Additional file 1 of CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison
Creators: name: Egli, Lukas
name: Kaulfuss, Meike
name: Mietz, Juliane
name: Picozzi, Arianna
name: Verhoeyen, Els
name: Münz, Christian
name: Chijioke, Obinna
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Supplementary Material 1: Figure S1: (A) Gating strategy for sorting of T and NK cells from PBMCs of healthy donors. (B) Purity of T and NK cells after 3 weeks of expansion and gating for further analysis of T and NK cells. Figure S2: Phenotype by flow cytometry of non-expanded T cells (baseline), expanded untransduced T cells and expanded CAR T cells purified from PBMC after expansion with K562mbIL21 and IL-2 supplementation for 3 weeks. (A) Representative contour plots showing marker expression in expanded untransduced T cells. (B) Bar graphs showing individual marker expression across T cell populations. (C) Bar graph showing individual expression of NK cell markers in T cells at baseline, expanded untransduced human T cells and expanded CAR T cells. Data from 1-3 experiments with n=3-9 donors. Significance by 2way ANOVA with Tukey’s multiple comparisons; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Symbols represent individual donors. Figure S3: (A) Timeline of the generation and expansion of anti-CD19 CAR T cells using CD3/CD28 costimulation. (B) Transduction rates of T cells after CD3/CD28 costimulation. (C) Flow cytometric analysis of CD4 and CD8 expression and percentages of CD4+ and CD8+ T cells within freshly isolated CD3+ T cells (baseline), untransduced T cells and CAR T cells, expanded with CD3/CD28 costimulation (D) Flow cytometric analysis of T cell differentiation and percentages of naïve (TN, CD45RA+CD62L+), central memory (TCM, CD45RA-CD62L+), effector memory (TEM, CD45RA-CD62L-) and TEMRA (CD45RA+CD62L-) cells within T cell populations before and after expansion with CD3/CD28 costimulation. (E) Bar graphs showing individual marker expression comparing CAR T cells generated with feeder-based expansion (CAR T) or with CD3/CD28 costimulation (CAR T (CD3/CD28stim)). (F) CAR T cells were cocultured with the indicated target cell lines at an effector to target ratio of 5:1 for 4 hours. Specific killing was assessed by flow cytometry using a membrane-impermeable DNA stain (TO-PRO-3) and PKH26 membrane-labeled target cells. Specific killing of NALM6 (left) and autologous LCL (right). Data for T cells expanded with K562mbIL21 feeder cells are from 1-3 experiments with n=7-9 donors and data for CD3/CD28 costimulation are from 1 experiment with n=3 donors. Significance by 2way ANOVA with Tukey’s multiple comparisons or two-tailed unpaired t-test as appropriate; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Symbols represent individual donors. Data shown as box and whiskers display the median as a line within the box; whiskers are shown from minimum to maximum data point. Figure S4: (A) Representative contour plots showing marker expression in expanded untransduced NK cells. (B) Bar graphs showing individual marker expression across NK cell populations. Data are from 3 independent experiments with n=5-9 donors. Significance by 2way ANOVA with Tukey’s multiple comparisons; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Symbols represent individual donors. Figure S5: (A) Flow cytometry plots of CD19 expression before (left) and after (right) CRISPR/Cas9-mediated knockout of CD19 in 721.221 cells (B-E) Expanded effector cells were cocultured with the indicated cancer cell lines at the indicated effector to target (E:T) ratios for 4 hours. Specific killing was assessed by flow cytometry using a membrane-impermeable DNA stain (TO-PRO-3) and PKH26 membrane-labeled target cells. Data are from 1 experiment with n=2-3 donors. Significance by 2way ANOVA with Tukey’s multiple comparisons (only CAR T vs. CAR NK is shown); **p&lt;0.01. Figure S6: (A) IFN-γ production of effector cells following stimulation with PMA and ionomycin. (B) Fold expansion of untransduced NK and CAR NK cells after 9 days in either the presence of an IFN-γ blocking antibody (clone B27) or recombinant 10 ng/ml IFN-γ. (C) Expression of IFN-γ-R1 (CD119) on CAR NK cells as assessed by flow cytometry. (D) Specific killing of 721.221 cells (left) and LCL (right) after single restimulation by effector cells in the absence of IL-2. Data are from 1 to 2 independent experiments with n=3-6 donors. Significance by RM one-way ANOVA with Tukey’s multiple comparisons; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Symbols represent individual donors. Data shown as box and whiskers display the median as a line within the box; whiskers are shown from minimum to maximum data point. Figure S7:Tumor-bearing NSG mice were adoptively transferred with 4 x 106 autologous CAR T or 4 x 106 allogeneic CAR NK cells at day 3 after tumor engraftment and presence of transferred cells in peripheral blood was assessed by flow cytometry on day 10 (A) and day 17 (B) after tumor engraftment. 5 mice per group, plots showing data from individual mice. Figure S8: Blood, spleen and tumors were harvested from NSG mice treated with 10 x 106 effector cells (autologous CAR T cells, autologous control T cells, allogeneic CAR NK cells or allogeneic control NK cells) at the end of the experiment and single-cell suspensions analyzed by flow cytometry. (A, B) Tumor growth curves of individual mice treated or not with ACT of 4 x 106 of the indicated effector cells (relates to Figure 7B) (A) and treated or not with ACT of 10 x 106 of the indicated effector cells (relates to Figure 7C) (B). Percentage or mean fluorescence intensity (MFI) of indicated markers within T cells (C-G). 5 mice per group. Significance by two-tailed unpaired t-test; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Data shown as box and whiskers display the median as a line within the box; whiskers are shown from minimum to maximum data point. Figure S9: Blood, spleen and tumors were harvested from NSG mice treated with 10x106 effector cells (autologous CAR T cells, autologous control T cells, allogeneic CAR NK cells or allogeneic control NK cells) at the end of the experiment and single-cell suspensions analyzed by flow cytometry. (A-D) Percentage or mean fluorescence intensity (MFI) of indicated markers. 5 mice per group. Significance by two-tailed unpaired t-test; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Data shown as box and whiskers display the median as a line within the box; whiskers are shown from minimum to maximum data point. Figure S10: (A) Schematic outline of in vivo NALM6 experiment. 5 x 105 NALM6-Luc cells were injected intravenously into NSG mice and four days after tumor engraftment, mice were treated with ACT of 2 x 106 (T and CAR T) or 10 x 106 (NK and CAR NK) effector cells from 2 donors each. Transferred cells were supported by supplementation of IL-2 (1 x 105 IU every second day, i.p.). Tumor growth was monitored weekly by IVIS. (B) Bioluminescence measurements of tumor-bearing NSG mice at indicated time points to assess in vivo functionality of adoptively transferred cells. (C) Tumor growth over time measured by IVIS (left) and overall survival (right). (D) Presence of adoptively transferred cells in peripheral blood was assessed by flow cytometry on day 8 after tumor engraftment, plots show individual mice. 4-6 mice per group. Significance by log-rank Mantel–Cox (survival) followed by Bonferroni correction. *p&lt;0.05, **p&lt;0.01.