Identifying new HIPs using by screening an antigen library
Since we identified the first epitope formed by posttranslational modification1, or neoepitope, It has becoming increasingly clear that the autoimmune CD4+ T-cell response that leads to type 1 diabetes (T1D) targets an array of neoepitopes2. Hybrid insulin peptides, HIPs, are a new class of neoepitope that we identified in 2016(3). These epitopes are formed by the fusion of two peptide fragments creating a new hybrid peptide which may be recognized by disease causing, or promoting, CD4+ T cells. To date very few HIPs have been identified that are recognized by human CD4+ T cells associated with T1D3-5.
HIPs can form, theoretically at least, from the fusion of fragments of any beta-cell granule proteins6. In addition, they can form from fragments of the same protein and they can form in either order, i.e ABC-HIJ, or HIJ-ABC. For these reasons there is a very large number of potential HIPs, which makes identifying new HIPs very challenging.
We have a large panel of CD4+ T-cell lines isolated from the pancreatic islets of deceased organ donors who had T1D7. For most of these T-cell clones the antigen/epitope that they recognize is unknown. Given the clear role that HIPs play in T1D, we believe that some, if not all, of these clones may recognize as-yet unidentified HIPs.
To facilitate identifying the antigens recognized by T cells in our islet-infiltrating T cell panel we have developed a T-cell line that expresses luciferase upon TCR-mediated antigen recognition. Over the past years we have cloned the TCR genes from over 50 human islet-infiltrating CD4+ T cells and expressed them in this T-cell line. As a result, we now have a large panel of T cell lines with a stable readout of TCR-mediated antigen recognition that is suitable for high-throughput analysis. More recently we have developed a protocol for screening large numbers, (~2,000-20,000) candidate HIPs. The genes for the candidate HIPs are cloned and expressed and screened for their capacity to stimulate TCR mediated responses in the T-cell lines. Using this approach, we have already identified several new HIPs recognized by four CD4+ T-cell lines.
The goals of this project are to:
(i) generate larger HIP libraries,
(ii) to use them to screen all of our CD4+ T-cell lines,
(iii) identify the HIP(s) that stimulate the T-cell response(s),
(iv) determine the HLA restriction and minimum epitope mapping for each HIP
(v) analyse CD4+ T-cell responses to the newly identified HIPs in the peripheral blood of people with and without T1D.
This project will allow the student to develop high level expertise and knowledge in: T-cell immunology of T1D, generating libraries and screening, functional T-cell assay, synthetic biology and sequencing.
Associate Professor Stuart Mannering: [email protected]
Dr Pushpak Bhattacharjee: [email protected]
A/Prof Stuart Mannering
Dr Pushpak Bhattacharjee
Type 1 diabetes
For further information about this project, contact: