Supplementary Figures In vivo generated human CAR T cells eradicate tumor cells   Shiwani Agarwal1, Tatjana Weidner1, Frederic B. Thalheimer1, Christian J. Buchholz1,2 1Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany 2Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany Correspondence Christian Buchholz, Paul-Ehrlich-Institut, Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany Email: christian.buchholz@pei.de Short title: Anti-tumoral activity of in vivo CARTs Keywords: cell therapy, CAR T cells, in vivo gene delivery, receptor targeting, T-cell targeting

Figure S1: Gating Strategy CD8 CD3 CD45 CD45RA FSC-A Live/Dead SSC-A SSC-W FSC-H CD19 CAR CD62L PBS CD8-LV Nalm-6 Figure S1: Gating Strategy Human cells were identified as living single lymphocyte population that expresses human CD45. Cells within the CD45+ gate were further gated for CD3 and CD19 populations. Cells within the CD3+ gate were gated for human CD8 and CAR expression via its myc tag. Further, from the CAR or CD8 gate the phenotype of the cells was characterized based on the expression of CD45RA and CD62L. Nalm-6-luc tumor cells were identified as CD19+ and CD45+ low population.

CD8 CD3 CD45 SSC-A CD56 CAR Figure S2: Gating Strategy for the identification of CAR+ NK and NKT cells Human cells were identified as living single lymphocyte population that expresses human CD45. Cells within the CD45+ gate were further gated for CD3+ and CD3- populations. Cells within the CD3+ gate were gated for human CD8 and CD56 expression. Further, from the CD8+CD56+ (NKT cells) and CD8+CD56- (CD8+ T cells) gates cells were gated for CAR expression. To identify CAR+ NK cells, the CD3- population was gated for CD56, CD8 and CAR in a hierarchical manner.

D E F A B C Figure S3: CAR expression on NK and NKT cells in spleen * D E F A PBS CD8-LV 0.01 CAR 1.21 0.0 2.10 5.18 B C CD3+CD56-CD8 T cells CD3+CD56+CD8 NKT cells CD3-CD56+CD8 NK cells CD8 Figure S3: CAR expression on NK and NKT cells in spleen Cells isolated from the spleen of PBS (grey symbols) or CD8-LV (black symbols) injected animals were evaluated by flow cytometry. Data depict frequencies of CAR+ cells within T cells (A), NKT cells (B), NK cells (C) and their percentage of total CD8 cells (D-F). A-C) Exemplary density plots showing different populations in the splenic cells of two representative mice at day 14. The gating strategy is represented in Fig S2. D-F) Frequencies of individual populations are calculated as count of individual cell populations normalized to total CD8 cell count. Percentages of D) CD3+CD56- T cells and T cell-derived CAR+ cells, E) CD3+CD56+ (NKT cells) and NKT-derived CAR+ cells as well as F) CD3-CD56+ (NK cells) and NK-derived CAR+ cells at 14 and 18 days post vector administration are shown. Donor 1 and Donor 2 are represented as open and filled circles, respectively. Data represent mean ± SEM for all groups. Statistical significance was determined using multiple t-test.