

Background: Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells.

Results: Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins.

Conclusions: Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing.
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http://dx.doi.org/10.1186/gb-2014-15-1-r26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053824PMC
January 2014

Rasgrp1 mutation increases naive T-cell CD44 expression and drives mTOR-dependent accumulation of Helios⁺ T cells and autoantibodies.

Elife 2013 Dec 12;2:e01020. Epub 2013 Dec 12.

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

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.
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http://dx.doi.org/10.7554/eLife.01020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858598PMC
December 2013

DOCK8 is critical for the survival and function of NKT cells.

Blood 2013 Sep 8;122(12):2052-61. Epub 2013 Aug 8.

Medical Research Council Human Immunology Unit, Weatherall Institute for Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom;

Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper-immunoglobulin E levels, eczema, and greater susceptibility to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1(+) NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.
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http://dx.doi.org/10.1182/blood-2013-02-482331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778549PMC
September 2013

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations.

PLoS Genet 2013 31;9(1):e1003219. Epub 2013 Jan 31.

Nuffield Department of Medicine and Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, United Kingdom.

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.
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http://dx.doi.org/10.1371/journal.pgen.1003219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561070PMC
May 2013

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects.

J Allergy Clin Immunol 2013 Feb;131(2):477-85.e1

Pediatric Hematology-Oncology and Bone Marrow Transplantation, Hadassah Hebrew University Medical Center, Jerusalem, Israel.

Background: Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history.

Objective: We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency.

Methods: Molecular, immunologic, and functional assays were performed.

Results: The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells.

Conclusion: Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.
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http://dx.doi.org/10.1016/j.jaci.2012.11.050DOI Listing
February 2013

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

J Exp Med 2013 Jan 24;210(1):31-40. Epub 2012 Dec 24.

Ramaciotti Immunization Genomics Laboratory, John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 2600, Australia.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.
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http://dx.doi.org/10.1084/jem.20121076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549710PMC
January 2013

ZBTB7B (Th-POK) regulates the development of IL-17-producing CD1d-restricted mouse NKT cells.

J Immunol 2012 Dec 26;189(11):5240-9. Epub 2012 Oct 26.

Ramaciotti Immunization Genomics Laboratory, Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 0200, Australia.

CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor γt positive, and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.
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http://dx.doi.org/10.4049/jimmunol.1201486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515659PMC
December 2012