Supplemental Materials Supplemental Methods Supplemental Figure 1. Immune phenotype of mCD19 targeted CAR T and dose titration of in vivo efficacy. Supplemental Figure 2. Gene expression of fluorescent-protein tagged CAR T cells. Supplemental Figure 3. Fluorescent protein tagged CAR T cells function similarly to non-tagged counterparts. Supplemental Figure 4. Transduction efficiency and immune phenotype of mCD19 targeted CAR T cells for survival study (Figure 2D). Supplemental Figure 5. Transduction efficiency and immune phenotype of CAR T cells used in irradiated CAR T study (Fig. 3B-C). Supplemental Figure 6. Differential gene expression of CD4+ m19-humBBz CAR T cells. Supplemental Figure 7. CAR expression and CD4/CD8 subsets of human CD19 targeted CAR T cells for Figure 5E-G. Supplemental Figure 8. Transduction efficiency and immune phenotype of mCD19 targeted wild type (WT) and TRAF1-/- CAR T cells used for in vivo study (Figure 6D). Supplemental Figure 9. Mutated m19-musBBz CAR T cells have increased NF-κB signaling, improved cytokine production, anti-apoptosis, and in vivo function. Supplemental Figure 10. TRAF and CAR co-expression in human CD19-targeted CAR T cells. Supplemental Figure 11. TRAF2 over-expressed h19BBz CAR T cells show similar in vivo efficacy to h19BBz CAR T cells in an aggressive leukemia model. S1 Supplemental Table 1. Probesets increased in m19z and m1928z vs m19-musBBz CAR T cells. Supplemental Table 2. Probesets increased in m19-musBBz vs m19z and m1928z CAR T cells. Supplemental Table 3. Probesets differentially expressed in m19z vs m19-musBBz CAR T cells. Supplemental Table 4. Probesets differentially expressed in m1928z vs m19-musBBz CAR T cells. S2 Supplemental Methods Gene Expression Microarray. For m19z, m1928z and m19-musBBz comparison, three million CAR T cells were incubated with 3×105 3T3-mCD19 cells overnight. The next day live CAR T cells were sorted into Trizol (Thermo Fisher Scientific, Waltham, MA). RNA was isolated according to manufacturer’s instructions and run on a MOE 430A 2.0 array Mouse Genechip (Affymetrix, Santa Clara, CA) at the Genomics Core Facility. Gene expression analyses and graphic representations were performed with the Partek Genomics Suite Software. RMA normalization was performed and values generated for each probeset for all samples. Differentially expressed genes were detected by ANOVA and probesets of statistical significance were defined by a-fold change > 2 and a FDR £ 0.05. RNA-SEQ. For m19z, m1928z and m19-humBBz comparison, three million CAR T cells were incubated with 1×106 3T3-mCD19 cells for 48 hr. Live CD4+ CAR T cells were sorted into Trizol. RNA was isolated according to manufacturer’s instructions and evaluated for quality. The Genomic Core performed mRNA enrichment and cDNA library preparation using the Illumina Tru-seq stranded mRNA sample prep kit. Final RNA-seq libraries were reviewed for size and quality on the Agilent TapeStation, followed by quantitative PCR-based quantitation with the Kapa Library Quantification Kit. The libraries sequenced on two NextSeq high-output 2x75 paired-end sequencing runs in order to generate approximately 40 million pairs of reads per sample. Sequence reads were aligned to the human reference genome in a splice-aware fashion using Tophat2 (1), allowing for accurate alignments of sequences across introns. Aligned reads were quantitated at the gene level using HTseq (2). Normalization, expression modeling, and difference testing were performed using DESeq (3). Quality control measures included custom scripts and RSeqC (4) to examine read count metrics, alignment fraction, chromosomal S3 alignment counts, expression distribution measures, and principle components analysis and hierarchical clustering. Differentially expressed genes were detected by ANOVA and probesets of statistical significance were defined by a-fold change > 4 and a FDR £ 0.01. Gene set enrichment analysis was performed (on the gene expression values) to analyze the enrichment of the gene sets using GSEA software (http://www.broadinstitute.org/gsea/index.jsp). C5 collection version v6.0 from the Molecular Signature Database (MSigDB v6.0 C5), which contains the expert-curated gene ontology (GO) gene sets, were used in the analysis. We used vertebrate homology resource (http://www.informatics.jax.org/homology.shtml) to convert between homologues human and mouse genes. For all comparisons, data was collapsed to gene symbols. 1000 permutations based on gene sets were performed. Gene sets were ranked according to false discovery rate (FDR) q-value. At the default FDR q-value cut-off within GSEA of 0.25, we identified 3 gene sets that are upregulated in m19z and 68 gene sets upregulated in m1928z. Microarray and RNA-SEQ data have been submitted to GEO (Gene Expression Omnibus) with the accession number GSE112567. CD33-targeted CARs Anti-CD33 antibodies were developed at the Vanderbilt Antibody and Protein Resource using standard methods (5). Briefly, after completing a series of immunizations splenocytes of immunized mice were isolated and fused to a non-Ig secreting myeloma cell line and grown in a semi-solid plate. Antibody- secreting clusters were identified in semi-solid plates and selected for clonal expansion in 96 well plates. S4 During expansion supernatant was collected and assayed for CD33 binding by ELISA as well as flow cytometry. Based on this screening hybridomas were selected for expansion and isolation of RNA, which was used to amplify IgH and IgL rearrangements. Based on the IgH and IgL rearrangements scFv were designed and cloned into the NcoI/NotI sites of our human CD19-targeted CAR in the SFG retroviral cassette. This allowed replacement of the anti-human CD19 scFv with anti-human CD33 scFv. These constructs were then used to produce gammaretroviral supernatant as described in Methods. In vivo NALM6 animal model of CD19-targeted CAR T cells The NALM6 leukemia mouse model has been described (6). Briefly, NALM6-GL cells were i.v. injected to NSG mice at 5x105 dose. Four days later, mice were treated with 3x105-1x106 human CD19 targeted CAR T cells. Human CD19 targeted CAR T cells with excess TRAF2 were made by CAR and mouse TRAF2 co-transduction or transduction with a bicistronic construct combining CAR and human TRAF2. Blood samples were collected weekly for flow cytometry. Leukemia burden was evaluated weekly using bioluminescence imaging on an IVIS system. Survival was monitored. Mice were sacrificed when they develop signs of progressive leukemia. S5 A m19∆z m1928z m19-musBBz CD8 CD4 CD44 CD62L B d d 5000 60 l bloo 4000 μ / l bloo 40 μ 3000 / 2000 cells T 20 1000 B cells 0 0 Thy1.1 B6 B6 z (1E6) z (1E6) m19zm19z (3E5) (1E6) m19zm19z (3E5) (1E6) m19 m19 m1928zm1928z (3E5) (1E6) m1928zm1928z (3E5) (1E6) m19-musBBzm19-musBBz (3E5) (1E6) m19-musBBzm19-musBBz (3E5) (1E6) Supplemental Figure 1. Immune phenotype of mCD19 targeted CAR T and dose titration of in 6 Supplementaryvivo efficacy figure. (A) 1. ImmuneImmune phenotypephenotype of of mCD19 transduced targeted T cellsCAR Tused and indose 5x10 titration.dose (A) in vivoImmune study pheno (Figure- type1C&D). of transduced Cells Twere cells pre used-gated in 5x10 on6 singledose in live vivo cells. study (B)(Figure In vivo 1C&D). B cell Cells killing were pre-gatedand T cell on persistence single live with T cells.cell (B) dose In vivo titration. B cell killing After and Eµ T-ALL cell persistence injection, withdifferent T cell dosesdose titration. of T cells After were Eμ-ALL given injection, to 300 differentmg/kg CTX pre- dosesconditioned of T cells were mice. given B (B220+CD19+) to 300 mg/kg CTX and pre-conditioned T (CD3+Thy1.1+) mice. B cells (B220+CD19+) in peripheral and bloodT (CD3+Thy1.1+) were quantitated 3 cellsweeks in peripheral after CAR blood T were injection. quantitated Each 3 dot weeks indicates after CAR one T mouse. injection. Data Each are dot from indicates one singleone mouse. experiment Data (n=34 are from one single experiment (n=34 total). total). S6 A B + GFP/ 80% m19 z 5’ LTR V G/S V EC TM G/S 3’ LTR H L mCherry SD SA m19z 60% + GFP/ 5’ LTR V V EC TM CD3 3’ LTR m1928z G/S G/S % H L mCherry SD SA 40% m19-musBBz + GFP/ CAR 5’ LTR V V EC TM CD3 3’ LTR H G/S L CD28 G/S mCherry SD SA 20% + 5’ LTR V G/S V EC TM m4-1BB CD3 G/S GFP/ 3’ LTR H L mCherry SD SA 0% CD8 Leader CD8 mCherryProtein mCherryL Protein mCherryL Protein mCherryL Protein L C D E m19-mBBz m1928z m19z Supplemental figure 2. Gene expression of fluorescent-protein tagged CAR T cells. (A) Schematic of genetic constructs for mCD19 targeted CARs. Shown are the long terminal repeats (LTR), packaging signal ψ, splice Supplemental Figure 2. Gene expression of fluorescent-protein tagged CAR T cells. (A) Schematic donor (SD), splice acceptor (SA), VH and VL regions of the scFv (single-chain variable fragment), the hinge (H), transmembraneof genetic constructs (TM), and for intracellularmCD19 targeted regions CARs. of the Shownretroviral are construct. the long G/S, terminal (Gly4Ser1)3 repeats linker(LTR), sequence. packaging (B) Comparisonsignal Ψ, splice of fluorescence donor (SD), protein splice and acceptor Protein (SA), L as aV methodH and V toL evaluateregions of CAR the expression.scFv (single One-chain million variable T cells transducedfragment), withthe extracellularmCherry-tagged hinge CARs (EC were), transmembrane incubated with (TM),1 µg Biotin-Protein and intracellular L and regions then of the retroviral fluochrome-conjugatedconstruct. G/S, (Gly4Ser1)3 streptavidin. linker Cells sequence. were subjected (B) Comparison to flow cytometry. of fluorescence Data are protein representative and Protein of 4 L as a independentmethod to evaluate experiments. CAR (C) expression.