Frances M. Brodsky

Monoclonal Antibodies for Analysis of the HLA System

III Antibodies A Monoclonal antibodies against determinants of the HLA-A,-B,-C glycoproteins and pjmicroglobulin 1. Antibodies against species specific, non-polymorphic determinants 2. Antibodies against polymorphic HLA-A,-B,-C determinants B Monoclonal antibodies against HLA-DRw determinants 1. Antibody against a species specific HLA-DRw determinants 2. Antibodies against polymorphic HLA-DRw determinants

Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission.

Transmission of HIV-1 via intercellular connections has been estimated as 100–1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.

EGF Receptor Signaling Stimulates SRC Kinase Phosphorylation of Clathrin, Influencing Clathrin Redistribution and EGF Uptake

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.

Partial purification and some properties of BB7.2 a cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28

The purification and properties of a cytotoxic mouse monoclonal anti-HLA-A2 antibody are described. This antibody, BB7.2, can be used as an HLA-A2 typing reagent with little modification of current typing techniques. It also recognizes a low frequency variant of HLA-A28. BB7.2 provides an example of an antibody which requires bivalent attachment to a polymeric antigen, e.g., a cell, to produce a readily detectable complex. This is due to a high rate of dissociation of the complex formed between a single BB7.2 combining sites and HLA-A2. The consequences of this property were investigated and some complications for potential uses of such monoclonal antibodies are discussed.

Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds

Significance Prion-like propagation of proteopathic seeds may underlie the progression of neurodegenerative diseases, including the tauopathies and synucleinopathies. Aggregate entry into the cell is a crucial step in transcellular propagation. We used chemical, enzymatic, and genetic methods to identify heparan sulfate proteoglycans as critical mediators of tau aggregate binding and uptake, and subsequent seeding of normal intracellular tau. This pathway mediates aggregate uptake in cultured cells, primary neurons, and brain. α-Synuclein fibrils use the same entry mechanism to seed intracellular aggregation, whereas huntingtin fibrils do not. This establishes the molecular basis for a key step in aggregate propagation. Recent experimental evidence suggests that transcellular propagation of fibrillar protein aggregates drives the progression of neurodegenerative diseases in a prion-like manner. This phenomenon is now well described in cell and animal models and involves the release of protein aggregates into the extracellular space. Free aggregates then enter neighboring cells to seed further fibrillization. The mechanism by which aggregated extracellular proteins such as tau and α-synuclein bind and enter cells to trigger intracellular fibril formation is unknown. Prior work indicates that prion protein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface to transmit pathologic processes. Here, we find that tau fibril uptake also occurs via HSPG binding. This is blocked in cultured cells and primary neurons by heparin, chlorate, heparinase, and genetic knockdown of a key HSPG synthetic enzyme, Ext1. Interference with tau binding to HSPGs prevents recombinant tau fibrils from inducing intracellular aggregation and blocks transcellular aggregate propagation. In vivo, a heparin mimetic, F6, blocks neuronal uptake of stereotactically injected tau fibrils. Finally, uptake and seeding by α-synuclein fibrils, but not huntingtin fibrils, occurs by the same mechanism as tau. This work suggests a unifying mechanism of cell uptake and propagation for tauopathy and synucleinopathy.

Interactions between HIV1 Nef and Vacuolar ATPase Facilitate the Internalization of CD4

CD4 is the primary receptor for the human immunodeficiency virus (HIV). Nef is an accessory protein of HIV that decreases the expression of CD4 on the surface of infected cells. In this study, we identified the Nef binding protein 1 (NBP1), which interacts specifically with Nef in vitro and in vivo. Since it shares sequence similarity with the catalytic subunit of the vacuolar ATPase (V-ATPase) and complements the loss of this VMA13 gene in yeast, NBP1 is the human homolog of Vma13p. Direct interactions between Nef and NBP1 were correlated with the ability of Nef to internalize CD4. The expression of the antisense NBP1 abrogated these effects. We conclude that NBP1 helps to connect Nef with the endocytic pathway.

A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.

Lipid Rafts Unite Signaling Cascades with Clathrin to Regulate BCR Internalization

A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.