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.

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‐DRlo) 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 Bhi PD‐1lo 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‐DRlo 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.

Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4+ T cells, CD8+ T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8+PD-1+ T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8+PD-1- T cells and non-Treg CD4+FOXP3- T cells; but increased exhausted CD8+PD-1+ T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1+CD8+ T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy.

BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.