Journal: Nature Communications

Article Title: A modular synthetic promoter toolkit for programmable gene control in activated and chronically stimulated T cells

doi: 10.1038/s41467-026-77730-7

Figure Lengend Snippet: A Experimental design. Primary human CD8⁺ T cells were co-engineered with a lentiviral construct encoding the NR4A2-responsive synthetic promoter SP73 driving mCherry and constitutive EGFP to identify transduced cells, together with a retroviral NY-ESO-1–specific TCR. Engineered T cells were expanded and co-cultured with NY-ESO-1–expressing SK-MEL23 melanoma cells. B Quantification of mCherry⁺ T cells over time during co-culture. SP73-engineered T cells show progressive reporter induction, whereas control cells transduced with the control promoter SP01 (lacking NR4A2 binding sites) remain at baseline levels. Data represent mean ± SEM of n = 3 independent donors. C Representative IncuCyte® live-cell images showing SP73-driven mCherry fluorescence during co-culture with SK-MEL23 melanoma cells; SP01 controls show negligible signal. D Expression of NR4A2 and activation/dysfunction-associated markers in engineered T cells during co-culture. Data represent mean ± SEM of n = 3 independent biological donors. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. P values: CD39, P < 0.0001 for R0vsR2 and R0vsR4, P = 0.0048 for R2vsR4. NR4A2, P = 0.0375 for R0vsR2. LAG-3, P = 0.0194 for R0vsR2, P = 0.0097 for R0vsR4. TIM-3, P = 0.008 for R0vsR2, P = 0.0433 for R2vsR4. TIGIT, P = 0.0082 for R0vsR4, P = 0.0433 for R2vsR4. E Functional assessment of engineered T cells prior co-culture (R0) and following persistent antigen exposure (R4). Left: Quantification of IL-2⁺, TNFα⁺, CD107a⁺, and IFNγ⁺ cells following Leukocyte Activation Cocktail (LAC) stimulation. Right: Representative flow cytometry histograms illustrating shifts in cytokine-producing populations. Data represent mean ± SEM of n = 3 independent biological donors. Statistical analysis was performed using a two-tailed paired t test comparing R0 vs R4. P values: IL-2, P = 0.0074; TNFα, P = 0.0068; CD107a, P = 0.0401; IFNγ, P = 0.0147. Source data for this figure is available in the file. Created in BioRender.

Article Snippet: The sequence of IL12p70 was obtained from pLN540 (FuGW-G8p-IL12-P2A-CCL21-1Pe) (Addgene #105201 ).

Techniques: Construct, Retroviral, Cell Culture, Expressing, Co-culture Assay, Control, Transduction, Binding Assay, Fluorescence, Activation Assay, Analysis, Functional Assay, Flow Cytometry, Two Tailed Test