Luca Gattinoni

Synthetic circuits that couple transgene expression to endogenous transcription factor (TF) activity enable cellular response to changing functional states. In T cells, antigen stimulation results in dynamic changes of transcription factor expression programs. Harnessing these endogenous TF dynamics for programmable gene control remains an important challenge in engineered T cells. Here, we develop a library of synthetic promoters (SPs) responsive to seven TFs identified through transcriptomic analysis of T-cell activation programs. These promoters couple endogenous TF activity to transcriptional output across a broad dynamic range of inducibility and basal expression. We quantitatively profile promoter performance in HEK293 and Jurkat cells, evaluate them for orthogonality, and integrate them into Boolean logic gate architectures and network topologies to minimize basal transcriptional leakiness and improve ON/OFF ratios. We test selected synthetic promoters in primary human CD8⁺ T cells, under endogenous TF regulation, and we further characterize the NR4A2-responsive promoter subset in vitro and in tumor-infiltrating engineered T cells in a melanoma xenograft model, demonstrating dynamic and reversible activation.Together, these results provide a modular SP toolkit of TF-responsive synthetic promoters for programmable transcriptional control in engineered T cells.

Immunoblot analysis of β-catenin in TWS119-treated or DMSO control T cells, NFAT activity assessed by eGFP fluorescence in non-activated or activated TPR cells treated with TWS119 or DMSO control, relative proteasome activity of T cells treated with 20SPA1 or DMSO control, relative protein aggregates accumulation in T cells treated with 20SPA1 or DMSO control, and relative expression of exhaustion markers in 20SPA1-treated or DMSO control T cells.

Fluorochrome-conjugated antibodies and fluorescent probes used in flow cytometry, primers used for mRNA quantification by quantitative PCR, antibodies used for immunoblotting, primers used for mouse genotyping, and differentially expressed oxidized proteins in T cells treated with TWS119 or DMSO control.

Maximal respiration, spare respiratory capacity, and ATP-production coupled respiration of T cells across time points and oxygen conditions of the exhaustion protocol, glucose dependence and FAO and AAO capacity in T cells, compensatory glycolysis of T cells, and MitoTracker Deep Red to Green ratio in chronically stimulated T cells under normoxic or hypoxic conditions.

Experimental workflow for sorting and treating distinct T cell phenotypes with TWS119 or DMSO.

Relative expression of exhaustion and activation markers on T cells across stimulations of the exhaustion protocol under normoxic or hypoxic conditions, gating strategy for T cell phenotype annotation, and gating strategy for the analysis of CD39+ and TCF7+ T cells.

Relative RNA expression of CCL3 and CRTAM across stimulations of the exhaustion protocol under hypoxic conditions, dysregulated GSEA pathways related to cell cycle progression and apoptosis between singly-stimulated and continuously stimulated T cells under hypoxia, and DEGs in chronically stimulated T cells under low glucose levels in hypoxic versus normoxic conditions.

Contribution of each study and cancer type to the total pool of CD8+ T cells analyzed in the single-cell dataset.

Normalized RNA expression levels of hypoxia markers and proteasome-related genes in T cells exposed to acute hypoxia or normoxic conditions, proteasome activity in non-activated, singly-stimulated or continuously stimulated T cells under hypoxia, expression of proteasome subunit-encoding genes in murine CD8+ TILs from B16-F10 bearing mice with different expression levels of PD-1 and TIM-3 exhaustion markers, and relative RNA expression of proteasome-specific chaperones in murine TILs classified according to the expression levels of PD-1 and TIM-3 exhaustion markers.

Mean percentage of positive cells for each panel and marker.