Islam, Farjana; (2025) Control of lymphocyte homeostasis by IKK-dependent regulation of extrinsic cell death and NF-κB signalling pathways. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The IKK complex is critical for inducing NF-κB signalling and has more recently been shown to repress CASPASE8-mediated extrinsic cell death through the repressive phosphorylation of RIPK1 kinase activity. CASPASE8 has a key function in inhibiting RIPK1-RIPK3-MLKL-mediated inflammatory necroptotic cell death. However, the role of IKK in regulating NF-κB and extrinsic cell death pathways for the homeostasis of conventional β T cells and ɣδ T cells is not clear. To dissect this, we examined CASPASE8 deficient huCD2iCre mice, inducing the Casp8 deletion from the earlier stages of double negative thymocytes. While the selection and maturation stage of single positive β thymocytes was normal, the development and expansion of double negative and double positive thymocytes were impaired. In the periphery, analysing intact mice and mixed bone marrow chimeras revealed that both CD4+ and CD8+ β T cell compartments exhibited survival defects following the ablation of CASPASE8. However, CASPASE8 deficient naive T cells were not apparently sensitive to Fas or TNFR-induced cell death. Furthermore, we found that kinase-dead RIPK1D138N also failed to rescue peripheral CD4+ and CD8+ β T cells from death in the absence of CASPASE8, indicating that cells were not susceptible to RIPK1-dependent necroptosis, with the sole exception of CD8+ effector memory T cells. CASPASE8 deficiency resulted in changes in the IL-7R expression, an NF-κB gene target. We asked whether CASPASE8 might regulate NF-κB activation by analysing mice lacking CASPASE8 and RELA expression in T cells. While this strain provided no evidence that CASPASE8 expression controls the induction of NF-κB regulation in T cells, we did find that co-expression of CASPASE8 and RELA is essential in the homeostatic development of B cells, which was otherwise normal in mice lacking one or other of CASPASE8 or RELA. The role of IKK signalling for the homeostasis of ɣδ T cells is unknown. My studies of IKK, CASPASE8 and REL deficient strains revealed that the development of adaptive and type 17 ɣδ thymocytes was normal in the absence of inflammatory signalling, but that generation of type 1 ɣδ thymocytes, was strictly NF-κB-dependent. In contrast, peripheral survival of ɣδ T cells required both IKK and NF-κB signalling. Additionally, while adaptive and type 1 ɣδ thymocytes were unaffected by the ablation of CASPASE8, their peripheral survival depended on CASPASE8 expression, in contrast to type 17 ɣδ T cells that were unaffected. Adaptive and type 1 ɣδ T cells appeared to be sensitive to RIPK1-dependent necroptotic cell death in the absence of CASPASE8. Therefore, our data suggest that β and ɣδ T cells exhibit diverse and distinct homeostatic requirements and responsiveness to inflammatory signals, that may reflect their functional distinctions in the adaptive immune system.

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