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Title: Exploring the role of CD8+ T-cells in chronic lymphocytic leukaemia
Author: Elston, Lauren
ISNI:       0000 0004 6349 2671
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2016
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Although chronic lymphocytic leukaemia (CLL) is a B-cell malignancy, T-cells from CLL patients often display abnormal characteristics when compared to age-matched healthy donors. One example is the preferential expansion of CD8+ T-cells within a subgroup of CLL patients resulting in an inverted CD4:CD8 ratio (CLLIR). This thesis describes a detailed analysis of the T-cell compartment with an emphasis on CD8+ T-cells. It confirms that CLL patients have abnormal memory T-cell subset distributions, with skewing to more differentiated memory subsets (EM/EMRA CD8+ T-cells) and an increase in a replicative senescent phenotype (CD57+). Furthermore, CLLIR patients demonstrated significantly inferior prognosis compared to their normal ratio counterparts (CLLNR). The aim of this thesis was to further characterise the expanded CD8+ T-cells within the CLLIR subgroup to define more precise prognostic markers and to understand the role of these T-cells in CLL disease. Polychromatic phenotyping of CLL patients (n = 99) was carried out using 8-colour flow cytometry, testing for markers associated with senescence (CD57/KLRG-1), exhaustion (PD-1) and activation (HLA-DR/CD38). This allowed the identification of multiple discrete subsets of T-cells, with a greater complexity of subsets seen in CLL patients compared to healthy donors. There was also an increase in senescent phenotypes within CLLIR patients, confirming previous results. However, the use of additional markers identified increased frequencies of an ‘activated senescent’ phenotype (CD8+CD57+HLA-DR+) within CLLIR patients. This phenotype was shown to have a prognostic effect on progression-free survival in both multivariate and univariate analysis, with higher frequencies conferring inferior prognosis. Multivariate analyses also revealed CD4+ subsets, including those with a CD4+HLA-DR+PD-1+ phenotype, to be of prognostic value. This prompted a phenotypic assessment of the CD4+ T-cell compartment. Like the CD8+ population, CD4+ T-cells showed increased frequencies of senescent phenotypes within CLLIR patients. There was also an exacerbation of CD57+HLA-DR+ and HLA-DR+PD-1+ phenotypes within the CLLIR subgroup, the latter being the strongest prognostic phenotype identified by multivariate analysis within the entire study. To further evaluate the prognostic potential and stability of the CD4:CD8 ratio in CLL, preliminary phenotypic analysis was performed on a small cohort of treated and untreated patients. These results showed a disproportionate number of patients with inverted ratio in the treated patient group. Furthermore, the prognostic phenotypes CD8+CD57+HLA-DR+ and CD4+HLA-DR+PD-1+ were over represented within the treated subgroup. Finally, single telomere length analysis (STELA) of CD8+ T-cells in CLL was performed to explore potential changes in the replicative history between CLLNR and CLLIR patients. Overall, the mean telomere lengths of T-cells from CLLIR patients was similar to that of CLLNR patients, suggesting that the expanded CD8+ compartment does not necessarily arise from a long-lived population that has undergone multiple rounds of division. The STELA did reveal two additional subgroups of CLL patients that had either “short” or “long” telomeres within their T-cell memory subsets. This observation may provide an additional molecular marker that could be investigated either in combination or alone for prognostic relevance. Overall the results from the phenotypic analysis and STELA suggest that there is an active ongoing T-cell response in CLL, but the nature of the stimulus is unclear. The expanded T-cells in CLL are unusual and do not follow the patterns of T-cell exhaustion and senescence seen in chronic viral diseases and other cancers. Importantly, this study has defined two phenotypes (CD8+CD57+HLA-DR+ and CD4+HLA-DR+PD-1+) that have greater prognostic power than the inverted CD4:CD8 ratio. These may be useful in identifying patients who are likely to develop more aggressive clinical disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH426 Genetics ; RC0254 Neoplasms. Tumors. Oncology (including Cancer)