Rusk, Rachel Aline2017-07-132017-07-132017-08-01http://hdl.handle.net/2097/35801The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis (M. bovis). γδ T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune system. Bovine γδ T cells have the capacity for multiple immune functions during infection with M. bovis. However, the alternative functions of γδ T cells as well as the responses of γδ T cells in vivo at the site of infection remain unclear. To identify novel functions for γδ T cells in response to M. bovis infections, RNA sequencing and transcriptomics analysis was completed on peripheral blood γδ T cells isolated from virulent M. bovis-infected cattle. Differentially expressed genes were confirmed with real-time PCR. In an attempt to model in vivo cell-to-cell interactions at the site of infection, γδ T cells were also isolated from naïve and M. bovis-infected calves and co-cultured with autologous, BCG-infected, monocyte-derived macrophages. γδ T cell chemokine and cytokine expression was analyzed via ELISA and real-time PCR. The characteristic lesions of bovine tuberculosis are well-organized pulmonary granulomas. To determine the relevance of the RNA-sequencing and in vitro co-culture results to in vivo infection, tissue samples from granulomatous lesions in the lungs and mediastinal lymph nodes of virulent M. bovis-infected cattle were collected 3 months after infection. mRNA transcripts for γδ T cells expression of-- IFN-γ, IL-17, IL-10, IL-22, and CCL2 were microscopically evaluated within the granulomas using an in situ hybridization system, RNAScope (Advanced Cell Diagnostics Inc.). Co-culture experiments and transcriptomics analysis revealed increased expression of chemokines and various cytokines by γδ T cells responding to M. bovis infection. The novel in situ hybridization assay revealed that cytokine expression by γδ T cells varied within the lesions, with significant levels of CCL2 and IFN-γ, and low expression of IL-10, IL-22, and IL-17 in situ at this time-point after infection. Co-culture experiments also revealed that γδ T cells from virulent M. bovis-infected cattle have the capacity to directly impact the viability of M. bovis in vitro. Our results suggest that γδ T cells accumulate within the granulomas, and influence host immunity to M. bovis by secretion of cytokines and chemokines, and direct cytotoxicity, in response to infected macrophages.en-US© the author. This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).http://rightsstatements.org/vocab/InC/1.0/Gamma delta T cellsMycobacterium bovisThesis