Fig 1: Antigen experience history directs multiple aspects of in vitro function of mouse CAR8 T cells.a, E2A–PBX mouse leukemia was engineered to knockout Cd19, followed by reintroduction of CD19 at different levels to generate a range of antigen density clones. b, Schematic showing the vaccine model for generating memory CD8+ OT-I T cells. In total, 5 × 103 OT-I T cells were transferred into congenically distinct hosts that were concurrently vaccinated with antigen and adjuvants. Three to 5 weeks later, CAR T cells were manufactured from memory OT-I T cells (CAR8MD T cells) or naive OT-I T cells (CAR8ND T cells). c, Intracellular cytokine staining of IFNγ and TNF after 6 h of coculture. d, Degranulation as measured by CD107a expression after 4 h of coculture. e–g, Quantification of cytokine data shown as the percentage of positive cells for IFNγ (e), TNF (f) and IFNγ and TNF (g); exact P values from left to right: P < 0.0001 (e) and P < 0.0001, P = 0.0001, P < 0.0001, P = 0.0019 and P = 0.0005 (g); NS, not significant. h, Quantification of CD107a data shown as the percentage of positive cells. i, Percent cytotoxicity, quantified after 6 h of coculture; exact P values from left to right: P = 0.0008, P = 0.0014, P = 0.0040, P = 0.0062 and P = 0.0030. j, Representative data for cell cycle entry as measured by Ki67 staining after 18 h of coculture. k, Representative data for proliferation as measured by dilution of CellTrace Violet dye after a 72-h coculture assay. All in vitro assays were performed with n = 3 technical replicates per experiment, which were averaged and compiled to perform quantifications and statistics on n = 3 independent experiments (CellTrace data are representative of two independent experiments). Flow plots show representative data from a single experiment. Statistical comparisons were performed using two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Statistical comparisons were performed between CAR8MD and CAR8ND T cell groups. Data are shown as mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. The schematic in b was generated using BioRender.com.Source data
Fig 2: E2A-PBX/mCD19 antigen density model and murine anti-CD19 CAR T cells, and additional comparisons of in vitro data.Data are related to Fig. 1. 1A: Schematic of the anti-mouse CD19 CAR contained in pMSCV backbone. 1B: Coexpression of CAR and EGFR on murine CAR T cells. 1C: Engineering of murine leukemia with lentiviral vectors containing hUbC or hEF1a promoters driving the CD19 transgene. 1D: Survival of mice after treatment with 1e6 EGFR+ (EGFR8, non-CAR expressing) naïve or memory-derived CD8 + T cells. 1E: Fold expansion of T cells during transduction protocol: During Stim (while on anti-CD3/CD28 beads, from day 1 to day 4), Post Stim (continued expansion from day 4 to day 6 after removing anti-CD3/CD28 beads), and Composite (total fold expansion from day 1 to day 6). (Exact p values, left to right: 0.0477, 0.0005, 0.0127). 1 F: Mean fluorescence intensity of IFNγ+ cell population. 1 G: Mean fluorescence intensity of TNF+ cell population. 1H: Mean fluorescence intensity of CD107a+ population. 1I: Statistical comparisons of Ki67Neg (% Ki67Neg of EGFR+), Ki67Lo (%Ki67Lo of EGFR+, MFI Ki67Lo of EGFR+) and Ki67Hi (%Ki67Hi of EGFR+, MFI Ki67Hi of EGFR+) populations. (Exact p value, far right plot: 0.0105) 1 J: Statistical comparisons of CellTraceLo (% CellTraceLo of EGFR+, MFI CellTraceLo of EGFR+) and total EGFR+ cells (GFMI CellTrace). Data in S1B is representative transduction data from 1 experiment. Data in 1 C is from 1 transduction experiment before single cell cloning. Data in S1D is from 1 experiment, total n = 5 mice per group. Data in S1E is compiled and quantified from 9 independent experiments. All in vitro assays in S1F-S1J were performed with n = 3 technical replicates per experiment, which were averaged and compiled to perform quantifications and statistics on n = 3 independent experiments (CellTrace data compiled from two independent experiments). Statistical comparisons were performed using two-way ANOVA with two-sided Tukey’s multiple comparisons test for comparisons of 3 groups or multiple two-sided t-tests or Welch t-tests with Holm-Sidak correction for multiple comparisons of 2 groups. Statistical comparisons are performed between CAR8MD and CAR8ND groups when not otherwise specified. Data represent mean +/− SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data
Fig 3: Metabolic health and function of Solupore-transfected cells(A) Untransfected, Solupore-transfected, or electroporation-transfected cell metabolic potential at 24 h post-thaw (cells were cryopreserved 3 days post-transfection) (n = 3 individual donors, two-way ANOVA statistical analysis, p < 0.05). (B) Representative OCR profile at 24 h post-thaw. (C) Cytokine profiling 4 days post-transfection. Data were normalized to cell counts on day 4 of the experiment to correlate with relative cytokine production per cell (paired t test, p = 0.0085 [IL-2], p = 0.0498 [IFN-γ]). (D) Surface marker phenotype data for CD8+ T cells (CTLA-4, LAG-3, PD-1, TIGIT, TIM-3) was 7 days post-transfection after three rounds of stimulation with PMA (10 ng/mL) and ionomycin (250 ng/mL) (stimulation on days 0, 3, and 5 post- transfection). Unpaired t test, untransfected versus Solupore p = 0.0267, untransfected versus electroporation p = 0.0047 (CTLA-4), Solupore versus electroporation p = 0.0461 (LAG-3), untransfected versus Solupore p = 0.0005, untransfected versus electroporation p = 0.005, Solupore versus electroporation p < 0.0001 (PD-1). Data represented as mean +/- SD.
Fig 4: Screening for the optimal conditions to obtain CD62L+iNKT cells(A and B) Frequency of iNKT cells on day 8 in the presence of α-GalCer (α-Gc) and multiple cytokines (IL-2, IL-7, IL-15, and IL-21), as determined by flow cytometry analysis and based on the statistical chart (α-Gc+IL-2 vs. α-Gc+IL-7: p = 0.0114, α-Gc+IL-2 vs. α-Gc+IL-15: p = 0.0084, α-Gc+IL-2 vs. α-Gc+IL-21: p = 0.0060, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(C and D) Flow cytometry analysis and statistical charts of α-Gc+IL-2/7, α-Gc+IL-2/15 and α-Gc+IL-2/21 induced iNKT cells on days 8 (NS: not significant, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(E) The number of iNKT cells induced by the combined use of cytokines compared to the single cytokine treated group (α-Gc+IL-2 vs. α-Gc+IL-2/7: p = 0.033, α-Gc+IL-2 vs. α-Gc+IL-2/15: p = 0.0491, α-Gc+IL-2 vs. α-Gc+IL-2/21: p = 0.0384, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(F and G) CD62L expression of iNKT cells was examined using flow cytometry on induction days 8 (α-Gc+IL-2 vs. α-Gc+IL-2/21: p = 0.0029, α-Gc+IL-2/7 vs. α-Gc+IL-2/21: p = 0.0025, α-Gc+IL-2/15 vs. α-Gc+IL-2/21: p = 0.0346, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(H and I) Flow cytometry analysis and statistical charts of α-Gc+IL-2/21 and α-Gc+IL-2/4/21+GM-CSF induced the frequency of iNKT cells on day 8 (α-Gc+IL-2/21 vs. α-Gc+IL-2/4/21+GM-CSF: p = 0.0013, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(J and K) The proportion of CD62L+iNKT cells by α-Gc+IL-2/21 and α-Gc+IL-2/4/21+GM-CSF as determined using flow cytometry analysis and statistical charts on day 8 (NS: not significant, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).(L and M) Counting statistics of iNKT cells expansion absolute numbers under the settings of α-Gc+IL-2/21 and α-Gc+IL-2/4/21+GM-CSF. (Day10: α-Gc+IL-2/21 vs. α-Gc+IL-2/4/21+GM-CSF, p = 0.0153, Fold change: α-Gc+IL-2/21 vs. α-Gc+IL-2/4/21+GM-CSF, p = 0.0130, Student’s paired t-test. Data are presented as the mean ± SD, n = 4).
Fig 5: RUNX2 overexpression enhances in vitro function of human CD4+ and CD8+ CAR T cells.a, Expression of the h1928z CAR in CD4+ or CD8+ cells cotransduced with hRUNX2, EGFR (control) or mock (untransduced). b, Intracellular staining for hRUNX2 in CD4+ or CD8+ cells cotransduced with hRUNX2, EGFR (control) or mock (untransduced). c, Degranulation as measured by CD107a expression and intracellular cytokine staining of IFNγ after 5.5 h of coculture; exact P values from left to right: P = 0.0112, P = 0.0076, P = 0.05, P = 0.01 and P = 0.033. d, Intracellular cytokine staining of IL-2 and TNF after 5.5 h of coculture; exact P values from left to right: P = 0.02, P = 0.005, P = 0.01 and P = 0.006. e, Proliferation as measured by dilution of CellTrace Violet (CTV) dye after 72 h of coculture. f, Quantification of data in e; exact P value of 0.02. MFI, mean fluorescence intensity. g, Percent cytotoxicity after 5.5 h of coculture. h, Indicated protein concentrations were measured in cell culture supernatants by multiplexed ELISA after 16 h of coculture; exact P values from left to right and then top to bottom: P = 0.004, P = 0.02, P = 0.007, P = 0.01, P = 0.02, P = 0.01, P = 0.02, P = 0.01, P = 0.04 and P = 0.03. All in vitro assays show data from three independent biological donors. Statistical comparisons were performed using multiple two-sided nonadjusted paired ratio t-tests or multiple two-sided nonadjusted paired t-tests whenever not possible (when a data point was at 0 or below); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.Source data
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