Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter‐observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell‐cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high‐throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost‐effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.

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.

Background and aims: Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics.Methods: We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays.Results: In-depth interrogation of the immune landscapes showed that regulatory T cells (T_REG) and CD8⁺ resident memory T cells (T_RM) were enriched in HBV-related HCC, whereas Tim-3⁺CD8⁺ T cells and CD244⁺ natural killer cells were enriched in non-viral-related HCC. NGS of isolated T_REG and T_RM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. T_REG and T_RM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1⁺ T_REG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that T_REG and T_RM contributed to overall patient survival: T_REG were associated with a poor prognosis and T_RM were associated with a good prognosis in HCC.Conclusion: We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.

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Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8⁺ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8⁺ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c⁺ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8⁺ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.