Protocol for the expansion of mouse immune effector cells for in vitro and in vivo studies.

Reproducible and efficient expansion of different immune effector cells is required for pre-clinical studies investigating adoptive cell therapies against cancer. Here, we provide a protocol for the rapid expansion of mouse T cells, natural killer (NK) cells, and bone-marrow-derived macrophages (BMDMs). We describe steps for αCD3/αCD8 plate coating, isolating splenocytes, and expanding T cells and NK cells. Further, we detail procedures for bone marrow isolation and BMDM differentiation.

Multifunctional mRNA-Based CAR T Cells Display Promising Antitumor Activity Against Glioblastoma.

PURPOSE: Most chimeric antigen receptor (CAR) T-cell strategies against glioblastoma have demonstrated only modest therapeutic activity and are based on persistent gene modification strategies that have limited transgene capacity, long manufacturing processes, and the risk for uncontrollable off-tumor toxicities. mRNA-based T-cell modifications are an emerging safe, rapid, and cost-effective alternative to overcome these challenges, but are underexplored against glioblastoma. EXPERIMENTAL DESIGN: We generated mouse and human mRNA-based multifunctional T cells coexpressing a multitargeting CAR based on the natural killer group 2D (NKG2D) receptor and the proinflammatory cytokines IL12 and IFNα2 and assessed their antiglioma activity in vitro and in vivo. RESULTS: Compared with T cells that either expressed the CAR or cytokines alone, multifunctional CAR T cells demonstrated increased antiglioma activity in vitro and in vivo in three orthotopic immunocompetent mouse glioma models without signs of toxicity. Mechanistically, the coexpression of IL12 and IFNα2 in addition to the CAR promoted a proinflammatory tumor microenvironment and reduced T-cell exhaustion as demonstrated by ex vivo immune phenotyping, cytokine profiling, and RNA sequencing. The translational potential was demonstrated by image-based single-cell analyses of mRNA-modified T cells in patient glioblastoma samples with a complex cellular microenvironment. This revealed strong antiglioma activity of human mRNA-based multifunctional NKG2D CAR T cells coexpressing IL12 and IFNα2 whereas T cells that expressed either the CAR or cytokines alone did not demonstrate comparable antiglioma activity. CONCLUSIONS: These data provide a robust rationale for future clinical studies with mRNA-based multifunctional CAR T cells to treat malignant brain tumors.