Prof Alain R Townsend FRS FRCP

Research Area: Cell and Molecular Biology
Technology Exchange: Cell sorting, Cellular immunology, Flow cytometry, Immunohistochemistry, Protein interaction and Vaccine production and evaluation
Scientific Themes: Immunology and Translational Medicine & Medical Technology
Keywords: Influenza, Live Attenuated Influenza Vaccine, Therapeutic Human Monoclonal Antibody, Class I Molecules and Influenza Evolution
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Most of my work has been concerned with the presentation of Influenza antigens with class I molecules of the Major Histocompatibility complex. In the past we identified the major targets for T cells as the conserved nucleoprotein and matrix protein components of the virus and demonstrated that a system of cytosolic antigen presentation exists that passes peptides derived from these proteins into the ER where they bind to class I MHC molecules. With the recent pandemic this interest continues with a practical extension into the issue of whether heterotypic immunity (between pandemic strains) can be induced in man with live attenuated strains of influenza. We have developed our own design of live attenuated virus called S-FLU, that relies on mutations in the haemagglutinin signal sequence that are permissive for infection but prevent replication of the virus. The advantage of this approach is that all of the viral proteins are expressed in their appropriate context in the lung, and thus can induce a full set of local T and B cell responses. We are deveoping methods to deliver the vaccine virus by aerosol in collaboration with Ronan Mac Loughlin at Aerogen. Preliminary results in collaboration with Dr Kanta Subbarao (NIH) show that our vaccine viruses are capable of preventing illness caused by the most virulent forms of influenza in a murine and ferret infection model, and we are studying responses in the pig as a relevant large animal (in collaboration with Elma Tchillian, Pirbright). We are presently investigating the mechanisms of this immunity in the pig . 

As part of a broader interest in the link between immunity and the evolution of seasonal influenza, we are isolating human monoclonal antibodies that neutralise the virus. We are finding that the human antibody response can often be focused on local regions of the haemagglutinin and neuraminidase molecules to such an extent that it may select point mutations and thus drive antigenic drift. In collaboration with John McCauley and Rod Daniels at the Crick Worldwide Influenza Centre we are developing sets of representative human monoclonal antibodies that select relevant mutations in the haemagglutinin and neuraminidase as reagents to monotor antigenic drift to help improve the selection of appropriate viruses for inclusion in the subunit vaccine.

An additional aim for this project is to build a library of neutralising antibodies that react with highly conserved regions of the haemagglutinin and neuraminidase that can be used as therapeutic agents. To date we have isolated very broadly inhibitory antibodies to the N1, N2 and N9 neuraminidase, and to the H1 and H5 stem regions. Crystal structures of antibodies to N1 and N9 will be used to enhance binding. This work is part of a collaboration with Prof Tao Dong and George Gao in China, and Kuan-Ying Arthur Huang in Taiwan.

 In recent years our interest has extended to the isolation of potentially therapeutic antibodies to Ebola from donors receiving experimental vaccines. We have isolated 82 antibodies to date to several regions of the Ebola Glycoprotein and will be testing cocktails of these as therapies in collaboration with Miles Carroll at Porton Down.

Students will have the opportunity of working with all of our collaborators.

Name Department Institution Country
Dr Ervin Fodor Dunn School of Pathology University of Oxford United Kingdom
Xiao J, Rijal P, Schimanski L, Tharkeshwar AK, Wright E, Annaert W, Townsend A. 2017. Characterization of an influenza virus pseudotyped with Ebolavirus glycoprotein. J Virol, | Show Abstract | Read more

We have produced a new Ebola virus pseudotype: E-S-FLU, which can be handled in biosafety level-1/2 containment for laboratory analysis. E-S-FLU is a single cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza haemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU production. Infection of cells by E-S-FLU was dependent on Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. E-S-FLU was neutralized specifically by anti-Ebola glycoprotein antibody and a variety of small drug molecules that are known to inhibit entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC®1280 Sigma) of 1280 pharmacologically active compounds for inhibition of virus entry. 215 compounds inhibited E-S-FLU infection, while only 22 inhibited the control H5-S-FLU virus coated in an H5 haemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action e.g. calcium channel blockers, estrogen receptor antagonists, anti-histamines, serotonin uptake inhibitors etc. and this correlates with inhibitor screening results with other pseudotypes or wild-type Ebola virus in the literature. E-S-FLU is a new tool for Ebola virus cell entry studies and is easily applied to high throughput screening assays for small molecule inhibitors or antibodies.Importance Ebola virus is from the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates of Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus, and screen for inhibitors. Here we characterized a new surrogate named E-S-FLU, that is based on a disabled influenza virus core coated with the Ebola virus surface protein, but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU in biosafety level-1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small molecule library of 1280 candidates for inhibitors of viral entry.

Huang K-YA, Rijal P, Schimanski L, Powell TJ, Lin T-Y, McCauley JW, Daniels RS, Townsend AR. 2015. Focused antibody response to influenza linked to antigenic drift. J Clin Invest, 125 (7), pp. 2631-2645. | Show Abstract | Read more

The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.

Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, Watson AS, Cerundolo V, Townsend AR, Klenerman P, Simon AK. 2014. Autophagy is a critical regulator of memory CD8(+) T cell formation. Elife, 3 | Show Abstract | Read more

During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

Armitage AE, Gileadi U, Stacey A, Giannoulatou E, Marshall E, Sturges P, Eddowes L, Cerundolo V, Townsend A, Webster C et al. 2013. HEPCIDIN REGULATION DURING ACUTE INFECTIONS AMERICAN JOURNAL OF HEMATOLOGY, 88 (5), pp. E135-E135.

Powell TJ, Silk JD, Sharps J, Fodor E, Townsend ARM. 2012. Pseudotyped influenza A virus as a vaccine for the induction of heterotypic immunity. J Virol, 86 (24), pp. 13397-13406. | Show Abstract | Read more

There is a need for vaccines that can protect broadly across all influenza A strains. We have produced a pseudotyped influenza virus based on suppression of the A/PR/8/34 hemagglutinin signal sequence (S-FLU) that can infect cells and express the viral core proteins and neuraminidase but cannot replicate. We show that when given by inhalation to mice, S-FLU is nonpathogenic but generates a vigorous T cell response in the lung associated with markedly reduced viral titers and weight loss after challenge with H1 and H3 influenza viruses. These properties of S-FLU suggest that it may have potential as a broadly protective A virus vaccine, particularly in the setting of a threatened pandemic before matched subunit vaccines become available.

Armitage AE, Eddowes LA, Gileadi U, Cole S, Spottiswoode N, Selvakumar TA, Ho L-P, Townsend ARM, Drakesmith H. 2011. Hepcidin regulation by innate immune and infectious stimuli. Blood, 118 (15), pp. 4129-4139. | Show Abstract | Read more

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6-dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β-dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

Cited:

125

Scopus

Armitage AE, Eddowes LA, Gileadi U, Cole S, Spottiswoode N, Selvakumar TA, Ho LP, Townsend ARM, Drakesmith H. 2011. Hepcidin regulation by innate immune and infectious stimuli Blood, 118 (15), pp. 4129-4139. | Show Abstract | Read more

Hepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6-independent hepcidin upregulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6- dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6-independent, but partially TGF-β- dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis. © 2011 by The American Society of Hematology.

Lakhal S, Schödel J, Townsend ARM, Pugh CW, Ratcliffe PJ, Mole DR. 2011. Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis. J Biol Chem, 286 (6), pp. 4090-4097. | Show Abstract | Read more

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.

Schimanski LM, Drakesmith H, Sweetland E, Bastin J, Rezgui D, Edelmann M, Kessler B, Merryweather-Clarke AT, Robson KJH, Townsend ARM. 2009. In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor. Blood Cells Mol Dis, 43 (2), pp. 180-193. | Show Abstract | Read more

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.

Lakhal S, Talbot NP, Crosby A, Stoepker C, Townsend ARM, Robbins PA, Pugh CW, Ratcliffe PJ, Mole DR. 2009. Regulation of growth differentiation factor 15 expression by intracellular iron. Blood, 113 (7), pp. 1555-1563. | Show Abstract | Read more

Growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor-beta superfamily and has been identified in different contexts as a hypoxia-inducible gene product and as a molecule involved in hepcidin regulation. The biology of iron and oxygen is closely related, and known regulatory pathways involving hypoxia-inducible factor (HIF) and iron-regulatory proteins (IRPs) are responsive to both these stimuli. We therefore sought to characterize the regulation of GDF15 by iron and oxygen and to define the involvement or otherwise of HIF and IRP pathways. Here we show that GDF15 is strongly up-regulated by stimuli that deplete cells of iron and that this response is specifically antagonized by the reprovision of iron. GDF15 exhibits greater sensitivity to iron depletion than hypoxia, and responses to hypoxia and iron depletion are independent of HIF and IRP activation, suggesting a novel mechanism of regulation. We also report significant induction of serum GDF15 in iron-deficient subjects and after administration of an iron chelator to normal subjects. These findings indicate that GDF15 can be induced by pathophysiologic changes in iron availability, raising important questions about the mechanism of regulation and its role in iron homeostasis.

Lee LY-H, Ha DLA, Simmons C, de Jong MD, Chau NVV, Schumacher R, Peng YC, McMichael AJ, Farrar JJ, Smith GL et al. 2008. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest, 118 (10), pp. 3478-3490. | Show Abstract | Read more

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Schimanski LM, Drakesmith H, Talbott C, Horne K, James JR, Davis SJ, Sweetland E, Bastin J, Cowley D, Townsend ARM. 2008. Ferroportin: lack of evidence for multimers. Blood Cells Mol Dis, 40 (3), pp. 360-369. | Show Abstract | Read more

Ferroportin is a multi-transmembrane glycoprotein that mediates iron export from cells. Mutations in ferroportin are linked to type IV hemochromatosis, a dominantly inherited disorder of iron metabolism. Multimers of ferroportin, whose existence may relate to the dominant inheritance pattern of disease, have been detected in some studies but not others. We looked for evidence of multimerization in several different types of experiment. We assayed the maturation of mutant and wild-type ferroportin and found that loss-of-function mutants had a reduced half-life but did not alter the stability of coexpressed wild-type. Using bioluminescence resonance energy transfer analysis, we tested how mature wild-type ferroportin behaved in intact live cell membranes. Ferroportin-ferroportin interactions gave the very low acceptor/donor ratio-independent energy transfer levels characteristic of random protein-protein interactions, consistent with ferroportin behaving as a monomer. Consistent with these experiments, we were unable to detect a dominant negative functional effect of mutant ferroportin on wild-type, even when expression of wild-type protein was titrated to low levels. These data suggest that dominantly inherited ferroportin disease does not result from the direct action of a mutated protein inhibiting a wild-type protein within multimers. We propose other possible mechanisms of disease.

Trowsdale J, Hanson I, Mockridge I, Beck S, Townsend A, Kelly A. 2008. Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters. 1990. J Immunol, 180 (5), pp. 2733-2736.

Townsend A, Ohlén C, Bastin J, Ljunggren H-G, Foster L, Kärre K. 2007. Pillars article: Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 1989. 340: 443-448. J Immunol, 179 (7), pp. 4301-4306.

Schimanski LM, Drakesmith H, Talbot C, Horne K, James JR, Sweetland E, Bastin JM, Cowley D, Davis SJ, Townsend ARM. 2007. Wild-type ferroportin is not impeded in maturation, cell surface expression or iron export function by mutant ferroportin, and bio-luminescence resonance energy transfer (BRET) experiments indicate monomeric form in cell membranes AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 529-529.

Drakesmith H, Eddowes L, Townsend A. 2007. Investigating the dependency of HIV-1 replication in macrophages on cellular iron AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 509-510.

Bastin J, Drakesmith H, Rees M, Sargent I, Townsend A. 2006. Localisation of proteins of iron metabolism in the human placenta and liver. Br J Haematol, 134 (5), pp. 532-543. | Show Abstract | Read more

Two anatomical sites that are important in human iron metabolism are the liver and placenta. Liver macrophages recycle iron from erythrocytes, and the placenta transfers iron from the mother to the fetus. The cellular distribution of proteins involved in iron transport in these two sites was studied. Transferrin receptor-1 (TfR1) and Ferroportin (FPN) expression was found on the placental syncytiotrophoblast (STB) and were polarised such that TfR1 was on the apical maternal-facing membrane and FPN was on the basal fetal-facing membrane, consistent with unidirectional iron transport from mother to fetus. Ferritin was strongly expressed in the stroma, suggesting that fetal tissue can store and accumulate iron. HFE was on some parts of the basal STB and, where present, HFE clearly colocalised with FPN but not TfR1. In the stroma, both HFE and FPN were present on CD68+ Hofbauer macrophage cells. In liver, the location of HFE is controversial. Using four mouse monoclonals and two polyclonal sera we showed that the pattern of HFE expression mirrored the distribution of CD68+ macrophage Kupffer cells. FPN was also most strongly expressed by CD68+ Kupffer cells. These findings contribute to understanding how iron is transported and stored in the human placenta and liver.

Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 2006. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides (reprinted from Cell, vol. 44, pg. 959-968, yr. 1986) JOURNAL OF IMMUNOLOGY, 176 (9), pp. 5141-5150.

Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 2006. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. 1986. J Immunol, 176 (9), pp. 5141-5150.

Drakesmith H, Chen N, Ledermann H, Screaton G, Townsend A, Xu X-N. 2005. HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis. Proc Natl Acad Sci U S A, 102 (31), pp. 11017-11022. | Show Abstract | Read more

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.

Drakesmith H, Schimanski LM, Ormerod E, Merryweather-Clarke AT, Viprakasit V, Edwards JP, Sweetland E, Bastin JM, Cowley D, Chinthammitr Y et al. 2005. Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin. Blood, 106 (3), pp. 1092-1097. | Show Abstract | Read more

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Schimanski LM, Drakesmith H, Merryweather-Clarke AT, Viprakasit V, Edwards JP, Sweetland E, Bastin JM, Cowley D, Chinthammitr Y, Robson KJH, Townsend ARM. 2005. In vitro functional analysis of human ferroportin (FPN) and hemochromatosis-associated FPN mutations. Blood, 105 (10), pp. 4096-4102. | Show Abstract | Read more

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.

Drakesmith H, Sweetland E, Schimanski L, Edwards J, Cowley D, Ashraf M, Bastin J, Townsend ARM. 2002. The hemochromatosis protein HFE inhibits iron export from macrophages. Proc Natl Acad Sci U S A, 99 (24), pp. 15602-15607. | Show Abstract | Read more

Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, reduces cellular iron by reducing iron uptake. However, in vivo, HFE is strongly expressed by liver macrophages and intestinal crypt cells, which behave as though they are relatively iron-deficient in HH. These latter observations suggest, paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialized cell types. Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the monocytemacrophage cell line THP-1, and extend these results to macrophages derived from healthy individuals and HH patients. In addition, we find that the HH-associated mutant H41D has lost the ability to inhibit iron release despite binding to TfR1 as well as wild-type HFE. Finally, we show that the ability of HFE to block iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive functions, binding to TfR1 in competition with Tf, or inhibition of iron release.

Townsend A, Drakesmith H. 2002. Role of HFE in iron metabolism, hereditary haemochromatosis, anaemia of chronic disease, and secondary iron overload The Lancet, 359 (9308), pp. 786-790. | Read more

Townsend A, Drakesmith H. 2002. Role of HFE in iron metabolism, hereditary haemochromatosis, anaemia of chronic disease, and secondary iron overload. Lancet, 359 (9308), pp. 786-790. | Show Abstract | Read more

Hereditary haemochromatosis is an iron overloading disorder caused by common mutations in the HFE gene. However, information with respect to the function of HFE protein does not explain how mutations in HFE lead to hereditary haemochromatosis. We propose a molecular model in which HFE has two mutually exclusive activities in cells: inhibition of uptake or inhibition of release of iron. The balance between serum transferrin saturation and serum transferrin-receptor concentrations determines which of these functions predominates. With this input, HFE enables the intestinal crypt cells and reticuloendothelial system to interpret the body's iron requirements and regulate iron absorption and distribution. In our model, mutations in HFE result in over absorption of dietary iron, and patterns of tissue iron deposition in agreement with clinical observations of hereditary haemochromatosis.

Drakesmith H, Townsend A. 2000. The structure and function of HFE. Bioessays, 22 (7), pp. 595-598. | Show Abstract | Read more

The iron overload disease hereditary haemochromatosis (HH) occurs in about 1 in 300 Caucasians; the protein mutated in this disorder is termed HFE.(1) HFE is homologous to major histocompatibility complex (MHC) class I proteins, but unlike MHC class I molecules, HFE does not present peptides to T cells.(2) The transferrin receptor (TfR) is a ligand for HFE, and the crystal structure of the HFE-TfR complex has been determined.(3) The many interesting features of this structure illustrate the diverse roles of the MHC fold in nature and clarify how HFE affects TfR function. Whether the interaction between HFE and TfR explains the pathogenesis of HH is not so clear.

Grandea AG, Townsend AR, Spies T, Van Kaer L, Ohlen C. 1999. Deficient peptide binding by MHC class I due to prolonged interaction with calnexin in a mutant cell line FASEB JOURNAL, 13 (5), pp. A1138-A1138.

Glithero A, Tormo J, Haurum JS, Arsequell G, Valencia G, Edwards J, Springer S, Townsend A, Pao YL, Wormald M et al. 1999. Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity. Immunity, 10 (1), pp. 63-74. | Show Abstract | Read more

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

Bastin JM, Jones M, O'Callaghan CA, Schimanski L, Mason DY, Townsend AR. 1998. Kupffer cell staining by an HFE-specific monoclonal antibody: implications for hereditary haemochromatosis. Br J Haematol, 103 (4), pp. 931-941. | Show Abstract

Hereditary haemochromatosis is an inherited disorder of iron absorption that leads to excessive iron storage in the liver and other organs. A candidate disease gene HFE has been identified that encodes a novel MHC class I like protein. We report the development of a monoclonal antibody (HFE-JB1) specific for recombinant refolded HFE protein. The antibody immunoprecipitates a 49 kD protein from the cell line U937, a histiocytic lymphoma. It binds HFE but does not recognize other recombinant non-classic MHC class I proteins (HLA-E, F and G), nor does it react with a variety of recombinant classic class I MHC molecules. COS cells transfected with HFE in culture are stained specifically. The immunohistochemical staining pattern in human tissues is unique and can be defined as a subset of the transferrin receptor positive cells. In the liver HFE protein was shown to be present on Kupffer cells and endothelium (sinusoidal lining cells), but absent from the parenchyma. Kupffer cells from an untreated C282Y HH patient failed to stain with the antibody. In the normal gut scattered cells in the crypts are stained. HFE was also present on capillary endothelium in the brain (a site of high levels of transferrin receptor) and on scattered cells in the cerebellum and cortex. These results raise interesting questions concerning the function of HFE in the control of body iron content and distribution.

Springer S, Doring K, Skipper JC, Townsend AR, Cerundolo V. 1998. Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding. Biochemistry, 37 (9), pp. 3001-3012. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Mukherjee S, Trivedi P, Dorfman DM, Klein G, Townsend A. 1998. Murine cytotoxic T lymphocytes recognize an epitope in an EBNA-1 fragment, but fail to lyse EBNA-1-expressing mouse cells. J Exp Med, 187 (3), pp. 445-450. | Show Abstract | Read more

Major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTLs) specific for epitopes within eight of the nine Epstein Barr Virus (EBV)-encoded latency-associated proteins have been recovered from EBV-infected human subjects by restimulation of lymphocytes in vitro. However, human class I-restricted CTL responses capable of recognizing EBNA-1 expressing cells were not detected in these studies. We have raised a murine CTL line that recognizes an epitope within EBNA-1 by immunizing mice with a vaccinia virus encoding a COOH-terminal EBNA-1 fragment. This novel CTL line was used to investigate whether the epitope (positions 509-517 in EBNA-1, presented through Kd) was presented to CTL by mouse cells expressing full-length EBNA-1 or a deletion mutant of EBNA-1, lacking the Glycine-Alanine (Gly-Ala)-rich region. Cells expressing full-length EBNA-1 are not lysed by the CTL line, whereas cells expressing the Gly-Ala deletion mutant are recognized. These results suggest that epitopes from full-length EBNA-1 are poorly presented, and that the Gly-Ala-rich region is responsible for this phenomenon. The inefficient presentation of EBNA-1-derived epitopes may explain the absence or rarity of EBNA-1-specific CTLs in vivo, a strategy that may allow EBV to maintain persistence within the immunocompetent host without being eliminated by CTLs.

Springer S, Poring K, Townsend ARM. 1997. Biophysical evidence for conformational change in MHC class I molecules upon peptide binding FASEB Journal, 11 (9), | Show Abstract

MHC class 1 molecules hind peptides of 8-12 amino acids in the endoplasmic reticulum of mammalian cells to present them at the reil surface to cytotoxic T lymphocytes. In crystal structures of the complex, peptides are deeply buried within a binding groove. We have expressed the murim1 class I molecule H'2Dh in soluble form, complexed with human beta-2 tnicroglobulin, in chinese hamster ovary cells. Purified peptide-free class I complexes are stable at 4 °(-, and are stabilised against thermal denaturation-by the binding of peptide. We have used these complexes to generate complete sets of kinetic association and dissociation as well as equilibrium binding constants of unmodified peptides using tritium labelled peptides and the natural tryptophan fluorescence of the protein. For the peptide FAPGNYPAL, the equilibrium binding constant of 0.2 x U)7 \ll and the kinetic dissociation constant of 7.1 x 10"6 s"1 (at 1 °C') predict a slow association rate, 650 Ms"'. for a simple one-step model of binding. Instead, we find fast association kinetics with 1.1 x 10b Ms"1 by stopped-flow fluorescence spectroscopy. Association is stower if the peptide is longer than optimal, modified by iodination, and also in the presence of detergent. This 'kinetic mismatch' suggests a multi-step binding mecha nism involving a conformational change of the class I binding groove in the poptide binding process. Therefore, the structure of a class I binding site at the time-point of peptide recognition might be different from what is seen in crystaliographic studies.

Cerundolo V, Benham A, Braud V, Mukherjee S, Gould K, Macino B, Neefjes J, Townsend A. 1997. The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells. Eur J Immunol, 27 (1), pp. 336-341. | Show Abstract | Read more

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.

Elliott T, Bodmer H, Townsend A. 1996. Recognition of out-of-frame major histocompatibility complex class I-restricted epitopes in vivo. Eur J Immunol, 26 (5), pp. 1175-1179. | Show Abstract | Read more

In the course of constructing a recombinant vaccinia virus encoding the influenza A nucleoprotein (NP) gene preceded by the hemagglutinin leader sequence, we isolated a single base-pair deletion mutant which gave rise to L+NP(1-159) in which only the first 159 amino acids were in frame. Despite this, when we infected target cells, we found that the point mutant was able to sensitize them for lysis not only by cytotoxic T cells recognizing residues 50-58 (the in-frame portion), but also by CTL to epitopes which are downstream of the mutation (366-374 and 378-386). Furthermore, normal C57BL/6 mice can be primed with the frameshift NP to recognize the immunodominant Db-restricted epitope 366-374 (which is out of frame). Experiments in which the mutant gene product was processed in the endoplasmic reticulum of target cells suggested that the apparent suppression occurred during polypeptide extension.

Mamalaki C, Murdjeva M, Tolaini M, Norton T, Chandler P, Townsend A, Simpson E, Kioussis D. 1996. Tolerance in TCR/cognate antigen double-transgenic mice mediated by incomplete thymic deletion and peripheral receptor downregulation. Dev Immunol, 4 (4), pp. 299-315. | Show Abstract | Read more

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H-2Kb promoter. Double-transgenic mice show negative selection of thymocytes at the CD4+8+TCRlo to CD4+8+TCRhi transition stage. A few CD8+ T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.

Blake NW, Kettle S, Law KM, Gould K, Bastin J, Townsend AR, Smith GL. 1995. Vaccinia virus serpins B13R and B22R do not inhibit antigen presentation to class I-restricted cytotoxic T lymphocytes. J Gen Virol, 76 ( Pt 9) (9), pp. 2393-2398. | Show Abstract | Read more

Vaccinia virus (VV) inhibits the presentation of certain epitopes from influenza virus nucleoprotein (NP), haemagglutinin (HA) and non-structural 1 (NS1) proteins to CD8+ cytotoxic T lymphocytes (CTL) by an unknown mechanism. We have investigated whether VV genes B13R and B22R, which encode proteins with amino acid similarity to serine protease inhibitors (serpins), are involved in this process. Recombinant VVs were constructed which express influenza virus proteins HA, NP or NS1 and which lack serpin gene B13R or both B13R and B22R. The lysis of cells infected with these viruses by influenza virus-specific CD8+ CTL was compared to the lysis of cells infected with viruses expressing both the influenza proteins and the serpin genes. Cytotoxicity assays showed that deletion of the VV serpin genes B13R and B22R and other genes between B13R and B24R did not increase the level of lysis, indicating that these genes are not involved in inhibition of antigen presentation of the epitopes tested.

Elliott T, Willis A, Cerundolo V, Townsend A. 1995. Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum. J Exp Med, 181 (4), pp. 1481-1491. | Show Abstract | Read more

We have introduced long precursor peptides directly into the endoplasmic reticulum (ER) of a mutant cell line (T2-Db) that lacks the ability to transport peptides from the cytosol to the ER in a transporter associated with antigen processing (TAP) dependent way. This was done by expressing various influenza A-derived peptides containing the naturally processed epitope ASNENMDAM (366-374) preceded by the influenza hemagglutinin ER translocation sequence. Peptides derived from these minigenes that became associated with Db were isolated and identified by combined reversed phase liquid chromatography and detection by cytotoxic T lymphocytes. Our results establish that NH2-terminal extensions of at least 40 residues can be trimmed from peptides entering the ER, but that proteolysis of larger proteins may be limited.

Clover LM, Sargent IL, Townsend A, Tampé R, Redman CW. 1995. Expression of TAP1 by human trophoblast. Eur J Immunol, 25 (2), pp. 543-553. | Show Abstract | Read more

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Anti-transporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G+ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.

Cerundolo V, Kelly A, Elliott T, Trowsdale J, Townsend A. 1995. Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport. Eur J Immunol, 25 (2), pp. 554-562. | Show Abstract | Read more

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.

van der Bruggen P, Bastin J, Gajewski T, Coulie PG, Boël P, De Smet C, Traversari C, Townsend A, Boon T. 1994. A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur J Immunol, 24 (12), pp. 3038-3043. | Show Abstract | Read more

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32% in Caucasian populations, since 49% of individuals carry the HLA-A2 allele and 65% of melanomas express MAGE-3.

Townsend A, Ohlén C, Rogers M, Edwards J, Mukherjee S, Bastin J. 1994. Source of unique tumour antigens. Nature, 371 (6499), pp. 662. | Read more

Kaklamanis L, Townsend A, Doussis-Anagnostopoulou IA, Mortensen N, Harris AL, Gatter KC. 1994. Loss of major histocompatibility complex-encoded transporter associated with antigen presentation (TAP) in colorectal cancer. Am J Pathol, 145 (3), pp. 505-509. | Show Abstract

Presentation of endogenous antigenic peptides to cytotoxic T lymphocytes (CTLs) is mediated by the major histocompatibility complex (MHC) class I molecules. These antigenic peptides derived from the cytoplasmic protein pool are transported by the recently described MHC-encoded transporters (TAP1 and TAP2) into a pre-Golgi region where they take part in the assembly of MHC class I molecules. Using an affinity-purified polyclonal antibody (AK1.7) for TAP1, we analyzed 81 colorectal carcinomas, 32 adenomas, and the respective nonneoplastic mucosa. Loss of the transporter molecule (TAP1) was observed in 14% (11 of 81) of the carcinomas, either complete (7 of 11) or focal (4 of 11), whereas adenomas and normal mucosa were always positive. This study adds further information to the understanding of the mechanisms related to the defective presentation of the MHC class I molecules by tumor cells.

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Townsend A, Öhlén C, Rogers M, Edwards J, Mukherjee S, Bastin J. 1994. Source of unique tumour antigens [7] Nature, 371 (6499), pp. 662.

BLAKE NW, KETTLE S, GOULD K, BASTIN J, TOWNSEND A, SMITH GL. 1993. DO VACCINIA VIRUS-ENCODED SERPINS INHIBIT ANTIGEN PRESENTATION TO CLASS I-RESTRICTED CTL JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 54-54.

Elvin J, Potter C, Elliott T, Cerundolo V, Townsend A. 1993. A method to quantify binding of unlabeled peptides to class I MHC molecules and detect their allele specificity. J Immunol Methods, 158 (2), pp. 161-171. | Show Abstract | Read more

A general method has been developed for measuring the stabilization of class I MHC molecules in extracts of the mutant cell lines .174/T2 and RMA-S. 35S-Met-labeled class I molecules which have been stabilized by peptides in vitro are immunoprecipitated with conformation dependent monoclonal antibodies and electrophoresed on polyacrylamide gels. The heavy and light chains are excised from the dried gel and quantified on a flat bed scintillation counter. The stabilizing effect of peptides on class I molecules in vitro correlates well with peptide binding measured by direct methods and can be therefore used to assess peptide binding affinity. We show that a peptide from HIV-1 gag (which has a high affinity for Db) is a CTL epitope restricted through Db, and also use the assay to analyse the effects of amino acid substitution on peptide affinity. In addition, the effect of a given peptide on a class I molecule within a mixture of human class I molecules can be distinguished by immunoprecipitation with the monomorphic antibody W6/32 and separation by 1-D isoelectric focussing. The technique therefore requires neither labeled peptide ligands nor allele-specific antibodies. It can be used to identify the peptide ligand of any human class I molecule, and gives a measure of peptide binding affinity. The technique should be of value in identifying epitopes recognized by CTL since we have found that these tend to bind with the highest affinities.

Townsend A, Trowsdale J. 1993. The transporters associated with antigen presentation. Semin Cell Biol, 4 (1), pp. 53-61. | Show Abstract | Read more

Two new members of the ABC superfamily of transporter genes have recently been identified within the Major Histocompatibility Complex in man, rat and mouse. Although the exact function of these genes is not known, they have been shown to be necessary for the presentation of peptides derived from the degradation of cytoplasmic protein antigens to the cellular immune system. For this reason they have been named TAP1 and TAP2 (for Transporter associated with Antigen Presentation). Each gene encodes one membrane spanning domain and one region homologous to the ATP binding domains that characterise the superfamily. The two proteins encoded by the TAP genes form a complex that is localised to the membranes of the endoplasmic reticulum and cis-golgi. Their most likely function is to transport short peptides, that lack signal sequences, from the cytoplasm to the endoplasmic reticulum, although the evidence for this is still indirect.

Mamalaki C, Elliott J, Norton T, Yannoutsos N, Townsend AR, Chandler P, Simpson E, Kioussis D. 1993. Positive and negative selection in transgenic mice expressing a T-cell receptor specific for influenza nucleoprotein and endogenous superantigen. Dev Immunol, 3 (3), pp. 159-174. | Show Abstract | Read more

A transgenic mouse was generated expressing on most (> 80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes alpha alpha 366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class I MHC Db (Townsend et al., 1986). The receptor utilizes the V beta 11 and V alpha 4 gene segments for the beta chain and alpha chain, respectively (Palmer et al., 1989). The usage of V beta 11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the V beta 11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly V beta 11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.

Hill AV, Elvin J, Willis AC, Aidoo M, Allsopp CE, Gotch FM, Gao XM, Takiguchi M, Greenwood BM, Townsend AR. 1992. Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature, 360 (6403), pp. 434-439. | Show Abstract | Read more

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component.

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HILL AVS, ELVIN J, WILLIS AC, AIDOO M, ALLSOPP CEM, GOTCH FM, GAO XM, TAKIGUCHI M, GREENWOOD BM, TOWNSEND ARM et al. 1992. MOLECULAR ANALYSIS OF THE ASSOCIATION OF HLA-B53 AND RESISTANCE TO SEVERE MALARIA NATURE, 360 (6403), pp. 434-439. | Show Abstract | Read more

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component. © 1992 Nature Publishing Group.

Elliott T, Cerundolo V, Townsend A. 1992. Short peptides assist the folding of free class I heavy chains in solution. Eur J Immunol, 22 (12), pp. 3121-3125. | Show Abstract | Read more

Previous experiments have shown that short peptides coresponding to naturally processed epitopes of viral antigens can induce a conformational change in the class I heavy chain (HC) to which they bind in the fully assembled molecule. Here, we present evidence that the mechanism for this conformational change may involve binding of peptide to a partially unfolded form of free HC, followed by its subsequent folding. These results may be important for understanding the way in which class I molecules are assembled in vivo, and how certain epitopes are selected for presentation to T cells.

Mamalaki C, Norton T, Tanaka Y, Townsend AR, Chandler P, Simpson E, Kioussis D. 1992. Thymic depletion and peripheral activation of class I major histocompatibility complex-restricted T cells by soluble peptide in T-cell receptor transgenic mice. Proc Natl Acad Sci U S A, 89 (23), pp. 11342-11346. | Show Abstract | Read more

Injection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.

Cerundolo V, Elliott T, Elvin J, Bastin J, Townsend A. 1992. Association of the human invariant chain with H-2 Db class I molecules. Eur J Immunol, 22 (9), pp. 2243-2248. | Show Abstract | Read more

We describe two proteins of 24 kDa and 33 kDa (p24 and p33) which associate with H-2 Kb and H-2 Db molecules, respectively, in human cells transfected with H-2 Kb and H-2 Db genes. This association is particularly clear in the mutant cell line T2, in which association of endogenous peptide with newly synthesized class I molecules may not occur (V. Cerundolo et al., Nature 1990. 345: 449). We show that p33 is the 33-kDa form of the human invariant chain which is resident in the endoplasmic reticulum of T2 cells (P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 1989. LIV:309). The stability of the invariant chain H-2 Db complex is critically dependent upon occupation of the class I binding site by peptide ligand. In the absence of peptide, the complex is stable at 4 degrees C whereas following exposure to peptide, the invariant chain dissociates rapidly from H-2 Db molecules (half-life of 30 min at 4 degrees C). Although the interaction between the human invariant chain and murine H-2 Db is unlikely to have any functional significance, the peptide-induced dissociation of the invariant chain is consistent with a conformational change in H-2 Db on peptide binding.

Elliott T, Elvin J, Cerundolo V, Allen H, Townsend A. 1992. Structural requirements for the peptide-induced conformational change of free major histocompatibility complex class I heavy chains. Eur J Immunol, 22 (8), pp. 2085-2091. | Show Abstract | Read more

In an attempt to define the structural features of peptides which are important for inducing the folding of free class I heavy chains in the absence of beta 2-microglobulin, and to determine whether they are the same as those required to form stable major histocompatibility complex (MHC): peptide adducts, we have used a panel of peptides related to the Db-binding nonamer ASNENMDAM (influenza nucleoprotein residues 366-374) with altered primary structures, and a number of other peptides which have the Db-binding "motif". In this way, we have shown that in addition to the "anchor" residues which define this motif, the alpha amino and carboxyl groups at the N and C termini also play a major role in both inducing the conformational change in free heavy chain (HC) and formation of a stable Db:peptide complex. We also show that the importance of the key residues is affected by the primary sequence "context" in which they appear. In addition, we have extended our original finding that naturally processed epitopes induce a conformational change in free HC to the H2Kb HC, and show that the effect does not require the presence of the class I alpha 3 domain.

Kleijmeer MJ, Kelly A, Geuze HJ, Slot JW, Townsend A, Trowsdale J. 1992. Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi. Nature, 357 (6376), pp. 342-344. | Show Abstract | Read more

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.

Townsend A. 1992. Antigen processing. A new presentation pathway? Nature, 356 (6368), pp. 386-387. | Read more

Kelly A, Powis SH, Kerr LA, Mockridge I, Elliott T, Bastin J, Uchanska-Ziegler B, Ziegler A, Trowsdale J, Townsend A. 1992. Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex. Nature, 355 (6361), pp. 641-644. | Show Abstract | Read more

Presentation of cytoplasmic antigens to class I-restricted cytotoxic T cells implied the existence of a specialized peptide transporter. For most class I heavy chains, association with peptides of the appropriate length is required for stable assembly with beta 2-microglobulin. Mutant cells RMA-S and .174/T2 neither assemble stable class I molecules nor present intracellular antigens, and we have suggested that they have lost a function required for the transport of short peptides from the cytosol to the endoplasmic reticulum. The genetic defect in .174 has been localized to a large deletion in the class II region of the major histocompatibility complex, within which two genes (RING4 and RING11) have been identified that code for 'ABC' (ATP-binding cassette) transporters. We report here that the protein products of these two genes assemble to form a complex. Defects in either protein result in the formation of unstable class I molecules and loss of presentation of intracellular antigens. The molecular defect in a new mutant, BM36.1, is shown to be in the ATP-binding domain of the RING11/PSF2 protein. This is in contrast to the mutant .134, which lacks the RING4/PSF1 protein.

Spies T, Cerundolo V, Colonna M, Cresswell P, Townsend A, DeMars R. 1992. Presentation of viral antigen by MHC class I molecules is dependent on a putative peptide transporter heterodimer. Nature, 355 (6361), pp. 644-646. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I molecules present peptides derived from the endogenous protein pool to cytotoxic T lymphocytes, which can thus recognize intracellular antigen. This pathway may depend on a transporter (PSF1) to mediate entry of the cytosolic peptides into a pre-Golgi compartment where they bind to class I heavy chains and promote their stable assembly with beta 2-microglobulin. There is, however, only indirect support for this function of PSF1. Here we show that PSF1 is necessary for the efficient assembly of class I molecules and enables them to present a peptide epitope derived from endogenously synthesized viral antigen. Immunochemical and genetic data demonstrate that the PSF1 polypeptide is associated with a complementary transporter chain, which is polymorphic and is encoded by the PSF2 gene, which is closely linked to PSF1.

Sibille C, Gould K, Hämmerling G, Townsend A. 1992. A defect in the presentation of intracellular viral antigens is restored by interferon-gamma in cell lines with impaired major histocompatibility complex class I assembly. Eur J Immunol, 22 (2), pp. 433-440. | Show Abstract | Read more

Surface expression of the majority of class I major histocompatibility complex (MHC) heavy chains is known to require assembly with beta 2 microglobulin (beta 2m). To define other factors involved in class I MHC assembly, we have studied two tumor cell lines that are deficient in cell surface class I (H-2) expression. The BC2 fibrosarcoma and the CMT lung carcinoma express only intracellular unassociated heavy chains despite the presence of beta 2m. As described previously, when these cell lines are treated with interferon-gamma (IFN-gamma), they are capable of assembling and transporting class I molecules to the cell surface. In this study, we have shown that in the absence of IFN-gamma these mutant cells are unable to present intracellular viral antigens, although they can be lysed by specific cytotoxic T lymphocyte (CTL) after pre-incubation with the corresponding synthetic peptide. Flow cytometric analysis demonstrated that extracellular peptide was capable of increasing twofold the surface expression of beta 2m-heavy chain complexes. Furthermore, immunoprecipitation experiments confirmed that peptide stabilizes chain association in the BC2 cell lysates. However, infecting these mutants with vectors expressing either pre-processed antigen or rapidly degraded antigen, failed to overcome their defect in the presentation of endogenous peptide to specific CTL or to mediate surface expression of class I MHC. Preincubation with IFN-gamma completely reversed the endogenous peptide presentation defect, even in mutant cells transfected with a vector encoding a cDNA for the H-2 molecule restricting CTL recognition. This last result suggests that IFN-gamma corrects the defect by a mechanism separate from simple enhancement of the number of class I molecules produced by the cell. Because there is growing evidence that endogenous peptides can participate in class I MHC assembly, the defect in these mutants could be ascribed to the lack of access to class I molecules by the endogenous peptide. This would prevent stable association of the heavy and light chains and their subsequent transport. Our data suggests that IFN-gamma reestablishes class I MHC surface expression by restoring access of endogenously synthesized peptide to class I molecules.

Aosai F, Ohlen C, Ljunggren HG, Höglund P, Franksson L, Ploegh H, Townsend A, Kärre K, Stauss HJ. 1991. Different types of allospecific CTL clones identified by their ability to recognize peptide loading-defective target cells. Eur J Immunol, 21 (11), pp. 2767-2774. | Show Abstract | Read more

Allospecific immune responses against the MHC of another individual are remarkably strong, due t a high number of responding T cell clones. Although it has been demonstrated that some allospecific cytotoxic T lymphocytes (CTL) recognize peptides presented by allogeneic MHC class I molecules, it has remained unclear whether MHC molecules can be recognized directly. We used the H-2b-derived murine lymphoma mutant RMA-S, which has a defect affecting peptide loading of class I molecules, to test whether recognition by allospecific CTL always requires the presence of peptides. Three types of anti-H-2Kb CTL clones can be distinguished by their ability to lyse RMA-S target cells. Type A CTL clones efficiently lyse these target cells, the lysis by type B CTL clones is inefficient, and type C clones fail to lyse RMA-S. Up-regulation of the levels of H-2Kb density improved lysis by type B clones, but did not lead to lysis by type C clones. Some type A and B CTL clones apparently can recognize class I molecules devoid of peptides, while others are likely to recognize peptides which are not affected by the presentation defect of RMA-S. We suggest that type C clones are specific for peptides which are not presented by the mutant cells. The results show that the majority of alloreactive CTL recognize peptide/MHC complexes, while some CTL behave as if they can recognize class I molecules in the absence of MHC-bound peptides.

Elvin J, Cerundolo V, Elliott T, Townsend A. 1991. A quantitative assay of peptide-dependent class I assembly. Eur J Immunol, 21 (9), pp. 2025-2031. | Show Abstract | Read more

We have developed a quantitative assay for the measurement of class I assembly induced by peptide. We have applied this assay to H-2Db, Kb and HLA-A2.1 with a panel of 49 overlapping peptides derived from HIV-1 gag protein. We find that the effects of peptide on assembly form a continuous distribution. By defining positives as those that increase the concentration of folded heavy chains more than three standard deviations from the control we show that 7/48 bind A2.1, 11/49 bind Db and 7/47 bind Kb. The assembly assay contrasts with solid-phase assays in being more discriminating (fewer peptides binding any given class I molecule), and showing less overlap in the patterns of peptides bound by the three class I molecules.

Cerundolo V, Elliott T, Elvin J, Bastin J, Rammensee HG, Townsend A. 1991. The binding affinity and dissociation rates of peptides for class I major histocompatibility complex molecules. Eur J Immunol, 21 (9), pp. 2069-2075. | Show Abstract | Read more

Peptides of various lengths derived from the influenza nucleoprotein (NP) bind to H-2Db class I molecules with affinities at 4 degrees C between approximately 3 x 10(5)- approximately 3 x 10(7) M-1. The peptide with the highest affinity corresponds to the sequence of nine amino acids (NP366-374) recently isolated from cells infected with influenza. This peptide forms stable complexes with half-lives greater than 110 h at 4 degrees C, 39 h at 22 degrees C and 3 h at 37 degrees C. Small increases in length of the peptide greatly reduce the stability of the complex (t1/2 approximately 1-10 h at 4 degrees C). These results may explain the homogeneous length of peptides isolated from class I molecules formed in vivo, and suggest that class I and II may differ in their dependence on the length of peptides for the formation of stable complexes.

Elliott T, Cerundolo V, Elvin J, Townsend A. 1991. Peptide-induced conformational change of the class I heavy chain. Nature, 351 (6325), pp. 402-406. | Show Abstract | Read more

There is evidence that peptide ligands take part in the assembly of class I molecules. In particular, addition of peptides to extracts of the mutant cells RMA-S and .174/T2, in which stable assembly of class I does not occur, results in a conformational change in the class I heavy chain and stable association of the heavy chain with beta 2-microglobulin (beta 2m). Thus specific peptides may stabilize or induce a conformational change in the class I heavy chain that results in a rise in the binding affinity of the heavy chain for beta 2m (Fig. 1a). Here we show that peptides have two cooperative roles in class I assembly. Specific short peptides (9-10 amino acids) can induce folding of the heavy chain in the absence of beta 2m. Both short (nine amino acids) and longer sequences (15 amino acids) can stabilize performed low-affinity complexes of heavy chain and beta 2m. To alter the conformation of free heavy chains, the peptides must be exactly the correct size, and they are found to correspond to the sequences isolated from infected cells. This property may therefore be the basis for selection of epitopes presented in vivo.

Cerundolo V, Tse AG, Salter RD, Parham P, Townsend A. 1991. CD8 independence and specificity of cytotoxic T lymphocytes restricted by HLA-Aw68.1. Proc Biol Sci, 244 (1310), pp. 169-177. | Show Abstract | Read more

The crystal structure of the HLA-Aw68.1 antigen binding site revealed a negatively charged pocket centred on aspartic acid 74 (Garrett et al. 1989). Access to this '74 pocket' is blocked in HLA-Aw68.2 and HLA-Aw69 by two substitutions at positions 97 and 116. This key feature suggests that the Aw68.1-peptide-specific interactions may involve salt bridges between oppositely charged residues. In this paper, the influenza epitope recognized by virus-specific HLA-Aw68.1-restricted cytotoxic T lymphocytes (CTL) has been defined in vitro with a synthetic peptide corresponding to residues 89-101 of the nucleoprotein (NP). Amino acid substitutions of the peptide NP 89-101 showed that the arginine at position 99 is an anchor point of the peptide within the Aw68.1 antigen binding site. Consistent with this we find that neither HLA-Aw68.2 nor HLA-Aw69 positive cells can present peptide NP 89-101 to Aw68.1-restricted CTL. Our results therefore suggest a model in which presentation of NP 89-101 by HLA-Aw68.1 is dependent upon interaction of the positively charged arginine residue at position 99 of the peptide, with the negatively charged aspartic acid in the '74 pocket' of HLA-Aw68.1. We also show that influenza-virus-specific HLA-Aw68.1-restricted CTL are CD8 independent. This result is consistent with the low affinity of HLA-Aw68.1 for CD8 (Salter et al. 1989) and reveals a unique example of CD8-independent priming of CTL by natural infection with a common pathogen in humans.

Elliott TJ, Cerundolo V, Ohlen C, Ljunggren HG, Karre K, Townsend A. 1991. Antigen presentation and the association of class-I molecules. Acta Biol Hung, 42 (1-3), pp. 213-229. | Show Abstract

We have identified two mutant cell lines which are not able to present epitopes of influenza virus synthesized in the cytoplasm but can present the same epitope when exposed to it as a peptide in the extracellular medium. The cell lines also have a defect in class-I assembly, with reduced expression of assembled alpha chain: beta 2M heterodimers at their cell surface. This led to the suggestion that the two traits were the result of the same mutation and that stable assembly of class-I molecules is dependent on peptide binding. Consistent with this idea was the finding that exposure to specific peptides in the extracellular fluid promotes stable association of class-I heavy chains with beta 2M and restores expression of class-I at the cell surface. We have gone on to show that stable assembly of class-I molecules can be supported in detergent extracts of the mutant cells when specific peptides are added. Peptides stabilized a conformational change in the class-I heavy chain and association with beta 2M by binding to the complexes. This effect is apparent at peptide concentrations around 100-fold lower than required in "peptide feeding" experiments with whole cells. We have also demonstrated that the conformational change induced in heavy chain is influenced by the concentration of beta 2M, and consequently have been able to demonstrate the formation of empty class-I molecules.

Trowsdale J, Hanson I, Mockridge I, Beck S, Townsend A, Kelly A. 1990. Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters. Nature, 348 (6303), pp. 741-744. | Show Abstract | Read more

Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.

Elliott T, Townsend A, Cerundolo V. 1990. Antigen presentation. Naturally processed peptides. Nature, 348 (6298), pp. 195-197. | Read more

LJUNGGREN HG, FRANKSSON L, HOGLUND P, OHLEN C, TOWNSEND A, PLOEGH HL, KARRE K. 1990. A METHOD TO MEASURE MHC CLASS-I-PEPTIDE INTERACTIONS AT THE CELL-SURFACE SCANDINAVIAN JOURNAL OF IMMUNOLOGY, 32 (4), pp. 403-403.

LJUNGGREN HG, STAM N, OHLEN C, NEEFJES JJ, HOGLUND P, HEEMELS MT, BASTIN J, SCHUMACHER T, TOWNSEND A, KARRE K, PLOEGH HL. 1990. EMPTY MHC CLASS-I MOLECULES COME OUT IN THE COLD SCANDINAVIAN JOURNAL OF IMMUNOLOGY, 32 (4), pp. 403-403.

Ljunggren HG, Stam NJ, Ohlén C, Neefjes JJ, Höglund P, Heemels MT, Bastin J, Schumacher TN, Townsend A, Kärre K. 1990. Empty MHC class I molecules come out in the cold. Nature, 346 (6283), pp. 476-480. | Show Abstract | Read more

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.

Townsend A, Elliott T, Cerundolo V, Foster L, Barber B, Tse A. 1990. Assembly of MHC class I molecules analyzed in vitro. Cell, 62 (2), pp. 285-295. | Show Abstract | Read more

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.

Cerundolo V, Alexander J, Anderson K, Lamb C, Cresswell P, McMichael A, Gotch F, Townsend A. 1990. Presentation of viral antigen controlled by a gene in the major histocompatibility complex. Nature, 345 (6274), pp. 449-452. | Show Abstract | Read more

We describe a mutant human cell line (LBL 721.174) that has lost a function required for presentation of intracellular viral antigens with class I molecules of the major histocompatibility complex (MHC), but retains the capacity to present defined epitopes as extracellular peptides. The cell also has a defect in the assembly and expression of class I MHC molecules, which we show can be restored by exposure of the cells to a peptide epitope. This phenotype suggests a defect in the association of intracellular antigen with class I molecules similar to that described for the murine mutant RMA-S (ref. 5), but in the present case the genetic defect can be mapped within the MHC locus on human chromosome 6.

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Elliott T, Townsend A, Cerundolo V. 1990. Naturally processed peptides Nature, 348 (6298), pp. 195-196. | Read more

Huet S, Nixon DF, Rothbard JB, Townsend A, Ellis SA, McMichael AJ. 1990. Structural homologies between two HLA B27-restricted peptides suggest residues important for interaction with HLA B27. Int Immunol, 2 (4), pp. 311-316. | Show Abstract | Read more

Recently we described an HLA B27-restricted peptide derived from HIV gag p24 protein. In this study we have isolated an HLA B27-restricted peptide from the nucleoprotein (NP) of influenza A virus. The shortest fragment recognized by cytotoxic T lymphocyte (CTL) is eight amino acids long, residues 384-391. Comparison of the sequence of these two HLA B27 restricted peptides reveals homologies which can be aligned from one peptide to the other. Of the eight residues, two are identical: tryptophan and isoleucine. Both peptides have a positively charged residue at the N terminus, lysine at position 265 of gag and arginine at position 384 of NP. Using modified peptides we have shown that lysine or arginine is crucial for the interaction with HLA B27. The wild-type gag peptide blocked CTL recognition of NP peptide by influenza-specific CTL, but removal of the lysine prevented inhibition of NP peptide recognition. The importance of these charged residues was confirmed by the observation that truncated NP and gag peptides where the lysine or arginine was removed were not recognized by specific CTL. Further studies showed that the tryptophan residue influenced the association of the gag peptide with HLA B27, because the affinity of the gag peptide for B27 was strongly increased after replacing this residue with a leucine or a tyrosine. However, these peptides were not recognized by gag-specific CTL, suggesting that the tryptophan may interact with both HLA B27 and T cell receptor. These observations should help in the identification of HLA B27-restricted peptides from other viruses or organisms.

OHLEN C, LJUNGGREN HG, FOSTER L, WOLPERT E, TOWNSEND A, KARRE K. 1989. MHC CLASS-I EXPRESSION IN TRANSPLANTATION TOLERANCE AND PEPTIDE PRESENTATION TO CTL - A MUTANT-CELL THAT FAILS TO PRESENT INTERNALLY DERIVED H-2 RESTRICTED ANTIGENS BUT REMAINS SENSITIVE TO ALLO H-2 SPECIFIC RESPONSES SCANDINAVIAN JOURNAL OF IMMUNOLOGY, 30 (5), pp. 644-644.

Gould K, Cossins J, Bastin J, Brownlee GG, Townsend A. 1989. A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. J Exp Med, 170 (3), pp. 1051-1056. | Show Abstract | Read more

A recombinant vaccinia has been designed to express amino acids 366-379 of influenza nucleoprotein, previously shown to be the minimal epitope recognized by a class I-restricted cytotoxic T cell clone. Target cells infected with the recombinant vaccinia virus expressing this peptide are recognized by CTL as efficiently as target cells expressing the complete nucleoprotein. The results imply the existence of a peptide transport system that constitutively passes the products of degraded proteins from the cytoplasm into a membrane-bound compartment of the cell.

Townsend A, Ohlén C, Bastin J, Ljunggren HG, Foster L, Kärre K. 1989. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature, 340 (6233), pp. 443-448. | Show Abstract | Read more

We describe a cell in which association of a major histocompatibility complex class I heavy chain with beta 2-microglobulin is induced by a peptide derived from influenza nucleoprotein. Association of antigenic peptides with the binding site of class I molecules may be required for correct folding of the heavy chain, association with beta 2-microglobulin and transport of the antigen-MHC complex to the cell surface.

Townsend A, Bastin J, Bodmer H, Brownlee G, Davey J, Gotch F, Gould K, Jones I, McMichael A, Rothbard J. 1989. Recognition of influenza virus proteins by cytotoxic T lymphocytes. Philos Trans R Soc Lond B Biol Sci, 323 (1217), pp. 527-533. | Show Abstract | Read more

Recombinant DNA techniques have been used to express the proteins of influenza virus individually. Target cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lymphocytes (CTLS). Results have shown that, contrary to expectation, the majority of the proteins recognized by class I major histocompatibility complex-restricted CTLS are not transmembrane glycoproteins. Experiments with deletion mutants of the nucleoprotein (NP) gene showed that transport of epitopes to the membrane for recognition by CTLS was independent of a definable signal sequence. In addition, the epitopes recognized were contained within short linear sequences of amino acids, and rapid degradation of large NP fragments within the target cell did not prevent recognition by CTLS. These results led to the suggestion that the epitopes recognized by class-I-restricted CTLS resulted from degradation of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro with short synthetic peptides. Five different epitopes of NP have now been demonstrated that can be defined with short peptides in vitro. Each peptide is recognized with a specific class I molecule (Db, Kk, Kd and HLA B37). This has been extended to the influenza matrix protein, and a peptide epitope defined that is recognized by human CTLS in association with HLA-A2. The question arose as to whether a similar phenomenon would be found with viral proteins which are naturally inserted in the target cell membrane. A mutant haemagglutinin has been produced that lacks a hydrophobic signal sequence. This protein is expressed as a short-lived, unglycosylated, intracellular protein. However, target cells expressing this molecule were recognized efficiently by CTLS raised to the wild-type haemagglutinin and vice versa. These and more recent results with non-viral glycoproteins are consistent with the existence of a mechanism for degrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association with class I molecules of the major histocompatibility complex.

Palmer MS, Bentley A, Gould K, Townsend AR. 1989. The T cell receptor from an influenza-A specific murine CTL clone. Nucleic Acids Res, 17 (6), pp. 2353. | Read more

Townsend A, Ohlën C, Foster L, Bastin J, Ljunggren HG, Kärre K. 1989. A mutant cell in which association of class I heavy and light chains is induced by viral peptides. Cold Spring Harb Symp Quant Biol, 54 Pt 1 pp. 299-308. | Show Abstract | Read more

Association of the Db heavy chain with beta 2-microglobulin and expression of Db and Kb at the surface of the cell are induced by specific peptides in the mutant RMA-S. Association of antigenic peptides with the binding site of class I molecules may be required for correct folding of the heavy chain, association with beta 2-microglobulin, and transport of the antigen-MHC complex to the cell surface.

Townsend A, Bodmer H. 1989. Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol, 7 (1), pp. 601-624. | Show Abstract | Read more

The work discussed here offers a unified view of T-cell recognition and suggests that class-I and class-II molecules have a closely related function in the presentation of peptides to T lymphocytes. The epitopes recognized by class I-restricted T cells that have been defined with peptides in the 4-6 hr lysis assay have all been derived from endogenously synthesized proteins expressed by virus infected or transfected cells. Evidence is accumulating that a cytoplasmic degradation system may be involved in the generation of these epitopes. The analysis of the specificity of CTL responses with synthetic peptides has demonstrated the control of immune responses to isolated epitopes by class-I genes and the great diversity of the receptor repertoire for individual class-I-restricted epitopes.

Nixon DF, Townsend AR, Elvin JG, Rizza CR, Gallwey J, McMichael AJ. 1988. HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides. Nature, 336 (6198), pp. 484-487. | Show Abstract | Read more

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.

Townsend A, Bastin J, Gould K, Brownlee G, Andrew M, Coupar B, Boyle D, Chan S, Smith G. 1988. Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen. J Exp Med, 168 (4), pp. 1211-1224. | Show Abstract | Read more

Vaccinia infection interferes with the presentation of influenza Haemagglutinin (HA) and Nucleoprotein (NP) to class I-restricted CTL. The inhibitory effect is selective for certain epitopes, and is more profound during the late phase of infection. For influenza A/NT/60/68 NP, the block is present during both early and late phases of infection, and is selective for the COOH-terminal epitope defined by peptide 366-379, having no detectable effect on the presentation of the NH2-terminal epitope 50-63. The presentation of HA is inhibited only during the late phase of vaccinia infection. For both proteins, presentation is partially (NP) or completely (HA) restored by expression of rapidly degraded protein fragments in the vaccinia infected target cell. For HA, deletion of the NH2-terminal signal sequence completely overcomes the block. For NP, either a large NH2-terminal deletion or the construction of a rapidly degraded ubiquitin-NP fusion protein partially restores presentation. These results illustrate the relationship between degradation of viral proteins in the cytoplasm of an infected cell and recognition of epitopes at the cell surface by class I-restricted T cells.

Gould K, Reay P, Townsend A, Brownlee GG. 1988. Studies on the nature and mechanism of influenza antigen recognition by murine cytotoxic T cells Virus Research, 11 (SUPPL. 1), pp. 65.

Townsend A, McMichael A. 1987. MHC protein structure. Those images that yet fresh images beget. Nature, 329 (6139), pp. 482-483. | Read more

Bastin J, Rothbard J, Davey J, Jones I, Townsend A. 1987. Use of synthetic peptides of influenza nucleoprotein to define epitopes recognized by class I-restricted cytotoxic T lymphocytes. J Exp Med, 165 (6), pp. 1508-1523. | Show Abstract | Read more

The conserved epitopes of influenza nucleoprotein (NP) recognized by class I MHC-restricted CTL from CBA (H-2k) and C57BL/10 (H-2b) mice have been defined in vitro with synthetic peptides 50-63 and 365-379, respectively. Two Db-restricted clones were described that recognize different epitopes on peptide 365-379. Finally, the recognition of complete NP was shown to be approximately 200-fold less efficient than peptide in the cytotoxicity assay. These phenomena are closely related to results with class II-restricted T cells and they strengthen the hypothesis that influenza proteins are degraded in the infected cell before recognition by class I-restricted CTL.

Gotch F, Rothbard J, Howland K, Townsend A, McMichael A. 1987. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature, 326 (6116), pp. 881-882. | Show Abstract | Read more

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.

Townsend AR. 1987. Recognition of influenza virus proteins by cytotoxic T lymphocytes. Immunol Res, 6 (1-2), pp. 80-100. | Read more

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Gould KG, Scotney H, Townsend ARM, Bastin J, Brownlee GG. 1987. Mouse H-2κ-restricted cytotoxic T cells recognize antigenic determinants in both the HA1 AND HA2 subunits of the influenza A/PR/8/34 hemagglutinin Journal of Experimental Medicine, 166 (3), pp. 693-701. | Read more

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Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 1986. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides Cell, 44 (6), pp. 959-968. | Show Abstract | Read more

A proportion of cytotoxic T lymphocytes (CTL) responding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I molecules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence. © 1986.

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Townsend ARM, Bastin J, Gould K, Brownlee GG. 1986. Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence Nature, 324 (6097), pp. 575-577. | Show Abstract | Read more

A surprising feature of most cytotoxic T lymphocytes (CTL) responding to influenza infection is that they recognize the unglycosylated (non-transmembrane) proteins of the virus, including the nucleoprotein 1-4 . Recognition of cells that express nucleoprotein by CTL does not depend on a definite signal sequence within the protein 5 , and the epitopes recognized can be defined with short synthetic peptides in vitro 6 . Haemagglutinin (HA), the major transmembrane protein of the virus, is recognized by a minor population of CTL from infected mice. We have deleted the sequence coding for the N-terminal signal peptide from a complementary DNA encoding HA of the HI subtype. The signal-deleted HA is detected with antibodies as a short-lived, unglycosylated, intracellular protein. However, CTL raised to the complete molecule recognize cells expressing the signal-deleted HA and vice versa. These results cast doubts on the assumption that CTL recognize the HA molecule only after its insertion into the plasma membrane. © 1986 Nature Publishing Group.

Townsend AR, Gotch FM, Davey J. 1985. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell, 42 (2), pp. 457-467. | Show Abstract | Read more

Recent work has shown that a major population of murine influenza A specific cytotoxic T lymphocytes (CTL) recognize the viral nucleoprotein. In order to investigate the mechanism by which this nonglycoprotein component of the virus is recognized by CTL, a series of deletion mutants of an A virus NP gene were studied. The results showed that CTL recognize three distinct epitopes of the NP molecule. Both N- and C-terminal fragments of the protein are transported, independently of each other, to the site of recognition by CTL. These findings imply that a mechanism may exist for transport to the cell surface and presentation to CTL, of viral proteins and protein fragments that lack defined signal sequences.

Townsend AR, McMichael AJ. 1985. Specificity of cytotoxic T lymphocytes stimulated with influenza virus. Studies in mice and humans. Prog Allergy, 36 pp. 10-43. | Show Abstract

Doherty et al. first demonstrated that cytotoxic T lymphocytes (CTLs) specific for virus-infected cells were restricted in their recognition of target antigens by class-I antigens of the major histocompatibility complex (MHC). Since that time this phenomenon has been thoroughly explored with the result that much is now known of the self-recognising properties of CTLs and rather less is understood about the recognition of virus antigen. Until the latter has been precisely characterised, an understanding of how CTLs recognise their target is unlikely. In this review we discuss the recognition of influenza virus antigens in association with MHC class-I molecules in both mouse and man. Precise mapping of the epitopes seen by CTL receptors is now possible with important practical implications for the development of vaccins.

Townsend A. 1985. Molecules at work on the T-cell surface. Immunol Today, 6 (3), pp. 68-70. | Show Abstract | Read more

Over the past two years a series of molecules on the T-cell surface have been defined using monoclonal antibodies. These have been grouped according to an agreed nomenclature (Table 1) and at a recent meeting(∗) there was conspicuous interest in the functional significance of some of these molecules. There was much new information on the T-cell antigen receptor which has been reviewed elsewhere(1).

Townsend AR, Skehel JJ, Taylor PM, Palese P. 1984. Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone. Virology, 133 (2), pp. 456-459. | Show Abstract | Read more

Cytotoxic T-cell clones raised against X-31 (H3N2) influenza virus in C57BL/6 mice can be directed against an influenza A virus subtype specific determinant (1). A representative T-cell clone (A3.1) has been used in combination with a set of genetically typed recombinant viruses, to show that the A/PR/8/34 nucleoprotein can be responsible for cytotoxic T-lymphocyte recognition of infected target cells.

Townsend AR, Skehel JJ. 1984. The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells. J Exp Med, 160 (2), pp. 552-563. | Show Abstract | Read more

Using genetically typed recombinant influenza A viruses that differ only in their genes for nucleoprotein, we have demonstrated that repeated stimulation in vitro of C57BL/6 spleen cells primed in vivo with E61-13-H17 (H3N2) virus results in the selection of a population of cytotoxic T lymphocytes (CTL) whose recognition of infected target cells maps to the gene for nucleoprotein of the 1968 virus. Influenza A viruses isolated between 1934 and 1979 fall into two groups defined by their ability to sensitize target cells for lysis by these CTL: 1934-1943 form one group (A/PR/8/34 related) and 1946-1979 form the second group (A/HK/8/68 related). These findings complement and extend our previous results with an isolated CTL clone with specificity for the 1934 nucleoprotein (27, 28). It is also shown that the same spleen cells derived from mice primed with E61-13-H17 virus in vivo, but maintained in identical conditions by stimulation with X31 virus (which differs from the former only in the origin of its gene for NP) in vitro, results in the selection of CTL that cross-react on target cells infected with A/PR/8/1934 (H1N1) or A/Aichi/1968 (H3N2). These results show that the influenza A virus gene for NP can play a role in selecting CTL with different specificities and implicate the NP molecule as a candidate for a target structure recognized by both subtype-directed and cross-reactive influenza A-specific cytotoxic T cells.

Townsend AR, McMichael AJ, Carter NP, Huddleston JA, Brownlee GG. 1984. Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells. Cell, 39 (1), pp. 13-25. | Show Abstract | Read more

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.

Bastin J, Drakesmith H, Rees M, Sargent I, Townsend A. 2006. Localisation of proteins of iron metabolism in the human placenta and liver. Br J Haematol, 134 (5), pp. 532-543. | Show Abstract | Read more

Two anatomical sites that are important in human iron metabolism are the liver and placenta. Liver macrophages recycle iron from erythrocytes, and the placenta transfers iron from the mother to the fetus. The cellular distribution of proteins involved in iron transport in these two sites was studied. Transferrin receptor-1 (TfR1) and Ferroportin (FPN) expression was found on the placental syncytiotrophoblast (STB) and were polarised such that TfR1 was on the apical maternal-facing membrane and FPN was on the basal fetal-facing membrane, consistent with unidirectional iron transport from mother to fetus. Ferritin was strongly expressed in the stroma, suggesting that fetal tissue can store and accumulate iron. HFE was on some parts of the basal STB and, where present, HFE clearly colocalised with FPN but not TfR1. In the stroma, both HFE and FPN were present on CD68+ Hofbauer macrophage cells. In liver, the location of HFE is controversial. Using four mouse monoclonals and two polyclonal sera we showed that the pattern of HFE expression mirrored the distribution of CD68+ macrophage Kupffer cells. FPN was also most strongly expressed by CD68+ Kupffer cells. These findings contribute to understanding how iron is transported and stored in the human placenta and liver.

Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. 2006. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. 1986. J Immunol, 176 (9), pp. 5141-5150.

Drakesmith H, Chen N, Ledermann H, Screaton G, Townsend A, Xu X-N. 2005. HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis. Proc Natl Acad Sci U S A, 102 (31), pp. 11017-11022. | Show Abstract | Read more

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.

Drakesmith H, Schimanski LM, Ormerod E, Merryweather-Clarke AT, Viprakasit V, Edwards JP, Sweetland E, Bastin JM, Cowley D, Chinthammitr Y et al. 2005. Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin. Blood, 106 (3), pp. 1092-1097. | Show Abstract | Read more

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Schimanski LM, Drakesmith H, Merryweather-Clarke AT, Viprakasit V, Edwards JP, Sweetland E, Bastin JM, Cowley D, Chinthammitr Y, Robson KJH, Townsend ARM. 2005. In vitro functional analysis of human ferroportin (FPN) and hemochromatosis-associated FPN mutations. Blood, 105 (10), pp. 4096-4102. | Show Abstract | Read more

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.

Drakesmith H, Sweetland E, Schimanski L, Edwards J, Cowley D, Ashraf M, Bastin J, Townsend ARM. 2002. The hemochromatosis protein HFE inhibits iron export from macrophages. Proc Natl Acad Sci U S A, 99 (24), pp. 15602-15607. | Show Abstract | Read more

Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, reduces cellular iron by reducing iron uptake. However, in vivo, HFE is strongly expressed by liver macrophages and intestinal crypt cells, which behave as though they are relatively iron-deficient in HH. These latter observations suggest, paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialized cell types. Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the monocytemacrophage cell line THP-1, and extend these results to macrophages derived from healthy individuals and HH patients. In addition, we find that the HH-associated mutant H41D has lost the ability to inhibit iron release despite binding to TfR1 as well as wild-type HFE. Finally, we show that the ability of HFE to block iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive functions, binding to TfR1 in competition with Tf, or inhibition of iron release.

Townsend A, Drakesmith H. 2002. Role of HFE in iron metabolism, hereditary haemochromatosis, anaemia of chronic disease, and secondary iron overload. Lancet, 359 (9308), pp. 786-790. | Show Abstract | Read more

Hereditary haemochromatosis is an iron overloading disorder caused by common mutations in the HFE gene. However, information with respect to the function of HFE protein does not explain how mutations in HFE lead to hereditary haemochromatosis. We propose a molecular model in which HFE has two mutually exclusive activities in cells: inhibition of uptake or inhibition of release of iron. The balance between serum transferrin saturation and serum transferrin-receptor concentrations determines which of these functions predominates. With this input, HFE enables the intestinal crypt cells and reticuloendothelial system to interpret the body's iron requirements and regulate iron absorption and distribution. In our model, mutations in HFE result in over absorption of dietary iron, and patterns of tissue iron deposition in agreement with clinical observations of hereditary haemochromatosis.

Townsend AR, Gotch FM, Davey J. 1985. Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell, 42 (2), pp. 457-467. | Show Abstract | Read more

Recent work has shown that a major population of murine influenza A specific cytotoxic T lymphocytes (CTL) recognize the viral nucleoprotein. In order to investigate the mechanism by which this nonglycoprotein component of the virus is recognized by CTL, a series of deletion mutants of an A virus NP gene were studied. The results showed that CTL recognize three distinct epitopes of the NP molecule. Both N- and C-terminal fragments of the protein are transported, independently of each other, to the site of recognition by CTL. These findings imply that a mechanism may exist for transport to the cell surface and presentation to CTL, of viral proteins and protein fragments that lack defined signal sequences.

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