Our Projects

Our researchers are focussed on discovering key immune markers and biological processes which will provide new diagnostic and therapeutic products for improving patient care.

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Searching for key immune markers

Learn More About Our Research

Please click below to explore our current research areas and individual research projects:
HONOURS PROJECTS 2017
PhD  PROJECTS 2017
BASIC DC DISCOVERY RESEARCH
DC IN HAEMATOLOGICAL DISEASES
GRAFT VERSUS HOST DISEASE

HONOURS PROJECTS 2017

The Dendritic Cell Research Group at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other cancers.

 

Dendritic cells (DC) and macrophages (Mφ) are specialized myeloid immune cells, which survey and capture antigens in their local tissue environment before transporting them to draining lymph nodes (LN). The cells express a range of surface receptors that guide their migration into and within specific sectors of lymphoid tissues, where they can interact and present antigens to lymphocytes to mount an appropriate adaptive immune response. Identifying and understanding the function of these “migratory” receptors is key to developing the next generation of improved immune therapies that will prevent infection or treat cancer.

 

The C-type lectin receptor family of molecules play important roles in the immune system through their ability to recognise conserved carbohydrate structures on foreign pathogens and altered structures on damaged cells. However, some members of the family have the ability to bind endogenous ligands and have been reported to mediate alternate functions such as cell adhesion, cell migration, or LN expansion and contraction. Our group has identified a new member of the C-type lectin receptor family, named DEC205 associated C-type Lectin-1 (DCL-1) or CD302. Our studies in humans and mice have shown that CD302 is highly expressed on dendritic cells, monocytes, macrophages, pointing to a significant role within the myeloid immune system. We have generated the first CD302 knockout mouse strain and shown that it has an important role in the migration of dendritic cells into the T cell areas of lymph nodes. The mechanisms by which it does so and the ligand/s bound by CD302 remain to be defined.

 

The aim of this project is to identify and characterize the DC and Μφ migratory mechanisms controlled by CD302. The project will involve generating a new monoclonal antibody to mouse CD302 to examine rare DC and Μφ populations expressing this molecule and how it changes with activation using flow cytometry. Confocal and intra-vital microscopy will be used to compare DC and Μφ migration within lymph nodes and other tissues when the cells can or cannot express CD302. Finally, the ligand for CD302 and the cells which express it will be identified using glycomic and proteomic screens or beads coated with the receptor. As we are a strong translational laboratory, discoveries of CD302 function in mouse DC and Μφ will be investigated in humans. This is an opportunity to gain experience in many different immunological techniques and contribute significantly to the understanding of an important new C-type lectin receptor. We see CD302 as an attractive target for potential therapeutic manipulation of immune responses to cancers, autoimmune diseases or infection.

 

Relevant Publications:

  1. Geijtenbeek, T. B., and S. I. Gringhuis. 2009. Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 9:465-479.
  2. Kato, M., S. Khan, E. d’Aniello, K. J. McDonald, and D. N. Hart. 2007. The novel endocytic and phagocytic C-Type lectin receptor DCL-1/CD302 on macrophages is colocalized with F-actin, suggesting a role in cell adhesion and migration. J Immunol 179:6052-6063.
  3. Kato, M., S. Khan, N. Gonzalez, B. P. O’Neill, K. J. McDonald, B. J. Cooper, N. Z. Angel, and D. N. Hart. 2003. Hodgkin’s lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1. J Biol Chem 278:34035-34041.
  4. Lo, T. H., Silveira, P. A., Fromm, P. D., Verma, N. D., Vu, P. A., Kupresanin, F., Adam, R., Kato, M., Cogger, V. C., Clark, G. J., and Hart, D. N. Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration. J Immunol. 197: 885-98.

PhD PROJECTS 2017

The Dendritic Cell Research Group at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other cancers.

 

Dendritic cells (DC) and macrophages (Mφ) are specialized myeloid immune cells, which survey and capture antigens in their local tissue environment before transporting them to draining lymph nodes (LN). The cells express a range of surface receptors that guide their migration into and within specific sectors of lymphoid tissues, where they can interact and present antigens to lymphocytes to mount an appropriate adaptive immune response. Identifying and understanding the function of these “migratory” receptors is key to developing the next generation of improved immune therapies that will prevent infection or treat cancer.

 

The C-type lectin receptor family of molecules play important roles in the immune system through their ability to recognise conserved carbohydrate structures on foreign pathogens and altered structures on damaged cells. However, some members of the family have the ability to bind endogenous ligands and have been reported to mediate alternate functions such as cell adhesion, cell migration, or LN expansion and contraction. Our group has identified a new member of the C-type lectin receptor family, named DEC205 associated C-type Lectin-1 (DCL-1) or CD302. Our studies in humans and mice have shown that CD302 is highly expressed on dendritic cells, monocytes, macrophages, pointing to a significant role within the myeloid immune system. We have generated the first CD302 knockout mouse strain and shown that it has an important role in the migration of dendritic cells into the T cell areas of lymph nodes. The mechanisms by which it does so and the ligand/s bound by CD302 remain to be defined.

 

The aim of this project is to identify and characterize the DC and Μφ migratory mechanisms controlled by CD302. The project will involve generating a new monoclonal antibody to mouse CD302 to examine rare DC and Μφ populations expressing this molecule and how it changes with activation using flow cytometry. Confocal and intra-vital microscopy will be used to compare DC and Μφ migration within lymph nodes and other tissues when the cells can or cannot express CD302. Finally, the ligand for CD302 and the cells which express it will be identified using glycomic and proteomic screens or beads coated with the receptor. As we are a strong translational laboratory, discoveries of CD302 function in mouse DC and Μφ will be investigated in humans. This is an opportunity to gain experience in many different immunological techniques and contribute significantly to the understanding of an important new C-type lectin receptor. We see CD302 as an attractive target for potential therapeutic manipulation of immune responses to cancers, autoimmune diseases or infection.

 

Relevant Publications:

  1. Geijtenbeek, T. B., and S. I. Gringhuis. 2009. Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 9:465-479.
  2. Kato, M., S. Khan, E. d’Aniello, K. J. McDonald, and D. N. Hart. 2007. The novel endocytic and phagocytic C-Type lectin receptor DCL-1/CD302 on macrophages is colocalized with F-actin, suggesting a role in cell adhesion and migration. J Immunol 179:6052-6063.
  3. Kato, M., S. Khan, N. Gonzalez, B. P. O’Neill, K. J. McDonald, B. J. Cooper, N. Z. Angel, and D. N. Hart. 2003. Hodgkin’s lymphoma cell lines express a fusion protein encoded by intergenically spliced mRNA for the multilectin receptor DEC-205 (CD205) and a novel C-type lectin receptor DCL-1. J Biol Chem 278:34035-34041.
  4. Lo, T. H., Silveira, P. A., Fromm, P. D., Verma, N. D., Vu, P. A., Kupresanin, F., Adam, R., Kato, M., Cogger, V. C., Clark, G. J., and Hart, D. N. Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration. J Immunol. 197: 885-98.

Dendritic Cell Research at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell (DC) research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other malignancies.

In mice, macrophage populations are routinely identified with the F4/80 monoclonal antibody (1,2) . The human homologue to F4/80 has been described as the ERM-1 molecule (3). ERM-1 and F4/80 are members of the epidermal growth factor seven span transmembrane family of adhesion molecules. There are very few reagents available and little is known about human ERM-1 function. Unlike mouse F4/80 which is primarily expressed on macrophage populations, human ERM-1 was found to be expressed on eosinophils. Our hypothesis is that the human ERM-1 molecule will be an important functional molecule on human myeloid cells playing a role in their migration or differentiation. This project will undertake a detailed analysis of the expression of ERM-1 in human myeloid lineage cells including eosinophil, monocyte, macrophage and antigen presenting cell populations and explore the role of the receptor in chemotaxis in vitro. The results will provide original data likely to lead to a high impact publication. The student will become proficient with flow cytometry, real-time PCR, cell culture, tissue staining, isolation of leucocyte populations through magnetic- and fluorescence-activated cell sorting, and molecular biology techniques to express and purify recombinant proteins.

References:

  1. Gordon S, Hamann J, Lin HH, Stacey M. F4/80 and the related adhesion-GPCRs. Eur J Immunol. 2011 Sep;41(9):2472-6.
  2. McKnight AJ, Macfarlane AJ, Dri P, Turley L, Willis AC, Gordon S. Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J Biol Chem.1996 Jan 5;271(1):486-9.
  3. Hamann J, Koning N, Pouwels W, Ulfman LH, van Eijk M, Stacey M, Lin HH, Gordon S, Kwakkenbos MJ. EMR1, the human homolog of F4/80, is an eosinophil-specific receptor. Eur J Immunol. 2007 Oct;37(10):2797-802.

Supervisor: Derek Hart

Dendritic Cell Research at the ANZAC Research Institute has forged a strong reputation in fundamental human dendritic cell (DC) research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other malignancies.

The LPS-responsive beige-like anchor protein (LRBA) molecule is a molecule linked with autoimmunity, lymphoproliferation, and humoral immune deficiency (1). It has been linked with multiple signalling pathways and intracellular vesicle trafficking (2). We have identified LRBA through our DC biomarker discovery program. There are very few reagents available to study this molecule in humans and is known about its function. We have demonstrated its expression in different populations of antigen presenting cells and identified a number of isoforms using a commercially available antibody.

This project will confirm the expression of LRBA in antigen presenting cells and to investigate the biology of LRBA in these cells. LRBA has some putative functional domains and is suggested to have a role in intra-vesicular trafficking. The results will provide original data likely to lead to a high impact publication. The student will learn several laboratory skills including flow cytometry, real-time PCR, cell culture, isolation of leucocyte populations through magnetic- and fluorescence-activated cell sorting, and various molecular biology techniques to express and purify recombinant proteins.

References:

  1. Lo B, Zhang K, Lu W, Zheng L, Zhang Q, Kanellopoulou C, et al. AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy. Science. 2015;349(6246):436-40.
  2. Wang JW, Howson J, Haller E, Kerr WG. Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. J Immunol. 2001;166(7):4586-95.

Supervisor: Georgina Clark

Dendritic Cell Research at the ANZAC Research Institute has forged a strong reputation in fundamental human Dendritic Cell (DC) research. Using its extensive clinical links and commercial collaborations, it is developing diagnostic and therapeutic antibodies for novel targets with application in clinical transplantation and the treatment of haematological and other malignancies.

DC are unique subsets of white blood cells, which are responsible for initiating and directing immune responses by priming and activating alloreactive T cells.  More importantly, DC can induce or suppress immune responses depending on their maturation status. Graft versus host diseases (GVHD) is a serious side effect after allogeneic hematopoietic stem cell transplantation (HCT) and graft-versus-tumor (GVT) is a major component of the overall beneficial effects of HCT in the treatment of tumors. CD83 is an important marker of activated dendritic cells (DC). We used a rabbit polyclonal anti-CD83 antibody and a potential therapeutic human anti-human CD83 monoclonal antibody, 3C12C, to deplete activated DC as a new approach to immunosuppression. Anti-CD83 prevented human PBMC induced graft versus host disease (GVHD) in mouse xeno graft model 1, 2. Whether CD83 antibody has any effect on the GVT effect in vivo needs to be clarified as an important issue.

This project will establish a new tumor graft model by injecting human leukemia cells into immune deficient NSG mice transplanted with human PBMC to induce GVHD. It follows a similar mouse model 1, 2 to our previous work. 3C12C antibody will be administrated to test whether: 1) human activated DC are depleted; 2) GVHD is prevented; 3) engrafted human T cells remain anti-tumor activity in vitro; 4) tumor growth in the mice is affected.

This project will generate valuable data to justify the clinical evaluation of the new human anti-CD83 mAb as a potential new immunosuppressive agent for clinical transplantation. The student will develop laboratory skills such as PBMC isolation, magnetic- and fluorescence-activated cell sorting, flow cytometry, T cell proliferation and function assays, mouse tissue dissection/staining and other immunological techniques.

References:

  1. Wilson J, Cullup H, Lourie R, Sheng Y, Palkova A, Radford KJ, Dickinson AM, Rice AM, Hart DN, Munster DJ: Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease. The Journal of experimental medicine 2009, 206:387-98.
  2. Seldon TA, Pryor R, Palkova A, Jones ML, Verma ND, Findova M, Braet K, Sheng Y, Fan Y, Zhou EY, Marks JD, Munro T, Mahler SM, Barnard RT, Fromm PD, Silveira PA, Elgundi Z, Ju X, Clark GJ, Bradstock KF, Munster DJ, Hart DN: Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation. Leukemia 2016, 30:692-700.

Supervisors: Xinsheng Ju and Derek Hart

BASIC DC DISCOVERY RESEARCH

Our program is based on understanding the membrane molecules found on the surface of DC. We generate monoclonal antibodies (mAb) to these biomarkers and when we understand the biology, we develop strategies for using these mAb to treat haematological malignancies. The pathway to using these agents in the clinic requires humanised products. Our approach is to use antibody engineering to produce humanised reagents which are used in our basic science studies. This will enable rapid translation of our findings into the clinic.

Team Members:  Georgina Clark, Derek Hart.

Our group discovered a family of biomarkers called CD300 molecules which are found on DC and some other white blood cells. It is clear from our own and others work, that these molecules may act to amplify or dampen wanted and unwanted inflammatory responses. Our effort has focused on their significant control of the human dendritic cells response to inflammation and their interactions with T cells. We are investigating the hypothesis that inflammatory environments alter the expression of CD300 family members. If this is correct, then reagents to them could be developed as biomarkers for monitoring inflammatory disease and potentially as agents to treat it. Products that independently manipulate CD300a or CD300c signalling have the potential to be turned into new drugs for controlling the response to transplants, other inflammatory diseases and possibly sepsis.

Team Members: Georgina Clark, Robin Gasiorowski, Derek Hart.

Whilst some leukaemias can be cured as a result of advances in treatment options, at least 60% of patients with the most common form of adult leukaemia, acute myeloid leukemia (AML), still die of the disease. Mylotarg, an antibody-toxin conjugate therapy for AML, was a promising drug but its recent removal from the market due to unacceptable side effects reinforces the need for other antibody targeted therapies. We have identified a new AML target called CD300f that may enable us to develop new antibody treatments for AML to replace Mylotarg. By defining how CD300f acts in AML and how to target it with antibodies, we hope to develop a less toxic treatment suitable for wide application.

 

Team Members: Robin Gasiorowski, Georgina Clark, Derek Hart, Ken Bradstock.

To understand the biology of the biomarkers that we have discovered, the DCBTG have developed preclinical models in which the relevant genes encoding the biomarkers have been deleted. One of these models has allowed us to gain insights into the role of CD302 in the migration of DC. This is crucial to understanding how DC based anti-cancer vaccines may move from the site of injection to the site of immune stimulation.

 

Team Members: Pablo Silveira, Kevin LoGeorgina Clark, Derek Hart.

DC IN HAEMATOLOGICAL DISEASES

The investigation of human blood DC subsets and their biology in malignant cancers is proving rewarding. We have continued to question why there are lower DC numbers in the peripheral blood of multiple myeloma (MM) patients whereas the disease site, the bone marrow, is enriched for some DC populations, and how this finding correlates with the disease status. We have focused our studies on the biology of a novel CD2 subset of plasmacytoid DC and how they contribute to the disease. These studies also direct our ongoing investigation of antibody selected DC populations for therapeutic DC vaccination trials.

 

Team Members: Phillip Fromm, Christian Bryant, Fiona Kupresanin, Jennifer Hsu, Derek Hart.
External Collaborators: Hayley Suen, Ross Brown, Douglas Joshua

Our work in collaboration with the Royal Prince Alfred Hospital investigating the immune mechanisms of disease control which may contribute to long-term survival in MM, has recently been published. We analysed relevant biomarkers and DC subsets in all current >10 year survivors and compared the results with a larger all-MM group. This analysis demonstrated a significant increase in many immunologic markers in the survivors. The conclusion that immune mechanisms contribute to long-term disease control encourages our efforts to generate new therapeutic immune approaches to treat MM.

 

Team Members: Christian Bryant, Derek Hart, Phillip Fromm.
External Collaborators: Harry Iland, John Gibson, Ross Brown, Shihong Yang, Hayley Suen, P Joy Ho

Together with our collaborators at the Royal Prince Alfred Hospital, we are investigating the dysfunctional effects of the malignant plasma cells of patients with MM on the immune system. A phenomenon occurs whereby cytotoxic effector T-cells in these patients acquire a range of molecules from malignant cells which alter their function resulting in the induction of regulatory T-cells. This phenomenon is more common in patients with MM than other chronic B cell disorders. These and other artefacts of malignant plasma cells are associated with poor prognosis and form the basis of our current research.

Team Members: Derek Hart, Phillip Fromm.
External Collaborators: John Gibson, Ross Brown, Shihong Yang, Hayley Suen, P Joy Ho

GRAFT VERSUS HOST DISEASE

DC are centrally involved in the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (alloHCT). Working with the Blood and Marrow Transplant Service at Westmead Hospital has enabled us to examine various DC biomarkers and DC activation status after alloHCT. GVHD in particular seems to be linked to the expression of the biomarker CMRF-44 on specific DC subsets. The DCBTG recently published work demonstrating that the biomarker, CCR5, on other DC subsets showed a positive correlation with acute GVHD. Preclinical studies are now underway to study whether the absence of CCR5 effects the development of GVHD in allotransplants.

 

Team Members: Derek Hart, Ken Bradstock.
External Collaborators: Mary Sartor, Stephen Larsen, Linda Bendall.

For patients with severe chronic GVHD, the use of expensive extracorporeal photophoresis (ECP) twice a week becomes the only option. This treatment serves to induce apoptosis in effector T-cells through a combination of intercalating agents and application of ultraviolet light. We have demonstrated that ECP treatment also serves to modulate activation markers of DC that we have previously shown to be prognostically associated with the onset of acute GVHD. Our current work is focussed on understanding how the induction of apoptosis serves to modulate DC function in these patients.

 

Team Members: Phillip Fromm, Fiona Kupresanin, Georgina Clark, Derek Hart.
External Collaborators: Hayley Suen, Ross Brown, Stephen Larsen, John Gibson, Douglas Joshua.

The pivotal role of DC in GVHD suggests that their depletion may control GVHD by preventing T-cells from being sensitised to host antigens without impairing immunity. Previously published work from Professor Hart’s group showed that a rabbit polyclonal IgG anti-human CD83 (RAH83) antibody prevented GVHD but maintained protective anti-viral and leukaemic activity in a xenogeneic model. We have been tracking the CD83 target on DC in the xenogeneic model to optimise potential therapeutic strategies. Our recent development of an anti-mouse CD83 antibody will allow us to determine how CD83 depletion controls GVHD in mouse allogeneic transplantation models – including solid organ models. We have re-established collaboration with the Cooperative Research Centre for Biomarker Translation reinvigorating our planning for a clinical trial to test a candidate anti-CD83 human monoclonal antibody as a novel immunosuppressive agent.

 

Team Members: Xinsheng Ju, Blake Hsu, Pablo Silveira, Phillip Fromm, Georgina Clark, Ken Bradstock, Derek Hart.

There is worldwide interest in developing immune therapies, including active vaccination with DC to treat cancer. Provenge, produced by the US based company Dendreon, was FDA approved after a phase 3 clinical trial showed that vaccination prolonged survival in anced prostate cancer. We are developing novel antibody based strategies to purify blood DC and this project is testing the optimal form of tumour target antigen to load into the DC product. Previous work suggested that RNA coding for tumour targets was processed effectively by DC and these generated anti-tumour responses in the test tube. We have shown that primary human DC isolated using a chimeric anti-human CMRF-56 monoclonal antibody, can effectively be loaded with mRNA and is able to generate robust anti-viral and anti-tumour T cell responses. We are testing the ability of different DC populations to take up and present mRNA-encoded tumour targets in preclinical work to determine the optimal clinically relevant strategies to allow this technique to be used for DC vaccination in both multiple myeloma and prostate cancer.

 

Team Members: Phillip Fromm, Jennifer Hsu, Michael Papadimitrious, Christian Bryant, Fiona Kupresanin, Georgina Clark, Derek Hart.
External Collaborators: Sebastien Anguille, Elizabeth Newman, Ilona Cunningham, Zwi Berneman, Lisa Horvath

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