1. 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 , Langen, Germany
Short title: Anti-tumoral activity of in vivo CARTs
Keywords: cell therapy, CAR T cells, in vivo gene delivery, receptor targeting, T-cell targeting

2. 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.

3. 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.

4. 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.