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Which epitopes do human islet-infiltrating CD8+ tTcells ‘see’?

Which epitopes do human islet-infiltrating CD8+ tTcells ‘see’?

PhD project

Introduction
Type 1 diabetes is an autoimmune disease caused by the CD4+ and CD8+ T-cell responses against the insulin-secreting beta cells found in the islets of Langerhans in the pancreas. While CD4+ T cells are the principal regulators of the (auto)immune response, CD8+ T cells are believed to be the primary ‘killers’ of beta cells in type 1 diabetes.  However, the antigens/epitopes that are ‘seen’ by pathogenic CD8+ T cells are not defined. This is an important question because knowledge of the targets of CD8+ T cells is essential for the development of new therapies to prevent type 1 diabetes. In addition, this is a major gap in our understanding of human autoimmunity in type 1 diabetes.

This question has not been addressed previously because human islet-infiltrating CD8+ T cells and HLA-matched beta cells have not been available. In our recent work (Pathiraja et al. Diabetes 2015) we isolated and characterised proinsulin specific CD4+ T-cell clones from within the pancreatic islets of an organ donor who suffered from type 1 diabetes. At the same time we isolated a large panel of CD8+ T cells. This gives us a unique panel of CD8+ T-cell clones strongly implicated in the pathogenesis of human T1D to study.

In collaboration with Prof Ed Stanley at the MCRI, we have generated induced pluripotent stem cells (iPSC) from the deceased organ donors from whom the islet-infiltrating T cells were isolated. These iPSC will be used to generate beta cells that express the same HLA class I molecules as the T cells. In this way we will be able to test the islet-infiltrating CD8+ T cells for responses to autologous beta cells. This will form the basis of our efforts to identify the epitopes ‘seen’ by human islet-infiltrating CD8+ T cells.  These reagents give us, for the first time, the tools required to dissect human CD8+ T-cell responses to beta cells and reveal the immune responses underlying this incurable disease.

Aims
The aim of this project is to identify the antigens and epitopes recognized by human islet-infiltrating CD8+ T cells. The specific aims are:

  1. To clone and sequence the TCR genes from islet-infiltrating CD8+ T-cell clones.
  2. Transduce CD8+ T cell reporter lines with TCRs derived from human islet-infiltrating clones.
  3. Test the transduced CD8+ T cells for recognition of primary human beta cells and autologous beta cells derived from iPSC.
  4. Identify the antigens/epitopes ‘seen’ by the transduced cells by screening a panel of peptides, Cos-7 cells transfected with plasmids encoding HLA class I molecules and beta-cell antigens, or Cos-7 cells transfected with a beta-cell derived cDNA library.

Methods
This project will use retroviral mediated gene transfer of TCR genes into primary human T cells to investigate the antigen specificity of the TCRs. A variety of laboratory techniques will be used, including: retroviral production and transduction, flow cytometry, molecular cloning, T-cell cloning and analysis of T-cell function, cytokine (IFN) ELISA and differentiation of iPSC-derived beta cells.

Conclusion
This project will give the student an in-depth training in immunology, particularly in CD8+ T-cell immunology of type 1 diabetes. This is an ambitious project that will reward an enthusiastic student.

Supervised by:

  • A/Prof Stuart Mannering
  • Disease Focus:

  • Type 1 diabetes
  • Research Unit:

  • Human Immunology