Monocyte And Dendritic Cell Development And Trafficking

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The ongoing investigations in my laboratory are focused on delineating immunomodulatory mechanisms of adaptive immune responses. We use the p47phox gene knockout mouse as the primary model for our ex vivo investigations. We also use a novel ex vivo system we developed for differentiating Sca-1+Lin- hematopoietic progenitor cells into functional myeloid precursor, immature and mature dendritic cells. The interrelated objectives of our research focuses on distinguishing phagocyte NADPH oxidase p47phox derived reactive oxygen species (ROS) dependent and ROS independent regulation of dendritic cell and T cell function. Work in progress in our group over the past year can be defined by the interrelated objectives: (I) NADPH oxidase p47phox ROS independent mediated survival of cytotoxic T cells, and (II) Phagocyte NADPH oxidase p47phox-/- dendritic cell regulated T cell function. We previously reported that T cell receptor (TcR) stimulated T lymphocyte blasts generate NADPH oxidase dependent hydrogen peroxide (H2O2), and that cytokine secretion is skewed towards a T helper 1 phenotype in NADPH oxidase deficient T lymphocytes. This year we expanded on our observations to define a role for p47phox as a critical regulator for CD8 lymphocyte survival. Our recent observations have shown that NADPH oxidase p47phox-/- CD8+ T cells have a previously unrecognized survival defect that is only partially corrected with exogenous pro-survival cytokines and restoration of H2O2. Ex vivo investigations resulted in our finding that NADPH oxidase p47phox-/- mice, without evidence of gross infection, develop reactive secondary lymphoid organs (SLOs) characterized by (1) LN hyperplasia with increased B cell and activated CD8+ cell division, (2) an increased accumulation of resting nave B and T lymphocytes, and (3) lower T:B cell ratios than wild type mice. Paradoxically, we also found that post-thymic CD8+ lymphocytes from p47phox-/- mouse LN and spleen undergo a rapid and profound post activation apoptosis in vitro. Using cytokine stimulation we discerned that the p47phox-/- lymphocyte apoptosis is associated in cultured p47phox-/- CD8+ lymphocytes with the induction of pro-apoptotic protein express ion, and the induction of the intrinsic death pathway. [unreadable] The NADPH oxidase enzyme complex is present in all professional phagocytes (macrophages, neutrophils, eosinophils) and also in dendritic cells. Using tissue derived and ex vivo generated myeloid dendritic cells we have characterized p47phox-/- dendritic cell maturation, antigen processing and presentation, and T cell activation. Results from in vitro antigen specific co-cultures using p47phox-/- dendritic cells to activate ovalbumin specific transgenic CD4+ T cells indicate that p47phox-/- dendritic cells significantly enhanced CD4+ T cell proliferation and T helper I cytokine secretion. We intend to further explore this enhancement by investigating immunological synapse interactions, and cytokine regulated pathways in p47phox-/- dendritic cells.