Figures S1-S11 from Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies

S1. Lack of functional exhaustion in expanding 28.z CD5 CAR T cells. S2. (A) Schematic representation of 28.z CD5 CARs with intermediate (CH3) and short spacer. (B) Expression level of CD5 CARs measured by flow cytometry using anti-mouse F(ab)-specific antibodies. (C) Expansion of T cells transduced with the 28.z CD5 CARs or BB.z CD5 CAR. Data are shown as mean and SD, n=3. S3. Relative frequency of CD45RA+ CCR7+ naïve-like (TNAIVE) and CD45RA- CCR7+ central memory (TCM) cells among T cells expressing indicated CD5 CAR constructs. Data are shown as mean and SD, n=3. S4. Reduced phosphorylation of IKKa/b in CD5 CAR T cells with mutant 4-1BB endodomain. S5. Relative frequency of CD45RA+ CCR7+ naïve-like (TNAIVE) and CD45RA- CCR7+ central memory (TCM) cells among T cells expressing indicated CD5 CAR constructs. Data are shown as mean and SD, n=3. S6. Morphology of T cells expressing indicated CAR constructs 5 days post-transduction. S7. (A) Expression of BB.z CD5 CAR in T cells transduced with retroviral (RV) or lentiviral (LV) vector. (B) Viability of BB.z CD5 CAR T cells 7 days post-transduction. (C) Expression of ICAM-1 on the surface of BB.z CD5 CAR T cells 7 days post-transduction. (D) In vivo control of systemic leukemia by 28.z and BB.z CD5 CAR T cells. (***, P<0.001 by one-way ANOVA with Bonferroni post-test correction). S8. Minimal CAR expression in the presence of DOX is insufficient to promote T-cell cytotoxicity. S9. Reciprocal expression of CD5 and CD5 CAR in Tet-OFF BB.z CD5 CAR T cells after DOX withdrawal. S10. Expansion (A), phenotype (B) and anti-tumor function of Tet-OFF 28.z CD5 CAR T cells in vitro (C). (D) CD5 CAR T cells were injected i.v. into mice with previously established systemic Jurkat leukemia (1x10^6 cells per mouse). Leukemia progression was monitored by IVIS imaging. S11. (A) CD5 CAR T cells with repressed (+DOX) or restored (-DOX) expression of BB.z CD5 CAR were injected i.v. into mice with previously established systemic Jurkat leukemia (1x10^6 cells per mouse). Leukemia progression was monitored by IVIS imaging. (B) Tumor levels in mice on day 34 post-tumor engraftment (*, P,0.05 by one-way ANOVA with Bonferroni post-test correction).