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NDORMS DPhil & MSc by Research

Ankylosing Spondylitis is a common inflammatory rheumatic disease of unknown pathogenesis.  The two strongest genetic associations are with the Human Leucocyte Antigen HLA-B27 (present in 96% of patients) and with polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) (Evans 2011).  The latter are only found in the B27+ patients.  Interestingly such an epistatic interaction has also been described for Behcet’s disease and for skin psoriasis, although in both cases the HLA molecules involved and precise ERAP1 haplotypes differ (Strange 2010).  We and others propose that AS, Behcet’s and psoriasis are “class-1-opathies” caused by aberrant class 1 antigen presentation (McGonagle 2015). The primary function of ERAP1 is to trim peptides for loading onto HLA class 1 molecules in the ER.  We hypothesize that ERAP1 polymorphisms alter HLA’s peptide repertoire, hence altering stability and export of pro-inflammatory forms to the cell surface.  Our group has recently shown that silencing or inhibition of ERAP1 reduces HLA-B27 heavy chain expression on the cell surface and subsequent Th17 cell generation (Chen 2016 & 2017).  Dr Liye Chen will co-supervise this project and has additionally developed key reagents and techniques for the current project, including CRISPR/Cas9 HLA class 1 and ERAP1 knockout Hela cells into which HLA-B51 has then been introduced.

Outline of research

We will ask the following questions:

  1. How does ERAP1 alter the peptidome presented by HLA-B51 and B27? And,
  2. How does ERAP1 alter the cellular transport of HLA-B51 and B27?

Specifically do HLA-B27 and B51 misfold or form more surface homodimers in the presence of ERAP mutants? Does ERAP interact directly with HLA-B27 and B51 and/or the peptide-loading complex? Is this altered for ERAP mutants? For some experiments we will also compare with HLA-C*0601, associated with psoriasis.

Experimental plan

1) How does ERAP1 alter the peptidome presented by HLA-B51 and B27?

In order to determine if HLA-B27 and B51 present peptide antigens differently in the absence of ERAP or in the presence of ERAP mutants associated with or protective against AS? The student will grow HLA class 1 knockout cell lines expressing HLA-B27 and HLA-B51 and HLA-C*0601, immunoprecipitate their HLA class 1 molecules and elute and sequence their bound peptides.  We have already set up the techniques and shown that absence of ERAP1 results in binding of longer peptides to HLA-B27 (Chen 2016). If clear effects are demonstrated comparing ERAP1-sufficient and -deficient cells, we will go on to determine the effect of the AS and Behcet’s disease-associated haplotypes on peptidome using ERAP1 knockout cell lines (Takeuchi 2016). This work will involve mass spectrometry analysis of the peptidome, which will be done in collaboration with Dr Nicola Ternette in Oxford University.

2) How does ERAP1 alter the cellular transport of HLA-B51 and B27?

We will carry out a preclinical assessment of ERAP/B27 inhibition and silencing/CRISPR using both human cell lines and AS patient-derived primary cells. We will use FACS to measure cell surface heavy chain expression (Chen 2016).  We will look for differences in different cell types including B cell lines (221, C1R), monocytic lines (U937 and THP1), epithelial cell lines (HeLa) and endothelial cell lines (HUVEC).  We will tag HLA and ERAP1 (e.g. HA or flag) and use these to follow cellular location and egress to the cell surface of both molecules biochemically and microscopically.

References

Chen L, and Bowness P. Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis. Ann Rheum Dis. 2016 May; 75(5): 916–923.

Chen L,.Bowness. Position 97 of HLA-B, a residue implicated in pathogenesis of ankylosing spondylitis, plays a key role in cell surface free heavy chain expression. Ann Rheum Dis. 2017 Mar;76(3):593-601.

Evans D et al Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat Genet. 2011 Jul 10;43(8):761-767.

McGonagle D et al. 'MHC-I-opathy'-unified concept for spondyloarthritis and Behçet disease. Nat Rev Rheumatol. 2015 Dec;11(12):731-40.

Strange A, et al A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet. 2010 Nov;42(11):985-90

Takeuchi M et al.  A single endoplasmic reticulum aminopeptidase-1 protein allotype is a strong risk factor for Behçet's disease in HLA-B*51 carriers. Ann Rheum Dis. 2016 Dec;75(12):2208-2211.

The research group

The Bowness group studies the immunology of Ankylosing Spondylitis and related diseases.  There are currently two DPhil students, one clinician scientist and 4 post-docs in the group. For informal enquiries please contact either Dr Chen or Prof Bowness.

Training

The Botnar Research Centre plays host to the University of Oxford's Institute of Musculoskeletal Sciences, which enables and encourages research and education into the causes of musculoskeletal disease and their treatment. Training will be provided in a variety of immunological, microscopic, proteomic and biochemical techniques.  A core curriculum of lectures will be taken in the first term to provide a solid foundation in a broad range of subjects including musculoskeletal biology, inflammation, epigenetics, translational immunology and data analysis.

Students will attend weekly seminars within the department and those relevant in the wider University.  Students will be expected to present data regularly to the department, the Bowness group and to attend external conferences to present their research globally. Students will also have the opportunity to work closely with the Wordsworth, Ternette and Knight collaborating groups. 

How to Apply

The department accepts applications throughout the year but it is recommended that, in the first instance, you contact the relevant supervisor(s) or the Graduate Studies Officer (samuel.burnell@ndorms.ox.ac.uk) who will be able to advise you of the essential requirements.

Interested applicants should have or expect to obtain a first or upper second class BSc degree or equivalent, and will also need to provide evidence of English language competence. The University requires candidates to formally apply online and for their referees to submit online references via the online application system.

The application guide and form is found online and the DPhil or MSc by research will commence in October 2018.  When completing the online application, please read the University Guide.

Project reference number #NDORMS-2018/5

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