Martino, Giovanna (2025) Synthetic Biology meets Immunology: Synthetic Promoters to Tune and Control the Functionality in T cell Exhaustion. [Tesi di dottorato]

Documento PDF 38_CQB_MARTINO_GIOVANNA_.pdf Visibile a [TBR] Amministratori dell'archivio Download (4MB)

Available Versions of this Item

• Methodologies and Techniques for Semantic Management of Documents in Dematerialization Processes. (deposited 24 Mag 2010 08:31)
  • Synthetic Biology meets Immunology: Synthetic Promoters to Tune and Control the Functionality in T cell Exhaustion. (deposited 07 Apr 2026 06:33) [Attualmente visualizzato]

Abstract

T cell exhaustion represents a major barrier to the success of cancer immunotherapies, particularly in solid tumors where persistent antigen exposure, hypoxia and immunosuppressive signals compromise effector function and cytokine secretion. Rather than treating exhaustion solely as a failure mode, this work explores the exhaustion-associated transcription factor upregulation as a controllable input to drive context-dependent therapeutic responses in engineered lymphocytes. We designed a modular library of synthetic promoters (SPs) composed of distinct transcription factor binding site (TF-BS) combinations targeting multiple TFs known to be upregulated during T cell dysfunction (including IRF4/BATF, MAF, NR4A2 and EOMES). The library was first functionally screened in HEK293 and Jurkat cell lines to evaluate inducibility, leakiness and dynamic range across TF inputs and minimal promoter backbones. SPs that displayed robust NR4A2 responsiveness in these screens (notably SP73 and SP78) were selected for detailed characterization in primary human T cells. Using an in vitro exhaustion model based on repeated CD3/CD28 stimulation and antigen-driven co-culture systems, we show that NR4A2-responsive SPs faithfully recapitulate endogenous NR4A2 dynamics: SP73 and SP78 become activated during chronic stimulation, with distinct kinetic profiles and tunable amplitudes determined by TF-BS composition, and they revert toward baseline upon stimulus withdrawal. Real-time imaging (IncuCyte®/xCELLigence) and flow cytometry analysis in dysfunctional contexts confirmed that promoter activity correlates with increasing NR4A2 expression and with classical exhaustion phenotypes (PD-1, TIM-3, TIGIT, LAG�3, CD39), while effector functions (IFN-γ, TNF-α, IL-2, CD107a, Granzyme B) decline. SP73 retained sensitivity and reversibility when deployed in CAR-T cells co-cultured with HER2⁺ HCC1954 or NY-ESO-1⁺ SK-Mel-23 tumor targets. Preliminary in vivo experiments in NSG NOG-IL15 mice engrafted with NY-ESO-1⁺ melanoma and adoptively transferred with SP73-transduced human T cells demonstrate detectable mCherry reporter activity in tumor-infiltrating and splenic lymphocytes, consistent with physiological T cell trafficking and activation. Overall, these results indicate that 6 the promoter remains responsive to T-cell activation and exhaustion both in the tumor microenvironment and in lymphoid tissues. Collectively, these data establish that synthetic promoters can be engineered to sense exhaustion-associated TF inputs with high specificity, tunability and reversibility. By coupling therapeutic transgene expression to intrinsic cellular states, such promoter circuits offer a promising route to adaptive, self-regulating cell therapies that restrict effector programs to precise physiological windows—enhancing both safety and efficacy in the treatment of solid tumors. This PhD project suggests how combining synthetic biology and immunology can help to turn a biological limitation, T-cell exhaustion, into a controllable design feature. By creating synthetic promoters that sense transcription factors such as NR4A2 activity, we can develop self-regulating therapeutic cells that adapt dynamically to their functional state through actuators or payloads. This approach opens exciting possibilities for more precise and robust cell therapies in the future.

Downloads

Actions (login required)

Modifica documento