Vector transfection and transduction CAR and CAR/CCR expression was induced by retroviral transduction. 10cm plates with a monolayer of HEK293t cells were transfected at 50–70% confluence using 20 µL of PEIpro with 10 µg construct vector DNA and 5 µg each of pCOLT-GAL-V and pHIT60 (MoMuLV) plasmids in 500 µL RPMI 1640. PEIpro was added onto the HEK293t cells cultured in 9mL of RPMI 1640 with 10% FCS, Pen/Strep and HEPES buffer. The culture medium containing transduction viruses was harvested after 10–24 h and added to 1.6e7 PBMC-derived T cells in a plate coated with poly-D-Lysin, centrifuged at 1600 x g for 90 min and cultured overnight. The process was repeated a second time with the same cells to improve transduction yields. During transduction T cells were kept stimulated overnight with 1000 U.mL^-1 IL-2 after the first, and 500–800 U.mL^-1 IL-2 after the second run. mCAR and mCAR/mCCR expression was also induced by retroviral transduction similar to the process detailed for human PBMC. The X-Vivo 15 media used were supplemented with 5% FCS and 10 mg.mL-1 protamine sulfate, and cells stimulated with 500 U.mL^-1 IL-2 and 10 ng.mL^-1 IL-15. The virus containing medium was centrifuged onto 6-well plates coated with Poly-D-Lysin at 1200 x g for 90 min after which the cells were added. GFP/tdTomato transduction of tumor cell lines Stable eGFP expression was induced in tumor cell lines via retroviral transduction. Two 10cm plates with a monolayer of HEK293t cells were transfected at 50–70% confluence using 20 µL of PEIpro with 10 µg DNA of plasmid pQCXI-P_eGFP and 5 µg each of pHIT60 (MoMuLV) and pMD2.G (VSV-G envelope) in 500 µL RPMI 1640. Culture supernatant containing transduction viruses was harvested after 10–24 h, added to 5x10^5 tumor cells, centrifuged at 1600 x g for 90 min and cultured for several days. Plasmid expressing cells were selected via puromycin treatment and fluorescence confirmed via flow cytometry on the Canto II platform. Stable tdTomato/fLuc expression was induced in Raji cells via retroviral transduction applying the same procedure, substituting the eGFP plasmid with 10 µg of pQCXIP_fLuc_tdTomato.

Chimeric antigen receptor T cell (CAR T) therapy is a potent treatment for relapsed/refractory (r/r) B cell lymphomas but provides lasting remissions in only ∼40% of patients and is associated with serious adverse events. We identify an upregulation of CD80 and/or CD86 in tumor tissue of (r/r) diffuse large B cell lymphoma (DLBCL) patients treated with tisagenlecleucel. This finding leads to the development of the CAR/CCR (chimeric checkpoint receptor) design, which consists of a CD19-specific first-generation CAR co-expressed with a recombinant CTLA-4-linked receptor with a 4-1BB co-stimulatory domain. CAR/CCR T cells demonstrate superior efficacy in xenograft mouse models compared with CAR T cells, superior long-term activity, and superior selectivity in in vitro assays with non-malignant CD19⁺ cells. In addition, immunocompetent mice show an intact CD80^-CD19⁺ B cell population after CAR/CCR T cell treatment. The results reveal the CAR/CCR design as a promising strategy for further translational study.