A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science / Alternaria alternata, is an airborne ubiquitous fungus, which is associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. While dendritic cells, which are known as the classical antigen presenting cells, play an important function in antigen recognition at the early stage of immune response and then pass the signal to other immune cells. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. As the results in this study, we found that BMDCs were highly sensitive to A. alternata spores and their cell wall components. Following stimulation with these molecules, the expression of major histocompatibility complex (MHC) molecules and other co-stimulators, were highly up regulated. In order to determine how different cell wall components affect other immune cells through BMDCs, we conducted co-culture experiments of BMDCs and different T cells population in this study. Both spores and cell wall component did not induce IL-4, a featured Th2 cytokine, in those co-culture studies. Interestingly, we found that cell wall component induced the switching of the T helper cell population to the Th17 type. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways.