Utilisation of an in vitro T-cell priming assay to characterise the effects of co-inhibitory signalling on the activation of antigen-specific T-cells.

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UTILISATION OF AN IN VITRO T-CELL PRIMING ASSAY TO

CHARACTERISE THE EFFECTS OF CO-INHIBITORY SIGNALLING ON THE

ACTIVATION OF ANTIGEN-SPECIFIC T-CELLS.

This thesis is submitted in accordance with the requirements of the University of

Liverpool for the degree of Doctor of Philosophy by

Andrew Gibson

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Declaration

I declare that the work presented in this thesis is all my own work and has not been

submitted for any other degree.

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Firstly, I am indebted to my supervisors Dr Dean Naisbitt and Professor Kevin Park for all of their help over the past three years. Their support and guidance has been crucial in my development from student to research scientist and for this I am truly grateful. I would also like to thank all of my friends and colleagues in the cell lab. In particular I would like to thank Dr Lee Faulkner who got me started in the lab. For her unrelenting patience, and willingness to guide and teach, I am very grateful. Thanks is also extended to John Farrell for his help with cell culture, his vast knowledge of science whilst in work, and not so scientific fields outside of work. Also I must send thanks to Dr Monday Ogese. Not only has he been of great help academically, but his creative use of the English language to form phrases only he knows the meaning of, and contagious enthusiasm for science has been truly motivational. Without doubt, my friends in the department have made my PhD a very enjoyable experience. Particular thanks goes to sully for his ‘upbeat’ demeanour, his skilled peer-pressuring to visit ‘just one more’ pub, and his ability to share a room with me during multiple conference trips (no mean feat!).

I reserve a special thanks to all of my family for their love and support over the past 25 years. While being born is of course a particular highlight, I must additionally thank my parents for their support of my decisions, encouragement, and their willingness to offer advice when sought. While I am not sure that they think that I take this advice on board, I do, and it’s really quite annoying how good it usually turns out to be. I am sure that my interest in science spurs from my dad, and although I can’t be sure that learning the difference between an agonist and antagonist aged 12 was the stimulus, it probably didn’t hurt! To my brothers, Daniel and David, thanks for what I can only call ‘brotherly love’ (mainly formed of sarcasm). I am honoured that you will both be best-men at my wedding next year, and I look forward to receiving abuse for the previous comment. As an extension, I thank my niece Keira and nephew Abe for their ability to entirely distract me when I visit, you help keep life’s problems in perspective; keep being so cool!

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Hypersensitivity denotes a form of immune-mediated adverse reaction associated with a high degree of morbidity and mortality. Due to a lack of animal models, research has focussed on patient lymphocytes ex vivo. However, such studies bypass investigation of naïve T-cell activation. Subsequently, in vitro assays to assess the priming of healthy donor naïve T-cells have been developed to facilitate the discrete analysis of primary and secondary T-cell responses. While these assays have been used to assess the association of specific HLA alleles with hypersensitivity, for most drugs, the majority of individuals who are positive for an HLA risk allele do not develop a reaction. Thus, predisposition to hypersensitivity is likely mediated by other parameters.

As polymorphisms in co-inhibitory pathways are associated with dysregulated immune responses, we first investigated the role of these pathways during the drug antigen (SMX-NO)-specific activation of T-cells. Antibody-mediated blockade of PD-L1 enhanced the activation of SMX-NO-primed naïve, but not memory T-cells. In comparison, inclusion of PD-L2 block had no effect, but in combination with PD-L1 block effectively dampened the enhanced T-cell activation seen with PD-L1-block alone. In comparison, inclusion of CTLA4 block enhanced the proliferative response of antigen-stimulated naïve, but also memory T-cells, suggesting a greater regulatory role for CTLA4 than PD-1 during secondary T-cell responses. Blockade of TIM-3 had no effect on T-cell activation. Further investigation focussed on the kinetics of receptor expression. While all receptors were upregulated on naïve and memory T-cells during the 3 week culture after antigen exposure, PD-1 was upregulated at earlier time points than CTLA4 and TIM-3 indicating a differential role for these receptors during early and late stage T-cell activation. Moreover, CTLA4 expression was induced in response to antigen far less on CD4+_{T-cells suggesting that CTLA4 has a greater regulatory role during the drug antigen-specific} activation of CD8+_{T-cells. High expression of individual co-inhibitory receptors including PD-1 has}

previously been associated with exhausted T-cells, while other studies indicate that these cells are highly functional. To address this, we compared receptor expression with T-cell clone function. To assess function, we investigated the role of the newly identified Th17 and Th22 subsets in these responses. A range of Th1/Th2 cytokines were secreted in response to SMX-NO including IFN-γ, IL-13, and IL-5. While no IL-17 was detected, 50% of clones secreted IL-22. Further analysis uncovered two distinct antigen-responsive T-cell subsets that secrete Fas-L/IL-22 or granzyme B, the presence of which was confirmed utilising cells from SMX-hypersensitive patients. This is the first data to show production of IL-22 alongside IFN-γ by antigen-specific T-cells from drug-hypersensitive patients. Analysis of the level of individual co-inhibitory receptor expression found no correlation with T-cell proliferative capacity or secretion of cytokines/cytolytic molecules.

Both SMX and SMX-NO, stimulate T-cells from hypersensitive patients. 9/10 healthy donors respond to SMX-NO, while only 30% respond to SMX in vitro. These observations were made using a whole lymphocyte population and thus we utilised an in vitro T-cell priming assay, whereby naïve or memory T-cells from healthy donors are stimulated with drug-antigen using mature dendritic cells, to assess the T-cell origins of antigen-responsive cells. Both naïve and memory T-cells were activated by NO in all donors. Although SMX failed to induce proliferative responses, 2/3 donors displayed SMX-induced IL-13 secretion. SMX and SMX-NO-responsive clones were subsequently generated from these donors from naïve and memory T-cell cultures indicating that the priming of naïve T-cells and the re-activation of memory T-cells has a role in the onset of SMX-induced hypersensitivity. As these responses were detected using cells from drug-naïve donors, the memory T-cell responses are likely a result of cross-reactivity with T-cells primed to an undetermined peptide antigen. Our data are the first to show that both hapten and parent drug can stimulate pre-existing memory T-cells from drug-naïve donors.

PPD, a compound found in dyes used for hair colouring, is associated with ACD. Both PPD and a downstream oxidation product, BB, activate T-cells in allergic patients. Thus we used an in vitro T-cell priming assay to assess the propensity of PPD and BB to activate naïve and memory T-cells from healthy donors, and compared responses to those characterised from allergic patients. 105 PPD- and 122 BB-responsive T-cell clones were generated from five allergic patients. More than 90% of patient BB-clones were CD4+_{, and all lacked cross-reactivity. In contrast, certain PPD-clones cross-reacted with}

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T-cells were activated by BB, which similarly to patients displayed a lack of cross-reactivity and secreted a similar panel of cytokines including IFN-γ, IL-13, and IL-22.

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