Publications by authors named "Lee Garner"

11 Publications

  • Page 1 of 1

T cell renaissance in COVID-19.

Nat Rev Immunol 2020 09;20(9):518

OxImmuno Literature Initiative, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1038/s41577-020-0401-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351537PMC
September 2020

CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action.

Immunology 2018 10 13;155(2):273-282. Epub 2018 Jun 13.

Sir William Dunn School of Pathology, Oxford, UK.

CD6 is a type I T-cell surface receptor that modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab™), which has reached the clinic for treatment of autoimmune disease. We characterized molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action. Epitope mapping using the crystal structure of CD6 to design mutants identified two distinct binding sites on different faces of domain 1, one containing residue R77, crucial for MT605 and T12.1 binding and the other, E63, which is crucial for itolizumab and MEM98. Analysis of binding kinetics revealed that itolizumab has a lower affinity compared with other CD6 domain 1 mAbs. We compared potential agonistic (triggering) and antagonistic (blocking) properties of CD6 mAbs in assays where the mechanism of action was well defined. CD6 domain 1 and 3 mAbs were equally effective in triggering interleukin-2 production by a cell line expressing a chimeric antigen receptor containing the extracellular region of CD6. CD6 domain 1 mAbs hindered binding of multivalent immobilized CD166 but were inferior compared with blocking by soluble CD166 or a CD6 domain 3 mAb. Characterization of CD6 mAbs provides an insight into how their functional effects in vivo may be interpreted and their therapeutic use optimized.
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http://dx.doi.org/10.1111/imm.12952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142284PMC
October 2018

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

Proc Natl Acad Sci U S A 2016 May 2;113(20):E2766-75. Epub 2016 May 2.

Tumour Growth Control Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.
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http://dx.doi.org/10.1073/pnas.1513023113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878476PMC
May 2016

A polymorphism in a phosphotyrosine signalling motif of CD229 (Ly9, SLAMF3) alters SH2 domain binding and T-cell activation.

Immunology 2015 Nov 13;146(3):392-400. Epub 2015 Sep 13.

Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome-wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val602 variant of the non-synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two-fold lower affinity compared with the SLE-associated Met602 variant for the small adaptor protein SAP. Comparison of the two variants in T-cell lines revealed the Val602 variant to be significantly more highly expressed than CD229 Met602 . Activation was diminished in cells expressing CD229 Val602 compared with CD229 Met602 as measured by up-regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine-tuning in the immune system with significant functional consequences.
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http://dx.doi.org/10.1111/imm.12513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610628PMC
November 2015

Structures of CD6 and Its Ligand CD166 Give Insight into Their Interaction.

Structure 2015 Aug 2;23(8):1426-1436. Epub 2015 Jul 2.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.
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http://dx.doi.org/10.1016/j.str.2015.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533223PMC
August 2015

Fine specificity and molecular competition in SLAM family receptor signalling.

PLoS One 2014 18;9(3):e92184. Epub 2014 Mar 18.

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092184PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958483PMC
December 2014

Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia.

Blood 2012 Jun 27;119(25):5955-62. Epub 2012 Mar 27.

Department of Pathology and Laboratory Medicine, Pearlman School of Medicine, University of Pennsylvania, 605A Stellar-Chance Labs, 422 Curie Blvd, Philadelphia, PA 19104, USA.

Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and platelet factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4(K50E) forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.
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http://dx.doi.org/10.1182/blood-2012-01-406801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383011PMC
June 2012

Crystal structure of leukocyte Ig-like receptor LILRB4 (ILT3/LIR-5/CD85k): a myeloid inhibitory receptor involved in immune tolerance.

J Biol Chem 2011 May 30;286(20):18013-25. Epub 2011 Mar 30.

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.

The myeloid inhibitory receptor LILRB4 (also called ILT3, LIR-5, CD85k), a member of the leukocyte immunoglobulin-like receptors (LILRs/LIRs), is an important mediator of immune tolerance. Up-regulated on tolerogenic dendritic cells, it has been shown to modulate immune responses via induction of T cell anergy and differentiation of CD8(+) T suppressor cells and may play a role in establishing immune tolerance in cancer. Consequently, characterizing the molecular mechanisms involved in LILRB4 function and in particular its structure and ligands is a key aim but has remained elusive to date. Here we describe the production, crystallization, and structure of the LILRB4 ectodomain to 1.7 Å using an expression strategy involving engineering of an additional disulfide bond in the D2 domain to enhance protein stability. LILRB4 comprises two immunoglobulin domains similar in structure to other LILRs; however, the D2 domain, which is most closely related to the D4 domains of other family members, contains 3(10) helices not previously observed. At the D1-D2 interface, reduced interdomain contacts resulted in an obtuse interdomain angle of ∼107°. Comparison with MHC class I binding Group 1 LILRs suggests LILRB4 is both conformationally and electrostatically unsuited to MHC ligation, consistent with LILRB4 status as a Group 2 LILR likely to bind novel non-MHC class I ligands. Finally, examination of the LILRB4 surface highlighted distinctive surface patches on the D1 domain and D1D2 hinge region, which may be involved in ligand binding. These findings will facilitate our attempts to precisely define the role of LILRB4 in the regulation of immune tolerance.
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http://dx.doi.org/10.1074/jbc.M111.221028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093875PMC
May 2011

Expression, purification, and refolding of the myeloid inhibitory receptor leukocyte immunoglobulin-like receptor-5 for structural and ligand identification studies.

Protein Expr Purif 2006 Jun 20;47(2):490-7. Epub 2005 Dec 20.

Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, UK.

The leukocyte immunoglobulin-like receptors (LIRs, also known as ILTs, CD85, and LILRs) comprise a family of related immunoregulatory receptors encoded within the leukocyte receptor cluster (LRC) on human chromosome 19. LIRs are transmembrane proteins containing either two or four extracellular immunoglobulin domains, and most family members are expressed predominantly on myeloid cell lineages. Although the inhibitory receptors LIR-1 and LIR-2 are known to bind to a broad range of class I MHC molecules and are thought to play important roles in immune regulation, the majority of LIRs are currently of unknown structure and their ligands remain unidentified. In this study, we describe recombinant production and characterisation of the extracellular portion of LIR-5 (ILT3), a poorly understood inhibitory receptor that transduces tolerising signals to dendritic cells. The two extracellular immunoglobulin domains of LIR-5 were expressed in Escherichia coli to a high level and were found to accumulate in inclusion bodies. Inclusion bodies were purified, solubilised, and receptor then renatured by dilution refolding, with acceptable yields. Size exclusion chromatography and SDS-PAGE analyses confirmed the extracellular portion behaved as a monomer in solution, and purified protein was antibody-reactive. LIR-5 is representative of a subset of LIR receptors that on the basis of structural and sequence comparisons with LIR-1 seem unlikely to bind class I MHC molecules. Successful prokaryotic generation of correctly folded LIR-5 in high levels has implications for production of other LRC receptors and should greatly facilitate attempts to define the structure and ligands of this important regulator of dendritic cell function.
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http://dx.doi.org/10.1016/j.pep.2005.11.020DOI Listing
June 2006

Virus-specific cytotoxic T lymphocytes differentially express cell-surface leukocyte immunoglobulin-like receptor-1, an inhibitory receptor for class I major histocompatibility complex molecules.

J Infect Dis 2005 Jun 26;191(11):1842-53. Epub 2005 Apr 26.

Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom.

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8+ T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection.
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http://dx.doi.org/10.1086/429927DOI Listing
June 2005

New texico?

Authors:
W Lee Garner

Science 1977 Sep;197(4307):940

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http://dx.doi.org/10.1126/science.197.4307.940-bDOI Listing
September 1977
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