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Title: 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
Author: Gibson, Andrew
ISNI:       0000 0004 6058 4001
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2015
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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 naive, 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 naive, 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 naive 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 SMX-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 BB, and 37.5% expressed CD8 promoting PPD as the central driving force behind the allergic reaction. However, upon utilising cells from healthy donors, neither naïve nor memory T-cells were activated by PPD suggesting the lack of important susceptibility factors. In comparison, both naïve and memory 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. In conclusion, the similar findings between drug and chemical antigen-responsive T-cells generated using an in vitro T-cell priming assay and those from patients highlight the promising use of this assay in the investigation of these responses. Indeed, the future definition and inclusion of (a) susceptibility factors and (b) the peptide-antigens responsible for naive T-cell priming will form a promising platform for the safe screening of drugs.
Supervisor: Naisbitt, D. J. ; Park, B. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral