Professor Richard J Cornall FMedSci FRCP

Research Area: Immunology
Technology Exchange: Cell sorting, Chromosome mapping, Immunohistochemistry, In vivo imaging, Protein interaction, Transcript profiling and Transgenesis
Scientific Themes: Immunology & Infectious Disease and Genetics & Genomics
Keywords: Autoimmune diseases, B Cell Development, Self Tolerance, T Cell Development, Nephrology and Adaptive Immunity
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Our aim is to understand how the immune system is formed and regulated and the causes of autoimmunity, particularly the systemic autoimmune diseases, and the development and selection of B cells. Adverse immunological reactions to self and foreign antigens that lead to autoimmune or inflammatory disease place a major economic and social burden on world health and individual quality of life. We are also interested in how people differ in their inherited susceptibility to these diseases and why these differences are sustained in human populations by natural selection. Advances in this area will have a large and impact on the management of human disease.

Our strategy involves research programmes in basic biology and in clinical medicine. In the first, we use transgenic models to investigate how lymphocytes function in health and in human disease and how our genes encode susceptibility to autoimmunity and immunodeficiency. In the second, which is a collaboration with Professor Simon Davis, we are developing ways to change the function of lymphocytes, turning them on in cancer and off during inflammation or autoimmunity.

Name Department Institution Country
Chris Goodnow FRS John Curtin School of Medicine Australian National University Australia
Jason Cyster University of California San Francisco United States
Dr Paul Love NIH United States
Professor Adrian Hayday FMedSci King's College London United Kingdom
Professor Steve Brown FMedSci MRC Harwell United Kingdom
Professor Moin Saleem FRCP University of Bristol United Kingdom
Dr Helen Su NIH United States
Professor Sophie Hambleton Newcastle University and the Great North Children's Hospital United Kingdom
Schwerd T, Bryant RV, Pandey S, Capitani M, Meran L, Cazier JB, Jung J, Mondal K, Parkes M, Mathew CG et al. 2017. NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease. Mucosal Immunol, | Show Abstract | Read more

Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.Mucosal Immunology advance online publication 1 November 2017. doi:10.1038/mi.2017.74.

Choi S, Cornall R, Lesourne R, Love PE. 2017. THEMIS: Two Models, Different Thresholds. Trends Immunol, 38 (9), pp. 622-632. | Show Abstract | Read more

THEMIS, a recently identified T-lineage-restricted protein, is the founding member of a large metazoan protein family. Gene inactivation studies have revealed a critical requirement for THEMIS during thymocyte positive selection, implicating THEMIS in signaling downstream of the T cell antigen receptor (TCR), but the mechanistic underpinnings of THEMIS function have remained elusive. A previous model posited that THEMIS prevents thymocytes from inappropriately crossing the positive/negative selection threshold by dampening TCR signaling. However, new data suggest an alternative model where THEMIS enhances TCR signaling enabling thymocytes to reach the threshold for positive selection, avoiding death by neglect. We review the data supporting each model and conclude that the preponderance of evidence favors an enhancing function for THEMIS in TCR signaling.

Deobagkar-Lele M, Anzilotti C, Cornall RJ. 2017. Themis2: setting the threshold for B-cell selection. Cell Mol Immunol, 14 (8), pp. 643-645. | Read more

Cheng D, Deobagkar-Lele M, Zvezdova E, Choi S, Uehara S, Baup D, Bennett SC, Bull KR, Crockford TL, Ferry H et al. 2017. Themis2 lowers the threshold for B cell activation during positive selection. Nat Immunol, 18 (2), pp. 205-213. | Show Abstract | Read more

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.

Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Crawford G, Anzilotti C, Steeples V, Ghaffari S et al. 2016. Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity. Proc Natl Acad Sci U S A, 113 (26), pp. E3706-E3715. | Show Abstract | Read more

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.

Siggs OM, Popkin DL, Krebs P, Li X, Tang M, Zhan X, Zeng M, Lin P, Xia Y, Oldstone MB et al. 2015. Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection. Proc Natl Acad Sci U S A, 112 (42), pp. E5706-E5714. | Show Abstract | Read more

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.

Taylor JC, Martin HC, Lise S, Broxholme J, Cazier JB, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Show Abstract | Read more

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

Iacovelli S, Hug E, Bennardo S, Duehren-von Minden M, Gobessi S, Rinaldi A, Suljagic M, Bilbao D, Bolasco G, Eckl-Dorna J et al. 2015. Two types of BCR interactions are positively selected during leukemia development in the Eμ-TCL1 transgenic mouse model of CLL. Blood, 125 (10), pp. 1578-1588. | Show Abstract | Read more

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Eμ-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL.

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Zhang Q, Dove CG, Hor JL, Murdock HM, Strauss-Albee DM, Garcia JA, Mandl JN, Grodick RA, Jing H, Chandler-Brown DB et al. 2014. DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity JOURNAL OF EXPERIMENTAL MEDICINE, 211 (13), pp. 2549-2566. | Read more

Petousi N, Copley RR, Lappin TR, Haggan SE, Bento CM, Cario H, Percy MJ, WGS Consortium, Ratcliffe PJ, Robbins PA, McMullin MF. 2014. Erythrocytosis associated with a novel missense mutation in the BPGM gene. Haematologica, 99 (10), pp. e201-e204. | Read more

Todd JA, Aitman TJ, Cornall RJ, Ghosh S, Hall JRS, Hearne CM, Knight AM, Love JM, McAleer MA, Prins J-B et al. 2014. Genetic analysis of autoimmune type 1 diabetes mellitus in mice (Reprinted from Nature, vol 351, pg 542-547, 1991) JOURNAL OF IMMUNOLOGY, 193 (1), pp. 7-12.

Todd JA, Aitman TJ, Cornall RJ, Ghosh S, Hall JR, Hearne CM, Knight AM, Love JM, McAleer MA, Prins JB et al. 2014. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. 1991. J Immunol, 193 (1), pp. 7-12.

Zhang Q, Dove CG, Hor JL, Murdock HM, Strauss-Albee DM, Garcia JA, Mandl JN, Grodick RA, Jing H, Chandler-Brown DB et al. 2014. DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity. J Exp Med, 211 (13), pp. 2549-2566. | Show Abstract | Read more

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

Cazier JB, Rao SR, McLean CM, Walker AK, Wright BJ, Jaeger EE, Kartsonaki C, Marsden L, Yau C, Camps C et al. 2014. Whole-genome sequencing of bladder cancers reveals somatic CDKN1A mutations and clinicopathological associations with mutation burden. Nat Commun, 5 pp. 3756. | Show Abstract | Read more

Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former are not mutually exclusive with TP53 mutations or MDM2 amplification, showing that CDKN1A dysfunction is not simply an alternative mechanism for p53 pathway inactivation. We find strong positive associations between higher tumour stage/grade and greater clonal diversity, the number of somatic mutations and the burden of copy number changes. In principle, the identification of sub-clones with greater diversity and/or mutation burden within early-stage or low-grade tumours could identify lesions with a high risk of invasive progression.

Bull KR, Mason T, Rimmer AJ, Crockford TL, Silver KL, Bouriez-Jones T, Hough TA, Chaudhry S, Roberts IS, Goodnow CC, Cornall RJ. 2014. Next-generation sequencing to dissect hereditary nephrotic syndrome in mice identifies a hypomorphic mutation in Lamb2 and models Pierson's syndrome. J Pathol, 233 (1), pp. 18-26. | Show Abstract | Read more

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.

Sharma VP, Fenwick AL, Brockop MS, McGowan SJ, Goos JA, Hoogeboom AJ, Brady AF, Jeelani NO, Lynch SA, Mulliken JB et al. 2013. Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet, 45 (10), pp. 1261. | Read more

Crawford G, Enders A, Gileadi U, Stankovic S, Zhang Q, Lambe T, Crockford TL, Lockstone HE, Freeman A, Arkwright PD et al. 2013. DOCK8 is critical for the survival and function of NKT cells. Blood, 122 (12), pp. 2052-2061. | Show Abstract | Read more

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.

Vitova A, Kuffova L, Klaska IP, Holan V, Cornall RJ, Forrester JV. 2013. Corrigendum Transplant International, 26 (8), pp. e74-e74. | Read more

James A, Blagojevic J, Benham SW, Cornall R, Frater J. 2013. Azathioprine hypersensitivity presenting as septic shock with encephalopathy. BMJ Case Rep, 2013 (mar18 1), pp. bcr2012008340-bcr2012008340. | Show Abstract | Read more

We present a case of azathioprine hypersensitivity presenting as septic shock with associated encephalopathy. The patient was presented with rapid onset of fever, hypotension, confusion and a rapidly declining conscious level. He was admitted to the intensive care unit where he received numerous invasive investigations and treatments with broad-spectrum antibiotics, antivirals and antifungals. All microbial cultures were negative. The patient-consistent with azathioprine hypersensitivity-made a spontaneous recovery after 7 days. The case shows that a time line of drug initiation is a key part of the medical history and consideration of azathioprine hypersensitivity could avoid unnecessary interventions and excessive antimicrobial use.

Vitova A, Kuffová L, Klaska IP, Holan V, Cornall RJ, Forrester JV. 2013. The high-risk corneal regraft model: a justification for tissue matching in humans. Transpl Int, 26 (4), pp. 453-461. | Show Abstract | Read more

Models of high-risk corneal graft rejection involve neovascularization induced via innate immune responses, e.g., suture-mediated trauma. We describe a model of high-risk corneal graft rejection using corneal graft donor-recipient pairing based on a single-antigen disparity. Donor corneas from transgenic mice on B10.BR (H-2k ) background, in which hen-egg lysozyme (HEL) as a membrane-bound antigen (mHEL) was expressed under the major histocompatibility complex (MHC) class I promoter (KLK-mHEL, H-2k), were transplanted into wild type B10.BR recipient mice. Unmanipulated wild type recipient mice rejected KLK-mHEL grafts (39%) slowly over 50-60 days. Graft rejection incidence was maximized (100%) and tempo accelerated (27 days) by priming with HEL-pulsed syngeneic dendritic cells and less so by increasing T-cell precursor frequency. Rejection also reached maximum levels (100%) and tempo (3-8 days) when mice which had rejected a first graft ('rejectors') were regrafted, and was associated with induction of HEL-specific memory T cells. In contrast, 'acceptors' rejected a second graft at rates and tempo similar to naïve mice. These data reveal the importance of (i) donor MHC antigens as alloantigens for indirect recognition, (ii) alloantigen-specific memory in high-risk graft rejection involving regrafts, and (iii) suggest a role for tissue matching in human corneal graft to avoid sensitization to donor MHC antigens.

Sharma VP, Fenwick AL, Brockop MS, McGowan SJ, Goos JA, Hoogeboom AJ, Brady AF, Jeelani NO, Lynch SA, Mulliken JB et al. 2013. Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet, 45 (3), pp. 304-307. | Show Abstract | Read more

Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ∼1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ∼21% of cases, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis. Using exome sequencing, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in individuals with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 have severe coronal synostosis. Hence, the dosage of TCF12-TWIST1 heterodimers is critical for normal coronal suture development.

James A, Blagojevic J, Benham SW, Cornall R, Frater J. 2013. Azathioprine hypersensitivity presenting as septic shock with encephalopathy. BMJ case reports, 2013 | Show Abstract

We present a case of azathioprine hypersensitivity presenting as septic shock with associated encephalopathy. The patient was presented with rapid onset of fever, hypotension, confusion and a rapidly declining conscious level. He was admitted to the intensive care unit where he received numerous invasive investigations and treatments with broad-spectrum antibiotics, antivirals and antifungals. All microbial cultures were negative. The patient-consistent with azathioprine hypersensitivity-made a spontaneous recovery after 7 days. The case shows that a time line of drug initiation is a key part of the medical history and consideration of azathioprine hypersensitivity could avoid unnecessary interventions and excessive antimicrobial use.

Vitova A, Kuffová L, Klaska IP, Holan V, Cornall RJ, Forrester JV. 2013. The high-risk corneal regraft model: A justification for tissue matching in humans Transplant International, 26 (4), pp. 453-461. | Show Abstract | Read more

Models of high-risk corneal graft rejection involve neovascularization induced via innate immune responses, e.g., suture-mediated trauma. We describe a model of high-risk corneal graft rejection using corneal graft donor-recipient pairing based on a single-antigen disparity. Donor corneas from transgenic mice on B10.BR (H-2 k ) background, in which hen-egg lysozyme (HEL) as a membrane-bound antigen (mHEL) was expressed under the major histocompatibility complex (MHC) Class I promoter (KLK-mHEL, H-2 k ), were transplanted into wild type B10.BR recipient mice. Unmanipulated wild type recipient mice rejected KLK-mHEL grafts (39%) slowly over 50-60 days. Graft rejection incidence was maximized (100%) and tempo accelerated (27 days) by priming with HEL-pulsed syngeneic dendritic cells and less so by increasing T-cell precursor frequency. Rejection also reached maximum levels (100%) and tempo (3-8 days) when mice which had rejected a first graft ('rejectors') were regrafted, and was associated with induction of HEL-specific memory T cells. In contrast, 'acceptors' rejected a second graft at rates and tempo similar to naïve mice. These data reveal the importance of (i) donor MHC antigens as alloantigens for indirect recognition, (ii) alloantigen-specific memory in high-risk graft rejection involving regrafts, and (iii) suggest a role for tissue matching in human corneal graft to avoid sensitization to donor MHC antigens. © 2013 The Authors Transplant International © 2013 European Society for Organ Transplantation. Published by Blackwell Publishing Ltd.

Bull KR, Rimmer AJ, Siggs OM, Miosge LA, Roots CM, Enders A, Bertram EM, Crockford TL, Whittle B, Potter PK et al. 2013. Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations. PLoS Genet, 9 (1), pp. e1003219. | Show Abstract | Read more

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.

Bergmann H, Yabas M, Short A, Miosge L, Barthel N, Teh CE, Roots CM, Bull KR, Jeelall Y, Horikawa K et al. 2013. B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A. J Exp Med, 210 (1), pp. 31-40. | Show Abstract | Read more

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.

Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Guarino E, Salguero I et al. 2013. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet, 45 (2), pp. 136-144. | Show Abstract | Read more

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

Chakera A, Bennett SC, Morteau O, Bowness P, Luqmani RA, Cornall RJ. 2012. The phenotype of circulating follicular-helper T cells in patients with rheumatoid arthritis defines CD200 as a potential therapeutic target. Clin Dev Immunol, 2012 pp. 948218. | Show Abstract | Read more

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting synovial joints in which the development of autoantibodies represents a failure of normal tolerance mechanisms, suggesting a role for follicular helper T cells (T(FH)) in the genesis of autoimmunity. To determine whether quantitative or qualitative abnormalities in the circulating T(FH) cell population exist, we analysed by flow cytometry the number and profile of these cells in 35 patients with RA and 15 matched controls. Results were correlated with patient characteristics, including the presence of autoantibodies, disease activity, and treatment with biologic agents. Circulating T(FH) cells from patients with RA show significantly increased expression of the immunoglobulin superfamily receptor CD200, with highest levels seen in seropositive patients (P = 0.0045) and patients treated with anti-TNFα agents (P = 0.0008). This occurs in the absence of any change in T(FH) numbers or overt bias towards Th1, Th2, or Th17 phenotypes. CD200 levels did not correlate with DAS28 scores (P = 0.887). Although the number of circulating T(FH) cells is not altered in the blood of patients with RA, the T(FH) cells have a distinct phenotype. These differences associate T(FH) cells with the pathogenesis of RA and support the relevance of the CD200/CD200R signalling pathway as a potential therapeutic target.

Randall KL, Chan SS, Ma CS, Fung I, Mei Y, Yabas M, Tan A, Arkwright PD, Al Suwairi W, Lugo Reyes SO et al. 2011. DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice. J Exp Med, 208 (11), pp. 2305-2320. | Show Abstract | Read more

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.

Chakera A, Bennett SC, Cornall RJ. 2011. A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response. J Transl Med, 9 (1), pp. 143. | Show Abstract | Read more

BACKGROUND: The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpots cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood. METHODS: Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot. RESULTS: Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 uL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing. CONCLUSIONS: A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation are required before processing.

Chakera A, Bennett S, Lawrence S, Morteau O, Mason PD, O'Callaghan CA, Cornall RJ. 2011. Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation. Clin Exp Immunol, 165 (3), pp. 401-409. | Show Abstract | Read more

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured.

Chakera A, Bennett SC, Cornall RJ. 2011. A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response Journal of Translational Medicine, 9 (1), | Show Abstract | Read more

Background: The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.Methods: Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.Results: Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.Conclusions: A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation is required before processing. © 2011 Chakera et al; licensee BioMed Central Ltd.

Cited:

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Chakera A, Bennett S, Lawrence S, Morteau O, Mason PD, O'Callaghan CA, Cornall RJ. 2011. Antigen-specific T cell responses to BK polyomavirus antigens identify functional anti-viral immunity and may help to guide immunosuppression following renal transplantation Clinical and Experimental Immunology, 165 (3), pp. 401-409. | Show Abstract | Read more

Infection with the polyoma virus BK (BKV) is a major cause of morbidity following renal transplantation. Limited understanding of the anti-viral immune response has prevented the design of a strategy that balances treatment with the preservation of graft function. The proven utility of interferon-gamma enzyme-linked immunospot (ELISPOT) assays to measure T cell responses in immunocompetent hosts was the basis for trying to develop a rational approach to the management of BKV following renal transplantation. In a sample of transplant recipients and healthy controls, comparisons were made between T cell responses to the complete panel of BKV antigens, the Epstein-Barr virus (EBV) antigens, BZLF1 and EBNA1, and the mitogen phytohaemagglutinin (PHA). Correlations between responses to individual antigens and immunosuppressive regimens were also analysed. Antigen-specific T cell responses were a specific indicator of recent or ongoing recovery from BKV infection (P < 0·05), with responses to different BKV antigens being highly heterogeneous. Significant BKV immunity was undetectable in transplant patients with persistent viral replication or no history of BKV reactivation. Responses to EBV antigens and mitogen were reduced in patients with BKV reactivation, but these differences were not statistically significant. The T cell response to BKV antigens is a useful and specific guide to recovery from BKV reactivation in renal transplant recipients, provided that the full range of antigenic responses is measured. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

Lambe T, Crawford G, Johnson AL, Crockford TL, Bouriez-Jones T, Smyth AM, Pham TH, Zhang Q, Freeman AF, Cyster JG et al. 2011. DOCK8 is essential for T-cell survival and the maintenance of CD8+ T-cell memory. Eur J Immunol, 41 (12), pp. 3423-3435. | Show Abstract | Read more

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.

Chakera A, Bennett S, Morteau O, Mason P, O'Callaghan C, Cornall R. 2010. QUANTIFICATION OF ANTIGEN-SPECIFIC T-CELL RESPONSES TO POLYOMA VIRUS BK FOLLOWING RENAL TRANSPLANTATION NEPHROLOGY, 15 pp. 41-41.

Randall KL, Lambe T, Johnson AL, Treanor B, Kucharska E, Domaschenz H, Whittle B, Tze LE, Enders A, Crockford TL et al. 2010. Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production (vol 10, pg 1283, 2009) NATURE IMMUNOLOGY, 11 (7), pp. 644-644.

Randall KL, Lambe T, Johnson A, Treanor B, Kucharska E, Domaschenz H, Whittle B, Tze LE, Enders A, Crockford TL et al. 2010. Corrigendum: Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production Nature Immunology, 11 (7), pp. 644-644. | Read more

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L et al. 2010. Erratum: Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection (Nature Immunology (2009) 10 (831-839)) Nature Immunology, 11 (1), pp. 97. | Read more

Morteau O, Blundell S, Chakera A, Bennett S, Christou CM, Mason PD, Cornall RJ, O'Callaghan CA. 2010. Renal transplant immunosuppression impairs natural killer cell function in vitro and in vivo. PLoS One, 5 (10), pp. e13294. | Show Abstract | Read more

BACKGROUND: Despite an increasing awareness of the importance of innate immunity, the roles of natural killer (NK) cells in transplant rejection and antiviral and cancer immunity during immunosuppression have not been clearly defined. METHODS: To address this issue we have developed a quantitative assay of NK cell function that can be used on clinical samples and have studied the influence of immunosuppression on NK cell function. NK cell degranulation and intracellular interferon (IFN)-γ production were determined by flow cytometry of peripheral blood samples. RESULTS: Overnight ex vivo treatment of peripheral blood cells from healthy controls with ciclosporin or tacrolimus inhibited NK cell degranulation and IFN-γ production in a dose-dependent manner. A similar impairment of function was seen in NK cells from patients treated in vivo with calcineurin inhibitors. In the early post-transplant period, there was a variable reduction of NK cell counts after treatment with alemtuzumab and basiliximab. CONCLUSIONS: The functional inhibition of NK cells in early transplant patients coincides with the period of maximum susceptibility to viral infections. The ability to assay NK cell function in clinical samples allows assessment of the impact of immunosuppression on these effector cells. This information may be helpful in guiding the titration of immunosuppression in the clinical setting.

Randall KL, Lambe T, Goodnow CC, Cornall RJ. 2010. The essential role of DOCK8 in humoral immunity. Dis Markers, 29 (3-4), pp. 141-150. | Show Abstract | Read more

The processes that normally generate and maintain adaptive immunity and immunological memory are poorly understood, and yet of fundamental importance when infectious diseases place such a major economic and social burden on the world's health and agriculture systems. Defects in these mechanisms also underlie the many forms of human primary immunodeficiency. Identifying these mechanisms in a systematic way is therefore important if we are to develop better strategies for treating and preventing infection, inherited disease, transplant rejection and autoimmunity. In this review we describe a genome-wide screen in mice for the genes important for generating these adaptive responses, and describe two independent DOCK8 mutant mice strains identified by this screen. DOCK 8 was found to play an essential role in humoral immune responses and to be important in the proper formation of the B cell immunological synapse.

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L et al. 2010. Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol, 11 (1), pp. 97-97. | Read more

Randall KL, Lambe T, Johnson AL, Treanor B, Kucharska E, Domaschenz H, Whittle B, Tze LE, Enders A, Crockford TL et al. 2009. Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production. Nat Immunol, 10 (12), pp. 1283-1291. | Show Abstract | Read more

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.

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L et al. 2009. Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol, 10 (8), pp. 831-839. | Show Abstract | Read more

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.

Nijnik A, Dawson S, Crockford TL, Woodbine L, Visetnoi S, Bennett S, Jones M, Turner GD, Jeggo PA, Goodnow CC, Cornall RJ. 2009. Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome. J Clin Invest, 119 (6), pp. 1696-1705. | Show Abstract | Read more

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.

Lambe T, Simpson RJ, Dawson S, Bouriez-Jones T, Crockford TL, Lepherd M, Latunde-Dada GO, Robinson H, Raja KB, Campagna DR et al. 2009. Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism. Blood, 113 (8), pp. 1805-1808. | Show Abstract | Read more

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.

Cornall RJ. 2008. HO-1 extends to stem cells. Blood, 112 (12), pp. 4363-4364. | Read more

Forrester JV, Xu H, Lambe T, Cornall R. 2008. Immune privilege or privileged immunity? Mucosal Immunol, 1 (5), pp. 372-381. | Show Abstract | Read more

Immune privilege is a concept that has come of age. Where previously it was considered to be a passive phenomenon restricted to certain specialized tissues, it is now viewed as comprising several mechanisms, both active and passive, shared in many aspects with emerging notions of the mechanisms of peripheral tolerance. The relative degrees of immune privilege vary from tissue to tissue depending on the number and strength of each of the mechanisms contained in that tissue. Immune privilege can be generated in non-privileged sites such as the skin and allografts, and is a property of the tissue itself. We therefore propose that, in addition to canonical central and peripheral tolerance mechanisms, there is a third route whereby the organism promotes self-antigen non-reactivity centered on the specific properties of each tissue and varying accordingly (relative degrees of immune privilege). This third mechanism of inducing immunological tolerance, as it is a local tissue phenomenon, might have particular therapeutic significance, for instance in devising strategies for induction of immunity to tumors by disrupting immune privilege or in preventing graft rejection by promoting immune privilege.

Lau AW, Biester S, Cornall RJ, Forrester JV. 2008. Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells. J Immunol, 180 (6), pp. 3889-3899. | Show Abstract

Dendritic cells (DC) are key regulators of immune responses. Mature DC are traditionally considered to be immunogenic, although there is accumulating evidence that they can also be tolerogenic and induce Ag-specific regulatory T cells (Tregs). However, the mechanism of this Treg induction and the site of Treg action in vivo are yet to be defined. In this study, using the experimental model of interphotoreceptor retinoid-binding protein peptide (1-20)-induced experimental autoimmune uveoretinitis, we show that s.c. inoculation of IRBP-peptide-pulsed IL-10-producing LPS-activated mature DC (IL-10-DC) at one site (the cervical region) suppresses autoimmunity induced at a separate site (the inguinal region). Our data show that s.c. IL-10-DC correlates with an increase in the number of CD4(+)CD25(+)Foxp3(+) Tregs at the DC-draining lymph nodes (DC-dLN). However, although MHCII(-/-) IL-10-DC also induces Treg expansion at this DC-dLN, they failed to suppress experimental autoimmune uveoretinitis. Furthermore, unlike wild-type IL-10-DC, MHCII(-/-) IL-10-DC did not correlate with an increase in the percentage of Tregs expressing CD62L at the DC-dLN, nor did they associate with an increase in Treg number at a distal site. Similar effects were also observed after s.c. hen egg lysozyme-pulsed IL-10-DC, which produced a strong reduction in the number and activation of proliferating Ag-specific CD4(+) 3A9 T effector cells. We therefore propose that IL-10-DC require MHCII-dependent Ag presentation, and hence TCR ligation, to promote CD62L-mediated trafficking of Tregs to the site of T effector cell priming, where they suppress autoimmunity.

Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Png CW, Crockford TL, Cornall RJ et al. 2008. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med, 5 (3), pp. e54. | Show Abstract | Read more

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.

Lambe T, Cornall RJ. 2008. Commmentary 7. Exp Dermatol, 17 (2), pp. 157-158. | Read more

Schallreuter KU, Bahadoran P, Picardo M, Slominski A, Elassiuty YE, Kemp EH, Giachino C, Liu JB, Luiten RM, Lambe T et al. 2008. Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else? Exp Dermatol, 17 (2), pp. 139-140. | Show Abstract | Read more

The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought--ranging, e.g. from the concept that vitiligo essentially is a free-radical disorder to that of vitiligo being a primary autoimmune disease--imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively--on the basis of sound experimental evidence, rather than by a war of dogmatic theories. Recognizing, however, that it is theories which tend to guide our experimental designs and choice of study parameters, the various pathogenesis theories on the market deserve to be critically, yet unemotionally re-evaluated. This Controversies feature invites you to do so, and to ask yourself: is there something important or worthwhile exploring in other pathogenesis scenarios than those already favoured by you that may help you improve your own study design, next time you have a fresh look at vitiligo? Vitiligo provides a superb model for the study of many fundamental problems in skin biology and pathology. Therefore, even if it later turns out that, as far as your own vitiligo pathogenesis concept is concerned, you have barked-up the wrong tree most of the time, chances are that you shall anyway have generated priceless new insights into skin function along the way.

Cited:

111

WOS

Schallreuter KU, Bahadoran P, Picardo M, Slominski A, Elassiuty YE, Kemp EH, Giachino C, Liu JB, Luiten RM, Lambe T et al. 2008. Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else? EXPERIMENTAL DERMATOLOGY, 17 (2), pp. 139-140. | Read more

Lambe T, Cornall RJ. 2008. Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else? Commentary 7 EXPERIMENTAL DERMATOLOGY, 17 (2), pp. 157-158. | Read more

Cited:

317

Scopus

Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Chin WP, Crockford TL, Cornall RJ et al. 2008. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis PLoS Medicine, 5 (3), pp. 0440-0460. | Show Abstract | Read more

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-1β, TNF-α, and IFN-γ 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-γ, TNF-α, 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. © 2008 Heazlewood et al.

Silver KL, Crockford TL, Bouriez-Jones T, Milling S, Lambe T, Cornall RJ. 2007. MyD88-dependent autoimmune disease in Lyn-deficient mice. Eur J Immunol, 37 (10), pp. 2734-2743. | Show Abstract | Read more

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.

Nijnik A, Woodbine L, Marchetti C, Dawson S, Lambe T, Liu C, Rodrigues NP, Crockford TL, Cabuy E, Vindigni A et al. 2007. DNA repair is limiting for haematopoietic stem cells during ageing. Nature, 447 (7145), pp. 686-690. | Show Abstract | Read more

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.

Cornall R. 2007. ENU mutagenesis: Unpicking the immune system INFLAMMATION RESEARCH, 56 pp. S344-S344.

Lambe T, Leung JC, Ferry H, Bouriez-Jones T, Makinen K, Crockford TL, Jiang HR, Nickerson JM, Peltonen L, Forrester JV, Cornall RJ. 2007. Limited peripheral T cell anergy predisposes to retinal autoimmunity. J Immunol, 178 (7), pp. 4276-4283. | Show Abstract

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.

Ferry H, Potter PK, Crockford TL, Nijnik A, Ehrenstein MR, Walport MJ, Botto M, Cornall RJ. 2007. Increased positive selection of B1 cells and reduced B cell tolerance to intracellular antigens in c1q-deficient mice. J Immunol, 178 (5), pp. 2916-2922. | Show Abstract

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.

Silver K, Bouriez-Jones T, Crockford T, Ferry H, Tang HL, Cyster JG, Cornall RJ. 2006. Spontaneous class switching and B cell hyperactivity increase autoimmunity against intracellular self antigen in Lyn-deficient mice. Eur J Immunol, 36 (11), pp. 2920-2927. | Show Abstract | Read more

IgG autoantibodies cause pathology due to their ability to bind self antigens. However, the extent to which the initial B cell activation and isotype switching is antigen-driven is unclear and it has been widely proposed that intrinsic B cell hyperactivity may be a contributing factor. To explore this issue we generated mice with B cell hyperactivity secondary to deficiency in the src kinase Lyn that also expressed a gene-targeted anti-hen egg lysozyme Ig construct (VDJkappa) capable of class switching to all isotypes. The B cell hyperactivity caused spontaneous hypersecretion of antibodies and class switching to IgM, IgA, IgG1 and IgG3 isotypes in the absence of self antigen, and this persisted as an autoimmune phenomenon in the presence of intracellularly expressed hen egg lysozyme. Exaggerated class switching was also unaffected by antigen in vitro. These findings show that systemic high-avidity intracellular self antigens do not induce self tolerance in the face of B cell hyperactivity. Under these circumstances, spontaneous activation of hyperactive B cells leads to isotype switching and the development of high titres of IgG autoantibodies against intracellular proteins.

Lambe T, Leung JC, Bouriez-Jones T, Silver K, Makinen K, Crockford TL, Ferry H, Forrester JV, Cornall RJ. 2006. CD4 T cell-dependent autoimmunity against a melanocyte neoantigen induces spontaneous vitiligo and depends upon Fas-Fas ligand interactions. J Immunol, 177 (5), pp. 3055-3062. | Show Abstract

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.

Nijnik A, Ferry H, Lewis G, Rapsomaniki E, Leung JC, Daser A, Lambe T, Goodnow CC, Cornall RJ. 2006. Spontaneous B cell hyperactivity in autoimmune-prone MRL mice. Int Immunol, 18 (7), pp. 1127-1137. | Show Abstract | Read more

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.

Ferry H, Crockford TL, Leung JC, Cornall RJ. 2006. Signals from a self-antigen induce positive selection in early B cell ontogeny but are tolerogenic in adults. J Immunol, 176 (12), pp. 7402-7411. | Show Abstract

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.

Silver K, Ferry H, Crockford T, Cornall RJ. 2006. TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells into the primary repertoire. Eur J Immunol, 36 (6), pp. 1404-1412. | Show Abstract | Read more

Toll-like receptors (TLR) have been shown to play an essential role in the generation of autoantibodies in mouse models of autoimmunity, but the timing and context of these effects are poorly understood. One hypothesis is that TLR ligands assist in the positive selection of self-reactive B cells into the primary repertoire and, in this way, distinguish between immunogenic and tolerogenic forms of self-antigen. To explore this idea we generated hen egg lysozyme-specific immunoglobulin (Ig(HEL)) and isotype class-switching anti-HEL mice deficient in MyD88, TLR4 or TLR9 signalling and studied B cell development and autoantibody secretion in the presence or absence of an intracellular form of self-antigen HEL that positively selects B1 cells. Our findings show that TLR4, TLR9 and MyD88 are not required for the positive selection of autoreactive B cells in the primary B cell repertoire, nor is MyD88 required for the generation of isotype-switched antibodies in the absence of antigen. These results suggest that the significant effects of TLR on autoimmunity occur in the established repertoire and not during B cell development.

Ferry H, Leung JC, Lewis G, Nijnik A, Silver K, Lambe T, Cornall RJ. 2006. B-cell tolerance. Transplantation, 81 (3), pp. 308-315. | Show Abstract | Read more

Autoreactive B cells are actively tolerized to more abundant self-antigens by a series of checkpoints involving receptor editing, deletion, anergy and competition for growth factors. In contrast, B cells reactive against rare, sequestered or tissue specific self-antigens remain functionally naïve. During an immune response, the autoimmune danger from these cells is countered by a variety of mechanisms comprising control of self-antigen presentation, limitation of immunogenic and tolerogenic costimuli including T cell help, homeostatic control of growth and strict regulation of germinal centre reactions. In this overview we consider how knowledge of these checkpoints may be used to gain a better understanding of transplant tolerance and the generation of alloantibodies.

Ferry H, Crockford TL, Silver K, Rust N, Goodnow CC, Cornall RJ. 2005. Analysis of Lyn/CD22 double-deficient B cells in vivo demonstrates Lyn- and CD22-independent pathways affecting BCR regulation and B cell survival (vol 35, pg 3655, 2005) EUROPEAN JOURNAL OF IMMUNOLOGY, 35 (12), pp. 3714-3714. | Read more

Ferry H, Crockford TL, Silver K, Rust N, Goodnow CC, Cornall RJ. 2005. 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, 35 (12), pp. 3655-3663. | Show Abstract | Read more

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.

Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, Hill KM, Yu D, Domaschenz H, Whittle B, Lambe T et al. 2005. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature, 435 (7041), pp. 452-458. | Show Abstract | Read more

Despite the sequencing of the human and mouse genomes, few genetic mechanisms for protecting against autoimmune disease are currently known. Here we systematically screen the mouse genome for autoimmune regulators to isolate a mouse strain, sanroque, with severe autoimmune disease resulting from a single recessive defect in a previously unknown mechanism for repressing antibody responses to self. The sanroque mutation acts within mature T cells to cause formation of excessive numbers of follicular helper T cells and germinal centres. The mutation disrupts a repressor of ICOS, an essential co-stimulatory receptor for follicular T cells, and results in excessive production of the cytokine interleukin-21. sanroque mice fail to repress diabetes-causing T cells, and develop high titres of autoantibodies and a pattern of pathology consistent with lupus. The causative mutation is in a gene of previously unknown function, roquin (Rc3h1), which encodes a highly conserved member of the RING-type ubiquitin ligase protein family. The Roquin protein is distinguished by the presence of a CCCH zinc-finger found in RNA-binding proteins, and localization to cytosolic RNA granules implicated in regulating messenger RNA translation and stability.

Ferry H, Crockford TL, Silver K, Rust N, Goodnow CC, Cornall RJ. 2005. Correction: Analysis of Lyn/CD22 double-deficient B cellsin vivo demonstrates Lyn- and CD22-independent pathways affecting BCR regulation and B cell survival. European Journal of Immunology, 35 (12), pp. 3714-3714. | Read more

Lewis G, Rapsomaniki E, Bouriez T, Crockford T, Ferry H, Rigby R, Vyse T, Lambe T, Cornall R. 2004. Hyper IgE in New Zealand black mice due to a dominant-negative CD23 mutation. Immunogenetics, 56 (8), pp. 564-571. | Show Abstract | Read more

Immunoglobulin E (IgE) plays a critical role in both resistance to parasitic infection and allergy to environmental antigens. The IgE response is in turn regulated by the B-cell co-receptor CD23, and CD23-deficient mice show exaggerated IgE responses and airway hyper-responsiveness. In this report, we show that New Zealand black (NZB) mice express a variant CD23 allele, with mutations in both the C-lectin-binding domain and stalk region, which fails to bind IgE at high affinity and has reduced expression on the cell surface. Expression of the variant CD23 chain interferes with trimerisation of the receptor and has a dominant-negative effect leading to reduced IgE binding in crosses between NZB and other strains. Genetic mapping shows that the variant CD23 leads to an exaggerated primary IgE response, which is independent of other strain-specific effects. These results suggest that NZB mice or mice carrying the variant allele will be useful models for studying both allergy and quantitative traits associated with atopy. The exaggerated IgE response provides an explanation for the natural resistance of NZB mice to parasitic infection by Leishmania.

Ferry H, Cornall RJ. 2004. Analysis of B-cell immune tolerance induction using transgenic mice. Methods Mol Biol, 271 pp. 239-260. | Show Abstract | Read more

Over the past 15 yr, the use of transgenic mice has led to significant advances in our understanding of immunological tolerance. In a normal repertoire the number of B cells with a single antigen receptor specificity is very small, making the study of their fate difficult. In contrast, animals that carry transgenes encoding rearranged immunoglobulin genes generate large numbers of B cells that, by the process of allelic exclusion, have an identical specificity. Exploitation of this effect has enabled the mechanisms involved in B-cell tolerance to be explored in some detail. In this review we use the hen egg lysozyme (HEL) model system to illustrate the generation and preparation of a transgene. In our example, we describe the generation of mice expressing HEL as a systemic, intracellular, membrane-bound self-antigen. The same principles and methods apply to immunoglobulin transgenes. We briefly discuss the techniques that could be used to explore mechanisms of tolerance to systemic intracellular antigens in these mice.

Ferry H, Jones M, Vaux DJ, Roberts IS, Cornall RJ. 2003. The cellular location of self-antigen determines the positive and negative selection of autoreactive B cells. J Exp Med, 198 (9), pp. 1415-1425. | Show Abstract | Read more

Systemic autoimmune disease is frequently characterized by the production of autoantibodies against widely expressed intracellular self-antigens, whereas B cell tolerance to ubiquitous and highly expressed extracellular antigens is strictly enforced. To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal. In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic. The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner. By analyzing the impact of differences in subcellular distribution independently from other variables, such as B cell receptor affinity, antigen type, or tissue distribution, we have established that intracellular localization of autoantigen predisposes for autoantibody production. These findings help explain why intracellular antigens are targeted in systemic autoimmune diseases.

Forrester JV, Cornall RJ. 2003. Tolerance and autoimmunity in the eye: a role for CD8 T cells in organ-specific autoimmunity in the retina. Immunology, 110 (3), pp. 293-295. | Read more

Silver K, Cornall RJ. 2003. Isotype control of B cell signaling. Sci STKE, 2003 (184), pp. pe21. | Show Abstract | Read more

The B cell receptor (BCR) consists of an antigen-binding membrane immunoglobulin (mIg) associated with the CD79alpha and CD79beta heterodimer. Naïve B cells express the IgM and IgD isotypes, which have very short cytoplasmic tails and therefore depend on CD79alpha and CD79beta for signal transduction. After antigenic stimulation, B cells undergo isotype switching to yield IgG, IgE, or IgA. Recent research suggests that the ability of the B cell coreceptor CD22 to regulate BCR signaling depends on the isotype of the mIg cytoplasmic tail. Cell lines that express a BCR with the cytoplasmic tail from IgG, the isotype found in memory B cells, are not subject to CD22 regulation, whereas cell lines that express BCRs with IgM cytoplasmic tails are subject to CD22 regulation. Moreover, stimulation through BCRs containing an IgG cytoplasmic tail causes increased numbers of antigen-specific clones to accumulate. These observations are a valuable step toward understanding the difference in B cell signaling between na ve and memory cells. Here, we discuss the implications of these findings for CD22 regulation and signaling through the mIgG-containing BCR.

Ngo VN, Cornall RJ, Cyster JG. 2001. Splenic T zone development is B cell dependent. J Exp Med, 194 (11), pp. 1649-1660. | Show Abstract | Read more

The factors regulating growth and patterning of the spleen are poorly defined. We demonstrate here that spleens from B cell-deficient mice have 10-fold reduced expression of the T zone chemokine, CCL21, a threefold reduction in T cell and dendritic cell (DC) numbers, and reduced expression of the T zone stromal marker, gp38. Using cell transfer and receptor blocking approaches, we provide evidence that B cells play a critical role in the early postnatal development of the splenic T zone. This process involves B cell expression of lymphotoxin (LT)alpha1beta2, a cytokine that is required for expression of CCL21 and gp38. Introduction of a B cell specific LTalpha transgene on to the LTalpha-deficient background restored splenic CCL21 and gp38 expression, DC numbers, and T zone size. This work also demonstrates that the role of B cells in T zone development is distinct from the effect of B cells on splenic T cell numbers, which does not require LTalpha1beta2. Therefore, B cells influence spleen T zone development by providing: (a) signals that promote T cell accumulation, and: (b) signals, including LTalpha1beta2, that promote stromal cell development and DC accumulation. Defects in these parameters may contribute to the immune defects associated with B cell deficiency in mice and humans.

Cutler AJ, Cornall RJ, Ferry H, Manderson AP, Botto M, Walport MJ. 2001. Intact B cell tolerance in the absence of the first component of the classical complement pathway. Eur J Immunol, 31 (7), pp. 2087-2093. | Show Abstract | Read more

A critical role for complement in the regulation of self tolerance has been proposed to explain the strong association between complement deficiency and autoimmunity. To elucidate the role of the classical pathway of complement in the maintenance of B cell tolerance, C1q-deficient (C1qa-/-) mice were bred with anti-hen egg lysozyme (HEL) immunoglobulin (Ig(HEL)) and soluble HEL (sHEL) transgenic mice. B cell tolerance was intact in C1qa-/- mice. In vivo, double-transgenic (Ig(HEL)/sHEL) C1qa-/- and wild-type control mice down-regulated surface immunoglobulin expression on splenocytes and equivalent numbers of HEL-binding B cells accumulated in the periphery. Maturation of B cells, evidenced by CD21 expression, was retarded to the same extent and at a similar time point. The frequency of anti-HEL-producing plasma cells and serum levels of anti-HEL immunoglobulin were comparably reduced in control and C1qa-/- double-transgenic mice compared to control Ig(HEL) and C1qa-/- Ig(HEL) mice. Furthermore, splenocytes from double-transgenic C1qa-/- or wild-type mice did not modulate intracellular calcium levels after stimulation with HEL in vitro. These data demonstrate that a stable form of B cell anergy persists in the periphery of C1qa-/- mice, suggesting that activation of the classical pathway by C1q is not essential for the maintenance of B cell tolerance in this transgenic model.

Townsend SE, Goodnow CC, Cornall RJ. 2001. Single epitope multiple staining to detect ultralow frequency B cells. J Immunol Methods, 249 (1-2), pp. 137-146. | Show Abstract | Read more

Here we describe a method for detecting ultralow frequency target cells from within a high background of irrelevant cells by a novel method, single epitope multiple staining (SEMS). Samples of murine splenocytes were seeded with a low number of splenocytes from mice transgenic for a hen eggwhite lysozyme (HEL)-specific immunoglobulin (Ig). These samples were stained with two reagents specific for the same epitope expressed by the transgenic B cells, which had been conjugated to two different detectable labels (FITC and biotin). This dual staining of a single epitope allowed us to reduce the background due both to non-specific binding of reagents and to probabilistic distribution of the cells. We also were able to detect the cells based on knowing only one thing about them, namely, their antigen specificity. The SEMS method allowed us to reproducibly detect transgenic cells at frequencies below one cell in one million cells. SEMS could be used to increase the sensitivity of numerous fluorescence-based applications in addition to the detection and isolation of antigen-specific lymphocytes, including the detection and highly specific isolation of genetically modified cells, transformed cells, stem cells, fetal cells, or infectious organisms.

Cornall RJ, Cheng AM, Pawson T, Goodnow CC. 2000. Role of Syk in B-cell development and antigen-receptor signaling. Proc Natl Acad Sci U S A, 97 (4), pp. 1713-1718. | Show Abstract | Read more

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles-promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79alpha/beta BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

Quek LS, Pasquet JM, Hers I, Cornall R, Knight G, Barnes M, Hibbs ML, Dunn AR, Lowell CA, Watson SP. 2000. Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway. Blood, 96 (13), pp. 4246-4253. | Show Abstract

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor gamma (FcR gamma chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn(-/-) platelets, tyrosine phosphorylation of FcR gamma chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn(-/-) platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and alpha-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn(-/-)lyn(-/-) double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLC gamma 2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway. (Blood. 2000;96:4246-4253)

Cutler AJ, Cornall R, Street H, Botto M, Walport MJ. 1999. B cell tolerance is intact in C1q deficient mice MOLECULAR IMMUNOLOGY, 36 (4-5), pp. 288-288.

Cornall RJ, Goodnow CC, Cyster JG. 1999. Regulation of B cell antigen receptor signaling by the Lyn/CD22/SHP1 pathway. Curr Top Microbiol Immunol, 244 pp. 57-68.

Cornall RJ, Cyster JG, Hibbs ML, Dunn AR, Otipoby KL, Clark EA, Goodnow CC. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1 are limiting elements of a biochemical pathway regulating BCR signaling and selection. Immunity, 8 (4), pp. 497-508. | Show Abstract | Read more

A B lymphocyte hyperactivity syndrome resembling systemic lupus erythematosus characterizes mice lacking the src-family kinase Lyn. Lyn is not required to initiate B cell antigen receptor (BCR) signaling but is an essential inhibitory component. lyn-/- B cells have a delayed but increased calcium flux and exaggerated negative selection responses in the presence of antigen and spontaneous hyperactivity in the absence of antigen. As in invertebrates, genetic effects of loci with only one functional allele can be used to analyze signaling networks in mice, demonstrating that negative regulation of the BCR is a complex quantitative trait in which Lyn, the coreceptor CD22, and the tyrosine phosphatase SHP-1 are each limiting elements. The biochemical basis of this complex trait involves a pathway requiring Lyn to phosphorylate CD22 and recruit SHP-1 to the CD22/BCR complex.

Cornall RJ, Goodnow CC, Cyster JG. 1995. The regulation of self-reactive B cells. Curr Opin Immunol, 7 (6), pp. 804-811. | Show Abstract | Read more

Self-reactive B cells are eliminated in a series of checkpoints that are triggered by antigen binding. Recent reports have shown that in addition to the processes of elimination at the immature B-cell stage, B-cell anergy and regulation of T-cell help, self-reactive cells are also controlled by follicular competition, Fas-mediated elimination by T cells and censoring in the germinal centres. Each checkpoint operates at a threshold that reflects the need to maintain immune diversity at the same time as suppressing autoimmune disease. Analysis of the motheaten mutation has given a direct demonstration of how such thresholds can be modulated by genetic effects.

Copeman JB, Cucca F, Hearne CM, Cornall RJ, Reed PW, Rønningen KS, Undlien DE, Nisticò L, Buzzetti R, Tosi R. 1995. Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33. Nat Genet, 9 (1), pp. 80-85. | Show Abstract | Read more

The role of human chromosome 2 in type 1 diabetes was evaluated by analysing linkage and linkage disequilibrium at 21 microsatellite marker loci, using 348 affected sibpair families and 107 simplex families. The microsatellite D2S152 was linked to, and associated with, disease in families from three different populations. Our evidence localizes a new diabetes susceptibility gene, IDDM7, to within two centiMorgans of D2S152. This places it in a region of chromosome 2q that shows conserved synteny with the region of mouse chromosome 1 containing the murine type 1 diabetes gene, Idd5. These results demonstrate the utility of polymorphic microsatellites for linkage disequilibrium mapping of genes for complex diseases.

Rodrigues NR, Cornall RJ, Chandler P, Simpson E, Wicker LS, Peterson LB, Todd JA. 1994. Mapping of an insulin-dependent diabetes locus, Idd9, in NOD mice to chromosome 4. Mamm Genome, 5 (3), pp. 167-170. | Read more

Ghosh S, Palmer SM, Rodrigues NR, Cordell HJ, Hearne CM, Cornall RJ, Prins JB, McShane P, Lathrop GM, Peterson LB. 1993. Polygenic control of autoimmune diabetes in nonobese diabetic mice. Nat Genet, 4 (4), pp. 404-409. | Show Abstract | Read more

Partial exclusion mapping of the nonobese (NOD) diabetic mouse genome has shown linkage of diabetes to at least five different chromosomes. We have now excluded almost all of the genome for the presence of susceptibility genes with fully recessive effects and have obtained evidence of linkage of ten distinct loci to diabetes or the prediabetic lesion, insulitis, indicative of a polygenic mode of inheritance. The relative importance of these loci and their interactions have been assessed using a new application of multiple polychotomous regression methods. A candidate disease gene, interleukin-2 (Il-2), which is closely linked to insulitis and diabetes, is shown to have a different sequence in NOD, including an insertion and a deletion of tandem repeat sequences which encode amino acid repeats in the mature protein.

Cornall RJ. 1993. Genetics of a multifactorial disease: autoimmune type 1 diabetes mellitus. Clin Sci (Lond), 84 (3), pp. 257-262. | Show Abstract | Read more

1. Three non-major histocompatibility complex genes, Idd-3, Idd-4 and Idd-5, that influence the onset of autoimmune type 1 diabetes in the non-obese diabetic mouse have been located on chromosomes 3, 11 and 1. 2. In undertaking this study, a new map of the mouse genome has been created from polymerase chain reaction-analysable mouse microsatellite markers. 3. The homologues of these genes may reside on human chromosomes 1 or 4 (Idd-3), 17 (Idd-4) and 2q (Idd-5).

Todd JA, Reed PW, Prins JB, Bain SC, Palmer SM, Cordell HJ, Pritchard LE, Ghosh S, Cornall RJ, Aitman TJ. 1993. Dissection of the pathophysiology of type 1 diabetes by genetic analysis. Autoimmunity, 15 Suppl (sup1), pp. 16-17. | Read more

McAleer MA, Aitman TJ, Cornall RJ, Ghosh S, Hall JR, Hearne CM, Love JM, Prins JB, Ramachandran S, Rodrigues N. 1992. Linkage analysis of 84 microsatellite markers in intra- and interspecific backcrosses. Mamm Genome, 3 (8), pp. 457-460. | Read more

Cornall RJ, Friedman JM, Todd JA. 1992. Mouse microsatellites from a flow-sorted 4:6 Robertsonian chromosome. Mamm Genome, 3 (11), pp. 620-624. | Show Abstract | Read more

Twenty microsatellites were generated from a previously characterized lambda gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.

Cornall RJ, Prins JB, Todd JA, Pressey A, DeLarato NH, Wicker LS, Peterson LB. 1991. Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1. Nature, 353 (6341), pp. 262-265. | Show Abstract | Read more

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.

Cornall RJ, Aitman TJ, Hearne CM, Todd JA. 1991. The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome. Genomics, 10 (4), pp. 874-881. | Show Abstract | Read more

Forty-three sequences containing simple sequence repeats or microsatellites were generated from an M13 library of total genomic mouse DNA. These sequences were analyzed for size variation using the polymerase chain reaction and gel electrophoresis without the need for radiolabeling. Seventy-two percent of the sequences showed allelic size variations between different inbred strains of mouse and the wild mouse, Mus spretus; and 53% showed variation between inbred strains. Thirty-seven percent were variant between B6/J and DBA/2J, and 81% of these were resolved using minigel agarose electrophoresis alone. This approach is a useful way of generating the large number of variants that are needed to create high resolution maps of the mouse genome.

Todd JA, Aitman TJ, Cornall RJ, Ghosh S, Hall JR, Hearne CM, Knight AM, Love JM, McAleer MA, Prins JB. 1991. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. Nature, 351 (6327), pp. 542-547. | Show Abstract | Read more

Two genes, Idd-3 and Idd-4, that influence the onset of autoimmune type 1 diabetes in the nonobese diabetic mouse have been located on chromosomes 3 and 11, outside the chromosome 17 major histocompatibility complex. A genetic map of the mouse genome, analysed using the polymerase chain reaction, has been assembled specifically for the study. On the basis of comparative maps of the mouse and human genomes, the homologue of Idd-3 may reside on human chromosomes 1 or 4 and Idd-4 on chromosome 17.

Hearne CM, McAleer MA, Love JM, Aitman TJ, Cornall RJ, Ghosh S, Knight AM, Prins JB, Todd JA. 1991. Additional microsatellite markers for mouse genome mapping. Mamm Genome, 1 (4), pp. 273-282. | Show Abstract | Read more

Mouse sequence information from the EMBL and GenBank databases, published sequences and genomic clones have been analyzed for simple repetitive elements or microsatellites. Each microsatellite has been amplified by the polymerase chain reaction (PCR) as a single locus marker. PCR primers were designed from unique sequence flanking each repeat. Size variation of PCR products less than 750 base pairs (bp) between mouse strains has been determined using ethidium bromide-stained acrylamide or agarose gels. A further 74 newly characterized microsatellites are presented in this paper, bringing to 185 the total we have analyzed. Of these, 157/185 (85%) have more than one allele, 143/178 (80%) vary in length between C57BL/6J and Mus spretus, and 82/168 (49%) vary between DBA/2J and C57BL/6J. Microsatellites provide informative single locus probes for linkage analysis in the construction of a genetic map of the mouse genome.

PRINS J, AITMAN T, CORNALL R, GOSH S, HALL J, HEARNE C, KNIGHT A, LOVE J, MCALEER M, RODRIGUES N et al. 1991. THE LOCALIZATION OF 2 SUSCEPTIBILITY GENES FOR INSULIN-DEPENDENT DIABETES IN THE NONOBESE DIABETIC MOUSE CYTOGENETICS AND CELL GENETICS, 58 (3-4), pp. 2137-2138.

Yavari A, Bellahcene M, Bucchi A, Sirenko S, Pinter K, Herring N, Jung JJ, Tarasov KV, Sharpe EJ, Wolfien M et al. 2017. Mammalian γ2 AMPK regulates intrinsic heart rate. Nat Commun, 8 (1), pp. 1258. | Show Abstract | Read more

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca(2+) release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

Bull KR, Rimmer AJ, Siggs OM, Miosge LA, Roots CM, Enders A, Bertram EM, Crockford TL, Whittle B, Potter PK et al. 2013. Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations. PLoS Genet, 9 (1), pp. e1003219. | Show Abstract | Read more

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.

Randall KL, Lambe T, Johnson AL, Treanor B, Kucharska E, Domaschenz H, Whittle B, Tze LE, Enders A, Crockford TL et al. 2009. Dock8 mutations cripple B cell immunological synapses, germinal centers and long-lived antibody production. Nat Immunol, 10 (12), pp. 1283-1291. | Show Abstract | Read more

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.

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L et al. 2009. Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol, 10 (8), pp. 831-839. | Show Abstract | Read more

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.

Nijnik A, Dawson S, Crockford TL, Woodbine L, Visetnoi S, Bennett S, Jones M, Turner GD, Jeggo PA, Goodnow CC, Cornall RJ. 2009. Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome. J Clin Invest, 119 (6), pp. 1696-1705. | Show Abstract | Read more

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.

Nijnik A, Woodbine L, Marchetti C, Dawson S, Lambe T, Liu C, Rodrigues NP, Crockford TL, Cabuy E, Vindigni A et al. 2007. DNA repair is limiting for haematopoietic stem cells during ageing. Nature, 447 (7145), pp. 686-690. | Show Abstract | Read more

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.

Lambe T, Leung JC, Ferry H, Bouriez-Jones T, Makinen K, Crockford TL, Jiang HR, Nickerson JM, Peltonen L, Forrester JV, Cornall RJ. 2007. Limited peripheral T cell anergy predisposes to retinal autoimmunity. J Immunol, 178 (7), pp. 4276-4283. | Show Abstract

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.

Ferry H, Crockford TL, Leung JC, Cornall RJ. 2006. Signals from a self-antigen induce positive selection in early B cell ontogeny but are tolerogenic in adults. J Immunol, 176 (12), pp. 7402-7411. | Show Abstract

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.

Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, Hill KM, Yu D, Domaschenz H, Whittle B, Lambe T et al. 2005. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature, 435 (7041), pp. 452-458. | Show Abstract | Read more

Despite the sequencing of the human and mouse genomes, few genetic mechanisms for protecting against autoimmune disease are currently known. Here we systematically screen the mouse genome for autoimmune regulators to isolate a mouse strain, sanroque, with severe autoimmune disease resulting from a single recessive defect in a previously unknown mechanism for repressing antibody responses to self. The sanroque mutation acts within mature T cells to cause formation of excessive numbers of follicular helper T cells and germinal centres. The mutation disrupts a repressor of ICOS, an essential co-stimulatory receptor for follicular T cells, and results in excessive production of the cytokine interleukin-21. sanroque mice fail to repress diabetes-causing T cells, and develop high titres of autoantibodies and a pattern of pathology consistent with lupus. The causative mutation is in a gene of previously unknown function, roquin (Rc3h1), which encodes a highly conserved member of the RING-type ubiquitin ligase protein family. The Roquin protein is distinguished by the presence of a CCCH zinc-finger found in RNA-binding proteins, and localization to cytosolic RNA granules implicated in regulating messenger RNA translation and stability.

Ferry H, Jones M, Vaux DJ, Roberts IS, Cornall RJ. 2003. The cellular location of self-antigen determines the positive and negative selection of autoreactive B cells. J Exp Med, 198 (9), pp. 1415-1425. | Show Abstract | Read more

Systemic autoimmune disease is frequently characterized by the production of autoantibodies against widely expressed intracellular self-antigens, whereas B cell tolerance to ubiquitous and highly expressed extracellular antigens is strictly enforced. To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal. In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic. The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner. By analyzing the impact of differences in subcellular distribution independently from other variables, such as B cell receptor affinity, antigen type, or tissue distribution, we have established that intracellular localization of autoantigen predisposes for autoantibody production. These findings help explain why intracellular antigens are targeted in systemic autoimmune diseases.

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