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1 supplemental Figure 1 A T cell T1 anti-ny-eso /hla-a*:1 CDζ CH/CH scfv B T cell T1 anti-ny-eso /hla-a*:1 CDζ CH/CH scfv C T cell BW1/6 anti-cea CDζ CH/CH scfv
2 supplemental Figure TEFF TEFF CDRA higg CDRA CD7 CCR7 CD late TEM early TEM TCM
3 supplemental Figure A B anti-ny-eso-1-t1-/cdζ anti-ny-eso-1-t1-cdζ CCR7+CD-CD7+-+ CCR7+CD-CD7+-- CCR7+CD-CD7+++ CCR7+CD-CD7++- CCR7+CD-CD7--+ CCR7+CD-CD7--- CCR7+CD-CD7-++ CCR7+CD-CD7-+- CCR7+CD+CD7+-+ CCR7+CD+CD7+-- CCR7+CD+CD7+++ CCR7+CD+CD7++- CCR7+CD+CD7--+ CCR7+CD+CD7--- T CM CCR7+CD+CD7-++ CCR7+CD+CD7-+- CCR7-CD-CD7+-+ CCR7+CD-CD7+-+ CCR7+CD-CD7+-- CCR7+CD-CD7+++ CCR7+CD-CD7++- CCR7+CD-CD7--+ CCR7+CD-CD7--- T CM CCR7+CD-CD7-++ CCR7+CD-CD7-+- CCR7+CD+CD7+-+ CCR7+CD+CD7+-- CCR7+CD+CD7+++ CCR7+CD+CD7++- CCR7+CD+CD7--+ CCR7+CD+CD7--- T CMRA CCR7+CD+CD7-++ CCR7+CD+CD7-+- CCR7-CD-CD7+-+ T RVE CCR7-CD-CD7+-- CCR7-CD-CD7+++ T RVE CCR7-CD-CD7++- CCR7-CD-CD7--+ CCR7-CD-CD7--- early T EM CCR7-CD-CD7-++ late T EM CCR7-CD-CD7-+- CCR7-CD+CD7+-+ T eff /T RVE CCR7-CD+CD7+-- CCR7-CD+CD7+++ T eff /T RVE CCR7-CD+CD7++- CCR7-CD+CD7--+ CCR7-CD+CD7--- T EMRA CCR7-CD+CD7-++ T EMRA CCR7-CD+CD7-+- T CMRA CCR7-CD-CD7+-- CCR7-CD-CD7+++ T RVE CCR7-CD-CD7-+- CCR7-CD+CD7+-+ CCR7-CD-CD7++- CCR7-CD-CD7--+ CCR7-CD-CD7--- T RVE CCR7-CD-CD7-++ early T EM late T EM CCR7-CD+CD7+-- CCR7-CD+CD7+++ T eff /T RVE CCR7-CD+CD7-++ CCR7-CD+CD7-+- CCR7-CD+CD7++- CCR7-CD+CD7--+ CCR7-CD+CD7--- T eff /T RVE T EMRA T EMRA % of non-transduced CD8+ T cells % of transduced CD8+ T cells
4 ****** ** supplemental Figure T CM CCR7+CD-CD7-++ T CMRA CCR7+CD+CD7-++ T RVE T RVE T eff /T RVE CCR7-CD+CD7++- CCR7-CD+CD7-++ early T EM CCR7-CD-CD7-++ late T EM CCR7-CD-CD7-+- T eff /T RVE CCR7-CD+CD7+-- T EMRA CCR7-CD+CD7-+- T EMRA CCR7-CD-CD7++- CCR7-CD-CD7+-- *** *** 1 MFI of CD8+ anti-ny-eso-1-t1-/cdζ
5 supplemental Figure A 8 T-1B LST-17 B 6 % lysis 6 IFNγ (pg/ml).:1.:1 1:1 :1 T-1B LS-17T ratio (anti-cea-bw-/cdζ : target) re-directed anti-cea-bw-/cdζ : target (:1)
6 supplemental Figure 6 A B x 6 6 x 6 6x human IgE (ng/ml) days after intravenious injection of U66 C D counts human IgG HLA class I 6. CCR
7 Supplementary Figures Figure S1: Schematic presentation of re-directed T cells including the different CARs: A: Re-directed T cells recognizing NY-ESO peptide without co-stimulatory domain (anti-ny-eso-1-t1-cdζ); B: Re-directed T cells recognizing NY-ESO peptide with co-stimulatory moiety (anti-ny-eso-1-t1-/cdζ); C: Re-directed T cells recognizing CEA with co-stimulatory domain (anti-cea-bw-/cdζ ). Figure S: Representative phenotypical characterization of CD8 + T cells expressing the anti-ny- ESO-1-T1-/CDζ CAR by multi-parametric flow cytometry. Pre-gated CCR7 + or CCR7 - / anti-ny-eso-1-t1-/cdζ re-directed T cells were arrayed in a CDRA vs. CD7 - parameter dot-plot. Cells in each of the four quadrants were then further subdivided by gating through / dot-plots. Figure S: Comparison of non-transduced (A) and transduced (B) subpopulations of anti-ny- ESO-1- CD8 + T cells. No statistically significant differences were detected between the subpopulations of CAR - and CAR + T cells (11 individual donors). T CM = central memory T cell, T EM = effector memory T cells, T EFF = effector T cells, T EMRA = effector memory T cell reexpressing CDRA, T eff = effector T cells. Figure S: Comparison of the anti-ny-eso-1-t1-/cdζ expression in different subpopulations of re-directed T cells. There was no statistically significant difference in the anti- NY-ESO-1-T1-/CDζ expression between central- and effector memory re-directed CD8+ T cells (11 individual donors). Figure S: Representative experiment showing antigen-specific cytolysis and IFN release of anti-cea-bw-/cdζ re-directed CD8 + T cells. Anti-CEA-BW-/CDζ re-directed T cells were cultured with stable-transfected T cells expressing HLA-A*:1 restricted NY- ESO-1 peptides (T-1B) and the tumor cell line LS-17T which expresses CEA at different effector to target ratios. Figure S6: Xenograft model of intravenously injected U66 HLA-A*:1/NY-ESO-1 positive myeloma cells ( =* 7 cells, = 7 cells =6* 6 cells). After injection of different doses of U66 cells sera concentrations of secreted human IgE were measured by ELISA (A). 6 days after injection U66 cells accumulated in the bone marrow (B). HLA class I positive cells were analysed by flow cytometry indicating the presence of U66 cells in the bone marrow (C). Anti-
8 NY-ESO-1-T1-/CDζ re-directed CD8 + T cells were found up to days after transfer in the peripheral blood of % of mice (representative dot plots of four individual mice, D).
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Supplementary Figure 1
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Appendix Figure S1 A B C D E F G H
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x Lymphocyte count /µl CD8+ count/µl 800 Calculated
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Supporting Information
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