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. 2019 Jun 27;10(10):764–769. doi: 10.1007/s13238-019-0643-y

Figure 2.

Figure 2

19BB-CAR-T cells cultured with IL-7/IL-15 show a superior antitumor phenotypein vitroand enhanced grafting efficiency after infusion into tumor-bearing mice. (A) 19BB-CAR-T cells cultured with IL-7/IL-15 generate a higher percentage of CD8+ T cells compared to cells cultured with IL-2. Bars show the distribution of CD4+ and CD8+ T cells in 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 3 (D3) and day 11 (D11). Data are presented as mean ± SEM from 4 independent experiments. (B) 19BB-CAR-T cells cultured with IL-7/IL-15 generate a higher percentage of CD8+ naïve cells (TN) compared to cells cultured with IL-2. Expression of CD45RA and CD62L was assessed by flow cytometry analysis of 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 5 (D5). The percentages of TN (CD45RA+CD62L+), TCM (CD45RA−CD62L+), TEM (CD45RA−CD62L−), and TRAEM (CD45RA+CD62L−) in CD8+ lymphocytes (left) and CD4+ lymphocytes (right) are shown. Results are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (C) 19BB-CAR-T cells expanded with IL-7/IL-15 generate a larger population of central memory T cells during culture. The percentages of TN (CD45RA+CD62L+), TCM (CD45RA−CD62L+), TEM (CD45RA−CD62L−), and TRAEM (CD45RA+CD62L−) in CD8+ lymphocytes (left) and CD4+ lymphocytes (right) in 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 are shown. Data are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (D) IL-7/IL-15 enhance CCR7 expression compared to IL-2. The expression of CCR7 on 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 was detected by flow cytometry. Results are presented as mean ± SEM from 4 independent experiments, *P < 0.05. (E) 19BB-CAR-T cells cultured with IL-7/IL-15 show higher migration ability compared to cells cultured with IL-2. Results are presented as mean ± SEM from 3 independent experiments, **P < 0.01. (F) IL-7/IL-15 decreases the Foxp3+CD4+ T cell population. The expression of Foxp3 in 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 at day 11 was detected by flow cytometry. Results are shown as mean ± SEM from 4 independent experiments, *P < 0.05. (G) The percentage of 19BB-CAR-T cells expressing the inhibitory receptor PD-1 is lower after culture with IL-7/IL-15 than with IL-2. The expression of PD-1, LAG-3 and TIM-3 on 19BB-CAR-T cells cultured with IL-7/IL-15 or IL-2 was determined by flow cytometry. Results are presented as mean ± SEM from 3 independent experiments, *P < 0.05. (H) IL-7/IL-15 increase the survival rate of 19BB-CAR-T cells in peripheral blood of tumor-bearing mice. CD3+GFP+ cells were detected in peripheral blood by flow cytometry in lymphoma-bearing mice at days 14 and 21 after infusion. Results are presented as mean ± SEM from 5 independent experiments, *P < 0.05. (I) IL-7/IL-15 enhance survival of 19BB-CAR-T cells in spleen of tumor-bearing mice after infusion. CD3+GFP+ cells were detected by flow cytometry in the spleen of lymphoma-bearing mice at day 21 after infusion. Results are shown as mean ± SEM from 5 independent experiments, *P < 0.05. (J) Copy numbers of 19BB-CAR vector per microgram genomic DNA in the peripheral blood of mice receiving 19BB-CAR-T cells at day 60 after infusion. Results are shown as mean ± SEM from 4 independent experiments, *P < 0.05. (K) IL-7/IL-15 maintain the CD8+ TCM (CD45RA-CD62L+) population in 19BB-CAR-T cells from peripheral blood in tumor-bearing mice. Flow cytometry results are presented as mean ± SEM from 4 independent experiments, *P < 0.05