Judy Bastin

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA‐F is a human non‐classical MHC molecule. Recombinant HLA‐F heavy chain was refolded with β2‐microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high‐affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA‐F. HLA‐F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA‐F could be immunoprecipitated from B cell lines and from HUT‐78, a T cell line. HLA‐F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA‐F tetramers stain peripheral blood monocytes and B cells. HLA‐F tetramer binding could be conferred on non‐binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA‐F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA‐F, suggest that HLA‐F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

The hemochromatosis protein HFE inhibits iron export from macrophages

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 monocyte/macrophage 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.

Kupffer cell staining by an HFE-specific monoclonal antibody : implications for hereditary haemochromatosis

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.

Localisation of proteins of iron metabolism in the human placenta and liver

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.

A mutant cell in which association of class I heavy and light chains is induced by viral peptides.

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.

Clinical applications of monoclonal antibodies

Monoclonal antibodies offer many distinct advantages over conventional antisera, the most obvious being their precise specificity for a single epitope on a single antigen and their potentially unlimited supply. The quantity and purity of available antibodies facilitates antigen purification by affinity chromatography. Monoclonal antibodies can also be used to characterize different parts of a macromolecule with regard to antigenicity, functional activity or genetic variability. Here Andrew McMichael and Judy Bastin concentrate on the potential value of monoclonal antibodies in clinical medicine, surveying papers published up to June 1980, and a number of preprints and personal communications, kindly made available to them by colleagues.

Vaccinia virus serpins B13R and B22R do not inhibit antigen presentation to class I-restricted cytotoxic T lymphocytes.

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.

In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor.

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.

Specific recognition of influenza virus polymerase protein (PB1) by a murine cytotoxic T-cell clone

Cytotoxic T-cell clones were raised in CBA mice that recognised both A/X31 and A/JAP/305/1957 influenza virus. Here, we describe one CTL clone that recognises target cells infected with a recombinant vaccinia virus expressing influenza PB1.

7 – The Epitopes of Influenza Nucleoprotein Recognized by Cytotoxic T Lymphocytes Can Be Defined with Short Synthetic Peptides

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.