CAR cellular immune reactivity is directed against V_H-CDR and V_K-FR epitopes in the CAIX-CAR protein. CAR protein spanning peptides, consisting of 84 11 aa overlapping 15-mer peptides, were generated to assess epitope specificity of the anti–CAIX-CAR cellular immunity. Responder cells were day 50 PBMCs of patient 4, and cocultivated for 5 weekly cycles with CAR T cells. (A) Design of the matrix peptide pools with numbers indicated in left column and top row. Individual 15-mer peptides in these pools correspond to the numbers in the respective columns and rows. (B) PBMCs were incubated in medium (negative control, left plot) or stimulated with CAIX-CAR⁺ T cells (middle plot) or the complete protein spanning peptide pool (84 15-mer peptides; right plot) as 2 positive stimulation controls and (C) 19 matrix pools, each containing 8-10 15-mer peptides and subsequently with the individual candidate peptides (D). (A) The gray shading in the matrix illustrates how candidate peptides were identified. Stimulated PBMC were assayed for CD137 expression by flow cytometry. Plots in panels B-D show CD137 versus CD8 expression within the CD3 T-cell population. Numbers in the top right quadrant of each dotplot indicate the percentage of cells that express CD137 in response to stimulation. Controls were stimulated by medium alone or by autologous CAR T cells (B).