Additional file 1 of LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells
Creators: name: Zhu, Shuxian
name: Zuo, Shiyu
name: Li, Chuo
name: You, Xingjie
name: Jiang, Erlie
name: Feng, Xiaoming
name: Luo, Yuechen
Publisher: name: figshare
Year: 2025
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 1. Supplementary Fig. 1: (A) Representative flow plots illustrate the percentage of Annexin V+/7-AAD+ cells in CAR-T cells without stimulation, (B) The fold plots illustrate the expansion of 38BBz CAR-T cells with and without CD38 knockdown in T-cell medium with 100 ng/ml IL-2 and 10�S within 7 days. (C) The gating strategy for cell sorting and flow cytometry immunophenotyping is as follows: we first gated the main cell population based on FSC/SSC to exclude debris, then used FSCH/A to exclude doublets and select singlet cells. Next, we gated for viable cells by excluding DAPI-positive dead cells. Using PCDH-T cells as a positive control, we identified cells negative for CD3, CD38, MHC-1, and MHC-2 expression. Finally, within the negative population, we gated cells positive for CAR expression. For 4KO-LLT1 CAR-T cells, only CAR-positive cells were sorted due to co-expression of the CAR epitope and LLT1. The same strategy was applied for 1KO, 2KO, and 3KO CAR-T cells. Supplementary Fig. 2: (A) Flow cytometric plots showing proportion of CD4+ T cells and CD8+ T cells in CAR-T/PCDH-T cells in unstimulated and antigen-stimulated condition. (B) The histogram shows the proportion of CD4+ T cells and CD8+ T cells in CAR-T/PCDH-T cells(n = 4) and proportion of CD4+ T cells and CD8+ T cells in CAR-T/PCDH-T cells which were stimulated with CCRF-CEM cells at ratio of 1:1(n = 4). Supplementary Fig. 3: (A) Surface overexpression of CD38 in CCRF-CEM, Raji, Molm13, Molt4, and Nalm6 cell lines (red histograms) versus FITC isotype (blue histograms) measured by flow cytometry. (B) Surface expression of GFP in CCRF-CEM, Raji and Molm13 cell lines (red histograms) versus isotype controls (blue histograms) measured by flow cytometry. (C) Surface expression of LLT1 in CCRF-CEM, Raji and Molm13 cell lines (red histograms) versus isotype controls (blue histograms) measured by flow cytometry. (D) Flowchart shows that mice (n = 4) were injected intravenously with 3 × 105 CCRF-CEM-Luc-GFP cells, followed by 2.5 × 106 CAR-T/PCDH-T cells seven days later. Tumor burden of mice was measured every week. (E) Bioluminescence imaging (BLI) results of tumor burden. Control group refers to PCDH-T cells treated mice. (F) The mean value of average radiance in each group of mice was monitored using bioluminescence (n = 4). (G) Survival curves of NSG ALL model (n = 4), the statistical significance was determined with log-rank test (Mantel-Cox). (H) The plot shows the quantity of CAR-T cells per 100 µl of mouse peripheral blood. Flow cytometry was conducted once a week to identify HuCD45+ CAR + cells within the peripheral blood of mice (n = 4). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student's t-test. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, and ****p &lt; 0.0001; ns, not significant. Supplementary Fig. 4 (A) Flow plots show the memory phenotype of 38BBz-LLT1 and 38BBz CAR-T cells, TN, naive, CD62L+CD45RA+; CM, central memory, CD62L+CD45RA−; EM, effector memory, CD62L−CD45RA−; TE, terminal effector, CD62L−CD45RA+. (B) The histogram shows frequency of memory phenotypic proportions of 38BBz-LLT1 and 38BBz CAR-T cells (n = 3). (C) The graph illustrates the cytotoxic activity of 38BBz-LLT1 and 38BBz CAR-T cells after a 12-h period of co-culture with target cells at a range of E:T ratios (n = 3). (D) Representative flow plots and (E) The histogram analysis illustrates the percentage of CD107a+CD3+cells in alloreactive PBMCs, following a period of 48 h of stimulation by irradiated CAR-T cells (n = 4). (F) The plots present the proportion of lysed 38BBz-LLT1 and 38BBz CAR-T cells when autologous primary NK cells and CAR-T cells were co-cultured at different E:T ratios over a 24-h period (n = 3). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student's t-test. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, and ****p &lt; 0.0001; ns, not significant. Supplementary Fig. 5 (A) Heatmap of the differential clustering of the RNA-seq results for 4KO CAR-T and 4KO-LLT1 CAR-T before and after tumor infiltration. Differential gene entry criteria were FDR p value &lt; 0.05 and [log2FoldChange] ≥|1|. (B) Representative flow plots illustrate the percentage of IL-10 expression in CAR-T cells with DMSO and 20 μmol JSH-23 in presence of CCRF-CEM. Supplementary Fig. 6: (A) The gating strategies and dot plot show the immunophenotyping of CD45+ CAR+ T cells from peripheral blood of mice models using flow cytometry. Data represent the mean ± SEM (n = 5). Supplementary Fig. 7: (A) Flow cytometric plots showing proportion of CD3−CD56+ NK cells of seven different donors after induced by IL-2 and IL-15 for a week. (B)&amp;(C) The plots present the proportion of lysed UCAR-T cells (n = 3) when co-cultured with primary NK cells from different donors at different E:T ratios over a 24-h period. Donor in (B) with approximately 20% CD161 expression. (D) The plots present the proportion of lysed UCAR-T cells (n = 3) every 24-h when autologous primary NK cells and donor UCAR-T cells were co-cultured at different 1:1 ratio over a 72-h period. (E) Surface expression of CD107 in NK cells of three different donors measured by flow cytometry, when co-cultured with 4KO CAR-T and 4KO-LLT1 CAR-T (n = 3). (F) The histogram shows the ratio represents the level of CAR-T cells expansion (n = 3), when mixed and cultured with onefold allogeneic NK cells and 30-fold CCRF-CEM-Luc-GFP cells for 48 h. (G) Representative flow plots showing the percentage of CAR-T cells and tumor cells in the well plate at the two time points of day 0 and day 2. (H) The histogram shows the ratio of GFP-positive tumor cells lysed after 48 h of reaction in comparison to the control group, which comprised NK cells and tumor cells without CAR-T cells (n = 3). Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student's t-test. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, and ****p &lt; 0.0001; ns, not significant. Supplementary Fig. 8: (A) The flow plots illustrate the proportion of T cells and NK cells in the primed PBMCs of four healthy donors. (B) Animal experimental timeline. Flowchart shows that mice (n = 5) were injected intravenously with 5 × 105 CCRF-CEM-Luc-GFP-B2MKO cells and 5 × 106 HLA-A2 mismatched alloreactive PBMC cells, followed by 2.5 × 106 CAR-T/PCDH-T cells and 2.5 × 106 NK cells 5 days later. Tumor burden of mice was measured every 1 week. (C) Bioluminescence imaging (BLI) results of tumor burden. (D) The mean value of average radiance in each group of mice was monitored using bioluminescence (n = 5). (E) Survival curves of humanized T-ALL model with alloreactive PBMCs (n = 5), the statistical significance was determined with log-rank test (Mantel-Cox). (F) The plot shows the quantity of CAR-T cells per 100 µl of mouse peripheral blood (n = 5). (G) Pathological analysis of the skin and lung of the representative four mice in every group at their terminal stage by HE staining. Magnification: 200 × . Data are presented as mean ± SEM and analyzed by two-tailed unpaired Student's t-test and log-rank test (Mantel-Cox). *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, and ****p &lt; 0.0001; ns, not significant.