Host response to mannosylated proteins from the pathogenic yeast, Cryptococcus neoformans

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The pathogenic yeast, Cryptococcus neoformans , is a leading cause of death among individuals with compromised T cell function. Mannoproteins (MP) are a set of functionally heterogeneous, heavily mannosylated glycoforms found in many fungal species including C. neoformans. Cryptococcal MP have been shown to stimulate cell-mediated immunity and pro-inflammatory cytokines, both vital for the clearance of this yeast. As it is unclear how MP elicit immunity, it was hypothesized that the extensive conjugated carbohydrate backbone present on MP plays an essential role in immune stimulation. Mannose receptors (MR), including the macrophage mannose receptor (MMR) and dendritic cell-specific ICAM-3-grabing nonintegrin (DC-SIGN), both present on antigen-presenting cells (APC) and known to recognize mannosylated pathogens were shown to bind MP. Deglycosylation of MP or MR blockade with competitive mannosylated ligands inhibited T-cell activation. The immunodominant APC responsible for immune stimulation was determined by incubating T cells with purified splenic dendritic cells (DC), B cells, and peritoneal macrophages. T cells responded to MP only in the presence of DC. In addition, whole splenocyte populations, but not DC-depleted splenocytes, stimulated MP-specific T cells. Comparing APC populations, only DC captured fluorescent MP in a MR-dependent process. The kinetics of MP capture appear to involve 2 processes, a major, rapid, saturable receptor-mediated process that is inhibited with mannan, and minor pinocytic uptake. Impressively, DC pulsed for short periods with MP were still able to functionally stimulate T cells in a MR-dependent mechanism. Confocal microscopy of MP in DC at early time points indicated co-localization with transferrin, MMR and DC-SIGN suggesting MP enters early endosomes via MR. Subsequently, MP enter degradative perinuclear compartments. In vivo, MP-immunization resulted in increased survival, as well as a decrease in organ fungal load in response to live C. neoformans challenge. This partial protection was dependent on T cells, and not B cells. Moreover, analysis of tissue pro-inflammatory cytokine levels showed an increase in tumor necrosis factor-α and interferon-γ in infected MP-immunized mice. Collectively, these studies begin to provide both a molecular and cellular basis for the immunogenicity of cryptococcal MP and support T cell vaccination strategies that target MR and DCs.2031-01-0