MOLECULAR IMMUNOLOGY

• Staff
  • Laboratory Head
  • Research Staff
  • Students
• Goals
• Research Projects
  1. Molecular identification of Regulatory T cells
  2. Cord Blood Stem cell differentiation in to regulatory T cells
  3. Lentiviral vectors for gene delivery and gene ablation
  4. High throughput genome wide function based gene discovery

STAFF

Laboratory Head

Associate Prof. Simon Barry

Ph: 08 8161 6562

Email: simon.barry@adelaide.edu.au

Research Staff

Students

GOALS

To improve our understanding of regulatory T cell function through studies of gene expression, transcription factors and surface molecules and to develop regulatory T cells for cell therapy.

RESEARCH PROJECTS

1. Molecular identification of Regulatory T cells

The recent identification of regulatory T cells (Tregs) as a key mediator of central and peripheral tolerance has led to an increase in our understanding of the cellular mechanisms.

The identification of a transcription factor named FoxP3 in both mouse and human Tregs defines a committed T cell subset that has regulatory capacity. There is however, very little known about the molecular basis of this process. This project aims to identify the genes directly regulated by FoxP3 and to determine their role in the regulatory phenotype. We are using a number of direct and indirect molecular approaches such as Chromatin Immunoprecipitation and microarray analysis to profile genes regulated by FoxP3, and we will validate their role in regulatory function by direct assays and by over expression or gene ablation studies. The candidate genes identified in this approach may lead to therapeutic approaches for intervention in the function of regulatory cells, and will also have application for diagnostic analysis of regulatory cell function.

2. Cord Blood Stem cell differentiation in to regulatory T cells

The clinical application of regulatory T cells is significantly hampered by the limited cell numbers that can be obtained from either cord or adult blood. Attempts to expand these purified Treg ex vivo have shown some promise, but there is some evidence that after extended culture ex vivo these cells loose their suppressive capacity. An alternative approach is to generate large numbers of T cells de novo from stem cells since these cells have the capacity to differentiate into all cells of the haemopoietic system. We have established an ex vivo differentiation assay that can expand cord blood stem cells and induce their differentiation along the lymphoid pathway using a cocluture system giving notch signals via the Notch ligand Delta like 1. In this system we robustly observe 5-600 fold expansion of cell numbers and the generation of T cell subsets as defined by CD4/CD8 staining.

Fig 1. Ex vivo differentiation and expansion of cord blood stem cells on feeder cells showing formation of CD4 CD25+ subsets with similar characteristics to natural Treg

3. Lentiviral vectors for gene delivery and gene ablation

Manipulation of primary cells has a key limitation in that these cells are refractory to standard transfection protocols. Also, since they are often of low mitotic index, they are only infected at low efficiency by murine retroviruses (RV), as these viruses require cell division for integration. The recent development of HIV1 based lentivectors (LV) provides an attractive option for gene delivery into T cell and stem cell populations, as these viruses carry the necessary cis elements to facilitate nuclear transport and integration in the absence of cell division.

We have developed a suite of lentiviral vectors for stable gene delivery into primary cells both for gene therapy and gene discovery applications, and more recently for gene ablation using RNA interference. This technology relies on the expression of a short hairpin RNA structure that is complimentary to the gene target, and that is processed by cellular machinery to generate an RNA oligo that can bind to and target the mRNA for destruction.

Fig 2. Lentiviral delivery of shRNAi showing ablation of FoxP3 expression that is inducible with doxacyclin and is reversible

4. High throughput genome wide function based gene discovery

As part of the Australian Retroviral expression cloning consortium (ARVEC) we are building the capacity to undertake large scale automated lentiviral expression cloning in a 96 well format, with the intention of screening all available full length clones in the human genome collection. This facility will allow function based gene identification in any cell line that can be cultured in plates, and will depend on screens that show gain or loss of function that can be measured either directly or indirectly by light/fluorescent microscopy using high content imaging. The facility will be open to any academic user on a cost recovery basis.