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Author's School

Graduate School of Arts & Sciences

Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

1-15-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Emil Unanue

Abstract

Exogenous antigen typically enters the major histocompatibility complex: MHC) class II system for processing and presentation to CD4+ T cells. Experiments conducted by Michael Bevan in the 1970s demonstrated that exogenous antigen can also enter the MHC class I system and cross-prime CD8+ T cells; the process by which antigen presentation occurs in this context is termed cross-presentation. We have found a CD8+ T cell epitope within hen egg-white lysozyme: HEL) that is presented on MHC class I via the cross-presentation pathway. The work in this thesis examines two issues: 1) CD8+ T cell cross-priming by subcutaneous immunization with exogenous HEL; and 2) cross-presentation of HEL to CD8+ T cell hybridomas by antigen presenting cells: APCs) from naïve and immunized mice. Non-obese diabetic: NOD) mice immunized with HEL in complete Freund's adjuvant: CFA) primed HEL 11-25-specific CD4+ T cells and HEL 20-35-specific CD8+ T cells; CD8+ T cell cross-priming was confirmed by several methods, including CD8+ T cell depletion, analysis of mice lacking CD8+ T cells and blockade of responses by MHC class I antibodies. Cross-priming by HEL-CFA immunization did not require CD4+ T cell help but was reduced in the absence of interferon-α/β: IFNα/β) or IFNγ signaling; Batf3-/- mice lacking CD8α+ dendritic cells: DCs) also showed reduced cross-priming. We examined the ability of draining lymph node: dLN) DCs to process and cross-present soluble HEL to CD8+ T cell hydridomas. DCs isolated from immunized, but not naïve, mice cross-presented soluble HEL ex vivo. Cross-presentation by dLN DCs was enhanced by IFNα/β, reduced by depletion of CD8α+ DCs and did not require the transporter associated with antigen processing: TAP). Naïve DCs treated with a variety of inflammatory stimuli, including IFNα/β and IFNγ, failed to cross-present soluble HEL. Finally, we assessed MHC class I and II presentation of HEL in liposomes targeted to early or late endosomal compartments. HEL in either early or late endosomal liposomes was presented on MHC class II; in contrast, only HEL in early endosomal liposomes was cross-presented on MHC class I. MHC class II presentation required nascent MHC molecules and endosomal acidification. Cross-presentation of HEL in early endosomal liposomes required endosomal acidification but not proteasomal activity, nascent MHC molecules or TAP. Blockade of endosomal acidification enabled cross-presentation of soluble HEL and HEL in late endosomal liposomes. Altogether, these data indicate that several forms of exogenous HEL enter the MHC class I pathway in vivo and in vitro. TAP is not necessary for HEL cross-presentation, implying that antigen processing occurs within endosomal compartments but not the cytosol; this feature distinguishes HEL from other cross-presented antigens, especially ovalbumin, and provides a model for investigating the intersection between the endosomal pathway and the MHC class I system.

DOI

https://doi.org/10.7936/K76Q1V76

Comments

Permanent URL: http://dx.doi.org/10.7936/K76Q1V76

Available for download on Friday, January 15, 2112

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