Fig 1: IL-21 promotes CAR-iNKT cells to inhibit subcutaneous tumors(A) Schematic of in vivo evaluation scheme for the effectiveness of B7H3.CAR iNKT cells. NSG mice were injected subcutaneously with 4 × 106 786-O renal tumor cells, which was recorded as day 0. CAR-iNKT cells were administered via tail vein injection starting from the 12th day, and the body weight and tumor volume were measured every 3–4 days. For three continuous weeks following treatment, the frequency of B7H3.CAR-iNKT cells was found in the peripheral blood of NCG mice (n = 5 mice).(B) Administration of B7H3.CAR-iNKT cells and B7H3-IL21.CAR-iNKT cells decreased the tumor volume. Data are represented as mean ± SD of five mice in each group, and statistical significance was calculated with ANOVA multiple comparison. (Saline vs. B7H3-IL21.CAR-iNKT: p = 0.0008, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNK: p = 0.0425, one-way ANOVA. Data are presented as the mean ± SD, n = 5 mice).(C) At days 7, 14, and 21 following treatment, CAR-iNKT cells was found by flow cytometry in the peripheral blood. (NS: not significant, Day14: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0046, Day21: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p < 0.001, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 5 mice).(D) Percentage change in body weight in tumor-bearing mice. Percentage weight change was calculated as: (weight at time X – weight at time 0)/(weight at time 0). (Saline vs. B7H3-IL21.CAR-iNKT: p < 0.001, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 5 mice).
Fig 2: Functional assessment of B7H3.CAR-iNKT cells co-expressing IL21 in vitro(A) Western blot analysis of the level of STAT1, STAT3, and STAT5 and the expression of phosphorylated STAT1 (pSTAT1), phosphorylated STAT3 (pSTAT3), and phosphorylated STAT5 (pSTAT5) in B7H3.CAR-iNKT and B7H3-IL21.CAR-iNKT cells co-cultured with or without 786-O cell.(B and C) B7H3.CAR-iNKT and B7H3-IL21.CAR-iNKT cells were stained with annexin-V and PI and cultured alone in complete medium without cytokine for 2 days. PI-positive/Annexin-V-positive cells were considered as necrotic cells, PI-negative/Annexin-V-positive cells were considered as apoptosis cells, and PI-negative/Annexin-V-negative cells were considered as viable cells, which were assessed by flow cytometry and represented as histograms (Necrotic cell: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0084, Apoptosis cell: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0025, Viable cell: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0271, Student’s paired t-test. Data are presented as the mean ± SD, n = 3).(D and E) B7H3.CAR-iNKT and B7H3-IL21.CAR-iNKT cells were counted and evaluated for the expression of the exhaustion markers after each round of tumor stimulation (NS: not significant, % of LAG-3+: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (III), p = 0.0311, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (IV), p = 0.0022, % of PD-1+: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (II), p = 0.0317, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (III), p = 0.0068, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (IV), p = 0.041, % of Tim-3+: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (IV), p = 0.017, iNKT cells fold expansion: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (I), p = 0.0252, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (II), p = 0.0056, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (III), p = 0.0037, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT (IV), p = 0.004, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 3).
Fig 3: IL-21 significantly prolonged the survival of CAR-iNKT cells in solid tumor-bearing mice(A) Overview of in vivo evaluation scheme for B7H3-IL-21.CAR-iNKT cells persistence. NSG mice were injected intravenously with 1×106 786-O renal tumor cell and then with 1 × 107 B7H3.CAR-iNKT cells with or without IL-21. iNKT cells were tracked by bioluminescence imaging every 3 days after treatment.(B) Bioluminescent monitoring of iNKT, B7H3.CAR-iNKT, and B7H3-IL21.CAR-iNKT cells injected into mice xenografts models of 786-O renal tumor cell (n = 5 mice).(C) Quantification of bioluminescence images in B (B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0394, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 5 mice).(D) At days 6 and 12 following CAR-iNKT cell treatment, CAR-iNKT cells were identified by flow cytometry in the peripheral blood (Day6: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0287, Day12: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0462. one-way ANOVA. Data are presented as the mean ± SD, n = 5 mice).(E) Quantification of iNKT (human CD45+/iNKT+) in lung total cells as determined by flow cytometry. (iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0253, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0269, one-way ANOVA. Data are presented as the mean ± SD, n = 3 mice).(F) Quantification of tumor cells (human CD45-/B7H3+) in lung total cells as determined by flow cytometry. (iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0012, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0021, one-way ANOVA. Data are presented as the mean ± SD, n = 3 mice).(G) Quantification of human iNKT cells in spleen total cells as determined by flow cytometry (iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0132, B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0177, one-way ANOVA. Data are presented as the mean ± SD, n = 3 mice).
Fig 4: Co-expression of IL-21 in CAR-iNKT treatment group effectively controlled tumor recurrence(A) Schematic illustration of treatment process in situ tumor model. In total, 2 × 105 luciferase-labeled 786-O renal tumor cells were injected in the renal capsule, followed by 8 × 106 B7H3.CAR-iNKT with or without IL-21. Tumor measurement was assessed weekly by bioluminescence imaging (n = 4 mice).(B) Bioluminescent monitoring of saline, B7H3.CAR, and B7H3-IL21.CAR-iNKT cells injected into the tumor mice models.(C) Quantification of bioluminescence images in B (B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0471, Student’s unpaired t-test. n = 4).(D) Assay of CAR iNKT cells in peripheral blood by flow cytometry (Week2: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0399, Week3: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0045, Week4: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0189, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 4 mice).(E) Kaplan–Meier curve generated from survival of animals in B (NS: not significant. Saline vs. B7H3-IL21.CAR-iNKT: p = 0.0084, Log rank (Mantel-Cox). n = 4 mice).(F) The quantification of human iNKT cells (human CD45+/iNKT+) collected from peripheral blood mononuclear cells (PBMNC) by flow cytometry (B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0218, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 3 mice).(G) The quantification of human iNKT cells (human CD45+/iNKT+) collected from spleen by flow cytometry (B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0119, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 3 mice).
Fig 5: Functional assay of CAR-iNKT cells generated by the induction system(A) Schematic of retroviral constructs encoding B7H3.CAR with and without IL21.(B and C) CAR expression in iNKT cells transduced (on day 3 after stimulation with CD3/28 antibody) as determined using retroviral vectors containing the indicated B7H3.CAR constructs as measured by flow cytometry (NS: not significant, Student’s paired t-test. Data are presented as the mean ± SD, n = 5).(D) Representative flow cytometric analysis of B7H3 on 786-O cell.(E) ELISA assessment of IL-21 releases from B7H3.CAR-iNKT and B7H3-IL21.CAR-iNKT cells cultured alone or at a 1:1 E:T ratio with B7H3-positive 786-O cells stimulated for 24h (B7H3.CAR-iNKT without 786-O vs. B7H3-IL21.CAR-iNKT without 786-O: p < 0.001, B7H3.CAR-iNKT with 786-O vs. B7H3-IL21.CAR-iNKT with 786-O: p < 0.001, B7H3-IL21.CAR-iNKT without 786-O vs. B7H3-IL21.CAR-iNKT with 786-O: p < 0.001, Student’s paired t-test. Data are presented as the mean ± SD, n = 3).(F and G) Representative flow cytometry analysis of CD62L expression on CAR-iNKT cells with or without IL-21 on day 7 after transduction (B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT: p = 0.0298, Student’s paired t-test. Data are presented as the mean ± SD, n = 3).(H and I) Graph of the real-time cell analysis results that show the cytotoxic effect of B7H3.CAR-iNKT cells with or without IL-21 against renal cells 786-O after co-culture for 50 h at E:T ratios of 5:1, 1:1, and 1:5 (NS: not significant, Student’s paired t-test. Data are presented as the mean ± SD, n = 3).(J) iNKT and B7H3.CAR-iNKT cells with or without IL-21 were stimulated by 786-O cell, supernatants were collected after 24 h, and concentrations of the indicated cytokines were quantified using a multi-analyte flow assay kit (NS: not significant, TNF-α: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0091, IFN-γ: B7H3.CAR-iNKT vs. B7H3-IL21.CAR-iNKT, p = 0.0128, Student’s unpaired t-test. Data are presented as the mean ± SD, n = 3).
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