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V gamma 9V delta 2 T cells is the dominant gamma delta T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for V gamma 9V delta 2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise V gamma 9V delta 2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly thawed human serum potentiated V gamma 9V delta 2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and consistently enabled V gamma 9V delta 2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallylmonophosphate stimulated V gamma 9V delta 2 T cell proliferation. We developed a method where the cytotoxicity of V gamma 9V delta 2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the V gamma 9V delta 2 T cell cytotoxicity towards colon cancer cells. In summary, we have developed an easily interpretable method to assess the cytotoxicity of V gamma 9V delta 2 T cells towards adherent cells, found that V gamma 9V delta 2 T cell proliferation can be potentiated by media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of V gamma 9V delta 2 T cell immunotherapy.

V gamma 9V delta 2 T cells, the dominant gamma delta T cell subset in human peripheral blood, are stimulated by phosphoantigens, of which (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, is produced in the apicoplast of malaria parasites. Cell-free media from synchronised Plasmodium falciparum asexual ring, trophozoite, and schizont stage-cultures of high purity as well as media from ruptured schizont cultures, all stimulated V gamma 9V delta 2 T cell proliferation, as did media from pure gametocyte cultures, whereas media from uninfected erythrocytes cultures did not. The media from ruptured schizont cultures and all the asexual and gametocyte stage cultures contained only background iron levels, suggesting that all erythrocyte haemoglobin is consumed as the parasites develop and supporting that the phosphoantigens were released from intact parasitized erythrocytes. The V gamma 9V delta 2 T cell-stimulating agent was not affected by freezing, thawing or heating but was sensitive to phosphatase treatment, confirming its phosphoantigen identity. In summary, phosphoantigens are released from parasitised erythrocytes at all developmental blood stages.

The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence.

Cholesterol is an essential component of mammalian cells. It is the major lipid constituent of the plasma membrane and is also abundant in most other organelle membranes. In the plasma membrane cholesterol plays critical physical roles in the maintenance of membrane fluidity and membrane permeability. It is also important for membrane trafficking, cell signalling, and lipid as well as protein sorting. Cholesterol is essential for the formation of liquid ordered domains in model membranes, which in cells are known as lipid nanodomains or lipid rafts. Cholesterol depletion is widely used to study the role of cholesterol in cellular processes and can be performed over days using inhibitors of its synthesis or acutely over minutes using chemical reagents. Acute cholesterol depletion by methyl-β-cyclodextrin (MBCD) is the most widely used method and here we describe how it should be performed to avoid the common side-effect cell death.

Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

Two related models for T cell signalling initiation suggest either that T cell receptor (TCR) engagement leads to its recruitment to ordered membrane domains, often referred to as lipid rafts, where signalling molecules are enriched or that ordered TCR-containing membrane nanodomains coalesce upon TCR engagement. That ordered domains form upon TCR engagement, as they do upon lipid raft marker patching, has not been considered. The target of this study was to differentiate between those three options. Plasma membrane order was followed in live T cells at 37 °C using laurdan to report on lipid packing. Patching of the TCR that elicits a signalling response resulted in aggregation, not formation, of ordered plasma membrane domains in both Jurkat and primary T cells. The TCR colocalised with actin filaments at the plasma membrane in unstimulated Jurkat T cells, consistent with it being localised to ordered membrane domains. The colocalisation was most prominent in cells in G1 phase when the cells are ready to commit to proliferation. At other cell cycle phases the TCR was mainly found at perinuclear membranes. Our study suggests that the TCR resides in ordered plasma membrane domains that are linked to actin filaments and aggregate upon TCR engagement.