Phagocytosis is a fundamental mechanism of innate immunity and its impairment is associated with severe chronic diseases, for example, chronic obstructive pulmonary disease. Investigating phagocytosis requires flexible tools and assay conditions, such as different fluorescent particle types, detection colors and readouts. We comprehensively evaluated and optimized phagocytosis assays using particles labeled with fluorescent pH-sensitive pHrodo® dyes, facilitating the specific detection of phagocytosed particles. Beads, bacterial and yeast particles labeled with pHrodo red and green were tested for their uptake by THP-1 cells and primary human macrophages by flow cytometry and high-content imaging. Whereas the latter allowed kinetic phagocytosis measurement, the former demonstrated the feasibility of using cell sorting for periods of up to 6 h, enabling downstream applications such as pooled genetic screens.

Although the human immune response to cancer is naturally potent, it can be severely disrupted as a result of an immunosuppressive tumor microenvironment. Infiltrating regulatory T lymphocytes contribute to this immunosuppression by inhibiting proliferation of cytotoxic CD8+ T lymphocytes, which are key to an effective anti-cancer immune response. Other important contributory factors are thought to include metabolic stress caused by the local nutrient deprivation common to many solid tumors. Interleukin-33 (IL-33), an alarmin released in reaction to cell damage, and sphingosine-1-phosphate (S1P) are known to control cell positioning and differentiation of T lymphocytes. In an in vitro model of nutrient deprivation, we investigated the influence of IL-33 and S1P receptor 4 (S1P4) on the differentiation and migration of human CD8+ T lymphocytes. Serum starvation of CD8+ T lymphocytes induced a subset of CD8Low and IL-33 receptor-positive (ST2L+) cells characterized by enhanced expression of the regulatory T cell markers CD38 and CD39. Both S1P1 and S1P4 were transcriptionally regulated after stimulation with IL-33. Moreover, expression of the chemokine receptor CXCR4 was increased in CD8+ T lymphocytes treated with the selective S1P4 receptor agonist CYM50308. We conclude that nutrient deprivation promotes CD8Low T lymphocytes, contributing to an immunosuppressive microenvironment and a poor anti-cancer immune response by limiting cytotoxic effector functions. Our results suggest that S1P4 signaling modulation may be a promising target for anti-CXCR4 cancer immunotherapy.

Background Cytotoxic T lymphocytes take on a leading role in many immune‐related diseases. They function as key effector immune cells fighting cancer cells, but they are also considerably involved in autoimmune diseases. Common to both situations, CD8 + T cells need to adapt their metabolism and effector function to the harsh and nutrient‐deprived conditions of the disease‐associated microenvironment. Methods We used an in vitro starvation as well as rapamycin treatment protocol mimicking nutrient deprivation to generate CD8 Low versus CD8 High T cells and performed FACS‐Sorting followed by transcriptomic profiling of the cytotoxic T cell subsets. Prominent markers identified in the CD8 Low versus the CD8 High T cells were then used to investigate the presence of these cell subsets in immune‐related human diseases. Employing cancer tissue microarrays and PhenOptics multispectral imaging as well as flow cytometry, we studied these CD8 + T cell subsets in cancer and relapsing‐remitting multiple sclerosis patients. Results Starvation induced a decreased expression of CD8, yielding a CD8 Low T cell subpopulation with an altered transcriptomic signature and reduced effector function. CD8 Low T cell showed enhanced ST2L and IL6ST (CD130) expression compared to CD8 High T cells which expressed elevated KLRD1 (CD94) and granzyme B levels within the tumour microenvironment (TME). Spatial analysis revealed the presence of CD8 High T cells in close proximity to tumour cells, while the CD8 Low T cells resided at the tumour boundaries. Importantly, the number of tumour‐infiltrating CD8 Low T lymphocytes correlated with a poor prognosis as well as with enhanced cancer progression in human mammary carcinoma. We also found a reduced frequency of CD8 Low T lymphocytes in a cohort of relapse (disease active) multiple sclerosis patients compared to healthy subjects during immune cell starvation in vitro. Conclusions In summary, our data show that functionally distinct cytotoxic T lymphocytes can be identified based on their expression of CD8. Indicating a more general role in CD8 T cell immunity, these cells may play opposing roles in the TME, and also in the pathophysiology of autoimmune diseases such as multiple sclerosis.