Fig 1: Screening of anti-CD70 CAR sequence (A) Schematic diagram illustrating the construction of the anti-CD70 CAR-T. (B) Luminescence intensity of CAR-expressing Jurkat reporter cells after co-culturing with 786-0 cells. (C) The selected CAR-expressing Jurkat reporter cells from (B) were co-cultured with either 786-0 or CD70 knockout 786-0 cells. The luminescence intensity ratio of Jurkat cells co-cultured with the two groups was calculated. (D) Cytotoxicity of CAR-T cells obtained from (C) was assessed using a short-term killing assay, with P8F8 serving as a positive control. (E) Results from membrane protein array (MPA) testing for the CAR sequences selected from (D). (F, G) The anti-tumor activity of various CAR-T cells was evaluated in ACHN (F) and 786-0 (G) tumor xenograft models. (H, I) Labeled and unlabeled CAR-T cells were co-cultured, and the downregulation of CD70 positive rate (H) and the apoptotic rate (I) of the CFSE-labeled CAR-T cells were measured to assess fratricide among the constructed CAR-T cells. Data are presented as means ± SD. Statistical significance was determined using Student’s t-test. ****p < 0.0001; ns, not significant.
Fig 2: Sodium citrate-pretreated CAR-T cells exhibited enhanced anti-solid tumor activity and reduce cancer relapse in mice. (A) Experimental setup: 786-0 tumor cells were subcutaneously injected into NKG mice on day 0. PBS, mock T cells, untreated anti-CD70 CAR-T cells, or sodium citrate-pretreated anti-CD70 CAR-T cells were injected via tail vein on day 16. (B, C) BLI images (B) and tumor growth quantification (C) at indicated time points (n = 5). (D) Tumor volumes over time for each group (n = 6). (E, F) Flow cytometry analysis of CAR-T cell number (E) and percentage (F) in peripheral blood at day 6 (n = 6). (G) CD62L expression in CAR-T cells from peripheral blood (n = 6). (H) PD-1 and LAG-3 expression in CAR-T cells from peripheral blood (n = 6). (I) Representative IHC staining for CD3 in tumor tissues from NKG mice treated with untreated or sodium citrate-pretreated CAR-T cells. Scale bar = 100 μm. (J) Body weight changes of mice throughout the experiment (n = 6). (K) Representative H&E staining of heart, liver, spleen, lungs, kidneys and brain from mock T, untreated CAR-T, and sodium citrate-pretreated CAR-T groups. Scale bar = 100 μm. Results were expressed as mean ± SD. Statistical significance was determined by t-test (E–G) and two-way ANOVA (H). ***p < 0.001.
Fig 3: Long-term culture induced CAR-T cell exhaustion, excessive activation and reduced cytotoxicity. (A) Representative flow cytometric profile showing the expression of CCR7 and CD45RA on CAR-T cells. (B) Proportions of Tscm (CCR7+CD45RA+), Tcm (CCR7+CD45RA-), Tem (CCR7-CD45RA-), and Teff (CCR7-CD45RA+) subsets in anti-CD70 CAR-T cells as measured by flow cytometry. (C) Representative flow cytometric profile showing the expression level of CD62L on CAR-T cells. (D) Histogram plot showing the percentage of CD62L+ CAR-T cells. (E, F) Mean fluorescence intensity (MFI) of CD25 (E) and CD69 (F) expression on CAR-T cells. (G) Levels of TNF-α and IFN-γ released by CAR-T cells at different time points, measured by ELISA. (H–J) MFI of exhaustion markers PD-1 (H), TIM-3 (I), and LAG-3 (J) in anti-CD70 CAR-T cells. (K–M) Cytotoxicity of anti-CD70 CAR-T cells against 786-0 cells after co-culture at the indicated effector-to-target (E:T) ratios for 24 hours (K), 48 hours (L), and 72 hours (M). Data were expressed as mean ± SD from at least 3 independent donors. Statistical significance was determined by t-test (D–F, H–J) and two-way ANOVA (G, K–M). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from ACROBiosystems for FITC-Labeled Human CD27 Ligand / CD70 Protein, His TagStar Staining