Comparable Overall Survival in Patients with Hepatocellular Carcinoma Diagnosed within and outside a Surveillance Programme:The Potential Impact of Liver Cirrhosis

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is rising. Mortality from HCC is predicted to increase by 140% by 2035. Surveillance of high-risk patients with cirrhosis or chronic liver disease may be one means of reducing HCC mortality, but the level of supporting evidence for international guidelines is low/moderate. This study explores the real-world experience of HCC surveillance at a tertiary referral centre. Electronic patient records for all new HCCs diagnosed between August 2012 and December 2021 were retrospectively reviewed. Patient and tumour characteristics were evaluated, including the co-existence of chronic liver disease, cancer treatment and survival, and categorised according to HCC diagnosis within or outside a surveillance programme. Patients with HCC who presented through surveillance had smaller tumours diagnosed at an earlier stage, but this did not translate into improved overall survival. All patients in surveillance had chronic liver disease, including 91% (n = 101) with cirrhosis, compared to 45% (n = 29) in the non-surveillance cohort. We propose that the immune dysfunction associated with cirrhosis predisposes patients to a more aggressive tumour biology than the largely non-cirrhotic population in the non-surveillance group

Isolation of human intrahepatic leukocytes for phenotypic and functional characterization by flow cytometry

With the growing appreciation of tissue-resident immunity, studying tissue-specific immune cells contributing to both homeostasis and disease is imperative. Here, we provide a protocol for the isolation of human intrahepatic leukocytes (IHL) maximizing viability, purity, and yield. Our protocol is scalable by tissue weight, allowing for reproducible and efficient IHL liberation suitable for functional characterization, cell isolation, and profiling by flow (or mass) cytometry. Furthermore, we provide a "guide" to determine an expected IHL yield per gram of tissue processed. For complete details on the use and execution of this protocol, please refer to Stegmann et al. (2016), Pallett et al. (2017), Easom et al. (2018), Swadling et al. (2020), Pallett et al. (2020), and Zakeri et al. (2022)

Characterisation and therapeutic harnessing of liver-resident gamma delta T cells to target hepatocellular carcinoma

More effective immunotherapeutic strategies are urgently needed for hepatocellular carcinoma (HCC). Gamma delta (γδ) T cells are attractive candidates for cancer immunotherapy with potential for HLA-unrestricted tumour reactivity. In this thesis, I characterise the phenotypic and functional profiles of tissue-resident memory (TRM) Vδ1 and Vδ2 T cells in human liver, examine their potential contributions to immunosurveillance in HCC, and explore therapeutic strategies to enhance the anti-tumour potential of Vγ9Vδ2 TRM cells for immunotherapy for HCC. I find that Vδ1 and Vδ2 T cells exhibit a bona fide TRM phenotype in human liver (CD69+CD49a+ and/or CD103+) with increased expression of the liver-homing chemokine receptors CXCR6 and CXCR3. Vδ1 and Vδ2 TRM cells show no egress from hepatic vasculature, demonstrate long-lived persistence in the liver for over a decade, and display superior anti-tumour cytokine production. Higher intratumoural γδ T cell counts in HCC are associated with smaller tumour size and longer patient survival. Vδ1 T cells display a more activated phenotype in HCC, while Vγ9Vδ2 T cells appear selectively depleted but maintain high IFN-γ production and equivalent capabilities to acquire an intratumoural γδ TRM phenotype. I demonstrate that intrahepatic and intratumoural Vγ9Vδ2 TRM cells efficiently target HCC cell lines (HepG2 or HuH7) sensitised to accumulate isopentenyl-pyrophosphate by the aminobisphosphonate Zoledronic acid (ZOL). In vitro expansion of blood Vγ9Vδ2 T cells using ZOL and IL-2 recapitulates a de novo TRM phenotype with enhanced cytotoxic potential. Using an in vivo murine model, adoptive cell transfer of ZOL-expanded Vγ9Vδ2 T cells combined with intratumoural ZOL sensitisation induces the greatest HepG2 tumour regression. In conclusion, intrahepatic γδ TRM cells demonstrate beneficial and long-lived immunotherapeutic properties. Future trials could explore a dual immunotherapeutic strategy using aminobisphosphonates to induce Vγ9Vδ2 TRM cells capable of replenishing the depleted pool in HCC, with additional intra-tumoral delivery of aminobisphosphonates to sensitise HCC for more efficient Vγ9Vδ2 TRM based targeting

Human Liver Memory CD8⁺ T Cells Use Autophagy for Tissue Residence

Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells. CD8+ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC