Structure of the O9 antigen from Burkholderia (Pseudomonas) cepacia

Abstract The O9 antigen of Burkholderia (Pseudomonas) cepacia has the following disaccharide repeating-unit: → 4)-α-D-Glc-p-(1 → 3)-α-L-Rha p-(1 → The same unit is present in the O antigens of Serratia marcescens strain S1254 and serogroup O4 (predominantly acetylated at O-2 of rhamnose in the latter case).     

Dendritic cell biology and cancer therapy

Dendritic cells (DCs) are natures best antigen-presenting cells. They possess attributes that allow them to effectively fulfill the requirements for priming/activating T cells and mediating tumor-specific immune responses. In this review, emphasis is placed on those aspects of DC biology that best illustrate their usefulness in immunotherapy of cancer. Culture, maturation, and polarization conditions for human DC are discussed, as are strategies for antigen-loading of DCs and for their delivery to patients with cancer. A concise recommendation for monitoring of DC-based vaccination trails is provided.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Identification of tumour antigens by serological analysis of cDNA expression cloning

The identification of antigens that distinguish normal cells from cancer cells is an important challenge in the field of tumour immunology and immunotherapy. The immunoscreening of cDNA expression libraries constructed from human tumour tissues with antibodies in sera from cancer patents (SEREX: serological identification of antigens by recombinant expression cloning) provides a powerful approach to identify immunogenic tumour antigens. To date, over 2,000 tumour antigens have been identified from a variety of malignancies using SEREX. These antigens can be classified into several categories, of which the cancer/testis (CT) antigens appear to be the most attractive candidates for vaccine development. The SEREX-defined tumour antigens facilitate the identification of epitopes (antigenic peptides) recognised by antigen-specific cytotoxic T lymphocytes (CTLs) and provide a basis for peptide vaccine and gene therapy in a wide variety of human cancers. Moreover, some of these antigens seem to play a functional role in the pathogenesis of cancer.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

How B cells and dendritic cells may cooperate in antigen purification

The specificity of the immunological responses is achieved through the cooperation of three classes of cells: B and T lymphocytes, and dendritic cells (DCs). A critical, intensely studied interaction is that between DCs and T cells, during which the DC presents MHC-bound antigenic fragments to the T cell receptor (TCR). There has been recent excitement about the possibility of increasing the signal-to-noise ratio in the detection of cognate antigen-TCR couples, by the use of kinetic proofreading mechanisms. We examine here the signal-to-noise problem in a broader perspective, and in particular, address the question of possible "antigen purification" mechanisms, prior to their presentation to the T cells. Ways in which the DCs might concentrate, purify and preserve their load of captured antigens are considered: (i) If antigens can be transferred from one DC to another, in such a way that the richer a DC in antigen, the more it captures antigens from other DCs, the antigens may end up concentrated in a small subset of DCs, (ii) antigen purification may be achieved through recycling interactions between DCs and B cells. A DC would transmit to a B cell antigen mixtures, and the DC would recapture only the antigens which can bind to the B cell's antibodies and (iii) dendrites, when they are present, may play an essential role in recapturing the antigens that were used in interactions of DCs with T cells, B cells, or other DCs, thereby reducing antigen losses. More generally, we provide a personal interpretation of cell-to-cell antigen transfers, in terms of a strategy in which there is a progressive emergence, through multiple interactions, of subsets of cells of each type better and better prepared for the subsequent rounds of interactions.     

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250-270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H-thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen-specific T responses, because of the low specific frequencies of the cells. Collagen II (250-270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250-270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3-color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.     

Activated mouse T-cells synthesize MHC class II, process, and present morbillivirus nucleocapsid protein to primed T-cells

A pivotal step in the initiation of T-cell immunity is the presentation of antigenic peptides by major histocompatibility complex (MHC) expressed on antigen presenting cells. The expression of MHC class II molecules by mouse T-cells has not been shown unequivocally. In the present work, we demonstrate that activated mouse T-cells synthesize MHC class II molecules de novo and express them on their surface. Further, we have demonstrated that in vitro activated T-cells take up extra-cellular soluble nucleocapsid protein of a morbillivirus. The internalized antigen goes to antigen processing compartment as shown by co-localization of antigen and LAMP-1 using confocal microscopy. We show that activated T-cells express H2M, a chaperone molecule known to have a role in antigen presentation. Further, we demonstrate that activated T-cells process and present internalized extra-cellular antigen to primed T-cells as shown by IL-2 secretion and in vitro proliferation. The presentation of antigen by T-cells may have implications in immuno-regulation, control of infection by lymphotropic viruses and maintenance of immunological memory.     

Antigen traffic pathways in dendritic cells

Dendritic cells (DC) are now believed to be the principal initiators of T cell-mediated immune responses. Their location in body tissues, migratory behaviour in response to inflammatory stimuli, endocytic properties, expression of MHC molecules and key T cell stimulatory molecules and many other attributes place these remarkable cells in a unique and influential position in the immune system. Progress in DC culture methods has recently allowed in-depth studies on the cell biological features that enable them to fulfil their crucial role in the immune response.