Alnabhan, RM; (2015) Study of the Activation of Peripheral Blood and Cord Blood Natural Killer Cells. Doctoral thesis , UCL (University College London).

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Abstract

Background: Natural killer (NK) cells are cytotoxic effectors providing a first line of defence against viruses and tumours. NK cells can be isolated from peripheral blood (PB) or cord blood (CB) for cancer immunotherapy. However, it was shown by our group and others, that CB NK cells express higher NKG2A and less killer immunoglobulin like receptors (KIRs) than PB NK cells indicating an immature phenotype. Also, CB NK cells require high doses of interleukin (IL)-2 for proliferation and activation. It was also shown that resting CB NK cells are poorly cytotoxic and produce less IFN-γ than PB NK cells after stimulation with IL-2. Hypothesis and aims: CB NK cells have an immature phenotype and could mediate different role in neonatal immunity than adult PB NK cells. Hence, the aim of this study was to explore whether differential mechanisms of activation exist between PB and CB NK cells. Methods: PB samples from healthy volunteers and CB samples were obtained with prior written consent and ethical approval from Anthony Nolan. Purified PB and CB NK cells were stimulated with cytokines including IL-2, IL-12, IL-15, IL-18, individually or in combination. Thereafter, comparative analysis was performed on their phenotype, signalling, proliferation, cytotoxicity, cytokine secretion and chemotaxis post-cytokine stimulation. Results: My results show that CB NK cells responded less to IL-2 activation than PB NK cells, which correlated with lower levels of IL-2 receptors and decreased phosphorylation of STAT5 pathway. CB NK cells activated with IL-15+IL-2 showed enhanced cytotoxicity while activation with IL-15+IL-18 promoted maximal proliferation, higher IFN-γ and TNF-α secretion. In contrast, optimal activation of PB NK cells was achieved by IL-2 stimulation. Interestingly, CB NK cells secreted substantial IL-8 concentrations following cytokine stimulation. IL-12 or IL-18 stimulation induced L- selectin expression on CB NK cells and promoted NK cell migration towards chemokines that induce homing to lymph nodes. I also generated long-lived memory- like NK cells from PB and CB using cytokines whereby IL-12+IL-15+IL-18 pre- activation led to substantial IFN-! production. Conclusions: CB NK cells are fully functional upon activation with IL-15+IL-2 or IL-15+IL-18 rather than IL-2 thereby providing a basis for activation of NK cells derived from different sources that may be utilised for future NK cell-based therapeutic purposes. In addition, CB NK cell cytokine secretion profile is suggestive for a role of neonatal NK cells in providing protective immunity against bacterial infections.

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