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Human Immunology

The Human Immunology Laboratory within SVI's Immunology Unit, is focussed on the autoimmune disease type 1 diabetes. Broadly, we have two goals. The first is to understand how and why the insulin-producing beta cells are attacked by the immune system’s T cells in people who develop type 1 diabetes. Our second goal is to use this information to develop safe and effective ways to stop progression of this disease in people at high risk. Ultimately, we hope to reverse type 1 diabetes in people who have been recently diagnosed.

Research overview

Currently the focus of our work is dissecting the specificity of the autoimmune responses that causes people to develop type 1 diabetes. We achieved a world first when we isolated and analysed human islet-infiltrating T cells from deceased organ donors who suffered from type 1 diabetes.  This invaluable resource has allowed us to understand the ‘nuts and bolts’ of the autoimmune response in the part of the body that is affected.  A second major focus of our work is to develop animal models that incorporate components of the human autoimmune response against beta cells and use these models to develop safe and effective antigen-specific therapies to prevent type 1 diabetes.

Research Themes

What antigens do human islet infiltrating CD4+ T cells see?

Group Leader: Dr Stuart Mannering
Team Members: Vimukthi Pathiraja, Eleanora Tresoldi, Prerak Trivedi
Collaborators: Prof Tom Kay and A/Prof Helen Thomas
Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Drs Maki Nakayama and Aaron Michels, Barbara Davis center for Childhood diseases, Colorado, USA.
Prof Jamie Rossjohn and Dr Hugh Reid, Monash University, Melbourne.
Dr Eddie James, Benaroya Research Institute, Seattle, USA.

Genome wide association studies (GWAS) have confirmed that the strongest genetic risk for T1D is mediated by HLA alleles, specifically HLA DQ8 and DQ2. The second strongest association maps to the promoter region of the insulin gene. While these associations are well accepted in the field it remains unclear how HLA DQ2/8 and insulin lead to type 1 diabetes. From our analysis of human islet-infiltrating CD4+ T-cell clones we have identified several HLA DQ8-restricted CD4+ T-cell epitopes derived from the C-peptide of proinsulin. This project will build upon this success and identified epitopes from other antigens (including GAD-65, IGRP, IAPP, ZnT8). The long-term goal is to identify common features of epitopes seen by islet infiltrating CD4+ cells. This project will examine the epitope’s affinity for the restricting HLA allele, the avidity of TCR binding and TCR usage.

Do islet infiltrating CD4+ T-cells recognize epitopes formed by posttranslational modification?

Group Leader: Dr Stuart Mannering
Team Members: Vimukthi Pathiraja, Eleanora Tresoldi, Prerak Trivedi
Collaborators: Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Drs Maki Nakayama and Aaron Michels, Barbara Davis center for Childhood diseases, Colorado, USA.
Prof Jamie Rossjohn and Dr Hugh Reid, Monash University, Melbourne.
Dr Eddie James, Benaroya Research Institute, Seattle, USA.

In 2005 we published the first description of an epitope formed by posttranslational modification to be implicated in T1D (Mannering et al JEM 2005). While the concept of autoimmune responses against self-antigens that have undergone posttranslational modification remains popular this area remains largely unexplored in human studies. Our panel of islet-infiltrating CD4+ T-cell clones are invaluable tools that allow us to determine if these cells recognize epitopes formed by posttranslational modification. Our approach is to test panels of synthetic peptides that incorporate modified amino acids and determine if any of these are recognized by islet infiltrating CD4+ T cells. To date we have identified one HLA-DQ8 restricted epitope that is formed by posttranslational modification. Future work will screen a larger panel of peptides from other beta-cell antigens to find new epitopes formed by posttranslational modification. Using our very sensitive CFSE-based proliferation assays (Mannering JIM 2003) we will measure responses to modified epitopes in PBMC from individuals with and without T1D.  Our biochemical/functional analysis will also include structural analysis of TCR–peptide-HLA complex to gain insights into how human T cells ‘see’ autoantigens.

Identifying the antigens and epitopes seen by human islet-infiltrating CD8+ T cells

Group Leader: Dr Stuart Mannering
Team Members: Prerak Trivedi & Eleanora Tresoldi,
Collaborators: Prof Tony Purcell and Dr Nadine Dudek, Monash University.
Prof Tom Kay, A/Prof Helen Thomas and Dr Bala Krishnamurthy.

CD8+ T cells that recognize beta-cell antigen derived epitopes presented by HLA class I molecules are believed to the ‘killers’ that destroy the insulin-secreting beta cells. The goal of this project is to identify the antigens and epitopes ‘seen’ by human islet-infiltrating CD8+ T cells. We have developed a Cos cell transfection system which allows us to screen CD8+ T-cell clones for responses to individual antigen/HLA combinations. This system has been validated using CD8+ T-cell clones specific for viral epitopes restricted by relevant HLA alleles. Antigens that stimulate the clones will be dissected to identify the epitope(s) and HLA restriction. This information will be used to generate HLA tetramers for characterizing beta cell antigen specific CD8+ T cells in human peripheral blood. 

Identifying the antigens and epitopes seen by human islet-infiltrating CD8+ T cells

Group Leader: Dr Stuart Mannering
Team Members: Eleanora Tresoldi
Collaborators: Dr Nadine Dudek, Monash University.
Dr Bala Krishnamurthy, SVI

Identification of antigens recognized by human T cells has been technically challenging. This is because relevant T-cell clones have been difficult to isolate and preparing antigen in a suitable format has been difficult. Hence the aim of this project is to develop improved techniques for identifying antigens recognized by human islet infiltrating T cells. We propose to genetically fuse the antigens to the Class II invariant chain that directs the protein to the lysosomes for loading into HLA Class II molecules. This project will investigate different antigen presenting cells, such as EBV transformed B cells and HLA transfected Cos cells. The capacity of the constructs to stimulate islet infiltrating CD4+ T-cell clones specific for proinsulin will be used to test the system. This project would be suitable for an Honors student. Successfully completing this project would be an excellent preparation for a PhD project using this system to characterize human islet-infiltrating T cells.

Student Projects

Staff

Publication Highlights

  1. So, M., Elso, C. M., Tresoldi, E., Pakusch, M., Pathiraja, V., Wentworth, J. M., . . . Mannering, S. I. (2018). Proinsulin C-peptide is an autoantigen in people with type 1 diabetes. Proc Natl Acad Sci U S A, 115(42), 10732-10737. doi:10.1073/pnas.1809208115
  2. Mannering, S. I., So, M., Elso, C. M., & Kay, T. W. H. (2018). Shuffling peptides to create T-cell epitopes: does the immune system play cards? Immunol Cell Biol, 96(1), 34-40. doi:10.1111/imcb.1015
  3. Mannering, S. I., Di Carluccio, A. R., & Elso, C. M. (2018). Neoepitopes: a new take on beta cell autoimmunity in type 1 diabetes. Diabetologia. doi:10.1007/s00125-018-4760-6
  4. Delong, T., Wiles, T. A., Baker, R. L., Bradley, B., Barbour, G., Reisdorph, R., . .Mannering, S ., Haskins, K. (2016). Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science, 351(6274), 711-714. doi:10.1126/science.aad2791