1) Investigation of the clathrin-dependent internalisation of Cytotoxic T Lymphocyte Antigen-4 2) Analysis of the antigen-specific cytokine responses of CD4+ T lymphocytes

Voice, Marie Ann (2011). 1) Investigation of the clathrin-dependent internalisation of Cytotoxic T Lymphocyte Antigen-4 2) Analysis of the antigen-specific cytokine responses of CD4+ T lymphocytes. University of Birmingham. M.Res.

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Abstract

Abstract 1) Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) is a key inhibitory regulator of T-cell function. Its expression is predominantly intracellular however, many theories surrounding it’s inhibitory action do not account for its endocytic behaviour. Recent reports have suggested a process of trans-endocytosis through which CTLA-4 internalises the ligand (CD80/CD86) from antigen presenting cells and prevents ligand binding to its stimulatory homologue CD28. The endocytic pathway of CTLA-4 is currently ill-defined and has a central role in this novel theory. Previous evidence suggests CTLA-4 is internalised via clathrin-dependent endocytosis (CDE). However observations in a transfected CHO cell model have suggested internalisation independent of this pathway. In this study CTLA-4 showed the same endocytic recycling behaviour when transfected into a HeLa cell line. Immunostaining showed a strong degree of co-localisation between internalising CTLA-4 and the transferrin receptor (which is classically associated with CDE). Co-localisation with Adaptor-Protein 1 suggested exocytosis from the trans-Golgi network to the plasma membrane was also clathrin-dependent, however, no association was observed with clathrin directly. Little CTLA-4 co-localisation to a lysosomal marker suggests that only a small proportion of the cytosolic receptor is targeted for degradation. CDE was inhibited by transfection of shRNA for Adaptor Protein 2 (AP-2). In CTLA-4+ CHO cells knock down of AP-2 resulted in decreased intracellular CTLA-4 but this was not statistically significant. This data supports but does not confirm that CTLA-4 can internalise independent of CDE.

Abstract 2)The phenotype of a T-lymphocyte can be defined by its expression of secretory cytokines. However, characterising the different T-cell lineages is challenging given the numerous subsets and significant overlap in cytokine profiles. Additionally the stability of a T-cell once fully differentiated is currently under review, with identifiable functional plasticity between different subsets. I investigated whether the recall cytokine response of CD4+ T-cells is influenced by the nature of the antigenic stimuli presented. PBMC were stimulated ex vivo with bacterial (SEB, PPD, Tetanus Toxoid), viral (EBV) and fungal antigens (yeast). IFNγ, TNFα, IL-17, IL-21 and IL-10 were stained intracellularly and expression analysed by flow cytometry. Results identified stimulus specific IFNγ, TNFα and IL-17 responses in the CD4+ CD45RO+ memory population for all antigens tested; however no detectable IL-21 and IL-10 secretion was established. There was variability in the ratio of IL-17+ : IFNγ + expression between different antigens indicating a potential Th17 or Th1 bias respectively. The ratio of TNFα+:IFNγ+ expressing cells was also variable, highlighting the potential significance of a TNFα+IFNγ- CD4+ population. Low number of responsive cells prevented comprehensive analysis of double positive cytokine secreting cells. However the proportion of IFNγ+ cells also TNFα+ following PPD stimulation was notably but not significantly higher than the positive control (SEB), whereas the IL-17+IFNγ+ proportions were similar. This study suggests but does not confirm that different antigenic stimuli cause an enrichment of differential T-cell subsets. Such findings are highly applicable to multiple disciplines involving cell-mediated immunity.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):