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Autologous platelet concentrates, like liquid platelet rich fibrin (iPRF), optimize wound healing; however, the underlying immunological mechanisms are poorly understood. Platelets, the main cellular component of iPRF, highly express the protein, Glycoprotein A repetitions predominant (GARP), on their surfaces. GARP plays a crucial role in maintaining peripheral tolerance, but its influence on the immune capacity of iPRF remains unclear. This study analyzed the interaction of iPRF with immune cells implicated in the wound healing process (human monocyte derived macrophages and CD4+ T cells) and evaluated the distinct influence of GARP on these mechanisms in vitro. GARP was determined to be expressed on the surface of platelets and to exist as a soluble factor in iPRF. Platelets derived from iPRF and iPRF itself induced a regulatory phenotype in CD4+ T cells, shown by increased expression of Foxp3 and GARP as well as decreased production of IL-2 and IFN-γ. Application of an anti-GARP antibody reversed these effects. Additionally, iPRF polarized macrophages to a “M0/M2-like” phenotype in a GARP independent manner. Altogether, this study demonstrated for the first time that the immune capacity of iPRF is mediated in part by GARP and its ability to induce regulatory CD4+ T cells.

The cellular composition of the tumor microenvironment, including tumor, immune, stromal, and endothelial cells, significantly influences responses to cancer therapies. In this study, we analyzed the impact of oxidative stress, induced by cold atmospheric plasma (CAP), on tumor cells, T cells, and macrophages, which comprise part of the melanoma microenvironment. To accomplish this, cells were grown in different in vitro cell culture models and were treated with varying amounts of CAP. Subsequent alterations in viability, proliferation, and phenotype were analyzed via flow cytometry and metabolic alterations by Seahorse Cell Mito Stress Tests. It was found that cells generally exhibited reduced viability and proliferation, stemming from CAP induced G2/M cell cycle arrest and subsequent apoptosis, as well as increased mitochondrial stress following CAP treatment. Overall, sensitivity to CAP treatment was found to be cell type dependent with T cells being the most affected. Interestingly, CAP influenced the polarization of M0 macrophages to a “M0/M2-like” phenotype, and M1 macrophages were found to display a heightened sensitivity to CAP induced mitochondrial stress. CAP also inhibited the growth and killed melanoma cells in 2D and 3D in vitro cell culture models in a dose-dependent manner. Improving our understanding of oxidative stress, mechanisms to manipulate it, and its implications for the tumor microenvironment may help in the discovery of new therapeutic targets.

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.

Glycoprotein A repetition predominant (GARP), a specific surface molecule of activated regulatory T cells, has been demonstrated to significantly contribute to tolerance in humans by induction of peripheral Treg and regulatory M2-macrophages and by inhibition of (tumorantigen-specific) T effector cells. Previous work identified GARP on Treg, and also GARP on the surface of several malignant tumors, as well as in a soluble form being shedded from their surface, contributing to tumor immune escape. Preliminary results also showed GARP expression on brain metastases of malignant melanoma. On the basis of these findings, we investigated whether GARP is also expressed on primary brain tumors. We showed GARP expression on glioblastoma (GB) cell lines and primary GB tissue, as well as on low-grade glioma, suggesting an important influence on the tumor micromilieu and the regulation of immune responses also in primary cerebral tumors. This was supported by the finding that GB cells led to a reduced, in part GARP-dependent effector T cell function (reduced proliferation and reduced cytokine secretion) in coculture experiments. Interestingly, GARP was localized not only on the cell surface but also in the cytoplasmatic, as well as nuclear compartments in tumor cells. Our findings reveal that GARP, as an immunoregulatory molecule, is located on, as well as in, tumor cells of GB and low-grade glioma, inhibiting effector T cell function, and thus contributing to the immunosuppressive tumor microenvironment of primary brain tumors. As GARP is expressed on activated Treg, as well as on brain tumors, it may be an interesting target for new immunotherapeutic approaches using antibody-based strategies as this indication.

Lipid exchange among biological membranes, lipoprotein particles, micelles, and liposomes is an important yet underrated phenomenon with repercussions throughout the life sciences. The premature loss of lipid molecules from liposomal formulations severely impacts therapeutic applications of the latter and thus limits the type of lipids and lipid conjugates available for fine-tuning liposomal properties. While cholesterol derivatives, with their irregular lipophilic surface shape, are known to readily undergo lipid exchange and interconvert, e.g., with serum, the situation is unclear for lipids with regular, linear-shaped alkyl chains. This study compares the propensity of fluorescence-labeled lipid conjugates of systematically varied lengths to migrate from liposomal particles consisting mainly of egg phosphatidyl choline 3 (EPC3) and cholesterol into biomembranes. We show that dialkyl glyceryl lipids with chains of 18–20 methylene units are inherently stable in liposomal membranes. In contrast, C16 lipids show some lipid exchange, albeit significantly less than comparable cholesterol conjugates. Remarkably, the C18 chain length, which confers noticeable anchor stability, corresponds to the typical chain length in biological membranes.