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Biomedical subjects

V Apostolopoulos

Publications and source records attributed to V Apostolopoulos.

At least 37 records · Page 2Linked to original sources
PubMed2000

Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.

Aldehydes↗
PubMed2000

A role for IL-5 in the induction of cytotoxic T lymphocytes in vivo.

IL-5 is generally regarded as a Th2 cytokine involved in eosinophil maturation and function and in B cell growth and antibody production, but without any well-established effects on T cells. Early reports suggested that IL-5 could stimulate the production of cytotoxic T lymphocytes (CTL) in vitro, but no evidence has been obtained to date for such a role in studies with IL-5-deficient (IL-5-/-) mice. Here we demonstrate that when oxidized mannan MUC1 fusion protein (M-FP) is used as an antigen in mice, IL-5 is required for the optimal generation of the CTL response. IL-5 was as effective as IL-2 for the induction of CTL from spleen cells in vitro and both CD4+ and CD8+ T cells from M-FP-immunized animals could be shown to secrete IL-5 in culture. In IL-5-/- mice, CTLp frequency was greatly diminished resulting in the inability to reject MUC1- tumors. Clearly, IL-5 is produced by functional T cells, especially the Tc1 type, after M-FP immunization and is required for an optimal CTL response to this antigen.

Amino Acid Sequence↗
PubMed2000

Generation of cellular immune responses to antigenic tumor peptides.

Tumor immunotherapy is currently receiving close scrutiny. However, with the identification of tumor antigens and their production by recombinant means, the use of cytokines and knowledge of major histocompatibility complex (MHC) class I and class II presentation has provided ample reagents for use and clear indications of how they should be used. At this time, much attention is focused on using peptides to be presented by MHC class I molecules to both induce and be targets for CD8+ cytolytic T cells. Many peptides generated endogenously or given exogenously can enter the class I pathway, but a number of other methods of entering this pathway are also known and are discussed in detail herein. While the review concentrates on inducing cytotoxic T cells (CTLs), it is becoming increasingly apparent that other modes of immunotherapy would be desirable, such as class II presentation to induce increased helper activity (for CTL), but also activating macrophages to be effective against tumor cells.

Antigen Presentation↗
PubMed2000

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Animals↗
PubMed2000

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1.

MUC1 is a mucin over-expressed in breast cancer and a proposed target for immunotherapy. By immunising mice with MUC1 conjugated to mannan (M-FP), CD8(+) MHC-class-I restricted cytotoxic T lymphocytes (CTL), of high CTL precursor (CTLp) frequency (1/8000) and with significant tumour protection, can be induced. The effect of various cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-7, interferon gamma (IFNgamma), and granulocyte/macrophage-colony-stimulating factor (GM-CSF)] on the MUC1 CTL immune response was investigated (a) by measuring the frequencies of CTLp in mice immunised with vaccinia virus constructs containing recombinant cytokines and M-FP, or (b) by immunising cytokine- or cytokine-receptor-knockout (-/-) mice with M-FP. Vaccinia virus (VV) constructs containing recombinant cytokines were used either individually or in combination in vivo with M-FP immunisation. M-FP immunisations combined with VV-IL-2, VV-IL-7 and VV-GM-CSF, and combinations of VV-IFNgamma + VV-IL-2, VV-IFNgamma + VV-IL-4 or VV-GM-CSF + VV-IL-7 increased CTLp frequencies up to threefold (1/17 666: M-FP + VV-GM-CSF + VV-IL-7) compared to M-FP (1/77 500) alone. By contrast, M-FP combined with VV-IL-4 decreased the CTLp frequency threefold whereas VV-IL-6 and VV-IFNgamma had no effect. Studies in cytokine- and cytokine-receptor-gene-knockout (-/-) mice demonstrated that mice that are IL-2 -/- and IL-7 receptor -/- produce the same CTLp response to M-FP as do control mice, whereas responses in the IL-6 -/-, IL-10 -/- and IFNgamma -/- mice were marginally improved and responses to M-FP in IL-4 -/- and tumour necrosis factor receptor 2 -/- mice were weaker. In spite of the increase in CTLp frequency, this was not reflected in an in vivo tumour model. Tumour challenges using MUC1(+) P815 cells, demonstrated that the addition of cytokines had little additive effect on the already effective tumour-regression capabilities of M-FP alone.

Amino Acid Sequence↗
PubMed2000

A functional hot spot for antigen recognition in a superagonist TCR/MHC complex.

A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.

Animals↗
PubMed2000

Selection and characterization of MUC1-specific CD8+ T cells from MUC1 transgenic mice immunized with dendritic-carcinoma fusion cells.

Mice transgenic for the human MUC1 carcinoma-associated antigen (MUC1.Tg) are tolerant to immunization with MUC1 antigen. Recent studies, however, have demonstrated that immunization of MUC1.Tg mice with fusions of MUC1-positive tumour and dendritic cells (FC/MUC1) reverses MUC1 unresponsiveness and results in rejection of established MUC1-positive pulmonary metastases. Here we demonstrate that lymph node cells from MUC1.Tg mice immunized with the FC/MUC1 fusion cells proliferate in response to MUC1 antigen by a mechanism dependent on the function of CD4, major histocompatibility complex (MHC) class II, B7-1, B7-2, CD28, CD40 and CD40 ligand. The findings demonstrate that stimulation of lymph node cells with MUC1 results in selection of MUC1-specific CD8+ T cells. We show that the CD8+ T cells exhibit MUC1-specific cytotoxic T lymphocyte (CTL) activity by recognition of MUC1 peptides presented in the context of MHC class I molecules Kb and Db. The MUC1-specific CD8+ T cells also exhibit antitumour activity against MUC1-positive metastases, but with no apparent reactivity against normal tissues. These results indicate that immunization of MUC1.Tg mice with FC/MUC1 reverses immunological unresponsiveness to MUC1 by presentation of MUC1 peptides in the presence of costimulatory signals and generates MHC-restricted MUC1-specific CD8+ T cells.

Animals↗
PubMed2000

Function and dysfunction of T cell receptor: structural studies.

The engagement of the T cell receptor (TCR) to its ligand, the major histocompatibility complex (MHC)-peptide complex, leads to T cell activation. The molecular mechanisms leading to this activation are still unknown. Dimerization or substantial conformational changes following TCR ligation have not been observed by classical biochemical methods or by X-ray crystallography of the TCR/MHC complex. However, most of these experiments have used reductionist approaches in which only MHC and TCR molecules were taken into account. In fact, the TCR is only one of many molecules forming the TCR complex (TCRC), and the interplay among the components of this larger complex have not been studied in depth. The reconstitution of a complete TCRC using recombinant molecules is our goal and will be the first step to new structural and functional studies.

Animals↗
PubMed2000

Structural implications for the design of molecular vaccines.

The major histocompatibility complex molecules bind and present short antigenic peptide fragments on the surface of antigen presenting cells to T-cell receptors. Recognition of peptide-MHC by cytotoxic T-cells initiates a cascade of signals to T-cells, which in turn destroy the antigen presenting cell. In the design of molecular vaccines for the treatment of diseases, an understanding of the 3-dimensional structure of MHC class I and is interaction with both peptide and T-cell receptor is an important prerequisite. In this review, we will discuss such crystal structures, as well as structures of glycopeptides and alternative T-cell antigens presented by MHC molecules.

Amino Acid Sequence↗
PubMed2000

The evolution of DNA vaccines.

Vaccination has eradicated smallpox and greatly decreased mortality and morbidity associated with a variety of viral and bacterial infectious diseases. However, conventional methodologies have failed to provide vaccines against many widespread deadly human diseases, among them HIV, malaria and cancer. Recombinant DNA vaccines have shown great promise in animal models in inducing protective immunity. In this review we will discuss their potential safe use in humans following recent advances in their use in animals, including non-human primates.

Animals↗
PubMed1999

Carbohydrate/peptide mimics: effect on MUC1 cancer immunotherapy.

Recent clinical studies with mannan mucin immunotherapeutic agents indicate that patients produce predominantly antibody responses while mice produce a high cytotoxic T lymphocyte response. In studying the reason for the 'immune deviation' occurring in mice to humans from cellular to antibody responses, it has been found that natural anti-Galalpha(1,3)Gal antibodies, present in all humans, react with the mucin component of the agent, providing an example of a carbohydrate-peptide mimic. The immune deviation can be overcome by in vitro sensitization of antigen-presenting cells in the absence of anti-Gal antibodies - at least in mice. The review examines the background of these observations and discusses other peptide carbohydrate mimics and immune deviation

Animals↗
PubMed1999

Mimics and cross reactions of relevance to tumour immunotherapy.

MUC1 has been used as a target for immunotherapy and with oxidised mannan in mice there is selective delivery into the class I pathway and the induction of a T1 response. We have also been working in pig-to-human transplantation and of particular interest is the description in humans of natural Galalpha(1,3)Gal antibodies (Abs) which react with pig tissues. A peptide mimic (DAHWESWL) to the Galalpha(1,3)Gal sugar was found in a phage display library and is also mimicked by MUC1 peptides. It was of interest to note that while mice make cytotoxic T cells (CTLs) and little Ab to MUC1, humans make the reverse immune response. It was found that the cross reaction of the natural Galalpha(1,3)Gal Abs in humans to MUC1 was likely to be responsible for the diversion. Cross reactions are therefore an important problem in tumour immunotherapy, although the problem can be overcome by in vitro immunisations.

Animals↗
PubMed1999

Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.

Animals↗
PubMed1999

MUC1 and breast cancer.

The development of an effective immunotherapeutic approach to cancer is now a major focus of research, and despite impressive progress over the last 10 years there are still many hurdles to overcome to elicit an effective immune response which will totally eradicate the cancer. Mucins (MUC1) have attracted interest as potential targets for immunotherapy of cancers of breast, pancreas, ovary and others, and we have demonstrated that mannan, a polymannose carbohydrate is an effective carrier for MUC1 in eliciting a cellular immune response. Several clinical trials are in progress to evaluate the immunogenicity of MUC1 and its suitability as to use for immunotherapy/vaccine for breast cancer.

Amino Acid Sequence↗
PubMed1998

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules.

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.

Amino Acid Sequence↗