Article Figures & Data
Figures
- Figure 1.
m1928z CAR T cells are less sensitive to antigen compared with m19hBBz. A, CAR T-cell numbers with the indicated domains in the bone marrow (BM) over time in vivo. Rag1−/− mice were i.v. injected with 1 × 106 CAR T cells. After 1, 2, 4, or 6 weeks, femurs were removed, and CAR T cells were counted by flow cytometry. B, Donor T cells in the BM after antigen challenge after 1 week. One week after Rag1−/− mice were i.v. injected with 1 × 106 CAR T cells, mice were challenged with 1 × 106 Eμ-ALL cells. One week after challenge, donor T cells in the femur were counted by flow cytometry. C–G, Rag1−/− mice were i.v. injected with m19dz, m19z, m1928z, or m19hBBz 1 × 106 CAR T cells. After 5 weeks, mice were challenged with 1 × 106 Eμ-ALL cells. One week after antigen challenge, CAR T (C) and donor (D) cells in the femur were counted by flow cytometry. CAR T cells were also analyzed for memory T-cell phenotype (E), PD-1 (F), and LAG3 (G). Data are representative of two independent experiments. For A–D, each group has 4 mice. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.
- Figure 2.
CD28 mutant CAR characterization. A, Schematic of CAR constructs. All included a 5′ long terminal repeat (LTR), CD8 signal peptide (black bar), scFv with a variable heavy chain connected with glycine–serine linker to a variable light chain (VH-G/S-VL), CD8 transmembrane and hinge domain, costimulatory and/or CD3ζ endodomain, glycine–serine linker (G/S), mCherry reporter, and 3′ LTR (33). B, CD28 mutant CAR transduction efficiency. A representative flow plot of CAR expression. FSC-A, forward-scatter area. C, CAR MFIs measured by mCherry. D and E, CD28 mutant CAR T-cell viability (D) and proliferation (E) compared with nonmutated CD28 CAR T cells prior to antigen stimulation. CAR T cells were counted after day 5 of production with trypan blue to determine viability and proliferation. For B and C, data are representative of 11 independent experiments. For D and E, each data point represents one CAR production. Error bars, SD.
- Figure 3.
CD28 mutant CAR T-cell in vitro function. A–D, Cytokine production of CD28 mutant CAR T cells compared with m1928z and m19z CAR T cells. CAR T cells were stimulated with 3T3-mCD19 cells at a 10:1 E:T ratio. After 24 hours, supernatants were harvested, and IFNγ (A), IL6 (B), IL2 (C), and TNFα (D) were measured by Luminex. E and F, Cytotoxicity of CD28 mutant CAR T cells compared with nonmutated CD28 CAR T cells. CAR T cells were cocultured with 3T3-mCD19 at either 10:1 (E) or 1:6 (F) E:T ratios. The xCelligence RTCA system monitored cytotoxicity. Arrows indicate the time point of CAR T-cell addition. Data are representative of three independent experiments. Error bars, SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA.
- Figure 4.
Mut06 CAR T cells support optimized in vivo function. C57BL/6 mice were injected i.p. with cyclophosphamide (300 mg/kg) 1 day prior to i.v. injection of 3 × 105 CAR T cells. Blood and bone marrow (BM) were collected at indicated time points and analyzed by flow cytometry. A, B-cell and CAR T-cell counts in the blood in weeks 1 to 6. B, B-cell and CAR T-cell percentage in BM after 8 weeks. C, CD28 mutant and nonmutated CAR T cells have similar memory phenotype counts at week 8. D, Mice with mut06 CAR T cells have better survival after Eμ-ALL challenge compared with m1928z CAR T cells. C57BL/6 mice were i.v. injected with 1 × 106 Eμ-ALL cells. On day 6, mice were injected i.p. with 300 mg/kg of cyclophosphamide. On day 7, mice were i.v. injected with 3 × 105 CAR T cells. Mice were then sacrificed after a 20% loss of body weight according to the IACUC-approved protocol. For A–C, each group has four mice and is representative of two independent experiments. For D, each group has eight mice. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.
- Figure 5.
Mut06 CAR T cells remain sensitive to antigen in vivo. CAR T-cell phenotype after antigen challenge in vivo. Rag1−/− mice were injected with 1 × 106 CAR T cells. After 1 week, mice were challenged with 1 × 106 Eμ-ALL antigen. Following 1 week of antigen challenge, mice were sacrificed, and bone marrow was collected. A, CAR T-cell phenotypes were determined using flow cytometry. B, Mut06 CAR T-cell cytokine expression compared with m1928z CAR T cells. Following 1 week of antigen challenge, mice were sacrificed and splenocytes were collected. Splenocytes were then cocultured with 3T3-mCD19/mPD-L1 in the presence of 1× protein transport inhibitor. After 4 hours, cells were stained for intracellular cytokines and analyzed by flow cytometry. Each group has five mice. Data are representative of two independent experiments. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.
- Figure 6.
Mut06 CAR T cells have decreased expression of exhaustion-related transcription factors. A, Schematic of CD28 signaling. B, Mut06 CAR T-cell Nur77 expression compared with m1928z CAR T cells. Spleens were harvested from Nur77GFP mice, and T cells were isolated. CAR T cells were produced and stimulated for 24 hours with 3T3-mCD19 cells. GFP was measured by flow cytometry. NFAT (C) and AP1 (D) in mut06 CAR T cells compared with m1928z cells. Jurkat cells with NFAT or AP1 firefly luciferase reporters were transduced with CARs and stimulated with 3T3-mCD19 cells. After 24 hours, cells were lysed and supernatants were analyzed with a luminometer. E, Phospho(p)-NFAT in mut06 compared with m1928z CAR T cells. CAR T cells were stimulated for 24 hours by 3T3-mCD19 cells or immediately lysed for Western blot and probed for p-NFAT, p-LCK, and S6. B–E, Data are representative of three independent experiments. Error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA.
- Figure 7.
Mut06 has less exhaustion-related gene expression and accessibility. A, RNA-seq heat map of 78 exhaustion-related T-cell genes. Cells were stimulated for 24 hours with 3T3-mCD19, and CAR T cells were sorted and lysed. RNA-seq was performed with 100-nt sequence reads using Illumina HiSeq 4000. B, RNA-seq heat map of 22 NFAT exhaustion-related T-cell genes (23, 33). C, Genome browser view of the Nfatc1, Tnf, Havcr2, Pdcd1, Nr4a1 (Nur77), Nr4a2, Lag3, Jun, and Fos loci after ATAC-seq. Cells were stimulated for 24 hours with 3T3-mCD19, and CAR T cells were sorted and lysed. Paired-end 42-bp sequencing reads generated by Illumina sequencing using NextSeq 500 were mapped to the genome using the BWA algorithm with default setting. Red stars denote the presence of the gene in ATAC-seq panel C.
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Volume 9, Issue 1
