Nowak, K;
(2016)
Why do children with severe combined immunodeficiency get warts? The role of the common γ-chain in skin immunity.
Doctoral thesis , UCL (University College London).
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Abstract
X-linked severe combined immunodeficiency, caused by mutations in the common γ-chain, is characterized by absence of T and NK cells which leads to life-threatening infections. The disease can only be cured by bone marrow transplantation or gene therapy. Even after successful replacement of the T-cell compartment, patients display a persistent susceptibility to severe cutaneous human papillomavirus (HPV) infections. We hypothesized that persistent HPV susceptibility is due to residual γ-chain deficiency in keratinocytes. The role of γ-chain in skin is unknown. In this thesis, we confirmed the expression of γ-chain in keratinocytes by reverse transcription PCR (RT-PCR) and flow cytometry and demonstrated that it is functional, mediating an increase in STAT5 and AKT phosphorylation in response to IL-2 and IL-15 stimulation. We then generated a γ-chain knock-down cell line which showed reduced levels of phopho-AKT after stimulation with IL-15 compared to control cells showing impaired signaling. Using HPV pseudovirions, an increase of 50% in infectivity in knock-down cells compared to control cells was observed indicating an increase in initial infection. In an organotypic raft model using keratinocytes transfected with HPV18, we observed increased suprabasal DNA synthesis in knock-down cells indicating dysregulation of keratinocyte proliferation even though expression of HPV life cycle markers was unchanged. Moreover, an increase in chemokine secretion after IL-15 stimulation was only observed in control but not in γ-chain deficient cells. These secreted levels of chemoattractants were able to induce migration of neutrophils, dendritic cells and CD4+ T cells. Using supernatants collected from keratinocytes transfected with HPV18 as chemoattractant, we saw altered migration towards supernatant from HPV18 positive γ-chain deficient cells compared to that of HPV18 positive control keratinocytes. Together the results presented here suggest that γ-chain deficiency in keratinocytes leads to an increased susceptibility to HPV infection, altered regulation of infection and impaired immune cell recruitment.
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