Biomedical subjects

Jean Davoust

Publications and source records attributed to Jean Davoust.

13 recordsLinked to original sources

PubMed2006

Simple conditioning with monospecific CD4+CD25+ regulatory T cells for bone marrow engraftment and tolerance to multiple gene products.

A major impediment to gene replacement therapy is immune elimination of genetically modified cells. In principle, this can be dealt with by inducing a strong, specific, and enduring tolerance through engraftment of transgene-modified autologous bone marrow (BM). Because usual myeloablation and/or immunosuppression are risk factors in most pathologies, we assessed the potential of monospecific CD4(+)CD25(+) regulatory T cells (Tregs) to engraft minor-mismatched BM without preconditioning. We found that as few as 5 x 10(4) Tregs directed to the male DBY protein promote the engraftment of foreign male BM into sex-mismatched female hosts, establishing sustained chimerism in all hematopoeitic compartments. We achieved concomitantly strong tolerance to all foreign antigens expressed in the BM, likely occurring through induction of anergy and/or deletion of antidonor T cells. Chimerism was obtained in thymectomized mice too, underlining the major role of peripheral tolerance mechanisms in our system. This allowed us to engraft gene-modified tissues while preserving full immunocompetence to third-party antigens. Our results demonstrate that very few donor-specific Tregs are effective as the sole conditioning to induce mixed molecular chimerism and long-term tolerance to multiple foreign antigens.

Animals↗

PubMed2006

Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay. Two mechanisms might explain the hyperthermia-induced enhanced cross-priming. First, heat-treated melanoma cells expressed increased levels of 70-kDa heat shock protein (HSP70), and enhanced cross-priming could be reproduced by overexpression of HSP70 in melanoma cells transduced with HSP70 encoding lentiviral vector. Second, hyperthermia resulted in the increased transcription of several tumor Ag-associated Ags, including MAGE-B3, -B4, -A8, and -A10. Thus, heat treatment of tumor cells permits enhanced cross-priming, possibly via up-regulation of both HSPs and tumor Ag expression.

Amino Acid Sequence↗

PubMed2005

Efficient migration of dendritic cells toward lymph node chemokines and induction of T(H)1 responses require maturation stimulus and apoptotic cell interaction.

Dendritic cells (DCs) have the unique ability to initiate primary immune responses, and they can be conditioned for vaccinal purposes to present antigens after the engulfment of apoptotic cells. To recruit the rare antigen-specific naive T cells, DCs require a maturation step and subsequent transport toward lymph node (LN). To date, prostaglandin E2 (PGE2) is the best-characterized compound inducing this LN-directed migration in vitro, but PGE2 may skew the immune responses in a T(H)2 direction. We demonstrate here that on incubation with apoptotic tumor cells and tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), human monocyte-derived DCs become fully mature and acquire high migratory capacities toward LN-directing chemokines. The migration of TNF-alpha-treated DCs occurs only after cotreatment with apoptotic cells but not with necrotic cells. DC migration requires CD36 expression and incubation with apoptotic cells in the presence of heat-labile serum components. Moreover, on treatment with apoptotic cells and LPS, the migrating DCs are able to recruit naive T cells to generate T(H)1 immune responses. Our results show that the cotreatment of DCs with apoptotic tumor cells and inflammatory signals is promising for the design of an antitumoral DC-based vaccine.

Apolipoproteins C↗

PubMed2005

Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells.

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Animals↗

PubMed2004

Imaging calpain protease activity by multiphoton FRET in living mice.

Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.

Animals↗

PubMed2004

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Adult↗

PubMed2004

CD208/dendritic cell-lysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes.

Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed2004

High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy.

Most of the human tumor-associated antigens (TAAs) characterized thus far are derived from nonmutated "self"-proteins. Numerous strategies have been developed to break tolerance to TAAs, combining various forms of antigens with different vectors and adjuvants. However, no study has yet determined how to select epitopes within a given TAA to induce the highest antitumor effector response. We addressed this question by evaluating in HLA-A*0201-transgenic HHD mice the antitumor vaccination efficacy of high- and low-affinity epitopes from the naturally expressed murine telomerase reverse transcriptase (mTERT). Immunity against low-affinity epitopes was induced with heteroclitical variants. We show here that the CTL repertoire against high-affinity epitopes is partially tolerized, while that against low-affinity epitopes is composed of frequent CTLs with high avidity. The high-affinity p797 and p545 mTERT epitopes are not able to protect mice from a lethal challenge with the mTERT-expressing EL4-HHD tumor. In contrast, mice developing CTL responses against the p572 and p988 low-affinity epitopes exhibit potent antitumor immunity and no sign of autoimmune reactivity against TERT-expressing normal tissues. Our results strongly argue for new TAA epitope selection and modification strategies in antitumor immunotherapy applications in humans.

Animals↗

PubMed2003

ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.

Induction of cytotoxic T-cell immunity requires the phagocytosis of pathogens, virus-infected or dead tumour cells by dendritic cells. Peptides derived from phagocytosed antigens are then presented to CD8+ T lymphocytes on major histocompatibility complex (MHC) class I molecules, a process called "cross-presentation". After phagocytosis, antigens are exported into the cytosol and degraded by the proteasome. The resulting peptides are thought to be translocated into the lumen of the endoplasmic reticulum (ER) by specific transporters associated with antigen presentation (TAP), and loaded onto MHC class I molecules by a complex "loading machinery" (which includes tapasin, calreticulin and Erp57). Here we show that soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, peptides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I molecules. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER-phagosome mix compartment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

CD4+CD25+ regulatory T cells inhibit immune-mediated transgene rejection.

Like cellular transplantation, gene therapy is often limited by immune rejection of the newly expressed antigen. In a model of gene transfer in muscle, delivery of the influenza hemagglutinin (HA) membrane protein by adeno-associated virus (AAV) is impaired by a strong immune response that leads to a rapid rejection of the transduced fibers. We show here that injection of HA-specific CD4+CD25+ T cells from T-cell receptor (TCR)-transgenic animals, concomitant with gene transfer, down-regulates the anti-HA cytotoxic and B-lymphocyte responses and enables persistent HA expression in muscle. This demonstrates for the first time that adoptive transfer of antigen-specific CD4+CD25+ regulatory T cells can be used to induce sustained transgene engraftment in solid tissues.

Animals↗

PubMed2003

Human germinal center B cells differ from naive and memory B cells by their aggregated MHC class II-rich compartments lacking HLA-DO.

To generate memory B cells bearing high-affinity antibodies, naive B cells first encounter antigen in the T cell-rich areas of secondary lymphoid organs. There, they are activated by antigen-specific T cells and become germinal center (GC) founder B cells. GC founders enter the GC to become centroblasts that proliferate and mutate their BCR. Centroblasts differentiate into centrocytes that undergo selection, which requires both the recognition/capture of antigen on follicular dendritic cells and the presentation of processed antigen to GC T cells. Because at each stage of differentiation B cells act as antigen-presenting cells, we analyzed their content of HLA-DR(+)-rich compartments (MIIC), as well as their expression of HLA-DM, which catalyzes peptide loading of class II molecules, and HLA-DO, which interacts with HLA-DM and focuses MHC class II peptide loading on antigens internalized by the BCR. Naive and memory B cells concentrate HLA-DR, -DM and -DO into compartments dispersed under the cell surface, which are identified by their expression of lysosome-associated membrane protein (Lamp)-1 as late endosomes/lysosomes. GC founders and GC B cells express larger Lamp-1(+)DR(+) compartments that are concentrated in the juxta-nuclear region. These compartments express lower levels of HLA-DM and virtually no HLA-DO. Upon induction of a GC founder phenotype through the prolonged (days) co-ligation of BCR and CD40, the naive B cell's peripheral DR(+)DM(+)Lamp-1(+) compartments aggregate in a polar fashion close to the nucleus. Furthermore, HLA-DO expression virtually disappears, whereas low levels of HLA-DM remain co-localized with HLA-DR. Anti-kappa/lambda antibodies, used as surrogate antigens, are promptly (minutes) endocytosed in naive, memory and GC B cells. Then, naive and memory B cells target the surrogate antigen to their peripheral HLA-DO(+) MIIC, while GC B cells target it to their HLA-DO(-) MIIC aggregates. Taken together, our results show that human GC B cells differ from naive and memory B cells by their aggregated MIIC that lack HLA-DO.

Animals↗

PubMed2002

The melanocytic protein Melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins.

To delineate the role of the melanocyte lineage-specific protein Melan-A/MART-1 in melanogenic functions, a set of biochemical and microscopical studies was performed. Biochemical analysis revealed that Melan-A/MART-1 is post-translationally acylated and undergoes a rapid turnover in a pigmented melanoma cell line. Immunofluorescence and immunoelectron microscopy analyses indicated that Melan-A/MART-1 is mainly located in the Golgi area and only partially colocalizes with melanosomal proteins. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular content of Melan-A/MART-1 was found in small vesicles and tubules throughout the cell, whereas the concentration was maximal in the Golgi region, particularly the trans-Golgi network. Substantial labeling was also present on melanosomes, endosomes, ER, nuclear envelope, and plasma membrane. In early endosomes, Melan-A was enriched in areas of the limiting membrane covered by a bi-layered coat, a structural characteristic of melanosomal precursor compartments. Upon melanosome maturation, Melan-A concentration decreased and its predominant localization shifted from the limiting membrane to internal vesicle membranes. In conjunction with its acylation, the high expression levels of Melan-A in the trans-Golgi network, in dispersed vesicles, and on the limiting membrane of premelanosomes indicate that the protein may play a role during the early stage of melanosome biogenesis.

Antigens, Neoplasm↗

PubMed1990

Lateral Mobility of the Cell Adhesion Molecule L1 Within the Surface Membrane of Morphologically Undifferentiated and Differentiated Neuroblastoma Cells.

Lateral mobility and localization in the surface membrane of the adhesion molecule L1 was studied in morphologically undifferentiated and differentiated neuroblastoma cells to gain insight into its possible association with the different molecular forms of N-CAM. In undifferentiated cells, the fraction of mobile L1 molecules is high and similar to that of N-CAM 140. Upon long-term morphological differentiation, the fraction of mobile L1 molecules is reduced by a factor of three and is similar to that of N-CAM 180, the predominant molecular form of N-CAM in differentiated neuroblastoma cells. Comparable to N-CAM 180, L1 is also preferentially accumulated at contact sites between these cells as seen by indirect immunofluorescence. These observations raise the question of whether at least part of the L1 molecules may be directly or indirectly (e.g. via N-CAM 180) linked to the cytoskeleton, thus stabilizing cell contacts between differentiated cells.

Journal Article↗