We co-cultured 10^5 DPP4-K562 cells with 10^5 anti-DPP4 CAR-T cells and 10^5 Jurkat cells, respectively, and added the appropriate amount of medium. And three sets of replicate experiments were set up. Afterwards 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h and 48h, respectively.

We co-cultured 10^5 DPP4-K562 cells with and without 3*10^5 anti-DPP4 CAR-T cells, respectively, and added the appropriate amount of culture medium. And three sets of replicate experiments were set up. After that 200 µl of the co-culture system (with the addition of 0.2 test DPP4 antibody) was counted by flow collection at 24h.

As can be seen from the figure, our constructed CAR-T killing system exhibited a significant killing effect in the target cells. This indicates that our killing system was engineered successfully. We will further test the release of cytokines.

We first transfected plasmids into RAW 264.7 cell line on the first day using Zetalife Advanced DNA RNA transfection reagent. The transfection efficiency was measured by flow assay on the third day. After the transfection efficiency test (>90%), we co-cultured 3*10^5 RAW 264.7 cells and 10^5 Raji cells in a total system of 200 µl. We set three concentrations (0, 1, 10 µl) depending on the amount of coffee (coffee was provided by ZJY Coffee). The number of Raji cells (Raji cells transfected with the luciferase gene) was detected by enzyme marker at 0h, 4h, and 24h after co-culture.

As the result, The CAR-COSMO-M killing system showed good lethality after the addition of caffeine, but the CAR-COSMO modulation system did not seem to completely inhibit the killing of Raji cell lines by CAR-M, which we believed is in error with the real result. After analysis, we thought that two biases in the experiment lead to erroneous experimental results. The first reason is that we used an enzymatic marker to measure the data but did not consider the effect of the color of the coffee itself on the data. In addition, we selected macrophages as chassis cells, which are not in full contact with the target cells, suspension cells of the Raji cell line. This may result in poor killing efficacy. Anyway, the result showed that the CAR-COSMO-M killing system exhibited good killing in the presence of caffeine, verifying the lethality of our product and the feasibility of the COSMO switch.

In humans, the concentration of IL6 is usually maintained at 0.00-2.90 pg/ml. With this in mind, we set up two sets of normal concentrations and three sets of abnormal concentrations to treat the cells that were then transferred. We found that the killing ability of Jurkat was enhanced after IL6 treatment, as shown in Figure 7. In the design concept of module1, we were expecting the expression of CAR to be inhibited at abnormal IL6 concentrations, thus reducing the side effects of killing; hence the problem with the system we designed. As the results of PD1 are shown in Figure 7, we can see that at high concentrations, PD1 successfully disrupts the signaling downstream of CAR-T, reducing the killing ability.

Although the experiment successfully proved that our system is problem-free, the activity of transfected Jurkat is not really high and the choice of dual promoter in the plot resulted in not high CAR expression, thus his killing is not very obvious. In addition, the concentration gradient in the normal group was set low and additional experimental groups should be set up in subsequent experiments.