Fig. 3: Moderate versus strong activation patterns are transferable to CD8 neoTCR-tg T cells from healthy donors during in vitro co-cultures with different cell lines.

Color code for neoTCRs in a–f indicated next to h: SYT-T1 red, KIF-sc1 light green, KIF-sc2 dark green, KIF-P2 blue. a–d Mel15 LCL were pulsed with titrated peptide concentrations (2 h, 37 °C) and co-incubated with TCR-tg T cells with subsequent ELISA-based assessment of IFN-γ-secretion within 24 h of co-culture (a). The cellular activation level was determined after 24 h by FACS staining of the extracellular level of CD137 (b), PD-1 (c) and LAG-3 (d) expression (reflected by geometric mean of all CD3⁺CD8⁺/TCRmu⁺ cells). Wildtype control depicts only the highest peptide concentration (100 µM wt peptide). The mean for ELISA data is depicted for technical triplicates of one representative of four donors; triplicates from the same donor have been pooled prior to EC FACS-staining. E:T = 1:1 (15,000 tg T cells:15,000 tumor cells). EC FACS staining at different time points after co-culture setup displays temporal dynamics of T cell activation marker CD137 (e, f) and inhibitory receptor LAG-3 (g, h) for TCR-tg T cells upon co-culture with JJN3-B27 peptide-pulsed target cells. A weak (0.01 µM for peptide pulsing; e, g) versus a strong (1 µM for peptide pulsing; f, h) stimulus were compared. E:T = 1:1 (10,000 tg T cells:10,000 tumor cells). gMFI-values of all TCRmu⁺ cells are shown. i Annexin-V/PI-staining was employed for detection of activation induced cell death (AICD) after 20 h of co-culture upon strong stimulation with 1 µM mut-peptide pulsed Mel15 LCL (early apoptotic = AnnexinV⁺PI⁻, late apoptotic = AnnexinV⁺PI⁺). E:T = 1:1 (30,000 tg T cells:30,000 tumor cells). j Representative FACS plot of a healthy donor of CTV-analysis for all TCRmu⁺ cells depicted after 4 days of co-culture with 1 µM mut-peptide pulsed Mel15 LCL (colors were chosen according to (b–e); representative wt mg-control depicted in gray). E:T = 1:1 (30,000 tg T cells:30,000 tumor cells). For all co-cultures, TCRmu⁺ rates were adjusted by addition of non-transduced T cells to equalize TCRmu⁺ cell frequencies for all neoTCRs. For all co-cultures in e–j technical triplicates per donor were pooled prior to staining; mean and SD for biological replicates from three different human donors are shown.