Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

Biomaterial-based scaffolds are promising tools for controlled immunomodulation. They can be applied as three dimensional (3D) culture systems in vitro, whereas in vivo they may be used to dictate cellular localization and exert spatiotemporal control over cues presented to the immune system. As such, scaffolds can be exploited to enhance the efficacy of cancer immunotherapies such as adoptive T cell transfer, in which localization and persistence of tumor-specific T cells dictates treatment outcome. Biomimetic polyisocyanopeptide (PIC) hydrogels are polymeric scaffolds with beneficial characteristics as they display reversible thermally-induced gelation at temperatures above 16°C, which allows for their minimally invasive delivery via injection. Moreover, incorporation of azide-terminated monomers introduces functional handles that can be exploited to include immune cell-modulating cues. Here, we explore the potential of synthetic PIC hydrogels to promote the in vitro expansion and in vivo local delivery of pre-activated T cells. We found that PIC hydrogels support the survival and vigorous expansion of pre-stimulated T cells in vitro even at high cell densities, highlighting their potential as 3D culture systems for efficient expansion of T cells for their adoptive transfer. In particular, the reversible thermo-sensitive behavior of the PIC scaffolds favors straightforward recovery of cells. PIC hydrogels that were injected subcutaneously gelated instantly in vivo, after which a confined 3D structure was formed that remained localized for at least 4 weeks. Importantly, we noticed no signs of inflammation, indicating that PIC hydrogels are non-immunogenic. Cells co-delivered with PIC polymers were encapsulated within the scaffold in vivo. Cells egressed gradually from the PIC gel and migrated into distant organs. This confirms that PIC hydrogels can be used to locally deliver cells within a supportive environment. These results demonstrate that PIC hydrogels are highly promising for both the in vitro expansion and in vivo delivery of pre-activated T cells. Covalent attachment of biomolecules onto azide-functionalized PIC polymers provides the opportunity to steer the phenotype, survival or functional response of the adoptively transferred cells. As such, PIC hydrogels can be used as valuable tools to improve current adoptive T cell therapy strategies.

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14 + DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system.

SummaryEndogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved.

Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.

Intracellular vesicle transport is essential to maintain cellular homeostasis and is known to be mediated by the endosomal pathway and SNARE proteins. Endosomal-mediated transport to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of effector immune responses. Despite the critical importance of this process, the specific molecular agents that regulate DC cytokine secretion are poorly characterised. Galectin-9, a ß-galactoside-binding protein, has emerged as novel modulator of numerous cellular processes although its exact functions remain to be elucidated. In particular, its intracellular roles in regulating (immune) cell homeostasis and cytosolic transport are virtually unknown. Here, we investigated galectin-9 function in cytokine secretion in primary human DCs, and report that galectin-9 is essential for intracellular trafficking of cytokines to the cell surface. Galectin-9 depletion resulted in the accumulation of cytokine-containing vesicles in the Golgi complex that eventually underwent lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using unbiased immunoprecipitation-mass spectrometry technology, and identified that galectin-9 was required for rerouting of Vamp-3-containing endosomes to the plasma membrane for cargo release upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle machinery and transport and shed new light into the multiple roles galectin and glycan-mediated interactions play in governing cell function.

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light into multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We developed a novel human, organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multi-cellular tissue architecture. By co-culturing keratinocytes, fibroblasts and immune cells with melanoma cells, onto a de-cellularized dermis, we generated a reconstructed TME that closely recapitulates tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC we observed the tumour-driven conversion of cDC2s into CD14 + DCs, characterized by a an immunosuppressive phenotype. The OMC provides a valuable complement to current approaches to study the TME.

Endogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved.