The manufacture and expansion of meso3 CAR-T cells has been described previously[22]. To generate meso3-CD40 CAR-T cells, T cells were coelectroporated with a pNB338B-meso3 CAR plasmid and a pS338B-CD40 plasmid using an electroporator (Lonza) and the Amaxa® Human T Cell Nucleofector® Kit (Lonza). After transfection, CAR-T cells were cultured in AIM-V medium containing 2% FBS and then transferred to six-well plates coated with the rhMSLN antigen (5 μg/mL) and anti-CD28 (5 μg/mL) antibody after 4 h. The cells were stimulated by coated plates for 3–4 days in medium containing 200 U/mL recombinant human interleukin-2 (rhIL-2), and the activated CAR-T cells were then cultured in 2% FBS-AIM-V medium containing 100 U/mL rhIL-2 for another 5–10 days. Mock T cells were generated by electroporation with the mock plasmid and then stimulated with anti-CD3 (5 μg/mL) and anti-CD28 (5 μg/mL) antibodies. Thereafter, the culture method for mock T cells was the same as that used for meso3 CAR-T cells. Subsequent experiments were typically conducted 10 days after electroporation.

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