Project 1: the role of PTPN22.6 in Intermediate Uveitis and Project 2: cord blood mononuclear cells as an alternative source for Chimeric Antigen Receptor-mediated cancer immunotherapy

Tubb, Vanessa (2013). Project 1: the role of PTPN22.6 in Intermediate Uveitis and Project 2: cord blood mononuclear cells as an alternative source for Chimeric Antigen Receptor-mediated cancer immunotherapy. University of Birmingham. M.Res.

Preview

Tubb13MRes.pdf
PDF - Accepted Version
Download (4MB)

Abstract

Project 1

BACKGROUND: Uveitis, an inflammatory disease with a putative autoimmune component, is a major cause of visual impairment in the UK. Lyp is a protein tyrosine phosphatase encoded by the gene PTPN22 which dampens TCR signalling. Mutations in PTPN22 are associated with autoimmune susceptibility. PTPN22.6 is a novel Lyp isoform suggested to correlate with rheumatoid arthritis. The role of this variant in uveitis pathology is therefore intriguing.

METHODS: Jurkat and CD4-T cells isolated from healthy and intermediate uveitis (IU) patients were stimulated using anti-CD3 and anti-CD28 antibodies. Lyp expression was detected by western blot and realtime quantitative PCR (rt-qPCR) in parallel. Expression of full-length PTPN22.1 and PTPN22.6 was determined in healthy and IU whole blood by rt-qPCR.

RESULTS: Jurkat and CD4-T cells expressed multiple Lyp isoforms which increased after TCR stimulation. Fulllength Lyp was most abundant in healthy patients, whereas smaller isoforms dominated in IU patients after stimulation. PTPN22.6 expression was detectable in all cells but variation was seen between healthy patients. Expression of PTPN22.1 and PTPN22.6 was not statistically different between control (n=26) and IU (n=39) patients, however interestingly the .1/.6 ratio was higher in control patients.

CONCLUSIONS: The balance between Lyp isoforms may alter T cell responsiveness and be important in IU.

Project 2

BACKGROUND: Adoptive T cell therapy (ACT) for cancer involves the ex vivo expansion and infusion of tumour-specific T cells. T cell specificity can be genetically engineered using chimeric antigen receptor (CAR) gene transfer. A CAR combines MHC-independent antibody recognition with downstream T cell signalling and cytotoxicity. Less differentiated T cells are more suitable for ACT because they show enhanced persistence and anti-tumour activity in vivo compared to effector T cells. Cord blood mononuclear cells (CBMCs) are dominated by naïve T cells, therefore are a potential source for CAR ACT.

METHODS: Peripheral blood mononuclear cells (PBMCs) (n=7) and CBMCs (n=9) were retrovirally transduced with a CAR targeting a tumour vasculature antigen. Cells were analysed for the differentiation markers CCR7 and CD45R using flow cytometry before and after transduction, and the IFNγ response to target antigen was assessed by ELISAs.

RESULTS: CBMCs achieved transduction rates indistinguishable to that of PBMCs but retained a significantly less differentiated phenotype after CAR transduction in CD4 and CD8 subsets. Some transduced CBMC donors produced significantly less IFNγ in response to target antigen while others produced none.

CONCLUSIONS: CBMCs are an attractive alternative source for CAR ACT.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):