The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonopsonized particles and bacteria by a pathway attenuated by extracellular ATP.

Phagocytosis of nonopsonized bacteria is central to innate immunity, but its regulation is less defined. We show that overexpression of the P2X(7) receptor greatly augments the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X(7) expression by siRNA significantly reduces the phagocytic ability of human monocytic cells. An intact P2X(7)-nonmuscle myosin complex is required for phagocytosis of nonopsonized beads because activation of P2X(7) receptors by adenosine triphosphate (ATP), which dissociates myosin IIA from the P2X(7) complex, inhibits this phagocytic pathway. Fresh human monocytes rapidly phagocytosed live and heat-killed Staphylococcus aureus and Escherichia coli in the absence of serum, but the uptake was reduced by prior incubation with ATP, or P2X(7) monoclonal antibody, or recombinant P2X(7) extracellular domain. Injection of beads or bacteria into the peritoneal cavity of mice resulted in their brisk phagocytosis by macrophages, but injection of ATP before particles markedly decreased this uptake. These data demonstrate a novel pathway of phagocytosis of nonopsonized particles and bacteria, which operate in vivo and require an intact P2X(7)-nonmuscle myosin IIA membrane complex. The inhibitory effect of ATP on particle uptake by the macrophage is regulated by the P2X(7) receptor and defines this phagocytic pathway.

Large-scale production and characterization of novel CD4+ cytotoxic T cells with broad tumor specificity for immunotherapy.

Immune-cell-based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4(+) cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer.

A quantitative method for routine measurement of cell surface P2X7 receptor function in leucocyte subsets by two-colour time-resolved flow cytometry.

The P2X(7) receptor is a ligand-gated cation channel activated by extracellular ATP and highly expressed on monocytes, macrophages and lymphocytes. Activation of this receptor by exposure to extracellular ATP opens a selective cation channel that allows Ca(2+) and Ba(2+) influx, and K(+) efflux. Over the first minute the channel adopts a second and larger permeability state allowing the uptake of ethidium(+), followed by a cascade of intracellular downstream effects. Current methods used to study the P2X(7) receptor function, do not give quantitative measurement in sub-populations of a mixed cell suspension. We describe a quantitative method to determine the P2X(7) receptor function using time-resolved two-colour flow cytometry by assessing ATP-induced ethidium(+) uptake. Practical factors such as ethidium bromide concentration, agonists, temperature and buffers are also studied. Moreover, the ATP-induced ethidium(+) uptake method is compared to ATP induced barium (Ba(2+)) influx with Fura-Red. These two compatible methods can be used to screen the channel/pore function of the cell surface P2X(7) receptor among individuals and the results may be useful to estimate susceptibility of subjects to certain infectious diseases.