RESEARCH ESSAYS
Introduction | Cell-Cell Interaction Group | Viral Immunology Group | Immunology Laboratory | Apoptosis Laboratory | Molecular Virology Laboratory | Molecular Immunology and Immunopathology Group | Experimental Haematology Group | Viral Engineering and Cytokine Group | Immune Regulation and Vaccine Development Laboratory | Mucosal Immunoregulation Laboratory | Synthetic Vaccines LaboratoryIntroduction
The Division of Immunology and Cell Biology is comprised of a number of research groups that investigate fundamental aspects of cell biology, with a major interest being the immune system. Research undertaken by the Division includes investigations of viral replication, analyses of the immune response to viral infections, development of HIV and cancer vaccines, studies of the processes involved in
programmed cell
differentiation and death, and research on the processes of haemopoiesis, inflammation,
tumour angiogenesis and tumour metastasis.The pursuit of long term basic science goals make up most of the Division's work but this is balanced by attempts to translate fundamental discoveries into clinical applications. The latter include the possible application of anionic sugar molecules as novel anti-inflammatory or anti-cancer drugs and naked DNA and recombinant poxviruses as vaccines for prevention of certain infectious diseases or treatment of cancer. More detail of these various themes is given in each Group or Laboratory report.
Professor Chris Parish, Head of Division
Recently the group has cloned the enzyme heparanase, a key enzyme involved in degradation of the extracellular matrix by invading tumour cells and by leukocytes entering inflammatory sites. Cloning and characterisation of this enzyme has eluded researchers for almost 20 years. Currently the CCI group is one of the leading research groups in the world in this research area.
The group has had considerable experience in designing sulfated oligosaccharide-based compounds as drug candidates, this part of the group's research being supported by a large R and D grant from Progen Industries, Brisbane. Sulfated oligosaccharide-based inhibitors of the heparanase enzyme have been synthesised and identified. Furthermore, based on a novel in vitro assay for human angiogenesis developed by the group, sulfated oligosaccharides have also been discovered which inhibit angiogenesis. An extensive drug screening program managed to identify a sulfated oligosaccharide, termed PI-88, which can simultaneously inhibit angiogenesis and heparanase activity. Preclinical testing has shown that PI-88 can inhibit primary tumour growth and tumour metastasis. The drug successfully completed a phase I clinical trial in healthy volunteers in 1999 and is now being tested in cancer patients.
A very productive collaboration has developed recently with Professor Martin Banwell, Research School of Chemistry, ANU in which sulfated pseudo-sugars are being synthesized as heparan sulfate mimetics. Although the initial aim of this collaboration is to produce better heparanase inhibitors, sulfated pseudo-sugars are also being examined as potential anti-angiogenics, anticoagulants, antiviral agents and antilipaemic drugs.
The group has been studying the plasma protein, histidine-rich glycoprotein (HRG), for many years, particularly regarding the ability of the protein to inhibit cell adhesion by masking cell surface carbohydrates. Recently, however, it became clear that HRG plays an important role in the immune system by preventing the insolubilisation of complexes between antibody and antigen (termed immune complexes). In fact HRG also assists in the uptake of these complexes by phagocytic cells. Thus HRG is probably a key molecule in aiding the elimination of immune complexes from the circulation. In fact, deficiencies in HRG may lead to immune complex-associated diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE). This is an extremely novel area of research which is being pioneered by the CCI Group.
A productive collaboration has developed with Dr Joe Altin, BaMBi, ANU in which a procedure has been devised to tether the extracellular domains of cell surface receptors to cell membranes. This technology has been used to graft costimulator molecules, such as CD40 and CD80, onto tumour cell surfaces such that the tumour cells can induce protective, tumour-specific, immunity. This approach has considerable promise as a simple means of producing cancer vaccines.
Results thus far show base changes concentrated in
complementarity-determining regions and no changes elsewhere in the gene
sequence.These data are incompatible with conventional neo-Darwinian evolutionary theory which predicts that random synonymous base changes should occur throughout the gene in addition to non-synonyomous changes (selectable) in complementarity-determining regions.
The mechanism of somatic hypermutation of immunoglobulin variable genes in B lymphocytes remains one of the major unsolved problems in immunology. We are testing a hypothesis which states that reverse transcription using pre-mRNA of rearranged variable regions as the template, thus producing cDNA which homologously recombines into the chromosomal rearranged DNA, is the primary agent of sequence variation. Production of a number of lines of single-copy transgenic mice is being undertaken to explore the role of the intronic enhancer/matrix attachment region in hypermutation.
The first project involves examining the effects of gold complexes on the rate of proliferation of tumour cells and normal cells. These toxins are synthetic complexes of the metal gold with varying phosphines. The latter substances are generally toxic in their own right but when combined with the (non-toxic) gold atom, they acquire a positive charge and selectively kill human tumour cells in culture and leave normal cells unaffected. This selectivity is due to the targeting of the toxin to the mitochondria or energy producing organelle of the cell. Mitochondria in tumour cells have different properties from those in normal cells, allowing enhanced uptake of toxins containing a particular type of positive charge. By synthesising new members of this class of toxin, we are optimising this selectivity and plan to extend these studies to animal tumour models.
The second type of toxin under study in our group are natural products produced by fungi as part of their chemical defence mechanisms. These toxins have probably been produced since the emergence of single celled animals on earth thousands of millions of years ago. They have therefore become selected to efficiently kill other single celled animals. The toxin gliotoxin is a typical member of this particular class of fungal metabolites and is bio-synthesised by the fungus from simple amino acids. One property of gliotoxin is its rapid accumulation in cells suggesting it is actively taken up rather than simply passively entering cells as many other toxins do. We believe that gliotoxin may be mistaken for an amino acid by the cell and thus actively taken into the cell. This strategy would mean that the producing fungus would only have to produce small quantities of the toxin to be effective. Since many cells become resistant to toxins because they actively extrude them to the outside of the cell, the particular mode of action of gliotoxin may suggest routes to new drugs which cannot be so easily removed by the cells defence mechanisms. Gliotoxin also induces a particular form of cell death called apoptosis which is triggered by the cells own biochemical pathways. We have shown that this toxin is at least as active as certain hormones in inducing apoptotic cell death.
A fundamental understanding of the different pathways underlying toxin action lays the groundwork for development of new drugs necessary to cope with the emergence of resistant pathogens and tumour cells.
(P Waring, T Davis, A Hurne, G Maxwell, D Bromhead, K Moerman)
Immunodominance of two peptide determinants in the cytolytic T cell response to flavivirus infection: Inverse correlation between peptide affinity for MHC class I and T cell precursor frequency
(M Regner, A Müllbacher, R Blanden, M Lobigs)
We have investigated the CD8+ cytotoxic T (Tc) cell immune response against the encephalitic flavivirus, Murray Valley encephalitis virus (MVE) restricted by the H-2Kk major histocompatibility complex (MHC) class I molecule. Split-clone limiting dilution analysis revealed almost exclusive recognition of two peptides derived from the viral NS3 protein. Both peptides were presented for recognition by Tc cells with a comparable kinetics during the latent period of infection. Binding affinity of the two determinants to H-2Kk was measured using a MHC class I cell surface stabilization assay. Affinity for H-2Kk and half-lives of the peptide/H-2Kk complexes were significantly different for the two peptides. However, there was an inverse correlation between their affinity for H-2Kk and the avidity of peptide-reactive Tc cell clones and Tc cell precursor frequency. This highlights the importance in the immunodominance phenomenon of avidity for MHC class I/peptide complexes of precursor Tc cells present in the repertoire. The precursor frequency of MVE-reactive Tc cells was determined by limiting dilution analysis for cytotoxic function and immunolabeling for interferon-gamma; the latter gave a 100-fold higher estimate of MVE-reactive Tc cell precursors. Accordingly, the phenotypes of virus-immune CD8+ lymphocytes induced by infection with MVE may be biased towards cytokine production and limited cytolytic effector function.
Mutagenesis of the Signal Sequence of Yellow Fever Virus PrM Protein: Enhancement of Signalase Cleavage, in vitro, is Lethal for Virus Production
(E Lee, CE Stocks, M Lobigs in collaboration with SM Amberg, and CM Rice)
Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced amino acid substitutions at the COOH-terminus of the yellow fever virus (YFV) prM signal sequence (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays using full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus despite efficient signal peptidase cleavage of prM in cell-free expression. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence, which disrupt the coordinated cleavages at the C-prM junction, can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by the regulated cleavages at this site.
Substrate selectivity of a peptide transporter associated with antigen processing (TAP) controlled by polymorphic residues in the TAP1
(M Lobigs, A Müllbacher, RV Blanden, in collaboration with GJ Hämmerling and F Momburg)
Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H-2 allelic products in two cell lines (BHK and NIL-2). These were overcome by transient expression of the rat TAP1 and TAP2 molecules. We could demonstrate that constitutive down-regulation of expression of accessory molecules of the MHC class I pathway in these cells reveal differences between H-2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes in the Syrian hamster cells two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. The polymorphic residues in TAP1 influence the substrate selectivity of the Syrian hamster peptidetransporters which was reflected in their differential preferences for C-termina l peptide residues as shown by an in vitro peptide transport assay.
The specific aims are to continue to investigate the basic principles which govern the mechanism of cytolytic leukocytes in particular CD8+ cytolytic T and natural killer cells and their role in recovery from infection. In this context, elucidation of the mode of action of the two dominant granzymes A and B which are major components of the cytolytic granules, will be of primary importance. Their role in recovery from ectromelia infection has been established but it is as yet not clear if granzymes are involved in other viral infections or recovery from bacterial and parasitic infections.
In addition, poxviruses encode serpins, which are thought to be involved in evasion from immune elimination, interfering with the caspase cascade leading to apoptosis. We aim to construct specific deletion mutants of one or more serpins of ectromelia and will analyze their virulence in the natural mouse host. Thus, we hope, to determine their true biological function as a possible evasion mechanism from cytolytic lymphocyte mediated control.
We also aim to gain an understanding as to the contribution of the Fas pathway of cellular cytotoxicity in recovery from a variety of viral infections. This is particularly relevant in the light of our recent findings which showed that in the absence of a functional granule exocytosis pathway, Tc cells induce Fas expression on previously Fas negative target cells. One further aim is to elucidate if this Fas induction is a general means of limiting antigen in a feedback loop to regulate Tc cell clonal expansion.
Immunological memory underpins all vaccine strategies. The vast majority of vaccines at present at use are protective as a result of B cell memory and antibody responses. The lack of efficient vaccines against a number of important human diseases (i.e.. HIV, influenza, malaria) suggest that B cell mediated protection is not attainable or inappropriate. One alternative is T cell memory; this has recently been attracting widespread attention in the immunological community especially cytotoxic T (Tc) cell memory . Although T cell memory was first described over fifty years ago much remains to be learned about the biochemical and cellular basis of the phenomenon. We do not have detailed knowledge of the cellular events which lead to the establishment of T cell memory. We are not even able to identify a memory Tc cell phenotype precisely. Most progress has been made in analyzing the general functional properties and activation requirements of memory T cells. We have identified two antigenic models, one a non-replicating and the other a virus model which allows induction of primary Tc cell responses but which does not lead to the generation of long lasting memory Tc cells which usually occurs with all other antigenic systems. These models may prove to be a valuable tool which may allow us to unravel the yet unknown aspects of T cell memory.
In order to generate reliable molecular data it is important to use homogeneous populations of cells. It is difficult to do this by isolating cells ex vivo so emphasis is placed on the generation of cell lines. Our primary experimental system involves the use of cell lines that we have made by transfecting progenitor cells that proliferate in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) with a retrovirus that contains a cDNA encoding a truncated form of c-Myb. Expression of this unregulated c-Myb allows the cells to grow indefinitely in GM-CSF. Expression of Myb is a prerequisite for normal proliferation of myeloid cells. As far as we know our myb-transformed haematopoietic cell lines (MTHC) differ from "normal" cells only in the unregulated expression of myb.
As is the case for many myelomonocytic cell lines we have tested, myb-transformed cell lines grown in GM-CSF can be induced to differentiate into macrophages in the presence of tumour-necrosis factor alpha (TNF-alpha) and interleukin-4 (IL-4), while the use of interferon gamma (IFN-gamma) instead of IL-4 forces the cells into the dendritic cell lineage. We are examining the novel genes that are expressed during commitment to these two different lineages using the technique of cDNA subtraction. This project was conducted by Dr. Joanne Banyer, then a post-doctoral fellow in the group. We have isolated a large number of genes that are selectively involved in either macrophage and/or dendritic cell differentiation and are now identifying and characterising these genes as a prelude to investigating their role in differentiation and cell function.
Instead of differentiating in response to TNF-alpha some of our cell lines undergo programmed cell death or apoptosis. Since the apoptotic (A) and differentiating (D) cell lines were sub-cloned from the same cultures we are trying to determine the key differences between them that result in such different outcomes of TNF-alpha signaling. During her thesis work in this group Ms. Peta O'Connell showed that one difference between the A and D cells was that when apoptosis occurred it did so as a result of ligand binding to both type I and type II TNF receptors whereas differentiation required binding to the type I receptor only. Another student, Mr. Hayden Henry, attempted to identify differences in the signaling pathways downstream from the receptors, and in collaboration with Dr. Banyer identified several genes that are selectively expressed or silenced in the A vs D cell lines. In parallel with these projects we are working with Dr. Waring's laboratory on cAMP-regulated pathways activated during apoptosis, proliferation and differentiation, and with Dr. Crouch's laboratory on the role of Gi alpha in proliferation and differentiation of myeloid cells.
At the level of cell biology we have shown that macrophages that differentiate in vitro from myb transformed cell lines are effective at phagocytosis but are poor presenters of antigen. In contrast, cells of the dendritic phenotype, induced by IFN-gamma have potent antigen presenting capacity. In an attempt to induce immune responses against tumour antigens we are transfecting selected genes into our progenitor cell lines and inducing differentiation with cytokines. Differentiated cells can then be used to immunise mice or to induce immune responses in vitro. This cell system provides a model for examining antigen processing and presentation, and for identifying phenotypic differences between antigen handling cells that influence immune class regulation.
Another program of work involves the application of gene targeting technology toward the analysis of certain aspects of cytokine signaling. We have made targeting constructs designed to knock out the receptor for M-CSF (c-fms) and the alpha chain of the interleukin-3 receptor. By combining gene targeting technology with the construction of myb-transformed cells lines from targeted ES cells, we can potentially circumvent the need for viable knock out mouse strains should any of these induced mutations prove lethal in the whole animal. As we identify and learn more about the genes that are involved in apoptosis, proliferation and differentiation of myeloid cells we will use targeting technologies to identify the biological role of targeted genes in vivo.
Marie Escourt, a PhD student in the Group, has recently shown why this vaccine strategy may be so effective. Using a sensitive tetramer staining technique she has shown that prime-boost immunisation can induce high numbers of epitope-specific CD8+ T cells, sometimes as many as 20% of all circulating CD8+ T cells. Prime-boost immunisation may therefore offer the best current prospects for preventive vaccination against HIV infection and a variety of other conditions against which no effective treatment is available.
A National Development HIV Vaccine Team has now been established to test these vaccines in clinical trials, firstly in Sydney and then Thailand. The agencies involved in this Team include the National Centre in HIV Epidemiology and Clinical Research at the University of New South Wales, the Department of Microbiology, University of Melbourne, CSIRO Animal Health and the University of Newcastle. Each of the institutions will be responsible for a particular aspect of the vaccine development from vector construction, manufacture to clinical trials.
Dr Joanne Banyer has recently joined the Viral Engineering Group. Joanne's focus of research will be to investigate how the immune system is triggered and controlled by the interaction of cells of the immune system, including dendritic cells and T and B lymphocytes both at the molecular and biological level. To target immunoregulatory factors, molecular analysis using subtractive hybridisation will be used to generate cDNA libraries that represent genes differentially expressed in cell lines stimulated under different conditions. This research may be useful for the identification of immunoregulatory factors that can enhance the protective capacity of vaccines or used in other forms of immunotherapy.
The focus of the Group is therefore to gain a greater understanding of the basic principles of immune regulation and to apply these findings to the development of more effective vaccines.
DNA vaccines have great potential for the prevention and treatment of a variety of diseases from infections to cancer and autoimmunity. A major problem with DNA vaccination, however, is the low efficacy of delivering DNA into cells and also low expression levels. We are attempting to improve the efficacy of our DNA vaccines by using cell signalling molecules that will target DNA into the nucleus.
Key molecular approaches to this work have involved the establishment, largely through international collaboration, of a number of strains of mice deficient for the production of individual immuno-modulatory factors, particularly cytokines or combinations of these factors. A further major approach has been the design and construction of different vector systems engineered to express genes encoding cytokines and other immuno-modulatory molecules for the delivery of these factors in vivo in attempts to study and selectively modulate immune responses.
A particular focus has been HIV infection. Our studies have contributed to the development of a prime-boost vaccination protocol involving the consecutive use of DNA vaccines and attenuated poxviruses vectors. This approach produces both strong systemic T cell-mediated immunity and disseminated mucosal immune responses against the virus, particularly in the reproductive tract. Our work is now directed particularly towards the development of HIV vaccine strategies for use in under-developed countries where the AIDS pandemic is devastating. The laboratory is now part of an Australian/USA consortium seeking to take this work, and further enabling research, through to clinical trials with the support of major international funding agencies.
In the case of allergic disease, we have recently shown that DNA and fowlpoxvirus vector vaccines, when administered to neonatal mice, are protective against the development of disease in adult life. We have also shown that inducible nitric oxide plays a significant role in the development of allergic airways pathology. Together, these findings have significant implications for the treatment and control of atopic diseases, including asthma.
Finally, the development of vectored fertility vaccines for some of Australia's feral pest species (mice, rabbits and foxes) has been another focus of our work, as members of the Vertebrate Biocontrol CRC. Our aim is to develop DNA and virally-vectored vaccines encoding fertility antigens, such as the ovarian zona pellucida glycoprotein, ZP3, which are of proven immunogenicity. We are also developing polyepitope antigens containing sequences from a variety of proteins associated with fertility. These immunogens are currently being tested for their immunogenicity and in fertility trials.
An integral part of our efforts to develop synthetic vaccines is to understand how immune responses are generated so that not only can the efficacy of such vaccines be enhanced but also so that they can be used to deviate the undesired immune responses which cause autoimmunity or allergy. With this in mind, we have a number of ongoing studies investigating the use of cytokine and chemokine genes like IFN-gamma, IL-4, IL-12, autocatylytic TGF-beta, and GM-CSF to alter the nature and strength of the immune responses generated by synthetic vaccines. Of particular interest is a new potent chemokine called I-TAC which attracts regulatory T cells to antigen presenting cells, called dendritic cells, which together initiate immune responses. Using these technologies we are currently developing vaccines for HIV, glandular fever and cervical cancer and therapies for multiple sclerosis and allergy.
STAFF - DIVISION OF IMMUNOLOGY AND CELL BIOLOGY
Cell-Cell Interaction Group
Viral Immunology Group
Immunology Laboratory
Apoptosis Laboratory
Molecular Virology Laboratory
Molecular Immunology and Immunopathology Group
Viral Engineering and Cytokine Research Group:
Experimental Haematology Group