Fig. 6

Characterization of the cellular composition of PB samples (n = 10) of patients after HD-CAR-1 treatment (n = 10) and PB composition of healthy donors (n = 3). A PBMC samples obtained from patients after CART administration were analyzed via high-parametric spectral flow cytometry and data were analyzed (see methods). UMAP visualization (bottom) showing a downsampled subset of PBMCs from ten CART recipients and additionally three healthy donor samples. After clustering, individual clusters were annotated based on surface marker expression and highlighted by different colors. B Boxplots indicating differential abundance of individual cell populations from PBMC samples collected after CART administration, comparing abundances in responders and non-responders. Positive log₂ fold changes indicate that a respective population is more abundant in responders (R), whereas negative log₂ fold changes indicate that the population is more abundant in non-responders (NR). C Scatterplot displays the gating strategy to define CAR + cells. CD8 + and CD4 + T cells from the PBMC samples were extracted, and fluorescence intensity levels of CD8/CD4 expression were plotted against the fluorescence intensity of the CAR targeting antibody. CAR + cells were determined by setting a CD8 + /CD4 + T cell-specific cutoff for downstream analysis and visualization. D UMAP visualizations of downsampled subsets from separately clustered CD8 + and CD4 + T cells identified in A. Dimensionality reduction and clustering were performed excluding the expression information of the CAR targeting antibody, to prevent CAR + specific clusters. After clustering, individual clusters were annotated based on surface marker expression and highlighted by different colors. E Density plots illustrating the distribution of CAR + cells within the CD8 + T cell (top) and CD4 + T cell (bottom) UMAP embedding. CAR + cells were identified and gated as displayed in C and as described in the material and methods section. F CD4 + and CD8 + T cells from D were used and binned into CAR- and CAR + CD8 + or CD4 + T cells, respectively, as described above (Fig. 5C). Boxplots display differential abundance of different CAR + CD8 + T cells (top) or CAR + CD4 + T cell phenotypes (bottom) of responders and non-responders. Positive log₂ fold changes indicate that a respective population is more abundant in samples of responders, whereas negative log₂ fold changes indicate that the population is more abundant in samples of non-responders. R responders, NR non-responders, CM central memory T cells, cDC conventional dendritic cells, EM effector memory T cells, hi high, TCR T cell receptor, NK natural killer, NKT natural killer T cells, pDC plasmacytoid dendritic cells, SCM memory stem cell-like T cells