Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Emil Unanue

Abstract

Peptides from the model antigen hen egg white lysozyme: HEL) are capable of binding MHC II in multiple conformations. Resting antigen presenting cells: APC) load peptide in early endosomal compartments where multiple conformations of a given pMHC pair are allowed, generating both type A and B pMHC complexes. Protein traffics to the late endosomal compartment and is loaded onto nascent MHC II in the presence of the editor H2-DM where type A pMHC conformers are selectively formed, generating exclusively type A pMHC from protein. Previous work demonstrated that type B pMHC are also generated when HEL is administered in vivo along with certain inflammatory stimulants. Using an in vitro system I established, I found that TLR ligands and type I interferons: IFN) act directly on dendritic cells: DC), allowing generation of type B pMHC from HEL. Both CD8alpha+ and CD8alpha- DC present type B pMHC with TLR stimulation, but the relative effectiveness varied based on the ligand used. Using a type I IFN receptor: IFNAR1) blocking antibody and DC from mice deficient in the receptor, I found that TLR-induced type I IFN production amplifies but is not required for TLR-induced type B presentation from HEL. While I have not determined a sub-cellular mechanism for TLR-induced type B presentation, I have excluded mechanisms and made several observations. DC deficient in H2-DM are capable of TLR-induced type B presentation, indicating that regulation of H2-DM is not a critical mechanism. DC do not release meaningful levels of peptides allowing generation of type A or B pMHC. Additionally, generation of type B pMHC from HEL is a delayed event, requiring eighteen hours for appearance at the cell surface. While surface MHC II levels only modestly increase, there is a significant increase in total pMHC complexes after TLR or type I IFN stimulation. I employed two-photon microcopy to observe type A and B T cells in intact lymph nodes. While there were no remarkable differences in T cell behavior between the two groups, I determined that DC present type A or both type A and B but never exclusively type B pMHC. Using mice that express HEL as a membrane-bound protein on APC: mHEL), I found that TLR stimulation increases type B presentation from membrane-bound protein. I found that while immunization of mHEL mice with CpG increases presentation of type B pMHC from pseudo-self HEL and primed anti-HEL CD4 T cells, no observable disease was detected. These results identified the cells and signals required for inflammation-associated type B presentation from HEL and established tools for future investigation of the sub-cellular mechanism controlling these events.

Comments

Permanent URL: http://dx.doi.org/10.7936/K75Q4T3T

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