Fig. 5
Characterization of the cellular composition of CART products of HD-CAR-1 patients (n = 10). A CART infusion products were analyzed via high-parametric spectral flow cytometry, and data were analyzed (see methods). Uniform manifold approximation and projection (UMAP) visualization display a downsampled subset of cells from all ten CART products (bottom). After clustering, individual clusters were annotated based on surface marker expression [66] and highlighted by different colors. B CD8 + and CD4 + T cell subsets from the CART product of ten patient samples were extracted and clustered separately. A representative subset of cells from all ten CART products is displayed in the UMAP visualizations. Density plots in the two lower panels indicate the differential distribution of cells between non-responders (NR) and responders (R) within the CD8 + and CD4 + T cell compartment, respectively. C Boxplots indicating differential abundance of individual clusters from CD8 + (left) and CD4 + T cell (right) subsets from the CART product of responders and non-responders. Positive log2 fold changes indicate higher levels in responders, whereas negative log2 fold changes indicate that a specific population is more abundant in non-responders. D Principal component analysis (PCA) of CD8 + T cells within the CART product. Cell-type frequencies of cell clusters from each sample were used as input for the PCA. Blue circles represent samples from responders, and green circles represent samples from non-responders. The two larger circles indicate the midpoint of the respective group. Gray arrows indicate the variables. E Boxplots indicating the abundance of CD39- effector memory (EM)-like and CD39 + EM-like cells within the CD8 + T cell population of the CART products (left). A generalized linear mixed model (GLMM) was used to compute significance between non-responders and responders. Adjusted p values are shown. Boxplot of CD39 expression levels in non-responders and responders within the CD8 + T cell subset of the CART product is displayed (right). Significance was assessed by applying a linear mixed model (LMM). F PCA of CD4 + T cells within the CART product. Cell-type frequencies of cell clusters from each sample were used as input for the PCA. Blue circles represent samples from responders, and green circles represent samples from non-responders. The two larger circles indicate the midpoint of the respective group. Gray arrows indicate the variables. G Boxplots showing the abundance of CD39- EM-like and CD39 + EM-like cells within the CD4 + T cell population of the CART product (left). A GLMM was used to compute significance between non-responder and responder. Adjusted p values are shown. Boxplot of CD39 expression levels in non-responder and responder samples within the CD4 + T cell subset of the CAR product (right). Significance was assessed by applying a LMM. R responders, NR non-responders, CM central memory T cells, cDC conventional dendritic cells, EM effector memory T cells, NK natural killer