Figure 3 from Nanobody-Engineered CLL-1 CAR T Cells: Optimizing Tumor-Specific Cytotoxicity and Minimizing Off-Tumor Toxicity

Primary human CAR T cells maintain viability, expansion capacity, and phenotype after transduction. A and B, Flow cytometry analysis confirming the surface expression of CLL-1 and CD33 CARs in human T cells. CAR expression was detected using Alexa Fluor 647 (AF647)–conjugated CD33 and PE-conjugated CLL-1 ECD proteins, demonstrating successful transduction. C, The proportion of CD4+ and CD8+ T cells was assessed after transduction to evaluate potential shifts in the T-cell subset composition. D, CAR T cells were evaluated for tonic signaling by detecting CD137 (4-1BB) expression in CAR+ cells compared with CD134 expression in all T cells for the untransduced (Unt.) control. No significant spontaneous activation was observed, indicating an absence of constitutive signaling. E and F, CAR T-cell expansion and viability were assessed after transduction using an automated cell counter, comparing CAR T cells to Unt. T cells. No significant differences in expansion rates or viability were observed, suggesting that CAR expression did not impair T-cell growth or survival. G, Memory phenotype analysis was performed to characterize CAR T cells after transduction, comparing them with Unt. T cells to determine whether the transduction process influenced differentiation into stem cell–like memory (TSCM), central memory (TCM), effector memory (TEM), or terminally differentiated effector T cells (TEMRA). TSCM were identified as CD95+CD45RA+CD45RO−CCR7+ cells, TCM as CD45RA−CD45RO+CCR7+ cells, TEM as CD45RA−CD45RO+CCR7− cells, and TEMRA as CD45RA+CD45RO−CCR7− cells. Data were generated from three independent experiments. A two-way ANOVA was performed to determine the statistical significance between groups based on two or more independent variables. The Tukey multiple comparisons test was used. ****, P < 0.0001; ns = not significant.