Publications by authors named "Tanya L Crockford"

22 Publications

  • Page 1 of 1

An ontogenetic switch drives the positive and negative selection of B cells.

Proc Natl Acad Sci U S A 2020 02 4;117(7):3718-3727. Epub 2020 Feb 4.

Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom;

Developing B cells can be positively or negatively selected by self-antigens, but the mechanisms that determine these outcomes are incompletely understood. Here, we show that a B cell intrinsic switch between positive and negative selection during ontogeny is determined by a change from Lin28b to let-7 gene expression. Ectopic expression of a Lin28b transgene in murine B cells restored the positive selection of autoreactive B-1 B cells by self-antigen in adult bone marrow. Analysis of antigen-specific immature B cells in early and late ontogeny identified Lin28b-dependent genes associated with B-1 B cell development, including and , and Lin28b-independent effects are associated with the presence or absence of self-antigen. These findings identify cell intrinsic and extrinsic determinants of B cell fate during ontogeny and reconcile lineage and selection theories of B cell development. They explain how changes in the balance of positive and negative selection may be able to adapt to meet the immunological needs of an individual during its lifetime.
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http://dx.doi.org/10.1073/pnas.1915247117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035474PMC
February 2020

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

Nat Immunol 2020 01 16;21(1):86-100. Epub 2019 Dec 16.

Wellcome Sanger Institute, Hinxton, UK.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
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http://dx.doi.org/10.1038/s41590-019-0549-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338221PMC
January 2020

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

J Clin Invest 2019 06 4;129(9):3640-3656. Epub 2019 Jun 4.

Nuffield Department of Medicine Research Building, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.
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http://dx.doi.org/10.1172/JCI124099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715380PMC
June 2019

An essential role for the Zn transporter ZIP7 in B cell development.

Nat Immunol 2019 03 4;20(3):350-361. Epub 2019 Feb 4.

Midwest Immunology Clinic, Plymouth, MN, USA.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn in modulating B cell receptor signal strength and positive selection.
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http://dx.doi.org/10.1038/s41590-018-0295-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561116PMC
March 2019

Themis2 lowers the threshold for B cell activation during positive selection.

Nat Immunol 2017 02 19;18(2):205-213. Epub 2016 Dec 19.

MRC Human Immunology Unit, Weatherall Institute for Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.
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http://dx.doi.org/10.1038/ni.3642DOI Listing
February 2017

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity.

Proc Natl Acad Sci U S A 2016 06 14;113(26):E3706-15. Epub 2016 Jun 14.

Medical Research Council Human Immunology Unit, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom;

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.
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http://dx.doi.org/10.1073/pnas.1607592113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932993PMC
June 2016

Next-generation sequencing to dissect hereditary nephrotic syndrome in mice identifies a hypomorphic mutation in Lamb2 and models Pierson's syndrome.

J Pathol 2014 May 6;233(1):18-26. Epub 2014 Feb 6.

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

The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson's syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease.
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http://dx.doi.org/10.1002/path.4308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241031PMC
May 2014

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

DOCK8 is essential for T-cell survival and the maintenance of CD8+ T-cell memory.

Eur J Immunol 2011 Dec 10;41(12):3423-35. Epub 2011 Nov 10.

Nuffield Department of Medicine, Henry Wellcome Building of Molecular Physiology, Oxford University, Oxford, UK.

Deficiency in the guanine nucleotide exchange factor dedicator of cytokinesis 8 (DOCK8) causes a human immunodeficiency syndrome associated with recurrent sinopulmonary and viral infections. We have recently identified a DOCK8-deficient mouse strain, carrying an ethylnitrosourea-induced splice-site mutation that shows a failure to mature a humoral immune response due to the loss of germinal centre B cells. In this study, we turned to T-cell immunity to investigate further the human immunodeficiency syndrome and its association with decreased peripheral CD4(+) and CD8(+) T cells. Characterisation of the DOCK8-deficient mouse revealed T-cell lymphopenia, with increased T-cell turnover and decreased survival. Egress of mature CD4(+) thymocytes was reduced with increased migration of these cells to the chemokine CXCL12. However, despite the two-fold reduction in peripheral naïve T cells, the DOCK8-deficient mice generated a normal primary CD8(+) immune response and were able to survive acute influenza virus infection. The limiting effect of DOCK8 was in the normal survival of CD8(+) memory T cells after infection. These findings help to explain why DOCK8-deficient patients are susceptible to recurrent infections and provide new insights into how T-cell memory is sustained.
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http://dx.doi.org/10.1002/eji.201141759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517112PMC
December 2011

Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production.

Nat Immunol 2009 Dec 8;10(12):1283-91. Epub 2009 Nov 8.

John Curtin School of Medical Research and Australian Phenomics Facility, Australian National University, Australia.

To identify genes and mechanisms involved in humoral immunity, we did a mouse genetic screen for mutations that do not affect the first wave of antibody to immunization but disrupt response maturation and persistence. The first two mutants identified had loss-of-function mutations in the gene encoding a previously obscure member of a family of Rho-Rac GTP-exchange factors, DOCK8. DOCK8-mutant B cells were unable to form marginal zone B cells or to persist in germinal centers and undergo affinity maturation. Dock8 mutations disrupted accumulation of the integrin ligand ICAM-1 in the B cell immunological synapse but did not alter other aspects of B cell antigen receptor signaling. Humoral immunodeficiency due to Dock8 mutation provides evidence that organization of the immunological synapse is critical for signaling the survival of B cell subsets required for long-lasting immunity.
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http://dx.doi.org/10.1038/ni.1820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437189PMC
December 2009

Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection.

Nat Immunol 2009 Aug 13;10(8):831-9. Epub 2009 Jul 13.

Nuffield Department of Clinical Medicine, Oxford University, UK.

T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.
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http://dx.doi.org/10.1038/ni.1769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908989PMC
August 2009

Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome.

J Clin Invest 2009 Jun 18;119(6):1696-705. Epub 2009 May 18.

Henry Wellcome Building of Molecular Physiology, Oxford University, Oxford, United Kingdom.

Hypomorphic mutations in DNA ligase IV (LIG4) cause a human syndrome of immunodeficiency, radiosensitivity, and growth retardation due to defective DNA repair by the nonhomologous end-joining (NHEJ) pathway. Lig4-null mice are embryonic lethal, and better mouse models are needed to study human LigIV syndrome. We recently identified a viable mouse strain with a Y288C hypomorphic mutation in the Lig4 gene. Lig4Y288C mice exhibit a greater than 10-fold reduction of LigIV activity in vivo and recapitulate the immunodeficiency and growth retardation seen in human patients. Here, we have demonstrated that the Lig4Y288C mutation leads to multiple defects in lymphocyte development and function, including impaired V(D)J recombination, peripheral lymphocyte survival and proliferation, and B cell class switch recombination. We also highlight a high incidence of thymic tumors in the Lig4Y288C mice, suggesting that wild-type LigIV protects against malignant transformation. These findings provide explanations for the complex lymphoid phenotype of human LigIV syndrome.
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http://dx.doi.org/10.1172/JCI32743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689126PMC
June 2009

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism.

Blood 2009 Feb 27;113(8):1805-8. Epub 2008 Oct 27.

Henry Wellcome Building for Molecular Physiology, Oxford University, Oxford, United Kingdom.

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.
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http://dx.doi.org/10.1182/blood-2007-11-120402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2947353PMC
February 2009

Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis.

PLoS Med 2008 Mar;5(3):e54

Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia.

Background: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.

Methods And Findings: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.

Conclusions: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
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http://dx.doi.org/10.1371/journal.pmed.0050054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2270292PMC
March 2008

MyD88-dependent autoimmune disease in Lyn-deficient mice.

Eur J Immunol 2007 Oct;37(10):2734-43

Henry Wellcome Building of Molecular Physiology, University of Oxford, Oxford, UK.

Recent evidence suggests that systemic autoimmune disease depends on signals from TLR ligands, but little is known about how TLR-dependent pathways lead to the loss of self tolerance in vivo. To address this, we have examined the role of TLR signaling in Lyn-deficient mice, which develop an autoimmune disease similar to SLE. We found that absence of the TLR signaling adaptor molecule MyD88 suppresses plasma cell differentiation of switched and unswitched B cells, and prevents the generation of antinuclear IgG antibodies and glomerulonephritis. In mixed chimeras the increased IgM and IgG antibody secretion in Lyn-deficient mice is at least partially due to B cell-independent effects of Lyn. We now show that MyD88 deficiency blocks the expansion and activation of DC in which Lyn is also normally expressed, and prevents the hypersecretion of proinflammatory cytokines IL-6 and IL-12 by Lyn-deficient DC. These findings further highlight the important role of TLR-dependent signals in both lymphocyte activation and autoimmune pathogenesis.
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http://dx.doi.org/10.1002/eji.200737293DOI Listing
October 2007

DNA repair is limiting for haematopoietic stem cells during ageing.

Nature 2007 Jun;447(7145):686-90

Henry Wellcome Building for Molecular Physiology, Oxford University, Oxford OX3 9DU, UK.

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.
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http://dx.doi.org/10.1038/nature05875DOI Listing
June 2007

Limited peripheral T cell anergy predisposes to retinal autoimmunity.

J Immunol 2007 Apr;178(7):4276-83

Henry Wellcome Building of Molecular Physiology, Roosevelt Drive, Oxford, UK.

Autoimmune uveoretinitis accounts for at least 10% of worldwide blindness, yet it is unclear why tolerance to retinal Ags is so fragile and, particularly, to what extent this might be due to defects in peripheral tolerance. To address this issue, we generated double-transgenic mice expressing hen egg lysozyme, under the retinal interphotoreceptor retinoid-binding promoter, and a hen egg lysozyme-specific CD4(+) TCR transgene. In this manner, we have tracked autoreactive CD4(+) T cells from their development in the thymus to their involvement in uveoretinitis and compared tolerogenic mechanisms induced in a variety of organs to the same self-Ag. Our findings show that central tolerance to retinal and pancreatic Ags is qualitatively similar and equally dependent on the transcriptional regulator protein AIRE. However, the lack of Ag presentation in the eye-draining lymph nodes results in a failure to induce high levels of T cell anergy. Under these circumstances, despite considerable central deletion, low levels of retinal-specific autoreactive CD4(+) T cells can induce severe autoimmune disease. The relative lack of anergy induction by retinal Ags, in contrast to the same Ag in other organs, helps to explain the unique susceptibility of the eye to spontaneous and experimentally induced autoimmune disease.
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http://dx.doi.org/10.4049/jimmunol.178.7.4276DOI Listing
April 2007

Increased positive selection of B1 cells and reduced B cell tolerance to intracellular antigens in c1q-deficient mice.

J Immunol 2007 Mar;178(5):2916-22

Henry Wellcome Building for Molecular Physiology, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.

Inherited deficiency of early components of the classical complement pathway is strongly associated with the targeting of intracellular self Ags in systemic lupus erythematosus, but the reasons for this association are debated. In this study, we show that C1q deficiency increases the positive selection of B1b B cells and IgM autoantibodies by an intracellular self Ag, which is exposed on dying cells, and decreases the negative selection of autoreactive conventional B cells by the same Ag. These effects are specific to intracellular Ag because C1q deficiency does not affect negative selection by extracellular self Ag or increase the positive selection of naive B cells. The B1-derived IgM autoantibody binds to the intracellular Ag when it is expressed on dying cells, leading to fixation of C1q and clearance of cells by phagocytosis. These findings suggest that the positive selection of autoreactive B1 cells by self Ags may contribute to the IgM and C1q-dependent clearance of dying cells in a feedback loop that limits exposure of conventional B cells to immunogenic self Ags. We show that exposure of intracellular Ag leads to the activation of conventional B cells, when there is a source of T cell help in vivo.
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http://dx.doi.org/10.4049/jimmunol.178.5.2916DOI Listing
March 2007

CD4 T cell-dependent autoimmunity against a melanocyte neoantigen induces spontaneous vitiligo and depends upon Fas-Fas ligand interactions.

J Immunol 2006 Sep;177(5):3055-62

Henry Wellcome Building of Molecular Physiology, University of Oxford, Oxford, United Kingdom.

Better understanding of tolerance and autoimmunity toward melanocyte-specific Ags is needed to develop effective treatment for vitiligo and malignant melanoma; yet, a systematic assessment of these mechanisms has been hampered by the difficulty in tracking autoreactive T cells. To address this issue, we have generated transgenic mice that express hen egg lysozyme as a melanocyte-specific neoantigen. By crossing these animals to a hen egg lysozyme-specific CD4 TCR transgenic line we have been able to track autoreactive CD4+ T cells from their development in the thymus to their involvement in spontaneous autoimmune disease with striking similarity to human vitiligo vulgaris and Vogt-Koyanagi-Harada syndrome. Our findings show that CD4-dependent destruction of melanocytes is partially inhibited by blocking Fas-Fas ligand interactions and also highlights the importance of local control of autoimmunity, as vitiligo remains patchy and never proceeds to confluence even when Ag and autoreactive CD4+ T cells are abundant. Immune therapy to enhance or suppress melanocyte-specific T cells can be directed at a series of semiredundant pathways involving tolerance and cell death.
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http://dx.doi.org/10.4049/jimmunol.177.5.3055DOI Listing
September 2006

Signals from a self-antigen induce positive selection in early B cell ontogeny but are tolerogenic in adults.

J Immunol 2006 Jun;176(12):7402-11

Henry Wellcome Building of Molecular Physiology, University of Oxford, UK.

Positive and negative signals from self-Ags shape the B cell repertoire and the development of distinct B cell subsets, but little is known about what distinguishes these signals. To address this question, we have studied the development of anti-hen egg lysozyme MD4 Ig transgene B cells while systematically varying the level, distribution, and timing of exposure to different forms of hen egg lysozyme as a self-Ag. This process has allowed us to explore the effects of Ag independent of BCR specificity. Our findings show how the selection of autoreactive B cells is a competitive process involving immunogenic and tolerogenic forms of self-Ags. Due to a developmental switch during B cell ontogeny, autoreactive anti-hen egg lysozyme MD4 Ig transgene B cells are negatively selected by self-Ags in adult bone marrow but susceptible to positive selection by some of the same self-Ags in fetal and neonatal life. However, the persistence of B1 cells and IgM autoantibodies from early ontogeny enables autoreactive B cells from the adult bone marrow to escape negative selection. Our data suggest that this rescue may be due to the clearance or masking of self-Ag by IgM autoantibody. We discuss the implications of these findings in terms of B cell selection and the maintenance of self-tolerance during early and adult life.
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http://dx.doi.org/10.4049/jimmunol.176.12.7402DOI Listing
June 2006

Analysis of Lyn/CD22 double-deficient B cells in vivo demonstrates Lyn- and CD22-independent pathways affecting BCR regulation and B cell survival.

Eur J Immunol 2005 Dec;35(12):3655-63

Henry Wellcome Building of Molecular Physiology, Oxford, UK.

B cell fate is determined by the strength of signals from the antigen receptor and from co-receptors that adjust the activation threshold and tune the B cell to its environment. These co-receptors have been broadly classified into inhibitory and enhancing groups, yet some, such as CD22, may have dual effects. CD22 recruits a variety of signal enhancers at the same time as Lyn-dependent phosphorylation leads to the binding of the inhibitory phosphatase SHP-1. To assess the relative importance of Lyn- and CD22-dependent and -independent pathways, we generated Lyn and CD22 single-deficient mice and Lyn/CD22 double-deficient mice expressing the MD4 immunoglobulin transgene against hen egg lysozyme (IgHEL). This genetic approach has enabled us to compare the contributions of Lyn and CD22 to B cell development in vivo, independent of BCR specificity and in the presence and absence of self-antigen. Our results show that although the effects of Lyn are dominant in negative regulation of B cell hyperactivity, Lyn and CD22 have independent and additive effects on B cell survival. These findings emphasize the subtle nature of regulation at the BCR and the usefulness of genetic complementation to dissect common and parallel pathways.
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http://dx.doi.org/10.1002/eji.200535247DOI Listing
December 2005
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