Human macrophages and dendritic cells can equally present MART-1 antigen to CD8(+) T cells after phagocytosis of gamma-irradiated melanoma cells

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8⁺ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8⁺ T cell clone. Confocal microscopy with Alexa Fluor®⁶⁴⁷-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8⁺ T cell cross-presentation thereafter.     

Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo

Download : Download video (68MB)     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Affinity enhancement bivalent morpholino for pretargeting: initial evidence by surface plasmon resonance

Pretargeting with bivalent effectors capable of bridging antitumor antibodies has been reported to provide superior results by affinity enhancement. Morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, likely to be a determinant of binding, may be adjusted simply by lengthening the chain. The goal of this investigation was to synthesize a bivalent MORF and to determine by surface plasmon resonance (SPR) whether the bivalent MORF exhibited bimolecular binding and whether the MORFs showed improved in vitro hybridization affinity in its bivalent form compared to its monovalent form. An 18 mer amino-derivitized MORF was made bivalent by dimerizing with disuccinimidyl suberate (DSS) in 1-methyl-2-pyrrolidinone (NMP) with N,N-diisopropylethylamine (DIEA) followed by purification by ion exchange chromatography. The in vitro hybridization affinity of bivalent compared to monovalent MORF was then measured by SPR. For these measurements, the complementary biotinylated cDNA was immobilized at coating densities that provided an average spacing of 20-100 angstroms and used to investigate the influence of this spacing on binding of the bivalent MORF with its binding regions separated by 25 A. The yield of bivalent MORF was as high as 45%, and the structure was confirmed by MALDI-TOF mass spectroscopy. When the sensograms obtained by SPR were analyzed using different binding models, the evidence was consistent with bimolecular binding of the bivalent MORF. The dissociation rate constant of the bivalent compared to monovalent MORF was more than 10-fold lower at 2.14 compared to 0.27 x 10(-5) (1/s) (p < 0.05), and since the association rate constants were similar at 8.53 and 5.64 x 10(5) (1/M.s) (p = 0.08), the equilibrium constant for hybridization to the immobilized cDNA of the bivalent compared to the monovalent MORF was almost 20-fold higher at 3.99 compared to 0.21 x 10(10) (1/M) (p < 0.05). In addition, qualitative evidence for bivalent binding of the bivalent MORF was apparent in the lower concentrations necessary to saturate the cDNA. Finally, the stoichiometry interpretation of the binding data provided estimates of the fraction of bivalent MORF binding bimolecularly. Under one set of conditions, this value was 20%. In conclusion, a bivalent MORF was easily synthesized by dimerization of a monovalent MORF. A lower dissociation rate constant and higher equilibrium constant was measured by SPR for the bivalent compared to monovalent MORF in their binding to an immobilized cDNA. These results show that bimolecular binding was occurring in the case of the bivalent MORF and suggest that bivalency may be superior to monovalency in MORF pretargeting applications.     

Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.     

The cost of growing large: costs of post‐weaning growth on body mass senescence in a wild mammal

Individual body mass often positively correlates with survival and reproductive success, whereas fitness costs of growing large are rarely detected in vertebrates in the wild. Evidence that adult body mass progressively declines with increasing age is accumulating across mammalian populations. Growing fast to a large body can increase the cellular damage accumulated throughout life, leading body growth in early life to be negatively associated with the rate of body mass senescence. Moreover, the onset of mass senescence may strongly depend on both sex‐specific reproductive tactics and environmental conditions. Assessing the timing and the rate of body mass decline with increasing age thus offers an opportunity to look for costs of having grown fast, especially after a poor start during early life, in both sexes and in different environments. Using a unique dataset including 30 years of longitudinal data on age‐specific body mass collected in two roe deer Capreolus capreolus populations subjected to contrasted environmental conditions, we looked for potential costs of high post‐weaning growth rate in terms of steeper rate of body mass senescence. Our analyses of body mass senescence accounted for the potential variation in the onset of senescence and allowed explicit comparisons of this variable between sexes and populations. Higher growth rates late in the growing period (after weaning) were associated with a steeper rate of body mass senescence, regardless of early mass (gained before weaning), but at different extents depending on sex and environmental conditions. Body mass senescence occurred earlier in males than in females, especially in the population facing limiting resources. In the wild, although heavy individuals generally survive better than small ones, the costs of growing large late in the growing period only became apparent late in life through mass senescence.     

3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.