Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19⁺ hematologic malignancies. 4-1BB–costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BBζ CAR T cells when compared to 28ζ CAR T cells. We showed that only BBζ CARs activated noncanonical nuclear factor κB (ncNF-κB) signaling in T cells basally and that the anti-CD19 BBζ CAR further enhanced ncNF-κB signaling after ligand engagement. Reducing ncNF-κB signaling reduced the expansion and survival of anti-CD19 BBζ T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BBζ and 28ζ CARs, they demonstrate the necessary and nonredundant role of ncNF-κB signaling in promoting the survival of BBζ CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.

嵌合抗原受体 (CAR) T 细胞疗法的临床反应与 CAR T 细胞的持久性相关，尤其是针对 CD19 ^{+ 的}CAR T 细胞血液系统恶性肿瘤。4-1BB 共刺激 CAR (BBζ) T 细胞在过继转移后表现出比 CD28 共刺激 CAR (28ζ) T 细胞更长的持久性。即使在 28ζ CAR 激活的情况下，4-1BB 信号也能改善 T 细胞的持久性，这表明由 4-1BB 细胞质结构域介导的不同促存活信号。为了专门研究 CAR 的信号转导，我们开发了一种用于 CD19 特异性 CAR T 细胞的无细胞、基于配体的激活和离体培养系统。与 28ζ CAR T 细胞相比，我们观察到 BBζ CAR T 细胞具有更高的离体存活率和随后的扩增。我们发现只有 BBζ CARs 激活了 T 细胞中的非经典核因子 κB (ncNF-κB) 信号，并且抗 CD19 BBζ CAR 在配体结合后进一步增强了 ncNF-κB 信号。减少 ncNF-κB 信号会降低抗 CD19 BBζ T 细胞的扩增和存活，并且与 Bim 最促凋亡同种型的丰度显着增加有关。尽管我们的研究结果不排除 BBζ 和 28ζ CAR 之间其他信号传导差异的重要性，但它们证明了 ncNF-κB 信号在促进 BBζ CAR T 细胞存活方面的必要和非冗余作用，这可能是使用该 CAR 观察到的植入持久性的基础设计。