Date of Award

3-18-2026

Embargo Period

5-1-2028

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Additional Department

Pharmacology

College

College of Graduate Studies

First Advisor

Leonardo Ferreira

Second Advisor

Christina Voelkel Johnson

Third Advisor

Wei Jiang

Fourth Advisor

Nathan Dolloff

Fifth Advisor

Shikhar Mehrotra

Abstract

A specialized CD4+ T cell subset, regulatory T cells (Tregs), maintain immune homeostasis through a diverse set of immunosuppressive mechanisms. Since their discovery in humans in 2001, adoptive transfer of Tregs has emerged as a promising strategy to suppress immune-mediated inflammatory diseases and prevent organ transplantation rejection. Prior reports have demonstrated that antigen-specific Tregs outperform polyclonal Tregs across multiple disease models. However, current approaches to confer antigen specificity, most notably chimeric antigen receptors (CARs), were optimized for conventional T cell (Tconv) signaling. Treg immunosuppressive function requires precise coordination between induction of regulatory gene programs and repression of pro-inflammatory programs. Whether second-generation CARs faithfully recapitulate the finely tuned signaling architecture essential for optimal Treg function remains unresolved. This dissertation tests the hypothesis that CAR signaling significantly alters human Treg functional outputs through differences in cell signaling interpretation compared to endogenous T cell receptor (TCR)/CD28 signaling.

Direct comparisons of endogenous TCR/CD28 stimulation and CAR activation were performed in primary human Tconv and Tregs using transcriptional profiling, flow cytometry, functional immunoassays, and xenogeneic graft-versus-host disease models. Despite preserved expression of Treg lineage-defining transcription factors, such as FOXP3 and HELIOS, CAR-activated Tregs exhibited diminished suppressive capacity relative to TCR/CD28-stimulated Tregs. Moreover, a subset of CAR Tregs displayed de novo induction of pro-inflammatory mediators, including IFN𝛾 and CD40L, without evidence of Treg destabilization.

Inflammatory mediator induction by CAR mediated activation in Tregs was stochastic, rather than a result of a predisposed population. Interestingly, reducing CAR affinity decreased inflammatory mediator production, indicating that inflammatory gene induction in human Tregs is sensitive to antigen receptor affinity. In parallel, transcriptomic and cell signaling interrogation of CAR and TCR/CD28 mediated Treg stimulation revealed higher canonical NFκB activity and upregulation of Immediate Early Response 3 (IER3) upon CAR activation, supporting altered signaling dynamics and feedback regulation. Single-cell RNA sequencing identified reduced expression of the transcription factor BATF within inflammatory CAR Treg subsets. Strikingly, enforced BATF expression in CAR Tregs constrained pro-inflammatory cytokine production and enhanced suppressive capacity in vitro and in vivo.

Collectively, these findings demonstrate that second-generation CAR Treg signaling can stochastically disrupt the balance between inflammatory and regulatory program induction without the loss of Treg lineage identity. By defining mechanisms of CAR-induced inflammatory gain-of-function in Tregs and demonstrating restoration through transcriptional reinforcement, this work provides a mechanistic framework for the design of next-generation CAR Treg therapies that account for Treg intrinsic signaling to achieve durable, balanced immunoregulation in patients.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Monday, May 01, 2028

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