Naked antigen-presenting molecules on dendritic cells.

Antigen-presenting cells work to present peptides derived from exogenous and endogenous antigens to circulating T cells, sparking off an immune response. Dendritic cells are unique amongst antigen-presenting cells, not least for their newly described ability to circumvent the need to internalize exogenous antigens before presenting them.

Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.

The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells.

Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rgamma chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a(+)HLA-DR(+)CD14(-)DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)alpha, and CD40L induce maturation of IL15-DCs to CD83(+), DC-LAMP(+) cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3alpha/CCL20. However, IL15-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.

Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin.

The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.

Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells.

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.

In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas.

We have analyzed the presence of immature and mature dendritic cells (DCs) within adenocarcinoma of the breast using immunohistochemistry. Immature DCs were defined by expression of CD1a-, Langerin-, and intracellular major histocompatibility complex class II-rich vesicles. Mature DCs were defined by expression of CD83 and DC-Lamp. Breast carcinoma cells were defined by morphology and/or cytokeratin expression. We demonstrate two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature CD1a(+) DCs, mostly of the Langerhans cell type (Langerin(+)), were retained within the tumor bed in 32/32 samples and (b) mature DCs, CD83(+)DC-Lamp(+), present in 20/32 samples, are confined to peritumoral areas. The high numbers of immature DCs found in the tumor may be best explained by high levels of macrophage inflammatory protein 3alpha expression by virtually all tumor cells. Confirming the immature/mature DC compartmentalization pattern, in vitro-generated immature DCs adhere to the tumor cells, whereas mature DCs adhere selectively to peritumoral areas. In some cases, T cells are clustering around the mature DCs in peritumoral areas, thus resembling the DC-T cell clusters of secondary lymphoid organs, which are characteristic of ongoing immune reactions.

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(-8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.

Low cytoplasmic pH inhibits endocytosis and transport from the trans-Golgi network to the cell surface.

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Immunodeficiency in protein kinase cbeta-deficient mice.

Cross-linking of the antigen receptor on lymphocytes by antigens or antibodies to the receptor results in activation of enzymes of the protein kinase C (PKC) family. Mice homozygous for a targeted disruption of the gene encoding the PKC-betaI and PKC-betaII isoforms develop an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B cells, which is similar to X-linked immunodeficiency in mice. Thus PKC-betaI and PKC-betaII play an important role in B cell activation and may be functionally linked to Bruton's tyrosine kinase in antigen receptor-mediated signal transduction.

Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

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.

Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes.

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.

Boundary lipids and protein mobility in rhodopsin-phosphatidylcholine vesicles. Effect of lipid phase transitions.

Purified rhodopsin from bovine retina has been incorporated into phospholipid bilayers. Dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine and egg phosphatidylcholine were used as host lipids, with ratio of lipid to protein of 120 : 1 (mol to mol). In order to probe the lipid-protein interface specifically, a spin-labeled fatty acid was covalently bound to rhodopsin via an isocyanate reacting group. A spin-labeled phospholipid was used to probe the bulk lipidic phase while a tightly bound maleimide spin label was used to obtain the protein rotational correlation time by the saturation transfer technique. The following results were obtained: (1) The kinetics of reduction by ascorbate of the spin-labeled fatty acid covalently bound to rhodopsin demonstrate that the alkyl chain attached to the protein is positioned in the membrane in the same way as the alkyl chains of a phospholipid. (2) The EPR spectra of the latter shows two components: a strongly immobilized component and a weakly immobilized component. The ratio of the two depends upon the temperature and on the nature of the phospholipids. (3) The signal of the weakly immobilized component is compared to that obtained in the corresponding pure lipids. The latter signal, assumed to represent non-bounded lipids, indicates a sharp transition at the phospholipid phase transition with dimytristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The former signal (corresponding to the lipid-protein interface) indicates only a broad transition extending over 7 degrees C with dipalmitoylphosphatidylcholine and almost no transition with dimyristoylphosphatidylcholine. (4) In a similar way, the rotational correlation time of the protein only changes progressively when the phase transition occurs. Our interpretation of the data can be summarized as follows: The immobilized component seen by the EPR technique in the hydrophobic environment of this intrinsic protein very probably reflects protein-protein contacts and thus corresponds to hindrance of the labeled chains, when they are trapped between neighbouring proteins. Below the phase transition lipid segregation whould increase the probability of protein contact. However, over a certain range of temperature, the contact with the protein interface probably at the same time prevents the non-segregated phospholipids from feezing. The differences in the results obtained with the various phosphatidylcholines above their transition temperature suggest that the solubility of rhodopsin in bilayers depends not only on the fluidity of the lipids, but also, to some extent, on the phospholipid chain length.

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Apical and basal Forssman antigen in MDCK II cells: a morphological and quantitative study.

We have studied the surface distribution of a glycosphingolipid (the Forssman antigen) in MDCK II and CCL39 cells. The Forssman antigen is mobile on the surface of both these cell lines. Its surface distribution is homogenous on non-polarized cells. Under conditions where MDCK II cells are well polarized, the Forssman antigen is present in equal amounts on the apical membrane and on the basal membrane and its processes. Very little Forssman antigen can be detected on the lateral membrane. The nature of the mechanism excluding the Forssman antigen from the lateral domain remains to be determined. This surface distribution is established within hours after plating and was observed with cells grown on different types of filters. The surface density of the Forssman antigen on the apical and on the basal domain has been estimated. No involvement of the basal Forssman antigen in cell attachment could be demonstrated. However, the apical Forssman antigen appears to be essential to the establishment of the cells in culture.

Dendritic cells from mice lacking the invariant chain express high levels of membrane MHC class II molecules in vivo.

We investigated in H-2k mice bearing a genetically disrupted invariant chain (Ii) gene, the MHC class II expression and antigen presentation ability of dendritic cells (DC) freshly purified from the spleen (SpDC) or derived from bone marrow precursors (BMDC) upon treatment with GM-CSF. In the absence of Ii, class II alpha/beta heterodimers are expressed on the DC membranes to a similar extent than in control mice, in contrast to splenic B cells. Class II molecules immunoprecipitated from the plasma membrane of Ii deficient DC are compact indicating that the dimers are stabilized by antigenic peptides. Furthermore DC from Ii mutant mice are able to present to CD4+ T lymphocytes, epitopes derived from the processing of the hen egg lysozyme (HEL) that normally require expression of the Ii molecule for presentation by B cells. All together, our results show that the antigen processing machinary of DC provides peptides that can reach class II molecules and stabilize their conformation in the absence of Ii mediated targeting of class II complexes.

Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.

We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.