Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Marko Z. Radic, Ph.D.

Committee

John Cox, Ph.D. Elizabeth Fitzpatrick, Ph.D. Linda Hendershot, Ph.D. Martha Howe, Ph.D. Monica Jablonski, Ph.D.

Keywords

B cells, Golgi, Autoreactivity, IgM secretion

Abstract

B cell receptors with certain heavy/light chain pairs predominate in the periphery of anti-DNA heavy chain transgenic mice while certain other B cell receptors are absent. We wanted to know why. To answer this, we re-constructed B cell receptors represented in the anti-DNA transgenic mice by cloning them as single chain variable fragments (scFv) to analyze their affinity to dsDNA. scFv representing receptors that were recovered from the spleens of transgenic mice had very little to no affinity to dsDNA. scFv representing receptors that were absent in the spleen had high affinity to dsDNA. We therefore concluded that receptors with low or no affinity to DNA successfully passed central tolerance in the bone marrow while receptors that had high affinity to DNA were likely altered by receptor editing or deleted. We also encountered B cell receptors that did not fit this conclusion. These were receptors that had anti-DNA heavy chains paired with the light chain Vk38c. They were an exception because, though they were present in the spleen, they bound DNA very well. How did these anti-DNA B cells escape central tolerance? In our effort to answer this puzzle, we discovered that receptors with the Vk38c light chain may have specificity to a Golgi antigen in addition to DNA. This suggested that such anti-DNA receptors may bind to an intracellular Golgi antigen and remain hidden inside the cell. Thus B cell receptor-cloistering in immature B cells, during development in the bone marrow, may allow the escape of anti-DNA B cells from central tolerance. After escaping from the bone marrow, these B cells become sequestered in the marginal zone of the spleen. Splenic B cells when immortalized as hybridomas show B cell receptor (IgM) accumulation in the Golgi. Receptor accumulates in the Golgi only when the heavy chain is paired with the Vk38c light chain. The location of B cell receptor accumulation in these hybridomas ranges from the cis-Golgi to medial- and trans-Golgi with the bulk of the receptor being concentrated in the medial-Golgi. Fragmenting the Golgi architecture with nocodazole, caused the intracellular IgM accumulation to disperse to the same locations as the Golgi, further confirming the location. A novelty about these B cells is that they have an uncommon way of antibody secretion. Instead of secreting IgM pentamers as B cells normally do, these cells extrude IgM as aggregates of up to 2µm. We named the aggregates “spherons” because of their appearance. The spherons do not have a membrane and are likely composed of the m heavy chain and the k light chain. In addition to extruding spherons, the B cells secrete soluble IgM as monomers and hemimers possibly as a consequence of IgM retention in the Golgi. Spherons have a high similarity to immune complexes deposited in kidneys of mice suffering from lupus nephritis. Spherons may be relevant in autoimmune nephritis and immunoglobulin deposition diseases. Understanding the mechanism of production of spherons could help design treatment strategies for patients suffering from autoimmune disease.

DOI

10.21007/etd.cghs.2008.0224

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