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.


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October 2018

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. All tissue samples with sufficient numbers of isolated viable lymphocytes were included in this study.
For phenotypic and functional assessment of human T-cells by flow cytometry or bioenergetic measurements, experiments were performed with samples from at least 6 donors (n numbers are stated in figure legends). Experimental variation between donors has been depicted graphically with all data points displayed in each figure. The experimental techniques used (e.g. leukocyte isolation, monoclonal antibody staining, flow cytometry, bioenergetic measurements, data analysis) were repeatedly performed on independent days. Experiments to measure the antiviral effect were performed in 4-6 replicates as stated in figure legend (Fig.5).
No randomization was performed. All tissue donors were pseudonymised.
During the analysis the investigators were blinded to all clinical patient data. There were no other experiments amenable to blinding.
Detailed information regarding all antibodies and other fluorescent agents used in this study are listed in Supp. Table 2 All antibodies were purchased from well established manufacturers and would have been validated by the vendor for species and target. Clones and catalogue numbers for each antibody have been included for cross-referencing of manufacturing company specification/validation processes in Supp. Table 2. We further validated antibodies by titration to optimal concentrations and by using positive controls where possible (e.g. using populations known to express a certain marker or by polyclonal stimulation). Antibody concentrations are stated in Supp Table 2.  The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided. To isolate IHL and TIL, liver/tumour tissue was cut into small pieces and incubated for 30min at 37C in 0.01% collagenase IV (Invitrogen) and 0.001% DNAse I (Sigma-Aldrich), followed by further mechanical disruption via GentleMACS (Miltenyi Biotech), filtration through a 70um cell strainer (BD Bioscience), removal of parenchymal cells on a 30% Percoll gradient (GE Healthcare) and lymphocyte isolation via density centrifugation as described above. IHL and TIL were used immediately after isolation.
The gating strategy used for PBMC is shown in Supp. Fig1a: cells were gated on their size and granularity to identify lymphocytes, single cells, live cells, CD3+, CD4-/CD8+ or CD4+/CD8-. The gating strategy used for IHL and TIL is shown in Supp. Fig1m: cells were gated on their size and granularity to identify lymphocytes and exclude residual hepatocytes/tumour cells, single cells, live