Protein Transfer of Glycosyl-Phosphatidylinositol (GPI)-Modified Murine B7–1 and B7–2 Costimulators

Brunschwig, Elaine; Fayen, John D.; Medof, M. Edward; Tykocinski, Mark L.

doi:10.1097/00002371-199909000-00002

Cited by 41 publications

(41 citation statements)

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“…The method described here to incorporate exogenous lipidanchored soluble proteins into cells is similar in some respects to the previously described technique of "cell painting" by which purified exogenous GPI-anchored proteins can be incorporated into cell surfaces (37)(38)(39)(40)(41)(42). However, for applications like those illustrated here it is advantageous to utilize proteins tethered at the membrane surface to lipid anchors that have simple and unnatural structures and, hence, are less likely to interact in a biospecific manner with other membrane components than is for example a GPI-anchor, which is native to all mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Wang¹,

Leventis

Silvius

2005

Journal of Biological Chemistry

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We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquidordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoylor dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaffolding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that crosslinking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.

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Section: Discussionmentioning

confidence: 99%

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Wang¹,

Leventis

Silvius

2005

Journal of Biological Chemistry

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“…Of relevance to this, the engraftment onto tumor cell membranes of the extracellular region of certain receptors (e.g., the extracellular region of B7.1) linked to a glycosylphosphatidylinositol anchor is often sufficient to render the tumor cells more effective as vaccines (6 -8). These observations suggest that techniques for modifying cell surfaces, for example, by anchoring molecules such as recombinant forms of the extracellular region of B7.1, may be a viable approach in the development of cell-based vaccines (6,7). Immobilized metal chelators, such as nitrilotriacetic acid (NTA), 3 have been used routinely for purifying recombinant proteins bearing polyhistidine tags by metal-ion affinity chromatography (9).…”

mentioning

confidence: 99%

Engrafting Costimulator Molecules onto Tumor Cell Surfaces with Chelator Lipids: A Potentially Convenient Approach in Cancer Vaccine Development

Broekhoven

Parish

Vassiliou

et al. 2000

The Journal of Immunology

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The genetic modification of cells to develop cell-based vaccines and to modulate immune responses in vivo can be risky and inconvenient to perform in clinical situations. A novel chelator lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA) that, via the NTA group has high affinity for 6His peptide, was used to directly anchor recombinant forms of T cell costimulatory molecules containing a C-terminal 6-His sequence onto tumor cell surfaces. Initial experiments using murine P815 tumor cells established the optimum conditions for incorporating NTA-DTDA onto the membranes of cells. P815 cells with incorporated NTA-DTDAbound hexahistidine-(6His)-tagged forms of the extracellular domains of murine B7.1 and CD40 (B7.1-6H and CD40-6H) at very high levels (fluorescence 200–300-fold above background), and both proteins could be anchored onto the cells simultaneously. Significant loss of the anchored or “engrafted” protein occurred through membrane internalization following culture of the cells under physiological conditions, but P815 cells with engrafted B7.1-6H and/or CD40-6H stimulated the proliferation of allogenic and syngeneic splenic T cells in vitro, and generated cytotoxic T cells when used as vaccines in syngeneic animals. Furthermore, the immunization of syngeneic mice with P815 cells engrafted with B7.1-6H or with B7.1-6H and CD40-6H induced protection against challenge with the native P815 tumor. The results indicate that the use of chelator lipids like NTD-DTDA to engraft costimulatory and/or other molecules onto cell membranes could provide a convenient alternative to transfection in the development of cell-based vaccines and for modulation of immune function.

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“…The efficiency of painting is high and reveals homogenous insertion of the glypiated protein into the plasma membrane. Painting is rapid and maximal insertion is reached after 90 min of incubation of the [26,[34][35][36]44]. Furthermore, the painted proteins are completely removed from the cell surface in the course of several cycles of cell division.…”

Section: Discussionmentioning

confidence: 99%

“…To achieve this, we equipped the plasma membrane of primary lymphocytes with KISS31-GPI. This strategy, called painting, is rapid, can be performed in standard medium and does not interfere with immune function [24,26,[34][35][36]. KISS31-GPI was constructed using the GPI anchor derived from the decayaccelerating factor (DAF).…”

Section: Kiss31-gpi: An > V I 3 Integrin-binding Moleculementioning

confidence: 99%

The α vβ 3 integrin as a tumor homing ligand for lymphocytes

Legler

Johnson‐Léger²,

Wiedle³

et al. 2004

Eur J Immunol

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Despite the presence of tumor‐specific effector cells in the circulation of cancer patients, the immune response of the majority of these patients is not sufficient to prevent the growth and spread of their tumors. That tumor cells can be killed in vitro by tumor‐reactive cytotoxic T cells is testimony to the fact that the tumors are not inherently resistant to T cell killing, but rather that there is a failure in immune recognition and effector cell activation. Many reasons for this failure of the body's defense system have been suggested, including the inability of tumor‐reactive lymphocytes to migrate to tumor tissue. Here we designed a strategy to improve homing of primary lymphocytes into vascularized tumors. As a homing molecule we selected the integrin α vβ 3 since it is expressed by angiogenic vascular endothelium in tumors. To promote lymphocyte adhesion to α vβ 3 we "painted" primary lymphocytes with a recombinant, glycosylphosphatidylinositol‐linked high‐affinity ligand for α vβ 3. These painted lymphocytes specifically bound to α vβ 3 in vitro and homed to vascularized, solid tumors in vivo. This novel strategy may provide a significant advance in anti‐tumor treatment such as adoptive immune therapy.

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Protein Transfer of Glycosyl-Phosphatidylinositol (GPI)-Modified Murine B7–1 and B7–2 Costimulators

Cited by 41 publications

References 0 publications

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association

Engrafting Costimulator Molecules onto Tumor Cell Surfaces with Chelator Lipids: A Potentially Convenient Approach in Cancer Vaccine Development

The α vβ 3 integrin as a tumor homing ligand for lymphocytes

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