Defense Date

1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Kathleen L. McCoy

Abstract

A Chinese hamster ovary (CHO) fibroblast, transfected with murine major histocompatibility complex (MHC) class II genes, inefficiently simulated CD4+ Th cells specific for ovalbumin (OVA), hen egg lysozyme (HEL), and pork insulin which contains disulfide bonds. However, the fibroblasts elicited a T cell response to λ-repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented, suggesting that the processing defect is a recessive trait. Three distinct processing phenotypes were observed among twenty-eight hybrid clones analyzed for their ability to process a suboptimal concentration of OVA suggesting that a limited number of genes mediates the defect of WAB4 cells. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL either as effectively or more efficiently than a B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed conjugate consisting of 125I-tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage for the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. The WAB4 cells had normal intracellular levels of cysteine, however they possessed diminished levels of intracellular glutathione (GSH). Buthionine sulfoximine (BSO) - mediated reduction of intracellular levels of GSH decreased the ability of the hybrid line WALC to process HEL. Conversely, treatment of WAB4 cells with N-acetyl cysteine increased their efficiency in the processing of HEl. These findings indicate that the intracellular level of GSH influences the capacity of cells to process antigens with the disulfide bonds. Thus, the antigen processing defect exhibited by transfected CHO cells is probably caused by their impaired ability to reduce disulfide bonds which may be related to the diminished intracellular GSH level.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

9-27-2017

Included in

Microbiology Commons

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