Clinical studies have shown that chronic liver disease, a major risk factor for hepatocellular carcinoma (HCC), is associated with the accumulation of senescent cells within the liver. Senescence is an acutely tumour-suppressive, but chronically pro-tumorigenic stress-responsive cell pathway. Senescent cells signal to immunocytes through a complex secretome, to trigger their own CD4+ T-cell dependent destruction, termed senescence surveillance. The form and functionality of this adaptive immune reaction and why it fails when cancer develops remain unclear.

In this work, I have studied the adaptive immune response to senescence using in vitro and in vivo models. I have used hydrodynamic tail vein (HDTV) delivery of NRASG12V-containing transposons in mice, to model hepatocyte oncogene-induced senescence (OIS) combined with multiple downstream assays to interrogate four elements of adaptive senescence surveillance: functionality of T-helper subsets; their transcriptomic profile; antigen-reactivity; and adaptive cell-in-cell mechanisms which are laid out in separate results chapters of this thesis.

We found that OIS hepatocytes were actively cleared at day 9 post NRASG12V-senescence induction. This clearance was dependent on functional adaptive immunity, evident from in vivo studies of immunodeficient Rag2(-/-) mice. Through analyses of lineage commitment of T-helper cells (functional studies of Th1, Th2, Th17 and T-regulatory cells) I demonstrated that Rorγt, a transcriptional regulator of Th17 cells, was more abundant in hepatic CD4+ T-helper subsets during senescence immune surveillance compared to the control. A Rorc(-/-) (the gene encoding Rorγt) murine model, lacking Th17 cells displayed blunted senescence clearance, implicating its functionality in the senescence surveillance of OIS hepatocytes.

I used Single-cell RNA sequencing of intrahepatic T-cells during senescence surveillance to understand potential functional and phenotypic correlates of successful anti-cancer immune response. This found that the Rorc+-expressing Th17 cluster of T-cells in the senescent condition downregulated 65 and upregulated 45 gene transcripts. This included Runx3 and Ccl5 involved in tissue residency and HCC-associated hepatic immunosurveillance, respectively.

I attempted to utilise the Antigen-Receptor Signalling Reporter (AgRSR) mouse, a novel lymphocyte-activation tracing mouse model that permits dual fluorescence-based lineage tracing of TCR-stimulated lymphocytes to understand adaptive immune cells that are specifically responding to senescence induction. We studied fluorescently labelled cells during the adaptive immune response to OIS hepatocytes in the AgRSR mouse. However, despite functional results linking adaptive immunity to successful senescence surveillance we were not able to identify a significant change in antigen-reactive intrahepatic T-helper cells within the senescent liver compared to the non-senescent liver. Similarly, we were not able to identify a specific enrichment within lineage committed T-helper subsets Th17, Th2, Th1 and T-regs.

Unexpectedly, I identified CD4+ punctate regions that co-localised uniquely with senescent hepatocytes. Flow cytometry, immunofluorescence, confocal and live-cell imaging techniques of hepatocyte and T-helper cell co-culture, suggested that senescent cells had the ability of heterotypic engulfment of T-helper cells. Single-cell sequencing of senescent hepatocytes found negligible levels of ectopic Cd4 transcripts. This implied that the Cd4 protein was likely transcribed and translated outside of the senescent cell before uptake into the senescent cell. I conducted further exploratory analyses of sc-RNAseq data from OIS hepatocytes that identified gene transcripts associated with three cell-in-cell mechanisms: cannibalism, emperipolesis, entosis.

The results that I have generated here have furthered our understanding of adaptive immunity during surveillance of hepatocyte senescence and led to further questions about the complexity of this process.