Patrick Machy

Class I‐restricted presentation of exogenous antigen acquired by Fcγ receptor‐mediated endocytosis is regulated in dendritic cells

We evaluated MHC class I‐ and II‐restricted presentation of exogenous antigen by mouse bone marrow‐derived dendritic cells (DC) and splenic B cells. DC presented to class I‐restricted transgenic T cells femtomolar concentrations of antigens from liposomes targeted to the IgG Fc receptor. Targeting these liposomes to surface immunoglobulin did not permit B cells to stimulate class I‐restricted responses. Nevertheless, both DC and B cells presented antigen from liposomes targeted to these same receptors with equivalent efficiency to class II‐restricted T cells. Acquisition of the capacity to present class II‐restricted antigens required shorter periods of differentiation of DC than presentation of exogenous class I‐restricted antigens. The latent period for class I‐restricted presentation of exogenous antigen by DC could not be shortened by exposing them to lipopolysaccharide, double‐stranded RNA or antibody to CD40. Class I presentation depended on expression of the TAP1 transporter. Our data are consistent with the existence of a regulated transport process present in DC which can convey exogenous antigen from endocytic vesicles to the cytosol.

Small liposomes are better than large liposomes for specific drug delivery in vitro

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 A, 2000 A or 4000 A, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2Kk monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 A vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.

Weak acid-induced release of liposome-encapsulated carboxyfluorescein

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form: 9 X 10(-9) cm X s-1), acetic acid (greater than 1 X 10(-7) cm X s-1) and glycine (cation: 6 X 10(-9) cm X s-1). These results are consistent with low effective proton permeability of liposomes (less than 5 X 10(-12) cm X s-1) and with the permeability coefficient of HCl (3 X 10(-3) cm X s-1) reported by Nozaki and Tanford ( (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324-4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.

Induction of MHC Class I Presentation of Exogenous Antigen by Dendritic Cells Is Controlled by CD4+ T Cells Engaging Class II Molecules in Cholesterol-Rich Domains

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-β-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.

MHC Class II-Peptide Complexes in Dendritic Cell Lipid Microdomains Initiate the CD4 Th1 Phenotype

We investigated differentiation of CD4 T cells responding to Ag presented by bone marrow-derived dendritic cells (DC) in association with MHC class II (MHC II) molecules. Peptides encapsulated in liposomes opsonized by IgG were taken up by endocytosis. MHC II-peptide-specific T cells responding to this Ag were polarized to a Th1 cytokine profile in a CD40-, CD28-, MyD88-, and IL-12-dependent manner. Th2 responses were obtained from the same transgenic T cell population exposed to the same DC on which MHC-peptide complexes had dispersed for 48 h following uptake of FcR-targeted liposomes. DC that took up the same FcR-targeted liposomes and then were exposed to methyl-β-cyclodextrin, which chelates cholesterol and dissociates lipid microdomains, also stimulated Th2 differentiation. Incubation of T cells with DC incubated with peptides directly binding to MHC II resulted in Th2 responses, whether or not the DC were coincubated with opsonized liposomes as a maturation stimulus. CD4 Th1 polarization thus appears to depend on MHC II-peptide complex clustering in DC lipid microdomains and the time between peptide loading and T cell encounter.

Antibody targeted liposomes containing poly(rI)•poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons α/β or γ

Double-stranded RNA can stimulate interferon production and mediate an antiproliferative effect on certain cell types. We evaluated the possibility of specifically targeting to cells in vitro the RNA duplex poly(rI) · poly(rC) in pharmacologically active form after its encapsulation in small, unilamellar liposomes, to which was covalently coupled protein A. These liposomes became bound to and were endocytosed by murine L929 cells in the presence of protein A-binding monoclonal antibodies specific for an expressed cell surface protein, the H-2K molecule. When L929 cells were preincubated in the presence of low doses of interferon α/β or γ, they could be activated to produce interferon following exposure to either free poly(rI) · poly(rC), or specifically bound liposome poly(rI) · poly(rC), but not the same liposomes in the presence of non-cell binding control antibodies. Specifically bound liposome-encapsulated poly(rI) · poly(rC) was toxic to L929 cells at dose levels at least three logs lower than free poly(rI) · poly(rC). This toxicity was also dependent on pre-treatment with interferon. These results indicate that liposome-encapsulated poly(rI) · poly(rC) can survive endocytosis and can be released in active form to specific cell populations, at concentration much lower than that required for pharmacologic effects of the same molecule in free form. They suggest that introduction into cells of other nucleic acids might benefit from the antibody-targeted liposome technology described here.

Immunoliposome-Mediated Delivery of Nucleic Acids: A Review of Our Laboratory's Experience

AbstractExperiments performed in our laboratory or in collaboration with other groups demonstrating the delivery to cells of mono-, oligo- and polynucleotides from antibody-targeted small liposomes are reviewed. Biologically-active molecules delivered into cells via these liposomes include: phosphorylated derivatives of dideoxyuridine, which have activity against the human immunodeficiency virus; the oligonucleotide (2-5) (A)n and various analogues, which have anti-viral and antiproliferative activity; and antisense phosphodiester and phosphorothioate oligodeoxynucleotides, which may have both gene-specific and nonspecific effects. Polynucleotides include: the RNA duplex poly (rI:rC), and related molecules, which are inducers of interferon and other cytokines; long RNA antisense molecules and plasmids. Advantages for delivery by liposomes, as compared to use of the same molecules free in solution include: protection against degradation, reduction of toxicity, improved pharmacokinetics and the possibilit...

Dendritic Cells Capture and Efficiently Present Antigen Encapsulated in Liposomes to T Cells In Vivo

For the last twenty-five years of the liposome era, liposomes have been modified in various ways to minimize their uptake by what used to be called the reticuloendothelial system (1). Cells of this system reduce the availability of liposomeassociated drugs to tumors or other intended target sites. Liposomes have been used as antigen (Ag) carriers for the generation of antibody (Ab) responses in vivo for about the same time (2). More recently, interest has extended to the use of liposomes as carriers for the generation of cytotoxic T cells. It now appears that some of those nasty reticuloendothelial cells that interfere with the circulation of liposomes injected by the drug deliverers are the desired targets of those interested in using liposomes for vaccination. These cells, now usually called dendritic cells (DC), are potent inducers of immune responses. Dendritic cells are widely distributed in the body and are specialized to take up and process Ag and to present it in association

Endocytosis of Cell Surface Molecules and Intracellular Delivery of Materials Encapsulated in Targeted Liposomes

The principle benefits for pharmacologic purposes of liposomes include the possibility of transporting highly concentrated reagents entrapped in the liposomes’ aqueous spaces or lipid bilayer and their protection against enzymatic degradation. In addition, certain molecules which may have little or no capacity to enter into cells because of their size or charge may be delivered in biologically active form inside cells by virtue of their association with liposomes. This capacity is enhanced when liposomes are targeted to the cell surface by the use of specific ligands such as monoclonal antibodies, which may additionally offer the possibility to concentrate material at the cell surface and to target sub-populations in a heterogeneous mixture of cells (for reviews see Leserman and Machy, 1987; Ostro, 1987).

Positive and negative liposome-based immunoselection techniques.

Publisher Summary This chapter highlights positive and negative liposome-based immunoselection techniques. Liposomes are vesicles composed of one or several phospholipid bilayers surrounding a closed aqueous compartment. They are useful for marking, killing, or rescuing cell populations. The use of liposomes as fluorescent reagents, notably for the cell sorter, offers high signal with low background. Liposomes are formed with the compound of interest encapsulated within the enclosed space or as part of the component phospholipids. After liposome formation, the liposomes are coupled to the freshly activated protein at room temperature, usually by mixing the protein and liposomes and dialyzing against buffered saline at pH 8–8.5 for several hours. High specificity of action is the case for the use of liposomes for negative selection, which requires that the target determinant is endocytosed by the cell, in contrast to the action of antibody and complement, for which expression of the molecule in question is sufficient for the cell to be killed. Positive selection with liposome-encapsulated protective reagents, though studied in a small number of model systems, is seriously challenged only by the fluorescence-activated cell sorter, which has been used for this application in only a few laboratories highly experienced in its use.