Publication Date
2020
Document Type
Thesis
Committee Members
Courtney Sulentic, Ph.D. (Advisor); Thomas Brown, Ph.D. (Committee Member); Nancy Bigley, Ph.D. (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Aryl hydrocarbon receptor (AhR) mediates the immunosuppressive effects of 2,3,7,8 -tetrachlorodibenzodioxin (TCDD) in murine B cells. The effects of AhR activation on the regulation of expression of human immunoglobulin isotypes (μ, γ1-4, α1-2 and ε) and Ig secretion is unclear. Our previous results using CL-01 cell-line originating from a Burkitt’s lymphoma patient, demonstrated an inhibitory effect of TCDD on IgG expression but a surprising and marked loss of IgG secretion when the AhR was knocked out by siRNA or CRISPR/Cas9 gene editing. To determine if the AhR is a critical mediator of IgG expression, current study is focused on characterizing IgG expression in another human B-cell line (SKW 6.4 or SKW WT) originating from a different, non-related Burkitt’s lymphoma. We confirmed that SKW WT cells do not have endogenous expression of AhR using PCR analysis and Western blotting. We also demonstrated that SKW 6.4 cells can be stimulated in-vitro using CD40L and IL-4 to produce more IgM antibodies as detected by ELISA assays. Further, we demonstrate that total IgG secretion induced by CD40L and IL-4 stimulation is severely impaired in SKW WT cells. Conversely, the Qt-PCR studies show that the expression of ε, γ2-4 transcripts that code for IgE and IgG2-4 respectively is significantly increased with stimulation as compared to un-stimulated SKW cell lines. The expression of γ1 was low in naive as well as stimulated SKW WT cells. The α1-2 transcripts coding for IgA1-2 respectively are not expressed at all in SKW cells regardless of stimulation. To further investigate, we used CL-01 AhRTA cells that express AhR with functional TAD, to compare the expression of different isotypes. It was found that the expression of γ1-4 and ε transcripts was significantly higher in AhR expressing CL-01 AhRTA cells as compared to SKW WT cells. Our observations imply that AhR plays a critical role in expression of the IgH gene.
Page Count
89
Department or Program
Microbiology and Immunology
Year Degree Awarded
2020
Copyright
Copyright 2020, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.
