Class I-restricted presentation of exogenous antigen acquired by Fcgamma 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.

Presentation of antigens internalized through the B cell receptor requires newly synthesized MHC class II molecules.

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their alpha- or beta-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak beta-chain is required for the presentation of the hen egg lysozyme determinant (46-61) following BCR internalization, but that dependence is not seen for the (34-45) determinant derived from the same protein. The tail of the beta-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II beta-chains control the loading of determinants produced after extensive Ag processing.

Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway.

The intracellular sites in which Ags delivered by the B cell receptor (BCR) are degraded and loaded onto class II molecules remain poorly defined. To address this issue, we generated wild-type and invariant chain (Ii)-deficient H-2k mice bearing BCR specific for hen egg lysozyme. Our results show that, 1) unlike Ags taken up from the fluid phase, Ii is required for presentation of hen egg lysozyme internalized through the BCR in a manner independent of the peptide analyzed; 2) BCR ligation induces intracellular accumulation of MHC class II molecules only in Ii-positive B cells; and 3) these class II molecules reach intracellular compartments where BCR targets exogenous Ag. No differences in expression of adhesion and costimulatory molecules or in the presentation of soluble peptides were detectable between Ii-positive and -negative B cells. Therefore, the BCR delivers its ligand to compartments containing MHC class II-Ii complexes and bypasses the Ii-independent presentation pathway. The linked roles of Ag internalization and B cell activation of the BCR leads to potent Ii-dependent presentation in splenic B cells.

Efficient presentation of multivalent antigens targeted to various cell surface molecules of dendritic cells and surface Ig of antigen-specific B cells.

To study the relation between the form of an Ag and the response to it, we compared presentation in vitro with hen egg lysozyme (HEL)-specific T cells from TCR transgenic mice of free HEL and liposome-encapsulated HEL by different APC. HEL-specific splenic B cells or bone marrow-derived dendritic cells were incubated with free HEL or HEL-containing liposomes targeted by Ab to either surface Ig, the Fc receptor, or MHC class I and II molecules. Ag presentation by HEL-specific B cells was at least 100-fold more efficient for HEL in surface Ig-targeted liposomes than free HEL taken up by the same receptor or HEL in liposomes targeted to class I or II molecules. Ag presentation by dendritic cells from Fc receptor-targeted vesicles was augmented 1,000-10,000-fold compared with free Ag or nontargeted liposomes, but presentation was also efficient when Ag was targeted to class I or II molecules. These results indicate that Ag-specific B cells and dendritic cells can be equally efficient in stimulating IL-2 production by Ag-specific T cells from unimmunized TCR transgenic mice when the Ag is multivalent and taken up by appropriate receptors. In contrast to B cells, which require engagement of surface Ig for optimal presentation, dendritic cells may present Ag by means of several different cell surface molecules.

MHC class I molecules are implicated in costimulatory signals during TCR/CD3-induced activation.

Mouse MHC class I-specific mAbs recognizing the alpha 1/alpha 2, but not those directed against the alpha 3 domain of the molecule, inhibited RNA, protein, and DNA synthesis of splenic T cells in response to stimulation through the TCR/CD3 complex. Similar inhibition was seen with LFA-1-specific mAbs under the same stimulation conditions. The effect of class I- and LFA-1-specific mAbs reflected a decrease of both IL-2 and IFN-gamma synthesis and IL-2 receptor alpha chain induction. IL-2, IL-2 receptor alpha chain, IFN-gamma, c-fos, c-jun, and c-myc mRNAs were not detected. Activation of AP-1 (c-Fos and c-Jun proteins) and NF-kappa B transcription factors were also inhibited. Inhibition was observed both after treatment of cells in culture and after intravenous injection of Abs in mice. Although bulk phosphorylation was inhibited, early tyrosine phosphorylation and calcium ion influx were normally induced. Protein phosphatase inhibitors did not reverse this inhibition, ruling out an enhanced activation of these enzymes in the observed inhibition. Cell surface expression of one of early PKC activation marker, CD69 was also inhibited. Phorbol esters that directly activate PKC prevented inhibition. Thus, class I molecules are implicated in signal transduction involved at an early stage for T cell activation in a manner that suggests their implication in accessory signal transmission that contributes to the regulation of PKC activity.

Influence of MHC class I molecules on T-cell proliferation induced by CD3 or Thy-1 stimulation.

We have reported that class I- [and lymphocyte function-associated antigen-1 (LFA-1-)] specific monoclonal antibodies (mAb) inhibit anti-CD3-mediated activation of naive T cells. The present study investigated the mechanism of this inhibition. CD28-specific mAb augmented stimulation induced by soluble CD3 mAb, but this costimulation was also inhibited by anti-class I or anti-LFA-1 mAb. However, stimulation of T cells was not inhibited when activated B cells were present. Neither B7-1- nor B7-2-specific blocking mAb or soluble CTLA-4, CD40 or gp39 restored the inhibition. Thus, other molecules expressed on activated B cells are implicated for T-cell activation, which could compensate blockade of class I or LFA-1 molecules. Inhibition induced by class I-specific mAb could potentially be mediated through extracellular, transmembrane or cytoplasmic domains of the target molecules. These possibilities were evaluated by the use of mice transgenic for the Qa-2 molecule, selected for expression of Qa-2 at levels equivalent to classical class I molecules. Qa-2 is inserted in the membrane through phosphatidylinositol linkages. Antibodies directed to Qa-2 inhibited CD3-induced stimulation, demonstrating that cytoplasmic and transmembrane protein sequences of class I molecules are not necessary for the inhibitory effect. Inhibition thus presumably depends on extracellular domains. Finally, T cells from beta 2-microglobulin knock-out mice responded to CD3-specific mAb as well as their class I-positive littermates. Nevertheless, stimulation of T cells from these mice with mitogenic anti-Thy-1 mAb was markedly reduced. Signalling by Thy-1 and the CD3 complex may normally occur through pathways in which class I molecules are implicated.

Specific in vitro labeling of cells with a fluorine-19 probe encapsulated in antibody-targeted liposomes: a F-19 NMR spectroscopy study.

Liposomes containing dexamethasone phosphate (DMp) were covalently coupled to protein A and then incubated with murine L929 fibroblast and RDM4 thymoma cells in the presence of monoclonal antibodies specific for the major histocompatibility complex. The detection of the specific F-19 labeling of cells is rapid (minutes). Such a strategy might be useful to study the kinetics of internalization processes of bound liposomes on cultured living cells.

Class I-specific antibodies inhibit proliferation in primary but not secondary mouse T cell responses.

Murine T and B splenocytes were incubated with antibodies that recognize CD3 or surface IgM. These antibodies induced proliferation of their respective target cells. Once stimulated via their receptors, the proliferation of both CD4+ and CD8+ T but not B lymphocytes was inhibited by class I-specific antibodies or their monovalent Fab' fragments. The inhibition of proliferation was dependent on the site on class I molecules recognized by the antibodies used, with the alpha 1/alpha 2 domains of H-2K molecules representing the major site for inhibition. Only soluble antibody-mediated proliferation could be inhibited by class I-directed antibodies; proliferation induced by CD3-specific antibody immobilized on plastic was not inhibited. Primary allogeneic MLR was also inhibited by class I-specific antibodies. In contrast, neither secondary allogeneic MLR, secondary Ag-specific responses, nor proliferation of CTL clones or tumor cell lines were inhibited by class I-specific antibodies. These results suggest a role for class I molecules in regulation of TCR/CD3- but not surface IgM-mediated cell signaling, which depends on the form of stimulation and the stage of differentiation of T cells.

Free and liposome-encapsulated double-stranded RNAs as inducers of interferon, interleukin-6, and cellular toxicity.

Poly(rI:rC) and Ampligen were entrapped in liposomes that were covalently coupled to Protein A, permitting binding to antibodies specific for the major histocompatibility complex-encoded H2K molecule of L929 cells, or to control antibodies. Free and encapsulated polynucleotides were compared for their capacity to stimulate secretion of interferon (IFN) and interleukin-6 (IL-6) and to induce cellular toxicity on L929 cells pretreated with IFN-alpha/beta. Free and encapsulated poly(rI:rC) or Ampligen (poly(rI:rC12-rU] induced similar levels of secretion of IFN over a broad dose range. The activity of the liposome-encapsulated polynucleotides was dependent on its binding to an antibody that permitted cell association and internalization; the same liposomes were inactive in the presence of control antibodies. IL-6 secretion was induced by double-stranded (ds) RNA in a dose-dependent manner, with a significantly greater effect seen for targeted, liposome-encapsulated material. The marked toxicity of targeted poly(rI:rC), as compared to free poly(rI:rC), was confirmed. Encapsulated Ampligen was less toxic than encapsulated Poly(rI:rC).

Endocytosis and recycling of MHC-encoded class II molecules by mouse B lymphocytes.

The movements of mouse MHC-encoded class II (H-2E) and class I (H-2K), transferrin receptor and surface Ig molecules of B lymphocytes were studied using radiolabeled mAb and electron microscopy. A total of 10 to 20% of antibodies specific for H-2E molecules were gradually internalized with a t 1/2 of 15 min, reaching a plateau after 30 min at 37 degrees C. Equivalent results were obtained either with the whole antibody or Fab' fragments, suggesting that the internalization of class II molecules was spontaneous. Similar results were obtained with antibodies specific for the transferrin receptor, of which 50% were internalized with t 1/2 of 5 min, reaching a plateau after 30 min. In contrast to antibodies specific for H-2E molecules and the transferrin receptor, antibodies specific for H-2K were not internalized. Reappearance of internalized H-2E-specific antibodies at the cell surface was observed at 37 degrees C. When compared to antibodies specific for surface Ig, degradation of antibodies specific for H-2E molecules was limited even after 5 h incubation. Neither ammonium chloride nor cycloheximide inhibited internalization and recycling. Electron microscopy showed that internalization of H-2E molecules occurred via coated pits/coated vesicles. These results indicate that class II molecules are spontaneously internalized and recycled by B lymphocytes.

Antibody-targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication.

Mouse L929 cells were incubated with antibody-targeted liposomes containing oligodeoxyribonucleotides (oligomers). When the oligomer was a 15-mer complementary to the 5'-end region of the mRNA encoding the N protein of vesicular stomatitis virus, the cells became less permissive for multiplication of that virus; greater than 95% reduction of viral multiplication was achieved. Protection was not seen for "empty" liposomes, liposomes containing a random oligomer sequence, or liposomes containing a sequence complementary to the 5' end of c-myc protooncogene mRNA targeted by the same antibody, nor was it seen when the liposomes containing the N-protein antisense oligomer were targeted by an antibody that does not bind to L929 cells. Antibody-bearing liposomes containing antisense oligomers thus have a double specificity: a particular cell selected by the targeting antibody on the liposome and a particular mRNA in the cell selected by sequence complementarity with the liposome-encapsulated oligomer. Nonencapsulated oligomers are sensitive to nucleases and usually must be administered to cells at high concentrations. Oligomers encapsulated in liposomes resist DNase and are active in amounts 1-2 orders of magnitude lower than for those reported for unencapsulated oligomer sequences.

Antibody targeted liposomes containing poly(rI).poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons alpha/beta or gamma.

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 alpha/beta or gamma, they could be activated to produce interferon following exposure to either free poly(rI).poly(rC), or specifically bound liposomes 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 concentrations 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.

Differential half-life of major histocompatibility complex encoded class I molecules in T and B lymphoblasts.

Major histocompatibility complex encoded class I molecules have been reported to be internalized in T lymphocytes but not in B lymphocytes. In order to better understand the physiology of these molecules, we investigated their kinetics of disappearance and the fate of antibody bound to them in T and B lymphoblasts. Metabolically labelled H-2K molecules were immunoprecipitated from the surface and from inside the cells after different periods of chase and analyzed by gel electrophoresis. In T lymphoblasts, there was a rapid disappearance of both surface and intracellular H-2K molecules with a half-life of about 5 hr. After a 20 hr chase, only lower mol. wt products were immunoprecipitated. In contrast, in B lymphoblasts, H-2K molecules were more stable with a half-life of greater than 20 hr. Bound anti-H-2K antibodies were degraded in T but not in B lymphoblasts. These results suggest that class I molecules and antibodies bound to them do not recycle back to the cell surface but are degraded after internalization in T lymphoblasts, whereas these molecules are less degraded in B lymphoblasts.

Gene transfer from targeted liposomes to specific lymphoid cells by electroporation.

Large unilamellar liposomes, coated with protein A and encapsulating the gene that confers resistance to mycophenolic acid, were used as a model system to demonstrate gene transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2a antibodies, was coupled to liposomes to target them specifically to defined cell types coated with IgG2a antibody. Protein A-coated liposomes bound human B lymphoblastoid cells preincubated with a mouse IgG2a anti-HLA monoclonal antibody but failed to adhere to cells challenged with an irrelevant (anti-H-2) antibody of the same isotype or to cells incubated in the absence of antibody. Transfection of target cells bound to protein A-coated liposomes was achieved by electroporation. This step was essential since only electroporated cells survived in a selective medium containing mycophenolic acid. Transfection efficiency with electroporation and targeted liposomes was as efficient as conventional procedures that used unencapsulated plasmids free in solution but, in the latter case, cell selectivity is not possible. This technique provides a methodology for introducing defined biological macromolecules into specific cell types.

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies.

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.

Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method.

We describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein-conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification. In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC-conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies (mAb) against H-2K and H-2D, respectively. When RDM4 cells were treated with anti-H-2K mAb followed by FITC-conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes.

Endocytosis of the major histocompatibility complex (MHC)-encoded class I and class II molecules has been the subject of recent investigations. Class I molecules, which are key elements in T cell-mediated cytotoxicity, are differentially endocytosed by different cell types. Fibroblasts internalize their class I molecules via uncoated cell surface vesicles and tubular invaginations when these molecules are cross-linked with multivalent ligands. T lymphocytes internalize their class I molecules spontaneously, but B lymphocytes do not internalize them at all. Here we describe a morphological investigation of the mechanism by which class I molecules are endocytosed by T lymphocytes. We show that, unlike fibroblasts, T lymphocytes spontaneously internalize 20-40% of their class I molecules in a process involving coated pits and coated vesicles. Thus, the endocytic pathway of class I molecules in T lymphocytes is similar to those of other more classical cell-surface receptors involved in receptor-mediated endocytosis. In contrast, the same class I molecules remained on the cell surface in B lymphocytes. These data show that class I molecules are differentially regulated in T and B lymphocytes and fibroblasts.

Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging.

Liposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2-5, the entrapped space inside liposome can contain several hundred to several thousand molecules of fluorochromes in a space chemically isolated from the outside environment, thus providing the potential for an amplified fluorescence signal. We have prepared small unilamellar liposomes containing the soluble fluorochromes carboxyfluorescein (CF), which fluoresces in the green and sulforhodamine (SR), which fluoresces in the red, and covalently coupled a series of monoclonal antibodies using a heterobifunctional reagent. We were able to detect, on an Epics 753 flow cytometer equipped with an argon ion and a dye laser and by fluorescence microscopy, both single and double labeled mouse spleen lymphocyte subsets, fibroblast L cells and Raji cells. Complete color separation was obtained with CF-labeled cells being detected only by the green photomultiplier and SR-labeled cells by the red photomultiplier. Cells labeled with both were detected by both photomultipliers. Liposomes bearing anti-Ia antibodies bound only to B lymphocytes whereas those with anti-H-2K antibody bound both to T and B lymphocytes. In another system, single and dual color immunofluorescence made possible the simultaneous detection of HLA and H-2K molecules on transfected murine fibroblast L cells. The signal-to-noise ratio was more favorable for the liposome-labeled reagents than reagents labeled with fluorescein isothiocyanate. Cells labeled with antibody-bearing liposomes could be fixed with paraformaldehyde or glutaraldehyde without adversely affecting the original staining patterns. Apart from the two fluorochromes described above, other markers of choice could be encapsulated without any adverse effect on the antibody-liposome coupling procedure or on the specificity of the conjugated antibody. Since the fluorochrome is not directly coupled to the protein, there is no requirement for protein conjugation sites in order for it be usefully encapsulated inside liposomes. Therefore, this system provides new opportunities to exploit different, as yet untapped fluorochromes for use in flow cytometry and imaging.