Supplementary Fig. 1. Nature Medicine: doi: /nm z Donor # BB-z. 28- IL2RB-z (YXXQ) 28-IL2RBz (YXXQ) Control MFI: 1866 MFI:

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1 Supplementary Fig. 1 a Donor # 28-IL2RB-z 28- IL2RB-z Control MFI: MFI: MFI: MFI: MFI: MFI: Protein L / streptavidin-pe 5 6 NGFR (Pacific Blue) P=.9 * 24 P=.8 18 * 12 6 Control 28-ΔIL2RBz 28-IL2RBz Mean fluorescence inteity of CAR expression b Supplementary Fig. 1. Surface expression of CAR cotructs. (a, b) Peripheral blood CD3+ T cells were individually traduced with a CAR gene linked to the truncated nerve growth factor receptor ( NGFR) or control NGFR and stained with biotin-labeled protein L and streptavidin-pe to analyze the surface expression of the CARs. FACS plots obtained from six different donor-derived T cells (a) and the mean fluorescence inteity (MFI) in the CD8+ NGFR+ T cell population (b) are shown (n=6 different donor samples). Horizontal lines indicate mean values. The MFI among different CAR cotructs was analyzed with repeated measures one-way ANOVA with Tukey s multiple compariso test; F=11.5; degree of freedom=23). * P<.1, not significant.

2 Supplementary Fig. 2 a 28-IL2RB-z z Control MFI: CD69 (PE) b MFI: perk (Alexa Fluor 647) MFI: MFI: MFI: IL2RBz MFI: MFI: MFI: MFI: c Mean fluorescence inteity of perk Mean fluorescence inteity of pakt P=.5 P=.45 n.s. pakt (Alexa Fluor 647) NALM-6 K IL2RBz 28- IL2RBz 28-IL2RBz 28- IL2RBz NALM-6 K562 Supplementary Fig. 2. Comparison of CAR signaling upon antigen recognition. (a) Peripheral blood CD3 + T cells were individually traduced with a CAR gene linked to the truncated nerve growth factor receptor ( NGFR) or control NGFR. CD69 expression in the CAR-traduced T cells was analyzed 24 hours after stimulation with the CD19 + cell line NALM-6. Representative FACS plots of three experiments are shown. (b, c) T cells traduced with the indicated CAR genes were stimulated with NALM-6 (CD19 + ) or K562 (CD19 - ) for 2 hours after resting overnight in cytokine-free media. Phosphorylated ERK and Akt within the CD8 + NGFR + T cell population were analyzed with intracellular flow cytometry. Representative FACS plots (d) and the MFI (e) within the CD8 + NGFR + T cell population are shown (n=4 different donor samples; repeated measures one-way ANOVA with Tukey s multiple compariso test; F=35.54 for perk and F=25.86 for pakt; degree of freedom=15 for each comparison). Horizontal lines represent mean values., not significant.

3 Supplementary Fig. 3 a NALM-6 K IL2RB-z z 28-IL2RB-z z IL-2 IL-21 pstat3 STAT3 pstat5 STAT5 -actin b pstat3 / STAT3 pstat5 / STAT5 Supplementary Fig. 3. JAK-STAT signaling induced by CAR-T cells. (a, b) T cells traduced with the indicated CAR genes shown in Fig. 1a were stimulated with NALM-6 or K562 after resting in cytokine-free media overnight. The NGFR + T cells were isolated 2 hours after stimulation, and phosphorylated or total STAT3/STAT5 was detected by immunoblotting. T cells mock-traduced and treated with IL-2 (3 IU/ml) or IL-21 (5 ng/ml) for 3 minutes were used as a control. Representative blots of three independent experiments (a) and the quantified band inteity (b) are shown. The ratio of phosphorylated to total protei was calculated and normalized to that of the CAR-T cells stimulated with NALM-6 (n=3). Horizontal lines indicate mean values ± s.d.

4 Supplementary Fig. 4 a 28-IL2RBz 28-IL2RBz NGFR (FITC) CD8 (Pacific Blue) NGFR NGFR b Mean fluorescence inteity pstat3 (Alexa Fluor 647) pstat3 P=.3 NGFR Mean fluorescence inteity NGFR pstat5 (Alexa Fluor 647) pstat5 P< IL2RBz 28-IL2RBz c d 3 pstat3/stat3 2 pstat3 STAT3 NGFR + NGFR - Relative band inteity 1 pstat5/stat5 pstat5 STAT5 -actin Supplementary Fig. 4. JAK-STAT pathway activation in CAR + and CAR - T cells. (a-d) CAR-T cells were stimulated with NALM-6 after resting overnight in cytokine-free media. The phosphorylation of STAT3 and STAT5 was analyzed 2 hours after stimulation by flow cytometry (a and b) and immunoblotting (c and d). (a) Representative FACS plots from the samples presented in Fig. 1b. (b) Mean fluorescence inteity of individual samples (n=4; paired two-tailed t-test; t=.2 for -pstat3, t=1.29 for -pstat3, t=8.97 for z -pstat3, t=.74 for -pstat5, t=4.61 for -pstat5, t=17.99 for 28- IL2RB-z -pstat5, degree of freedom=3). Horizontal lines indicate mean values. For immunoblotting, NGFR +/- CD8 + T cells were isolated after stimulation by flow cytometry. Representative blots of four experiments (c) and the quantified band inteity normalized to that in CAR-T cells (d) are shown. Horizontal lines indicate mean values ± s.d., not significant. 28-IL2RBz 28- IL2RBz 28-IL2RBz NGFR + NGFR IL2RBz

5 Supplementary Fig. 5 a 28- IL2RB (FLSL)-z z b 28- IL2RB (FLSL) -z z pstat3 MFI pstat3 (Alexa Fluor 647) STAT3 pstat5 STAT5 -actin MFI pstat5 (Alexa Fluor 647) c Relative ratio of phosphorylated/total protein 28- IL2RBz (Y XXQ) 28- IL2RB (FLSL)- z (Y XXQ) 28- IL2RB-z 28- IL2RBz (Y XXQ) 28- IL2RB (FLSL)- z (Y XXQ) 28- IL2RB-z pstat3/stat3 pstat5/stat5 Supplementary Fig. 5. Differential roles of tyrosine residues in inducing STAT3 and STAT5 phosphorylation. (a-c) T cells traduced with the indicated CARs were stimulated with NALM-6. The phosphorylation levels of STAT3 and STAT5 were analyzed 2 hours after stimulation by intracellular flow cytometry (a) or immunoblotting (b and c). Immunoblotting was performed three times, and the quantified band inteity normalized to that of the z CAR-T cells is shown (c). Horizontal lines indicate mean values ± s.d.

6 Supplementary Fig. 6 a Fold expaion * CD4 + P=.4 * P=.1 * Fold expaion * * CD8 + * P=.1 P=.3 P=.1 * * * * 28- IL2RBz 28- IL2RB (FLSL)-z 28- IL2RB-z 28- IL2RBz 28- IL2RB (FLSL)-z 28- IL2RB-z b 28- IL2RB (FLSL)-z z CD4 + MFI CD8 + CFSE MFI Supplementary Fig. 6. Proliferation of CAR-T cells. (a) T cells traduced with the indicated CAR genes were stimulated with NALM-6 or K562 and cultured in the absence of exogenous cytokines. Fold expaion of the CAR-T cells within CD4 + or CD8 + T cell population was analyzed 7 days after stimulation (n=8 different donor samples; repeated measures oneway ANOVA with Tukey s multiple compariso test for the NALM-6 data, F=4.47 for CD4 + T cells, F=3.84 for CD8 + T cells, degree of freedom=39; two-tailed paired t-test for comparison between the NALM-6 and K562 data in the individual CAR-T cells, t=6.79 (CD4 + -), t=5.32 (CD4+-), t=5.97 (CD4 + -z ), t=5.58 (CD IL2RB (FLSL)-z ), t=8.29 (CD4 + -z), t=8.73 (CD8 + -), t=7.36 (CD8 + -), t=1.8 (CD8 + -z ), t=7.45 (CD IL2RB (FLSL)-z ), t=7.93 (CD8 + -z), degree of freedom=7). Horizontal lines denote mean values. * P<.1. (b) CAR-T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with NALM-6. CFSE dilution within CD4 + /CD8 + NGFR + T cell population was analyzed three days after the stimulation. Representative FACS plots of four samples are shown.

7 Supplementary Fig. 7 CD62L (PE) IL2RBz IL2RB (FLSL)-z IL2RB-z CD45RA (FITC) CD27 (APC-Cy7) CCR7 (Pacific Blue) CD28 (APC) CCR7 (Pacific Blue) CD28 (APC) CD45RA (FITC) CD62L (PE) CCR7 (Pacific Blue) CD95 (PerCP/Cy5.5) Supplementary Fig. 7. Expression of memory T cell markers in the CAR-traduced T cells. The expression of CD45RA, CD62L, CCR7, CD27, CD28 and CD95 was analyzed in the CAR-traduced CD8+ T cells 7 days after stimulation with NALM-6. Representative FACS plots of the nine different donor samples presented in Fig. 2d are shown.

8 Supplementary Fig. 8 a c MFI: pstat3 (Alexa Fluor 647) CD45RA + CD62L + CCR7 + cells within CD8 + CAR-T cells (%) DMSO P=.3 S3I-21 MFI: pstat5 (Alexa Fluor 647) Pimozide S3I-21+ Pimozide DMSO S3I-21 Pimozide b Mean fluorescence inteity P=.12 DMSO S3I-21 pstat3 Pimozide DMSO P=.4 S3I-21 pstat5 Pimozide Supplementary Fig. 8. Specific inhibition of STAT3 and STAT5 phosphorylation. (a, b) The z CAR-T cells were stimulated with NALM-6 in the presence or absence of 25 M of S3I-21 or 5 M of pimozide and were analyzed for the phosphorylation of STAT3 and STAT5 in the CD8 + NGFR + T cell population 12 minutes after the stimulation. Representative FACS plots (a) and mean fluorescence inteity of pstat3 and pstat5 are shown (b, n=4 different donor samples; repeated measures one-way ANOVA with Tukey s multiple compariso test; F=54.54 for pstat3 and F=115.8 for pstat5; degree of freedom=11). (c) The z CAR-traduced T cells were stimulated with NALM-6 and were treated with 25 M of S3I-21 and/or 5 M of pimozide for three days. The frequency of CD45RA + CD62L + CCR7 + cells within the CD8 + NGFR + T cell population was analyzed on day 7 (n=7 different donor samples, repeated measures one-way ANOVA with Tukey s multiple compariso test agait the DMSO control; F=7.71; degree of freedom=27). In b and c, horizontal lines indicate mean values., not significant.

9 Supplementary Fig. 9 a 28- IL2RBz 28- IL2RB (FLSL)-z 28- IL2RB-z Control NGFR K562 No cytokine NALM-6 IL-2 K562 No cytokine NALM-6 IL-2 IL-21 PD-L1 (PE) IL-21 TIM-3 (APC-Cy7) PD-1 (Alexa Fluor 488) LAG-3 (PerCP/Cy5.5) TIM-3 IL-21 Control NGFR IL-2 IL-21 IL-2 Control NGFR none 28- IL2RB-z 28- IL2RB (FLSL)-z 28- IL2RB-z NALM-6 K562 none IL2RB-z 28- IL2RB (FLSL)-z LAG-3 P= IL2RB-z 6 P= Mean fluorescence inteity PD-L1 PD-1 b Supplementary Fig. 9. Expression of immunoinhibitory molecules in CAR-T cells upon antigen stimulation. The indicated CAR-T cells were cocultured with NALM-6 or K562, or T cells traduced with the control NGFR were treated with IL-2 (3 IU/ml) or IL-21 (5 ng/ml). The expression of PD-1, PD-L1, LAG-3 and TIM-3 was analyzed 24 hours later. Representative FACS plots (a) and the mean fluorescence inteity (b) are shown (n=4; repeated measures one-way ANOVA with Tukey s multiple compariso test for the data for activated CAR-T cells; F=22.8 for PD-1, F=1.52 for PD-L1, F=16.66 for LAG-3, F=2.28 for TIM-3; degree of freedom=19)., not significant. Horizontal lines indicate mean values.

10 Supplementary Fig. 1 a NALM-6 NALM-6 NALM-6 Cytokine production Day b Fold expaion of CD8 + CAR-T cells P=.3 P=.8 P=.2 P=.13 P=.2 P=.7 P=.3 P=.19 P<.1 P=.19 P=.39 P=.33 CAR-T cells Proliferation IL2RB (FLSL)-z 28- IL2RB-z 28- IL2RB (FLSL)-z 28- IL2RB-z 28- IL2RB (FLSL)-z 28- IL2RB-z c IL-2 production (%) IFN- production (%) Day 7 P=.5 P=.7 P=.2 P<.1 Day 14 Day 21 TNF- production (%) P=.3 P=.18 IL-2 + IFN- + TNF- + cells (%) P= IL2RB (FLSL)-z 28- IL2RB-z 28- IL2RB (FLSL)-z 28- IL2RB-z P=.13 P=.2 P< IL2RB (FLSL)-z P= IL2RB-z Stimulation 1st 2nd 3rd Supplementary Fig. 1. Proliferation and cytokine production in the CAR-traduced T cells upon repeated antigen stimulation. (a-c) The CAR-traduced T cells were stimulated weekly with NALM-6 (a). The fold expaion of the CD8 + CAR-T cells at 7, 14 and 21 days after the initial stimulation was calculated (b; n=5 different donor samples, repeated measures one-way ANOVA with Tukey s multiple compariso test; F=26.41 for Day 7, F=52.27 for Day 14, F=83.83 for Day 21; degree of freedom=24). (c) The production of IL-2, IFN- and TNF- was analyzed by intracellular flow cytometry after each stimulation. The frequency of the cells producing individual cytokines and those positive for all cytokines within the CD8 + NGFR + T cell population are shown (n=7 different donor samples, repeated measures one-way ANOVA with Tukey s multiple compariso test for each round of stimulation; F=2.1 (IL-2-1st), 2.57 (IL-2-2nd), 27.4 (IL-2-3rd), 2.54 (IFN- -1st), 2.57 (IFN- -2nd), 2.66 (IFN- -3rd), 6.95 (TNF- -1st), 7.17 (TNF- -2nd), 3.9 (TNF- -3rd), 4.44 (IL-2 + IFN- + TNF- + -1st), 4.87 (IL-2 + IFN- + TNF- + -2nd)), (IL-2 + IFN- + TNF- + -3rd); degree of freedom=34)., not significant. Horizontal lines indicate mean values.

11 Supplementary Fig. 11 a 28- IL2RB (FLSL)-z z Control NGFR PD-1 (Alexa Fluor 488) Mean fluorescence inteity PD-1 PD-L1 LAG-3 TIM-3 P=.4 P=.1 P< IL2RB-z 28- IL2RB (FLSL)- z 28- IL2RB-z Control 28- IL2RB-z 28- IL2RB (FLSL)- z 28- IL2RB-z TIM-3 (APC-Cy7) PD-L1 (PE) LAG-3 (PerCP/Cy5.5) b Control 28- IL2RB-z 28- IL2RB (FLSL)- z 28- IL2RB-z Control 28- IL2RB-z 28- IL2RB (FLSL)- z P= IL2RB-z Control Supplementary Fig. 11. Expression profiles of exhaustion markers in CAR-T cells after repeated antigen stimulation. (a, b) CAR-T cells were stimulated weekly with NALM-6. Expression profiles of PD-1, PD-L1, LAG-3 and TIM-3 in CD8 + CAR-T cells 7 days after the third stimulation were analyzed by flow cytometry. Representative FACS plots (a) and mean fluorescence inteity (b) are shown (n=5 different donor samples, repeated measures one-way ANOVA with Tukey s multiple compariso test; F=16.17 for PD-1, F=2.97 for PD-L1, F=31.35 for LAG-3, F=56.36 for TIM-3; degree of freedom=29)., not significant. Horizontal lines indicate mean values.

12 Supplementary Fig. 12 a IL-2-induced genes IL-7-inducd genes IL-15-induced genes P=.178 FDR=.511 P=.649 FDR=.796 P=.444 FDR=.662 P=.313 FDR=.24 P=.143 FDR=.218 P=.59 FDR=.24 b 4 hours z z IL-21-induced genes 4 hours 72 hours 72 hours P=.545 FDR=.656 P=.66 FDR=.676 P<.1 FDR=.49 P<.1 FDR=.11 c 4 hours STAT3 target genes 4 hours 72 hours 72 hours P=.596 FDR=.615 P=.326 FDR=.395 P<.1 FDR=.43 P<.1 FDR=.21 Supplementary Fig. 12. Gene expression profiles of the stimulated CAR-T cells. (a) Gene set enrichment analysis (GSEA) comparing the expression profiles of the z CAR-T cells versus the or CAR-T cells stimulated with NALM-6 for 24 hours (n=4 different donor samples for each group). induced by IL-2, IL-7 or IL-15 treatment were used as the gene sets. (b, c) GSEA of IL-21-induced gene (b) and STAT3 target genes (c) comparing 28- IL2RB-z CAR-T cells and / CAR-T cells stimulated for 4 or 72 hours. A nominal P value for enrichment was calculated by a permutation test, and a false discovery rate (FDR) was calculated to adjust for multiple hypothesis testing.

13 Supplementary Fig. 13 % tumor cells IL2RB (FLSL)- z 28- IL2RB-z 28- IL2RB (FLSL)- z 28- IL2RB-z 28- IL2RB (FLSL)-z 28- IL2RB-z Target cells NALM6-GL K562-CD19 K562 Supplementary Fig. 13. Cytotoxic activity of CAR-T cells. The CAR-traduced T cells were cocultured with the indicated target cells at a ratio of 1:1. The frequency of the residual target cells was determined by flow cytometry (n=4 technical replicates; ordinary one-way ANOVA with Tukey s multiple compariso test for each target cell; F=.48 for NALM6-GL,.49 for K562-CD19,.9 for K562; degree of freedom=19)., not significant. Horizontal lines indicate mean values ± s.d.

14 Supplementary Fig. 14 Weeks after T cell infusion No T cells z Supplementary Fig. 14. Antileukemic effects of the CAR-T cells in vivo. NOD-scid IL2r null (NSG) mice were intravenously injected with the CD19 + leukemia cell line NALM-6 traduced with EGFP-luciferase (NALM6-GL) and were treated with 5x1 6 CAR-T cells 14 days later. In vivo bioluminescent imaging of luciferase activity following the infusion of the CAR-T cells is shown.

15 Supplementary Fig. 15 a CAR traduction aapc/mokt3 NALM-6 stimulation Day CD3 + T cells Day -14 NALM6-GL i.v. b Weeks after T cell infusion 1 4 CAR-T cell i.v. 2 2 c Total photon counts (log1) Weeks after T cell infusion No T cell No T cells z 6 8 d Leukemia-related survival (%) g 1 IL-2 (pg/ml) Weeks after leukemia infusion P=.1 P=.9 No T cell No T cell IFN- (ng/ml) e CD8 + CAR-Tcells in the peripheral blood (%) Days after T cell infusion P=.4 P<.1 P= Supplementary Fig. 15. Improved therapeutic effects of CAR-T cells by dose escalation. (a) NOD-scid IL2r null (NSG) mice were intravenously infused with the NALM6-GL and treated with two infusio of the 5x1 6 CAR-T cells. (b, c) Leukemia progression was monitored by in vivo bioluminescent imaging of luciferase activity. Individual images (b) and the quantified total photon counts (c) are shown (n=6 mice for each group). (d) Kaplan Meier curve for leukemia-related survival of the mice (n=6 mice for each group, log-rank test). The mice that died of xenogeneic graft-versus-host disease (GVHD) were ceored at the time of death. See Supplementary Table 3 for detailed information. (e) The frequency of CD8 + CAR-T cells in the peripheral blood (n=6; ordinary one-way ANOVA with Tukey s multiple compariso test; F=1. for Day 7, F=3.92 for Day 21; degree of freedom=17). (f) The absolute number of CAR-T cells persisting within the spleen was analyzed at the time of lethal GVHD or on 9 days after leukemia infusion (n=4 for 28z, n=5 for, n=6 for z ; ordinary one-way ANOVA with Tukey s multiple compariso test; F=5.72; degree of freedom=14). The data in d-f are representative of two experiments. (g) Serum was collected from the mice 4 days after the first CAR-T cell infusion, and the concentratio of IL-2, IFN- and TNF- were measured using an enzyme-linked immunosorbent assay (n=6 for each CAR group; ordinary one-way ANOVA with Tukey s multiple compariso test; F=11.16 (IL-2-one), 3.3 (IL-2-two), 2.1 (IFN- -one), 2.57 (IFN- -two), 6.84 (TNF- -one), 3.59 (TNF- -two); degree of freedom=17). IL-2, IFN-, and TNF- were not detected in 8, 12, and 1, respectively, of the 12 No-T cell samples., not significant; nd, not detected. In e-g, horizontal lines indicate mean values ± s.d. P=.3 P=.8 nd No T cell P=.4 TNF- (pg/ml) f Absolute number of CAR-Tcells in the spleen P=.9 CAR-T cell infusion One Two One Two One Two P= P=.3 P=.4 No T cell

16 Supplementary Fig. 16 a CAR-T cell infusion Day: 5 x 1 6 Day : 5 x 1 6 Day 2: 5 x z * * * * Weeks after T cell infusion Weeks after T cell infusion Weeks after T cell infusion b NGFR (Pacific Blue) CD45 (APC) Supplementary Fig. 16. The development of xenogeneic graft-versus-host disease (GVHD) by CAR-T cells after leukemia eradication. (a) Serial weight monitoring of NALM-6-bearing mice infused with the indicated number of CAR-T cells (n=6 mice for each group). Relative body weight compared with the weights prior to T cell infusion are shown. The development of lethal GVHD is denoted by *. (b) Persistence of CAR-T cells within the spleen of the mice that received two infusio of 5x1 6 CAR-T cells was analyzed at the time of lethal GVHD or at 9 days after leukemia infusion. Representative FACS plots analyzing CD45 + NGFR +/- cells within the spleen of 6 mice for each group are shown.

17 Supplementary Fig. 17 a Infusion 5 million 1 million % body weight z Weeks after T cell infusion % body weight * 8 ** * * * * * % body weight * * * * * * * * * * * * * * Weeks after T cell infusion Weeks after T cell infusion * * * * * b Overall survival (%) c 5 million 1 million Absolute number of CAR-Tcells in the spleen Weeks after T cell infusion Overall survival (%) z Supplementary Fig. 17. The development of xenogeneic graft-versus-host disease by CAR-T cells in the absence of antigen. Tumor-free NSG mice were irradiated with 1.5 Gy, and traplanted with 5 or 1 million of CAR-T cells. (a) Serial weight monitoring of the mice. Relative body weights compared with the weights prior to T cell infusion are shown. The development of lethal GVHD is denoted by * (n=6 mice for each group). (b) Overall survival of the mice traplanted with CAR-T cells (n=6 mice for each group, log-rank test). (c) Tumor-free NSG mice were irradiated with 1.5 Gy and adoptively traferred with 5x1 6 CAR-T cells. The mice were sacrificed 14 days after T cell infusion, and the absolute number of the CAR-T cells within the spleen was analyzed (n=6 mice; ordinary one-way ANOVA with Tukey s multiple compariso test; F=2.54; degree of freedom=17). Representative of two experiments., not significant. Horizontal lines indicate mean values ± s.d.

18 Supplementary Fig. 18 a No T cells 28- IL2RB-z SSC Debris exclusion FSC FSC-W Doublets exclusion SSC-W FSC-H CD19 (PE) SSC-H HLA-A2 (FITC) 1 * * 1.1 Day IL2RB-z CD45 (APC) NGFR (Pacific Blue) b CD8+ CAR-Tcells in the peripheral blood (%) c Time of tumor progression Supplementary Fig. 18. Relapse of the melanoma cell line A375-CD19 after treatment with CAR-traduced T cells. (a, b) NSG mice were subcutaneously injected with A375 melanoma cells traduced with CD19 (A375-CD19) (day -21) and infused with 5x15 CAR-T cells on day and 4. The mice were sacrificed when the tumor size exceeded 3 mm3. (a) Progressing tumor cells were analyzed for CD19 expression. Cells were co-stained with HLA-A2 to discriminate the A375CD19 tumor cells. (b) Persistence of the CAR-T cells within the tumor was analyzed when the tumor size exceeded 3 mm3. In a and b, representative FACS plots from seven mice in each group are shown. (c) The frequency of CD8+ CAR-T cells in the peripheral blood 7 days after CAR-T cell infusion or at the time the tumor mass exceeded 3 mm3 (n=7 mice; ordinary one-way ANOVA with Tukey s multiple compariso test for each time point; F=4.19 for Day 7, F=.71 for Time of tumor progression; degree of freedom=2). * P<.1., not significant. Horizontal lines indicate mean values ± s.d.

19 Supplementary Table 1. Amino acid sequencing of the CAR signaling domai used in this study. IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR Blue: CD28 Black: CD3z 28-IL2RB-z z 28- IL2RB (FLSL)-z z TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE Black: CD8 EEGGCELPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR Green: 4-1BB DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY Black: CD3z QGLSTATKDTYDALHMQALPPR IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQ Blue: CD28 GYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSG Red: IL2RB EDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERV Black: CD3z with PRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFP YXXQ WSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAYRHQA LPPR IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA Blue: CD28 AYRSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS Red: IL2RB with SSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRVK internal deletion FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK Black: CD3z with NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY YXXQ DAYRHQALPPR IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC Blue: CD28 YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA Red: IL2RB with AYRSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS internal deletion SSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAFLSLQELQGQDPTHLVRVK and FLSL FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK Black: CD3z with NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY YXXQ DAYRHQALPPR IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA Blue: CD28 AYRSNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS Red: IL2RB with SSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRVK internal deletion FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK Black: CD3z NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR

20 Supplementary Table 2. Differentially expressed genes in different CAR-T cells at the indicated time points (P<.1 with repeated measures one-way ANOVA, and fold change >1.5 or <-1.5). IL-21-induced genes, STAT3 target genes and genes included in both gene sets are highlighted in red, blue and green, respectively. 4 hours after NALM-6 stimulaiton (356 genes) BBz vs 28z vs BBz BBz z vs BBz BBz z vs BBz BATF CLECL ABCB TWSG KCNA IL MFSD2A MIR548C CTDSP PRDM FIGNL CATSPERB NKG KAT2B WRAP CTLA ELL SKA SEC61A POFUT TTK METRNL ST8SIA LINC GPR ADAM FSCN ABTB INPP4B RMI CYTIP OAS ALG JAK MIR GOLGA8DP MLLT MYO1G IL RORA PIK3R AGFG BATF ANTXR NUDT ICOS HAPLN HLA-DRA KIF3A MT1CP CHEK IL SEC24D CD LOC MDFIC FAM9A1P SH3BP PIM PRIM MAF SEC31B PTTG3P APOL CD CBX CD MX HLA-DPA CCR LOC C7orf MSC SLC7A GPR FAM46C SGPL RFC RCAN ACOX CENPU IL TRPS IL1R APOL LOC DHCR SYTL CST NCAPH TOX LOC CTTNBP2NL GZMH MAML ANLN DDX LMNB E2F C1orf TPI1P FOXM TNFRSF ADRB VTRNA FCRL LOC MIR NUGGC SLC41A BRIP IL6R UBXN ITGAV LY SOD MTX LAMP LOC TPX INSIG ITGA LGALS9B HERC MCOLN GTSE PDE4DIP LOC HIST1H3G PROK FNDC ECT FGFBP MIR GCNT LTA IL MIR ADGRG KLF NDC STRIP TGFBR CDC F TNF CCL GALNT NIPA PLK GSAP PLEK CKAP TRGV B3GNT KIAA EOMES LOC BIRC RASA LINC NUSAP LIMA CCL3L SCCPDH TMEM PIGV UBE2C MIR ZNF28D ACAT MIR518A CDC42EP DTL LOC MIR CENPE CD BCL9L MTBP EPAS PTPN NCAPG PFKFB DLGAP GINS SETBP LINC ADRBK ST6GAL MYCBP CCNB

21 vs 28z vs BBz z vs BBz z vs BBz KIF ADRBK RASGEF1B PRC CD ANKRD3B SQLE SMOX PIP5K1B RAPH ABHD BTLA UBE2T FGFR CD ATAD IKZF CD INSL SERPINE MTSS FANCI SLAMF TREML5P MKI TRAV NR4A USP17L9P FAM213A INSM DLGAP LOC FGL TICRR ZG16B ID CCNA INPP5F PHEX TOP2A GREB VCAM YPEL TSPAN ARMC FAM111B CD FAM72A COL6A FLNB ARHGAP CCNB HHLA IGLC HLA-DQA ARHGAP NCEH EIF1AX DUSP COL6A TNFSF MYO1E KIAA NCAPG DGKA CCL CCR UPRT DHRS CDK KIT NHS NCAPD RSPH4A CD NEK SGK SERPINE THYN SNORD ISM MCM EVI2A CCL LOC GJB IL23A HSD17B ASPM CCND PDCD DOCK FLJ MYADM CCP LOC HSPB RILPL LEF TLE CAMK2D CD P2RY CENPH SPTBN EXO CCDC LOC DRAM NELL RRM ENOX E2F KIF ASB SPRY BNIP3L TMEM14C HMGB XCL PRR PIKFYVE C12orf PTPN CHST TESK TBC1D HVCN AFF RALGAPA CXorf FCMR FAT SLC35E LCK LAYN MIR IL TRAF SESTD CENPF LOC HMMR TCF POLR3C GPR MELK NLRP SMC FGF CENPV PLXNC SUOX ZCWPW TYMS FAM12A NT5E MGAT GPATCH CD ARMCX PRCP CDKN JAG

22 24 hours after NALM-6 stimulaiton (429 genes) vs 28z vs BBz vs 28z vs BBz vs 28z vs BBz IL MIR LOC IL1R CD PCCA KLRC SLC2A MTM F2R LOC SETBP GZMB PALLD CASP PRDM AIM FCMR CCR TNIP PWARSN IL1R LOC CLK KSR ABCA RASGRF GZMA RGS PGAM NKG ANKRD2A5P LAG GCNT TMED1P GLUD BMPR1A MIR PBXIP IL XRRA CIITA ABLIM ACVR LOC GALNT TC2N CST CTLA ARRDC TMEM14EP SBNO MIR MAP3K CD TANC TMEM SPEF LINC-PINT NIPAL MFAP BCAT SERPINI SOX RTKN LGALS NFIA RNF LOC ITGA PGAP CCL4L RFX LOC ZNF BCL KLRAP SNORD BATF SNORD11B VAMP SOCS CD MYO FOSL TMEM SLC7A5P MCTP SIK3-IT FKBP GPR MIR CLU OSTF IL1RAP C1orf TNFRSF ZC3H12D SLC25A LOC NUGGC ZNF IL IGANRP ARHGEF EPAS SERPINB LOC COL6A LOC ADAMTS RCAN CCR ADCY LOC ADD F TRERF HLA-DRA EMP SLCO4C TXK TP53INP IL7R NARF MYOF GLUL FAM184A BNIP ZNRF RRAGC ALOX5AP ZBED RPRD1A LOC ANKRD2A PARP HERC SULT1B KLRD GZMK DDIT ATP2B SIRPB SATB CRYZ PAM STRIP SNORD GPSM SH3BP HNRNPU RHOU FAM46C LINC SAMD9L GIMAP GAB ENPP GPR SPATS2L ERP GIMAP TRIB VDR PRKAR2B LOC EVI2B FURIN TCAF MVB12B STOM ARID3B CNN KDSR PLGLA TJP SLC27A SLC9A GS GPR RNF SNORD35B MAF GALNT DKFZP586I LUCAT CYP4F35P DYNC2LI PIM SLC39A SLFN FNIP LOC SAMD GZMH GPR LOC

23 vs 28z vs BBz vs 28z vs BBz vs 28z vs BBz CTH CYLD ETS CINP KIAA TNFSF FAM65B OR51A FAM69A SMAD NFIX FOXP NCKAP TNFAIP FUT KIF PDE7A SERTAD SNORA71A UBASH3B LOC GVINP SNTB NFE2L GNPDA CLIC INPP5F APOBEC3H LOC PDCD CCNG UNQ ARHGAP LRMP HIVEP P2RY CCR RAB9A CRIM IQCF LAX ATXN NFATC CD SYPL TRGJP LOC AMICA KCCAT LITAF NEK GLIPR IL1RA TLR RGCC XIRP TNFSF LOC SNX MAL MCOLN KIAA PSTPIP MYBL MIR IKZF TNFSF13B DNAJC5B COL6A SIRPG ARHGEF SNX RHOB OAS ISM MIR155HG KRT NCF GPR RAB SGPP C12orf PTPN RDH DUSP LOC RAB8B CCL3L ADO SH2B CHRNA TIGIT CXorf MFSD2A STAP P2RY XAF STAT5A MPZL IGKV3D RILPL CD GSTA LOC FSCN CCDC LOC TIAM IRF PHLDA CD IFNG NELL ACTN MDFIC EBI SESN LOC FAM15A CD B3GNT EGR BCL2L ARHGEF NINJ TRAF LOC NRIP DENND5A IL9R PPFIBP PRRT ADAP LINC MIR146A PIKFYVE GPR LOC EED LRIG PELO RAB11FIP IGFLR ZNF ACSL FGFR AICDA DOK NFKB CCL4L ADRBK CYSLTR RGS IQCF STARD CCR EPB41L4B ADGRE MIR2C TP53I AGK CRTAM C16orf NFKBIA CD GCNT CAV LAYN BMF GUCY1B BACH SIAH PLPP PTGIR ZBTB TNF TAGAP CD ZG16B TBC1D LINC TRGV SPRY LOC SERPINE MYO1E TTN TRIP REL COL6A AHI CD VIM SGK PHEX GBP CD LOC IFNAR HVCN NHS TSPAN SHC CCL

24 vs 28z vs BBz KIAA VCAM ABTB IL23A XCL DHRS PDE7B IGF IL TNFRSF SDC IL1A NR4A DUSP IER NR4A HNF1B AFAP1L CD CD TNFSF XCL CSF CCL IL CCL IL

25 72 hours after NALM-6 stimulaiton 1351 genes) vs 28z vs BBz z vs BBz z vs BBz IL18RAP LINC HIST1H4B GZMB ITGA LOC IL CATSPERB HYKK NEAT FGFBP LOC IL12A VSIG NUCB MXD LOC KLRC GAS2L GIMAP MUC CCR TNFRSF SEC31B NKG LINC JUN IFNG CXCR HELB LPCAT GLUL GNLY KLRC CEP KLRD F2R BCL RNF PLEKHF IGKV3OR LGMN CD TJP GIMAP CCNE LINC LINC GLIPR PDLIM CCDC PLAC LY PLSCR BMPR1A MAP3K SPATS2L PRDM NUAK IGKV3D SEMA7A ELOVL LOC ASXL FBXO FAM228B ZFP GIMAP ADD CD IL ABCB SBNO TRPS PITPNC ABLIM LOC STRIP TIAM IKZF MVP PPP1R3B SLC9A ARRDC ITGA TC2N FURIN CD2R STOM GCA TBC1D PIK3AP GRAMD1B METRNL TRIB HIST1H2BC RAPGEF KCTD LOC ARHGAP GIMAP TXK GPSM ZFYVE SLC27A GALNT TP53INP ISG JAK SELL CAPRIN NPC ABCA GUSBP IMPA GZMH PDGFRB PGS SDK SETBP CFLAR GAB PTPN CTSA MIR LINC SIGIRR AIM SH3BP IQGAP GIMAP CSTF2T IL17RA ANKRD2A CD3A PRPF38B CLU GSDMB SOCS KDSR LINC BCL HSBP1L CD8B JUNB RARRES MX BATF MIR JHDM1D DOCK LOC HIVEP SUSD FASLG ACVR BCR NUGGC CASP DUSP STYK SLAMF DPY19L C8orf TMEM MCTP PARP ANKRD2A NUP LOC KLF TIPARP FAM184A CMTM TANC PPIAL4E GBP SULT1B LUCAT CEP KIAA226L COL6A LOC LOC RFX PHF ZNRF INTS LOC TRERF RASA LINC LOC RRAGD PHOSPHO RCAN DUSP CD

26 vs 28z vs BBz z vs BBz z vs BBz CLEC2B ZNF658B UBL PLGLB SYCP LDAH OGFRL FAM8A PFKFB CCDC GPR SGMS SMIM LOC FKBP C17orf ZNRF GDPD GLUD BLVRB AGO LOC IGFBP BCAT SLC2A ZFP36L HEATR5A CEP CLK TIMD LOC GRAMD1C ADD TCAF LOC OMA WDR METTL LDOC1L SETDB MKRN LOC ZNF OARD MTERF GIMAP CXorf OPN INADL DPY19L ZDBF AGTPBP GPRIN ZNF LAG ZNF ZNF FAM46C ATP2B KLF PCED1B HENMT FAM16B PHYKPL LOC MAN1A NARF LOC PQLC FLJ ZNF PTPRCAP KLHDC LOC PRG RGS STK17A AQP VWA5A RAB SLC16A GPR ADGRE FAM89B LOC JAKMIP GSAP MT1L UHRF TLR RWDD2A TRIM ZNF IRAK ACVR2A DHRS PAPD RNFT GZMM RNF ST6GALNAC JMJD1C ENPP HMGB TPP MT1F NOTCH CHST FOSB CST F PAFAH1B LOC KDM7A LOC DENND6A MYO1F ARIH INPP4B OASL NEMP LOC FYB NMRK ATF7IP HIST1H2BE CCNL CYTH LZTFL BIN SLC46A SENP BNIP CD ITGAX SATB ERMP ATP8B HS3ST3B IFI TMEM SNORD111B MSL EZH GSDMA ATP6VA MYLIP GSTM C CRIP ZNF CREBZF HIST1H2BN THADA OLFM KLHDC7B IFT PDK MALAT PICALM FASTKD MXRA GNAI BFSP TAS2R IFT DHX ABCA ERI DTX3L NRSN CDC25B AAED POLG PCCA HIST1H2AL LOC LRRC8C SNTB ZNF37BP SERPINB TPT ZNF APPL GOLGA2P MGAT4A VPS LRRC CFL FAM214A ORMDL LUC7L CLUAP LONP PPP2R5C SSH LINS CECR DTNBP SIAE MAN2A MFGE DKFZP586I PRMT