Fig. 2: CAR-T cells derived from healthy and patient samples show differential effector functions following in vitro antigen exposure in a stimulation-dependent manner.

a PCA of cytokine expression in CAR-T cell products following 1.25:1 (effector: target) stimulation with Raji cells, colored by disease state. Arrows represent PCA loadings and magnitude of contribution to PC1 and PC2. b Frequency of IL-2-expressing CD8⁺ CAR-T cells generated from healthy and patient samples across varying stimulation doses following 1.25:1 (effector: target) stimulation with Raji cells. In vitro cytotoxicity of CAR-T cell products generated across a range of APC-ms (c) or Dynabead (d) stimulation following co-culture with Raji-luc cells. Left: healthy donor CAR-T, right: patient-derived CAR-T. e Schematic representation of workflow linking CAR-T cell cytotoxicity and phenotype. Single cell fluorescence data from healthy donors or patient-derived samples (a patient sample is shown) were aggregated and clustered using K-means clustering with progressively increasing k to maximize the clustering explained variance, then visualized in a heatmap. Cytotoxicity was assigned to each single cell within each CAR-T cell product, normalized and thresholded at mean ± 1 s.d. to represent low, moderate, and high cytotoxicity, as well as the average expression of CAR-T cell phenotypic markers associated with these levels. The % cytotoxicity was taken at the 1.25:1 effector: target (E:T) ratio. f Heatmaps of CD8⁺ CAR-T cell markers associated with either high, moderate, or low levels of cytotoxicity in healthy and patient-derived CAR-T cells. g Fraction of cells classified as high, moderate, or low cytotoxicity mapped to APC-ms stimulation dose. Data represent median ± interquartile range. Data in b–d, g represent CAR-T cell products derived from n = 5 healthy and n = 5 patient samples. For the D3:1 group, n = 4 patient samples were assessed. Comparisons in b were calculated using either two-sided Mann–Whitney or Wilcoxon signed-rank tests for comparisons between healthy vs patient CAR-T or within either healthy or patient CAR-T, respectively. Comparisons in c, d were calculated using two-sided Wilcoxon signed-rank tests.