Biology subjects

Layzell, S.

Publications and source records attributed to Layzell, S..

4 recordsLinked to original sources

bioRxiv2025

Evidence for a RIPK1-independent survival mechanism for CASPASE-8 in αβ T cells

CASPASE8 promotes both cell death and survival by acting as a trigger of apoptosis but also a repressor of necroptosis. In T cells, CASPASE8 is required for FAS induced apoptosis, but protects activated T cells from necroptosis. The broader role and mechanisms of CASPASE8 in the wider T cell compartment is less certain. Here, we analysed mice in which Casp8 was conditionally deleted in the lymphoid compartment by huCD2iCre. In the thymus, we found evidence of a modest impairment of early thymic progenitors and a striking absence of NKT cell development. Amongst mature peripheral T cells, there was a substantial and specific reduction in the CD8 T cell compartment, that included naive, central memory and virtual memory subsets. While life spans and turnover of CD8 T cells appeared largely normal, we did identify an acute requirement for continued CASPASE8 expression for survival of a fraction of CD8 T cells, since induced Casp8 deletion by tamoxifen induced CD8CreERT resulted in an acute loss of CD8 T cells. CASPASE8 deficient T cells were resistant to FAS or TNF induced cell death in vitro. Generating Casp8 deficient mice that express a kinase dead RIPK1 confirmed that necroptosis contributed to death of thymic progenitors and some peripheral CD8 T cell subsets in the absence of CASPASE8. However, kinase dead RIPK1 failed to restore NKT cell development and only partially rescued CD8 VM T cells, while analysing mixed bone marrow chimeras suggested that CASPASE8 deficient CD4 and CD8 T cells were less competitively fit than WT T cells, even in the absence of RIPK1 kinase activity. These latter observations suggest the existence of a novel mechanism by which CASPASE8 promotes T cell survival that is independent of its established role in repressing necroptosis.

immunology↗

bioRxiv2023

NF-κB dependent expression of A20 controls IKK repression of RIPK1 dependent cell death in activated T cells

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of canonical NF-{kappa}B activation. More recently, RIPK1 has also been identified as a phosphorylation target of the IKK complex, resulting in repression of extrinsic cell death pathways. Our previous work shows that normal thymocyte development is exclusively reliant on repression of TNF triggered cell death pathways by IKK, and that NF-{kappa}B signalling is in fact redundant for development. The role of IKK signalling in activated T cells is unclear. To investigate this, we analysed activation of IKK2 deficient TCR transgenic T cells with cognate peptide. While early activation events were normal, proliferation of blasts was impaired. Surprisingly, cell cycle progression in IKK2 KO T cells was unperturbed. Instead, dividing cells were more sensitive to apoptosis triggered by extrinsic cell death pathways, since inhibition of RIPK1 kinase activity almost completely rescued cell survival. Transcriptomic analysis of activated IKK2 deficient T cells revealed defective expression of several NF-{kappa}B targets, including Tnfaip3, that encodes A20, a negative regulator of NF-{kappa}B in T cells. To test whether A20 expression was required to protect IKK2 deficient T cells from cell death, we generated mice with T cells lacking both A20 and IKK2. Conditional deletion of both Ikk2 and Tnfaip3 in T cells resulted in near complete ablation of peripheral naive T cells, in contrast to mice lacking one or other gene. Strikingly, this phenotype was completely reversed by inhibition of RIPK1 kinase activity in vivo. Therefore, our data suggests that IKK signalling in T cells protects against RIPK1 dependent death, both by direct phosphorylation of RIPK1 and through NF-{kappa}B mediated induction of A20, that we identify for the first time as a modulator of RIPK1 function in T cells.

immunology↗

bioRxiv2021

Tonic IKK signalling regulates naive T cell survival in vivo by both repressing RIPK1 dependent extrinsic cell death pathways and independent activation of NF-κB

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-{kappa}B activation. In addition, IKK has recently been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1. Our previous work shows that normal thymopoiesis relies on IKK exclusively for repression of TNF triggered cell death pathways, and that NF-{kappa}B activation by IKK is redundant for development. The role of these pathways in mature naive T cells has not previously been reported. Here, we show that, like thymocytes, naive peripheral T cells require continued IKK1/2 expression for survival. In contrast, however, cell loss is only partially prevented by blocking extrinsic cell death pathways by either deleting Casp8 or inhibiting RIPK1 kinase activity. Inducible deletion of Rela in mature CD4+ T cells also results in a significant loss of naive CD4+ T cells and loss of IL7R expression, revealing an additional reliance upon NF-{kappa}B for long term survival of mature T cells. Together, these data show that IKK dependent survival of naive T cells depends upon both repression of extrinsic cell death pathways and activation of NF-{kappa}B survival programme. One sentence summaryIKK regulates naive T cell survival

immunology↗

bioRxiv2020

Epithelial Cell Regulation of Type 2 Cytokine Release by Peripheral Blood Mononuclear Cells

BackgroundType 2 cytokines such as IL-13 and IL-5 are important drivers of pathophysiology and exacerbation in asthma. Defining how these type 2 cytokine responses are regulated is a research priority. Epithelial cells promote type 2 responses by releasing alarmins including IL-25, IL-33 and TSLP, but much less is known about inhibitory factors. MethodsIL-13 release was measured from peripheral blood mononuclear cells (PBMC) cultured with Interleukin (IL)-2 for five days. Epithelial cell lines or human bronchial epithelial cells (HBEC) isolated from healthy or asthma donors were added to these PBMC cultured with IL-2 and release of IL-13 or IL-5 measured. To characterise the mechanisms, we assessed the effect of mechanical disruption of epithelial cells, addition of the COX inhibitor indomethacin and the G-protein inhibitor pertussis toxin. ResultsPBMC cultured with IL-2 secreted type 2 cytokines in a cell number and time dependent manner. Epithelial cell lines inhibited IL-13 and IL-5 release after co-culture with PBMC in the presence of IL-2, directly, across a transwell and using epithelial cell supernatant. Cells or supernatant from HBEC from healthy or asthma donors also inhibited the cytokine release. Trypsin treatment of conditioned media indicated that inhibitory factor(s) are trypsin insensitive. Mechanical disruption of epithelial cells or indomethacin treatment had no effect, but pertussis toxin reduced epithelial cell inhibition of IL-2 driven type 2 cytokine release. ConclusionEpithelial cells regulate cytokine release by soluble factor(s) and this could be an important immunoregulatory function of the airway epithelium.

immunology↗