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International audience; The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Reaching long term immune tolerance to therapeutic transgenes represents an important goal for gene therapists, particularly for the treatment of monogenic muscular pathologies, such as Duchenne muscular dystrophy. Indeed, when the transgene contains sequence elements divergent from endogenous ones, a collection of lymphocytes that have not been purged during their development is available to initiate rejection of transduced cells. The ensuing recruitment and activation of transgene-specific lymphocytes generates adverse humoral and cellular responses, which are prominent in tissues subjected to vector transduction and inflammation. To overcome this obstacle, we harnessed the tolerogenic properties of the liver and explored how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment. We found that concurrent rAAV transduction of muscle and liver promotes a state of transgene-specific tolerance, resulting in the absence of CD8+ T cell responses combined with lower humoral response to the transgene. This tolerance is achievable in muscle weeks after liver transduction and is equally able to override preexisting immunity to the transgene. Regarding the mechanism, we found that dual muscle-liver transduction converts preexisting polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 expression and low IFN-γ production. Our results complemented with muscle confocal imaging demonstrate that transduction of muscle tissue can be protected from immune attack by liver-mediated control of humoral and cytotoxic T cell responses. Importantly, this liver-based tolerance induction process applies even in the presence of preexisting immunity to the transgene of interest.

Unilamellar liposomes can be fused at low pH with the plasma membrane of cells that express the hemagglutinin glycoprotein of influenza virus on their surface [van Meer, G., & Simons, K. (1983) J. Cell Biol. 97, 1365-1374]. Here, we have resolved this fusion process into two kinetically distinct steps. The first and more rapid step converts the bound liposome to a form that can no longer be released by neuraminidase. The second step is the actual membrane fusion as measured by the loss of resonance energy transfer between two liposomal fluorescent phospholipids, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolami ne (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In contrast to the first step, the rate of the second one was highly dependent on the liposomal lipid composition and the cell type used. The replacement of 50% of the phosphatidylcholine (PC) in egg PC-cholesterol liposomes by unsaturated phosphatidylethanolamine (PE) species increased the rate of fusion at least 2-fold. Of the PE-containing liposomes that were associated with Madin-Darby canine kidney (MDCK) cells after 30 s of fusion, 80% had actually fused with the plasma membrane. Fringe pattern fluorescence photobleaching experiments showed that after fusion a fraction of the cell-associated N-Rh-PE diffused laterally in the plasma membrane. Without fusion, the N-Rh-PE was completely immobile. Under optimal conditions, the mobile fractions were 65% on MDCK cells and 78% on baby hamster kidney cells. The mobility was acquired simultaneously with the dilution of the fluorescent phospholipids as measured from the loss of resonance energy transfer. The mobile fraction of N-Rh-PE on the cell surface can therefore be used as a second independent measure of actual membrane fusion. Finally, we observed that upon fusion up to 80% of the nonexchangeable liposome markers cholesterol [14C]oleate and glycerol tri[14C]oleate became accessible to cellular hydrolases. The results showed that this hydrolysis assay can also be used to monitor the second step of the fusion process.

Programmed death via apoptosis is the metazoan physiologic mode of cell death. Apoptotic cells are recognized by scavenger phagocytes via a number of membrane receptors and engulfed. Thereafter, little is known of their fate, or that of phagocytes. Here, we have traced apoptotic cells upon their engulfment by macrophages. After 3 h, apoptotic cells were contained in discrete well-defined vacuoles. Upon overnight chase, several small vesicles, possibly originating from the fragmentation of original vacuoles, were evident all over the macrophage body. Furthermore, Ags were diffused in the cytosol of some cells, which raises the possibility that epitopes from engulfed apoptotic cells may associate with macrophage MHC class I molecules and be recognized by T lymphocytes. Indeed, Ag-specific CTLs recognize and specifically lyse syngeneic macrophages upon phagocytosis of MHC class I-positive or -negative apoptotic cells, provided that they contain the relevant Ags. Synthesis and membrane expression of class I molecules by macrophages, together with functional transporters associated with Ag presentation, were necessary for recognition and lysis. The indirect presentation of epitopes from engulfed apoptotic cells by scavenger Ag-presenting phagocytes may, in the absence of "danger" signals, have implications for the establishment of central and peripheral self-tolerance.

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.

B lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig α/Ig β heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig α/Ig β components in BCR constitutive endocytosis. The role of Ig α subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig α, surface BCR lacking Ig α cytoplasmic domain were not constitutively internalized. The respective roles of Ig α and Ig β subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig α cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig α chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig α, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.