Author ORCID Identifier

0000-0001-7772-2456

Date of Graduation

8-2025

Document Type

Dissertation (PhD)

Program Affiliation

Therapeutics and Pharmacology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Greg Lizee, PhD

Committee Member

Alexandre Reuben, PhD

Committee Member

Katy Rezvani, MD, PhD

Committee Member

Michael Davies, MD, PhD

Committee Member

William Plunkett, PhD

Committee Member

Mark Bedford, PhD

Abstract

CD40 ligand (CD40L) is a transmembrane protein expressed on activated CD4 T cells, traditionally known for its role in forward signaling by engaging CD40 on antigen- presenting cells to promote immune activation. While this pathway is well characterized, there is evidence that CD40L also mediates reverse signaling, acting as a signaling receptor on T cells which enhances T cell function. However, the extent, consequences, and mechanism of CD40L-mediated reverse signaling in CD4 T cells remain poorly defined. This dissertation investigates the mechanisms and effects of CD40L reverse signaling on T cell function using both primary human T cells and the Jurkat T lymphoblastoid cell line. Crosslinking CD40L with monoclonal antibody 5c8 induced upregulation of activation markers CD69 and CD25, ERK1/2 phosphorylation, and secretion of key cytokines including IFN-a, IL-2, and TNF-a in primary CD4 T cells. In contrast, reverse signaling in Jurkat cells elicited phosphorylation of pERK1/2 but little to no cytokine production, limiting their usefulness as a model for studying downstream consequences of reverse signaling. A synthetic SH2-domain binding array identified Abl2, CRK, and CRKL as candidates that interact with the highly conserved phosphorylated tyrosine residue at position 5 (Y5) in the CD40L cytoplasmic domain. To assess structural requirements, CD40L constructs with either a cytoplasmic domain deletion or a Y5F point mutation were evaluated. Surprisingly, neither mutation impaired ERK1/2 vi phosphorylation, suggesting that this domain might not be essential for initiating some intracellular signaling events. Moreover, neither mutation affected the ability of costimulation to mobilize intracellular CD40L to the cell surface. However, the deletion of the cytoplasmic domain reduced overall protein expression, indicating a potential role for the cytoplasmic domain in CD40L stability or translation efficiency. Together, these findings contribute to the growing body of knowledge that CD40L can function as a signaling receptor in CD4 T cells, triggering intracellular pathways that influence activation and cytokine production. These studies also suggest that the cytoplasmic domain is dispensable both for mobilization of CD40L to the cell surface, and for induced phosphorylation of ERK1/2.

Keywords

CD40L, CD154, CD4 T cell, Cytokine, T cell function

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