Investigating and Manipulating Major Histocompatibility Complex (MHC) Class I-Restricted Peptide Epitopes and Cognate T-cell Responses

The major histocompatibility complex class I (MHCI) molecules are restrictive elements for responses by natural killer (NK) and cluster of differentiation (CD)8+ T cells, which are cytotoxic immune cell effectors that assist in the containment of intracellular pathogens and the destruction of foreign tissues. While studies have shown the existence of antigen-specific CD8+ T-cell responses in cats and dogs, data regarding the capability of putative classical MHCI allomorphs to restrict such responses are lacking. Thus, one objective of this dissertation is to characterize genes critical to normal peptide:MHCI (pMHCI) expression, the transporters associated with antigen processing (TAP1/2), and to specifically knock out (KO) these genes in order to create a tool kit useful for studying peptide interactions with putatively classical feline and canine MHCI molecules. Having such a tool kit will allow for studies to detect CD8+ T-cell responses restricted by pathogen-derived epitopes presented by newly defined, classical MHCI allomorphs in these species. Herein, we determined the coding sequences of the canine TAP1/2 genes, and also confirmed the TAP2 locus in the cat by targeted KO using a biotechnological scissorlike mechanism, called transcription activator-like effector nucleases (TALENs). Furthermore, we created two novel feline cell lines (CRFK-PoBoy1 and 1A3- PoBoy2) that possess TALEN-mediated genomic modifications at the putative feline TAP2 locus, which resulted in reduced MHCI expression that is restored by exogenous TAP2 gene complementation. Future studies will determine the capability of these cells to stabilize feline leukocyte antigen class I (FLAI) complexes at the surface following exposure to peptides that match the feline MHCI-E*02001 allelic peptide-binding motif, and to bulk peptides eluted from FLAI complexes on parental cells.;We further utilized TALEN technology in our second objective; the intentional downregulation of classical MHCI protein on murine cell lines. For this objective, we selectively targeted the heavy chain genes of MHCI and not other genes involved within the peptide loading complex, as other studies have shown that KO of nonheavy chain genes may still result in surface MHCI expression. Following transfection of a murine beta cell line (NIT-1) with TALENs and a green fluorescence protein (GFP) reporter, we enriched for GFP positive cells possessing high TALEN activity and characterized the resulting clones. By this technique, we created a novel MHCI-deficient insulinoma cell line, NIT-KG, which possess targeted mutations in both the classical murine H2 loci (H2-K1 and --D1), and determined the ability of these cells to escape recognition by H2-Kb-restricted, autoreactive CD8+ T cells. We show that preventing surface expression of murine MHCI by way of TALEN technologies results in protection of these insulin producing cells from transgenic, autoreactive CD8+ T cells, which are major effectors in type 1 diabetes onset and recurrence.;Overall, this body of work contributes to the understanding of antigen presentation in feline and canine species by providing new data on TAP genes in these species and by creating tool kits useful for studying CD8+ T-cell responses. It further provides data indicating the usefulness of TALEN technology in creating genetically engineered cell lines in multiple species. Finally, TALEN-mediated genetic engineering strategies that prevent surface MHCI expression may result in tissues that can evade anti-graft responses, providing for a possible new treatment option for type 1 diabetes patients.