Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

5-24-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Emil Unanue

Abstract

ABSTRACT OF THE DISSERTATION Presentation of citrullinated peptides derived from unmodified protein by antigen presenting cells. By Jamie Michelle Rimer Doctor of Philosophy in Biology and Biomedical Sciences: Immunology) Washington University in St. Louis 2010 Professor Emil R. Unanue, Chair Autoimmune responses to citrullinated proteins have been associated with rheumatoid arthritis; however, little is known of the mechanism by which tolerance to citrullinated proteins is established or the biology of citrullination by cells of the immune system. We find that immunization of B10.BR mice with HEL in CFA gives rise to T cells that specifically recognize citrullinated epitopes. We generated T cell hybridomas by fusing these T cells. T cell hybridomas that recognize citrullinated peptides from hen egg white lysozyme: HEL) were used to detect presentation of citrullinated peptides. Using this technique, we show that antigen presenting cells: APC) present citrullinated peptides after processing whole unmodified protein. As expected, APC isolated from the draining lymph node of mice immunized with HEL in complete Freund's adjuvant elicit a response from the citrullinated peptide specific T cell hybridomas. Examination of APC from HEL transgenic mice revealed that splenic dendritic cells, macrophages and thymic dendritic cells presented citrullinated peptides constitutively. Conversely we did not detect a response from B cells pulsed with HEL or from HEL transgenic mice or from a B lymphoma line. The reported localization of the enzymes that convert peptidylarginine to peptidylcitrulline is either the cytosol or nucleus and not in antigen processing and loading compartments. The role of autophagy, or the mechanism by which cytosolic proteins and senescent organelles are taken up in to vesicles for degradation in lysosomes, in citrullination of antigen was examined. Treatment of dendritic cells and macrophages with 3-Methyladenine: 3MA), a class III PI3 kinase inhibitor known to inhibit autophagy, blocked presentation of citrullinated peptides but presentation of unmodified peptides was not affected. B cell receptor engagement has been shown to induce autophagy in primary B cells. We examined presentation of HEL by B cells from anti-HEL transgenic mice, in which all B cell receptors bind HEL, and found that, in contrast to B cells from B10.BR mice, they presented citrullinated peptides. B cells from mHEL mice present citrullinated peptides after BCR engagement with anti-IgM or anti-IgG antibodies. Induced presentation of citrullinated peptides was blocked by 3MA. In addition, we find that C3.F6.mHEL B lymphoma cells present citrullinated peptides after serum starvation but blocked by 3MA treatment. C3.F6.mHEL cells that stably expressed shRNA targeting Atg5, a protein essential for autophagy, did not present citrullinated peptides after serum starvation. Higher levels of citrulline in naturally processed peptides from these cells were detected biochemically after culture in serum starved conditions. Our findings demonstrate a role for autophagy in citrullination of antigen during processing by antigen presenting cells.

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Permanent URL: http://dx.doi.org/10.7936/K7J10156

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