Competitive benefits to TCR-stimulated TCL1A-expressing Tcells. (A) The experimental set-up for panels A-E is illustrated above the x-axis. Earlier and more pronounced outgrowth of TCR-stimulated T cells in the presence of TCL1A. Blood was taken every 4 weeks from unstimulated (green, w/o stim) and repeatedly stimulated (red, OVA stim) recipients, of either TCL1^neg/GFP⁺ or TCL1A⁺/GFP⁺OT-1 cells. Mean percentages (with SEM) of GFP⁺ cells (flow cytometry, CD3⁺ gating) were compared between the cohorts throughout the observation period (5 mice per group). (B) Unstimulated and stimulated recipient mice of TCL1A-Luc or T-Sapphire-Luc (control) transduced OT-1 cells were imaged 12 weeks after the first OVA/vehicle injection. Pseudocolor images were adjusted to the same threshold. (C) Quantified bioluminescence corroborates the data of panels A and B. Signal intensities (average radiance [photons/s/cm²/sr]) are shown as relative values (untreated controls set to 100). (D) Accelerated T-cell tumor induction and shorter OS upon provision of OVA peptide (vs unstimulated) in mice transplanted with TCL1⁺/GFP⁺OT-1 cells (log-rank test). (E) T-cell tumors (5 animals per cohort) induced by TCL1A-transduced OT-1 cells showed medium-sized lymphoid cells with a scant basophilic cytoplasm (spleen and PB) and a memory-T immunophenotype. (F) System of CARs as TCR surrogates: splenocytes from CAR^CEA, Lck^pr-hTCL1A^tg, and CAR^CEAxLck^pr-hTCL1A^tg mice were transplanted into lympho-depleted CEA^tg mice. (G) Blood samples from CEA^tg recipients of CAR^CEA (blue), Lck^pr-hTCL1A^tg (green), or CAR^CEAxLck^pr-hTCL1A^tg (red) cells were taken every 2 to 4 weeks and were analyzed by flow cytometry for repopulation of GFP⁺ (CAR) or TCL1A⁺ cells (CD3⁺ gated). Statistical significances for recipients of Lck^pr-hTCL1A^tg and CAR^CEAxLck^pr-hTCL1A^tg cells (descriptive, nonparameteric 95% confidence bands computed on the basis of a normal distribution assumption with smoothed conditional bounds on the basis of local polynomial regression fitting). Insets: activation (CD69) and proliferation (Ki-67) was enhanced in T cells of CAR^CEAxLck^pr-hTCL1A^tg, particularly over CAR^CEA mice (example at 42 weeks post-transplantation; unpaired Student t test, SEM). (H) Quantification of splenic CD8⁺ T-cell content by IHC on fresh-frozen tissues. In each section 3 representative areas were used for signal quantification (QuPath software; percentage of positive cells; unpaired Student t test with SEM. (I) Immunofluorescence on spleen sections (representative images, 3 mice/cohort) at 621 days post transplantation. Quantified cytoplasmic pCD3ζ signal intensities (3 areas per section; unpaired Student t test, SEM) implicate additive contributions by CAR and genuine TCR signals (single fluorescence channels in supplemental Figure 9E). Similar surface expression of CD3 in all 3 lines (MFIs, bottom right).