Publication Date

2025

Document Type

Dissertation

Committee Members

Courtney Sulentic, Ph.D. (Committee Chair); Thomas L. Brown, Ph.D. (Committee Co-Chair); Richard L. Salisbury Jr., Ph.D. (Committee Member); Weiwen Long, Ph.D. (Committee Member); Mike Kemp, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Antibody production is an essential component of the immune response against pathogens. The immunoglobulin heavy chain (IgH) gene codes for the heavy chain of antibodies. The IgH constant regions Cμ, Cδ, Cγ1-4, Cα1-2, and Cε encode the five major classes of antibodies, i.e., IgM, IgD, IgG1-4, IgA1-2, and IgE, respectively. The transcription of the IgH gene and class switch from IgM to other isotypes is regulated by two 3’ IgH regulatory regions (3’IgHRRs), each of which is a cluster of three enhancer regions (hs3, hs1.2 and hs4). The genetic variations in the hs1.2 enhancer have been identified; a ~53 bp invariant sequence is repeated one to four times among human populations. The effects of genetic variations in AhR and hs1.2 enhancers among the human population on the regulation of antibody production are unknown and represent a significant gap in knowledge. The current study has three specific aims that collectively evaluate the effects of AhR and hs1.2 genetic variations on the transcriptional regulation of the IgH gene in human B cells. We utilized the human Burkitt lymphoma cell line CL-01, which represent mature B cells and can switch from IgM to other antibody isotypes, enabling us to evaluate the effect of AhR ligands on antibody production. We identified three AhR SNPs (P517S, R554K, V570I) linked to low AhR transactivation in CL-01WT cells. In CL-01WT cells, AhR activation by two agonists (exogenous 2,3,7,8 -Tetrachlorodibenzo-p-dioxin (TCDD) and naturally derived indirubin) significantly inhibited IgG secretion but did not affect IgM or IgA. The AhRA treatment reversed these effects. AhR agonists had little to no impact on Cμ and Cα2 transcripts at the transcript level but significantly inhibited Cγ1-4 and Cε transcripts. Interestingly, with improved AhR transactivation function in CL-01TA+ cells, the level of inhibition of Cγ1-4 and Cε transcripts by AhR activation did not change compared to that in CL-01WT cells. However, the AhRA treatment significantly inhibited the expression of Cα2 in CL-01TA+ cells compared to CL-01WT cells. These results show that AhR activation regulates the IgH transcription via a non-canonical AhR signaling. For the second aim, we utilized another Burkitt lymphoma cell line, SKW 6.4 cells, that can only produce IgM and are ideal for investigating IgH gene germline transcription, a prerequisite to class switch. The SKW WT cells also lack endogenous AhR expression (SKW AhR-). Using lentiviral transduction, we introduced an AhR transgene generating SKW AhR+ clone to study the effect of AhR on IgH germline transcription. TCDD treatment significantly inhibited germline transcription of Cγ2-4 and Cε in SKW AhR+ cells but not in SKW AhR- cells, and AhRA significantly increased the expression of Cγ2-4 but not Cε. These results indicate that AhR targets the IgH germline transcription. Lastly, hs1.2-edited CL-01 variants were used to evaluate the impact of hs1.2 genetic variation on IgH expression. The hs1.2 genotype and transcription factor binding sites were evaluated by DNA sequencing. The IgH expression was altered in the hs1.2-edited clones. Taken together, these results suggest that the genetic variations in the AhR and hs1.2 enhancer influences the IgH expression.

Page Count

139

Department or Program

Biomedical Sciences

Year Degree Awarded

2025

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