Objective: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. Design: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. Results: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. Conclusions: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

Objectives: Yttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response. Design: Time-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE. Results: TILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8 ⁺ T cells, CD56 ⁺ NK cells and CD8 ⁺ CD56 ⁺ NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB ⁺ CD8 ⁺ T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8 ⁺ and CD4 ⁺ T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1 ⁺ /Tim-3 ⁺ CD8 ⁺ T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs. Conclusion: High-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8⁺ T cells (T_RMs), resident natural killer cells (NK_Rs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3⁺CD4⁺ and PD-1⁺CD8⁺ T cells. Interestingly, Tregs and T_RMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and T_RMs isolated from the NTME. We found PD-1⁺ T_RMs were the predominant T-cell subset responsive to anti–PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8⁺ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NK_Rs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed. Significance The roles of tumor-infiltrating leukocytes in mediating cancer progression are well recognized, but a multidimensional analysis of the entire cancer immune system is lacking. Here, we dissected the cancer-immune landscape in hepatocellular carcinoma (HCC) across tumor, nontumor, and peripheral blood cells using time-of-flight mass cytometry, multiplex immunofluorescence tissue staining, and NanoString analysis. We identified various immune subsets that were enriched in the tumor microenvironment and their potential impact on the tumor immunity based on their detailed phenotypes. This study has validated the concept of a cancer-immune gradient and demonstrated in primary HCC that immune-cell subsets become progressively suppressive as they traverse the nontumor to tumor microenvironment. These data have opened avenues for the design of immunotherapeutics in HCC.

Background and Aims: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. Approach and Results: We analyzed the immune landscapes of hypoxia‐low and hypoxia‐high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen–DR isotype (HLA‐DR^lo) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia‐high tumor regions. On the other hand, the abundance of active granzyme B^hi PD‐1^lo CD8⁺ T cells in hypoxia‐low tumor regions implied a relatively active immune landscape compared with hypoxia‐high regions. The up‐regulation of cancer‐associated genes in the tumor tissues and immunosuppressive genes in the tumor‐infiltrating leukocytes supported a highly pro‐tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C‐C motif chemokine ligand 20) and CXCL5 (C‐X‐C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA‐DR^lo cDC2 to hypoxia‐high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen‐presenting HLA‐DR on cDC2. Conclusions: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg‐mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune-ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune-ITH is associated with tumour transcriptomic-ITH, mutational burden and distinct immune microenvironments. Tumours with low immune-ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks contributing to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.

The systemic treatment landscape for advanced hepatocellular carcinoma (HCC) has experienced tremendous paradigm shift towards targeting tumor microenvironment (TME) following recent trials utilizing immune checkpoint blockade (ICB). However, limited success of ICB as monotherapy mandates the evaluation of combination strategies incorporating immunotherapy for improved clinical efficacy. Radiotherapy (RT) is an integral component in treatment of solid cancers, including HCC. Radiation mediates localized tumor killing and TME modification, thereby potentiating the action of ICB. Several preclinical and clinical studies have explored the efficacy of combining RT and ICB in HCC with promising outcomes. Greater efforts are required in discovery and understanding of novel combination strategies to maximize clinical benefit with tolerable adverse effects. This current review provides a comprehensive assessment of RT and ICB in HCC, their respective impact on TME, the rationale for their synergistic combination, as well as the current potential biomarkers available to predict clinical response. We also speculate on novel future strategies to further enhance the efficacy of this combination.