Publications by authors named "Edward M Bertram"

26 Publications

  • Page 1 of 1

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

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

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Nature 2015 Oct 16;526(7575):666-71. Epub 2015 Sep 16.

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

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
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http://dx.doi.org/10.1038/nature15541DOI Listing
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

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

LIGHT (TNFSF14/CD258) is a decisive factor for recovery from experimental autoimmune encephalomyelitis.

J Immunol 2013 Jul 29;191(1):154-63. Epub 2013 May 29.

Department of Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia.

The TNF superfamily ligand LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator [HVEM], a receptor expressed by T lymphocytes) has been shown to play a role in T cell costimulation and be involved in apoptosis of mononuclear cells. As both T cells and monocytes are key components in the development and progression of experimental autoimmune encephalomyelitis (EAE), we studied the role of LIGHT in EAE. Following immunization with myelin oligodendrocyte glycoprotein peptide (35-55), LIGHT-deficient mice developed severe EAE that resulted in an atypically high mortality rate. Histological examinations revealed intensive activation of microglia/macrophages in the CNS and higher numbers of apoptotic cells within the CNS parenchyma of LIGHT-deficient mice. However, myelin oligodendrocyte glycoprotein peptide-specific CD4(+) T cells from LIGHT-deficient mice showed reduced IFN-γ and IL-17 production and migration. Serum levels of reactive nitrogen intermediates and CNS transcripts of several proinflammatory cytokines and chemokines were also substantially decreased in the absence of LIGHT. EAE adoptive transfer experiments and bone marrow chimeras indicated that expression of LIGHT on donor cells is not required for disease induction. However, its expression on CNS host cells is a decisive factor to limit disease progression and tissue damage. Together, these data show that LIGHT expression is crucially involved in controlling activated macrophages/microglia during autoimmune CNS inflammation.
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http://dx.doi.org/10.4049/jimmunol.1203016DOI Listing
July 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

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

Breakdown in repression of IFN-γ mRNA leads to accumulation of self-reactive effector CD8+ T cells.

J Immunol 2012 Jul 8;189(2):701-10. Epub 2012 Jun 8.

Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, Australia.

Tight regulation of virus-induced cytotoxic effector CD8(+) T cells is essential to prevent immunopathology. Naturally occurring effector CD8(+) T cells, with a KLRG1(hi) CD62L(lo) phenotype typical of short-lived effector CD8(+) T cells (SLECs), can be found in increased numbers in autoimmune-prone mice, most notably in mice homozygous for the san allele of Roquin. These SLEC-like cells were able to trigger autoimmune diabetes in a susceptible background. When Roquin is mutated (Roquin(san)), effector CD8(+) T cells accumulate in a cell-autonomous manner, most prominently as SLEC-like effectors. Excessive IFN-γ promotes the accumulation of SLEC-like cells, increases their T-bet expression, and enhances their granzyme B production in vivo. We show that overexpression of IFN-γ was caused by failed posttranscriptional repression of Ifng mRNA. This study identifies a novel mechanism that prevents accumulation of self-reactive cytotoxic effectors, highlighting the importance of regulating Ifng mRNA stability to maintain CD8(+) T cell homeostasis and prevent CD8-mediated autoimmunity.
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http://dx.doi.org/10.4049/jimmunol.1102432DOI Listing
July 2012

DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice.

J Exp Med 2011 Oct 17;208(11):2305-20. Epub 2011 Oct 17.

Department of Immunology, The John Curtin School of Medical Research , Australian National University, Canberra, Australian Capital Territory 0200, Australia.

In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA(+)CCR7(-) phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell division. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells.
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http://dx.doi.org/10.1084/jem.20110345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201196PMC
October 2011

Decreased T-cell receptor signaling through CARD11 differentially compromises forkhead box protein 3-positive regulatory versus T(H)2 effector cells to cause allergy.

J Allergy Clin Immunol 2011 May 12;127(5):1277-85.e5. Epub 2011 Feb 12.

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

Background: Allergy, the most common disease of immune dysregulation, has a substantial genetic component that is poorly understood. Although complete disruption of T-cell receptor (TCR) signaling causes profound immunodeficiency, little is known about the consequences of inherited genetic variants that cause partial quantitative decreases in particular TCR-signaling pathways, despite their potential to dysregulate immune responses and cause immunopathology.

Objective: We sought to elucidate how an inherited decrease in TCR signaling through CARD11, a critical scaffold protein that signals to nuclear factor κB (NF-κB) transcription factors, results in spontaneous selective accumulation of large numbers of T(H)2 cells.

Methods: "Unmodulated" mice carry a Card11 single nucleotide variant that decreases but does not abolish TCR/CD28 signaling to induce targets of NF-κB. The consequences of this mutation on T-cell subset formation in vivo were examined, and its effects within effector versus regulatory T-cell subsets were dissected by the adoptive transfer of wild-type cells and by the examination of forkhead box protein 3 (Foxp3)-deficient unmodulated mice.

Results: Unlike the pathology-free boundary points of complete Card11 sufficiency or deficiency, unmodulated mice have a specific allergic condition characterized by increased IgE levels and dermatitis. The single nucleotide variant partially decreases both the frequency of Foxp3(+) regulatory T cells and the efficiency of effector T-cell formation in vivo. These intermediate effects combine to cause a gradual and selective expansion of T(H)2 cells.

Conclusions: Inherited reduction in the efficiency of TCR-NF-κB signaling has graded effects on T-cell activation and Foxp3(+) regulatory T-cell suppression that result in selective T(H)2 dysregulation and allergic disease.
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http://dx.doi.org/10.1016/j.jaci.2010.12.1081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189857PMC
May 2011

T cell costimulatory molecules in anti-viral immunity: Potential role in immunotherapeutic vaccines.

Can J Infect Dis 2003 Jul;14(4):221-9

T lymphocyte activation is required to eliminate or control intracellular viruses. The activation of T cells requires both an antigen specific signal, involving the recognition of a peptide/major histocompatibility protein complex by the T cell receptor, as well as additional costimulatory signals. In chronic viral diseases, T cell responses, although present, are unable to eliminate the infection. By providing antigens and costimulatory molecules together, investigators may be able to increase and broaden the immune response, resulting in better immunological control or even elimination of the infection. Recent progress in understanding the function of costimulatory molecules suggests that different costimulatory molecules are involved in initial immune responses than are involved in recall responses. These new developments have important implications for therapeutic vaccine design. In this review the authors discuss the function of T cell costimulatory molecules in immune system activation and their potential for enhancing the efficacy of therapeutic vaccines.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094939PMC
http://dx.doi.org/10.1155/2003/214034DOI Listing
July 2003

Exploiting 4-1BB costimulation for enhancing antiviral vaccination.

Viral Immunol 2006 ;19(4):593-601

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

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is emerging as an important costimulatory molecule, particularly in the regulation of CD8(+) T cell responses. Costimulation through 4-1BB, such as by utilizing agonistic anti-4-1BB monoclonal antibodies, has been well studied in various tumor models. However, 4-1BB is also an important regulator of antiviral CD8(+) T cell responses. This review summarizes these findings and describes how 4-1BB is beginning to be exploited in terms of boosting antiviral vaccine responses.
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http://dx.doi.org/10.1089/vim.2006.19.593DOI Listing
February 2007

4-1BBL coexpression enhances HIV-specific CD8 T cell memory in a poxvirus prime-boost vaccine.

Vaccine 2006 Nov 22;24(47-48):6867-74. Epub 2006 Jun 22.

Department of Immunology and Genetics, The John Curtin School of Medical Research, Canberra City, Australia.

We have constructed a recombinant fowlpox virus expressing HIV antigens and the costimulatory molecule 4-1BBL. When included in the boost, but not the prime of a poxvirus prime-boost strategy, 4-1BBL significantly enhanced the anti-HIV T cell response generated to this vaccination in BALB/c mice, as detected by ex vivo IFNgamma ELISPOT responses, intracellular cytokine staining to HIV Gag antigens, and enumeration of Gag-reactive CD8 T cells. 4-1BBL however, is not capable of modulating the CD4 T cell response, nor the antibody response to this vaccination strategy. Enhancement of the T cell response by 4-1BBL continues into the memory phase, as detected 2 months post vaccination. This data is the first to show modulation of the immune response to a viral vaccine by coexpression of 4-1BBL and supports this strategy as an exciting approach for enhancement of T cell memory in prime-boost vaccines.
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http://dx.doi.org/10.1016/j.vaccine.2006.06.007DOI Listing
November 2006

4-1BB and OX40 act independently to facilitate robust CD8 and CD4 recall responses.

J Immunol 2004 Nov;173(10):5944-51

Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada.

Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL(-/-) mice show less impairment in CD4 secondary responses than OX40L(-/-) mice. The 4-1BBL(-/-) and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L(-/-) and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.
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http://dx.doi.org/10.4049/jimmunol.173.10.5944DOI Listing
November 2004

Role of T cell costimulation in anti-viral immunity.

Semin Immunol 2004 Jun;16(3):185-96

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

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.
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http://dx.doi.org/10.1016/j.smim.2004.02.006DOI Listing
June 2004

Studying host immune responses against duck hepatitis B virus infection.

Methods Mol Med 2004 ;96:3-25

Department of Molecular Biosciences, Adelaide University, SA, Australia.

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http://dx.doi.org/10.1385/1-59259-670-3:3DOI Listing
April 2004

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo.

J Immunol 2004 Jan;172(2):981-8

Department of Immunology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.
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http://dx.doi.org/10.4049/jimmunol.172.2.981DOI Listing
January 2004

The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses.

Nat Immunol 2003 Sep 17;4(9):899-906. Epub 2003 Aug 17.

Advanced Medical Discovery Institute, Ontario Cancer Institute, and Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario M5G 2C1, Canada.

We investigated the in vivo function of the B7 family member B7-H3 (also known as B7RP-2) by gene targeting. B7-H3 inhibited T cell proliferation mediated by antibody to T cell receptor or allogeneic antigen-presenting cells. B7-H3-deficient mice developed more severe airway inflammation than did wild-type mice in conditions in which T helper cells differentiated toward type 1 (T(H)1) rather than type 2 (T(H)2). B7-H3 expression was consistently enhanced by interferon-gamma but suppressed by interleukin 4 in dendritic cells. B7-H3-deficient mice developed experimental autoimmune encephalomyelitis several days earlier than their wild-type littermates, and accumulated higher concentrations of autoantibodies to DNA. Thus, B7-H3 is a negative regulator that preferentially affects T(H)1 responses.
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http://dx.doi.org/10.1038/ni967DOI Listing
September 2003

A novel cytotoxicity assay to evaluate antigen-specific CTL responses using a colorimetric substrate for Granzyme B.

J Immunol Methods 2003 May;276(1-2):89-101

Department of Biochemistry, University of Alberta, 463 Medical Sciences Bldg., T6G 2H7, Edmonton, Alberta, Canada.

We have utilized the unique enzymatic properties of a key cytotoxic mediator in target cell destruction, Granzyme B (GrB), to establish an attractive alternative to 51Cr-release assays for the assessment of antigen-specific CTL responses. A number of potential colorimetric peptide substrates were compared to evaluate levels of GrB activity in cytolytic cells. The most specific and sensitive substrate for GrB was Ac-IEPD-pNA, as shown by the minimal enzymatic hydrolysis in apoptotic Jurkat cells and strong hydrolysis in human NK cells. When human peripheral blood lymphocytes were stimulated in vitro, elevated GrB levels were detected by both Ac-IEPD-pNA and a GrB ELISA. Analysis of allo-antigen-specific murine CTLs revealed that GrB exocytosis was only detectable upon challenge with appropriate allogeneic target cells and strongly correlated to 51Cr-release data. The validity of using Ac-IEPD-pNA in vaccine trials was demonstrated in mice immunized with allogeneic P815 cells, where GrB enzymatic activity was measurable in ex vivo splenocytes cell cultures only upon co-incubation with P815 targets. Additionally, influenza-infected mice were also assessed for GrB activity following in vitro peptide-stimulation of splenocytes and strongly reflected both peptide-specific tetramer staining and 51Cr-release results. The novel cytotoxic assay presented here should give investigators a sensitive, cross-species, nonradioactive alternative to 51Cr-release assays as a means to assess antigen-specific CTL responses in vaccine trials.
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http://dx.doi.org/10.1016/s0022-1759(03)00073-5DOI Listing
May 2003

Role of ICOS versus CD28 in antiviral immunity.

Eur J Immunol 2002 Dec;32(12):3376-85

Department of Immunology, University of Toronto, Toronto, Canada.

The costimulatory protein ICOS is inducibly expressed on activated T cells. Previous results have shown that ICOS(-/-) mice are defective in germinal center formation, antibody (Ab) production and class switch as well as Th1 and Th2 cytokine production in response to protein or parasite antigens. However, ICOS-Ig failed to block antiviral Ab responses. To date the immune response to viruses has not been examined in ICOS(-/-) mice. In this report we compared antiviral Ab responses to LCMV, VSV and influenza virus in ICOS(-/-) versus wild-type mice. Our results show that ICOS is important in the Ab response to all three viruses, with greater effects on primary as compared to secondary responses. Although ICOS(-/-) mice are impaired in some immune responses following influenza infection, the effects were less severe than for CD28(-/-) mice. There was no defect in initial influenza-specific CD8 T cell expansion in ICOS(-/-) mice or in cytotoxic effector function. However, ICOS was important in maintaining CD4 cytokine production and CD8 T cell numbers late in the primary response. Upon secondary infection, ICOS(-/-) mice show wild-type levels of influenza-specific CD8 T cells, whereas CD28(-/-) mice show greatly impaired secondary CD8 T cell expansion. Overall, our results show that ICOS plays a clear role in the primary response to viruses at the level of Ab production, germinal center formation and Th cytokine production, but has diminished effects following secondary viral challenge.
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http://dx.doi.org/10.1002/1521-4141(200212)32:12<3376::AID-IMMU3376>3.0.CO;2-YDOI Listing
December 2002

Cutting edge: profound defect in T cell responses in TNF receptor-associated factor 2 dominant negative mice.

J Immunol 2002 Sep;169(6):2828-31

Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

TNFR-associated factor 2 (TRAF2) is an adapter protein that links several members of the TNFR family to downstream signaling pathways. Mice expressing a dominant negative form of TRAF2 in their lymphoid cells (TRAF2.DN mice) have a profound defect in T cell responses to allogeneic APC. In contrast, APC from wild-type or TRAF2.DN mice show an equivalent level of stimulation in a MLR. Ab production and class switch are unimpaired in TRAF2.DN mice. Thus, defects in the TRAF.DN mice appear to be limited to T cells. TRAF2.DN mice demonstrate an impaired T cell response to influenza virus, including decreased secondary expansion of IFN-gamma-secreting T cells as well as a decrease in CTL activity. CD4 T cell production of IL-2 was also dramatically impaired in TRAF2.DN mice. These studies suggest an essential role of TRAF2-linked receptors in secondary CD4 and CD8 T cell responses and have important implications for transplantation.
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http://dx.doi.org/10.4049/jimmunol.169.6.2828DOI Listing
September 2002

Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection.

J Immunol 2002 Apr;168(8):3777-85

Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

In this report, we demonstrate that CD28(-/-) mice are severely impaired in the initial expansion of D(b)/NP366-374-specific CD8 T cells in response to influenza virus infection, whereas 4-1BB ligand (4-1BBL)(-/-) mice show no defect in primary T cell expansion to influenza virus. In contrast, 4-1BBL(-/-) mice show a decrease in D(b)/NP366-374-specific T cells late in the primary response. Upon secondary challenge with influenza virus, 4-1BBL(-/-) mice show a decrease in the number of D(b)/NP366-374-specific T cells compared to wild-type mice such that the level of the CD8 T cell expansion during the in vivo secondary response is reduced to the level of a primary response, with concomitant reduction of CTL effector function. In contrast, Ab responses, as well as secondary CD4 T cell responses, to influenza are unaffected by 4-1BBL deficiency. Thus, CD28 is critical for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the response and is essential for the survival and/or responsiveness of the memory CD8 T cell pool.
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http://dx.doi.org/10.4049/jimmunol.168.8.3777DOI Listing
April 2002

Overexpression of rab7 enhances the kinetics of antigen processing and presentation with MHC class II molecules in B cells.

Int Immunol 2002 Mar;14(3):309-18

Department of Immunology, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

rab7 is an intracellular GTPase involved in early to late endosome fusion. By overexpressing rab7 in a B lymphoma we show that the rate of antigen presentation with MHC class II molecules is increased for four different peptide-MHC combinations, under conditions where levels of other components of the antigen-processing pathway remained constant. Resting B cells were shown to express significantly lower levels of rab7 when compared to adherent macrophages or to 'immature' or 'mature' dendritic cells. rab7 expression was up-regulated by stimulation of B cells with lipopolysaccharide or CD40 ligand. Other components of the endocytic pathway were also up-regulated in activated B cells, suggesting that B cell activation leads to a general enlargement of the endocytic compartment, correlating with the increased ability of activated B cells to process antigen. Taken together, our results suggest that rab7 levels regulate the rate of antigen presentation in B cells, and that rab7 and late endocytic compartments are important in MHC class II-restricted antigen presentation in B cells.
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http://dx.doi.org/10.1093/intimm/14.3.309DOI Listing
March 2002