CD19-重定向嵌合抗原受体（CAR）T细胞对B细胞癌表现出显著的活性。尽管第二代CAR在急性淋巴细胞白血病患者中诱导完全缓解的比例超过80％，但类似的单一疗法仅在慢性淋巴细胞白血病（CLL）患者中诱导长期缓解的比例仅为26％。这种差异归因于自体来源的CLL T细胞中效应物缺陷的细胞内本质。然而，白血病细胞影响CAR T细胞效力的机制尚不清楚。在此，我们描述了一种体外检测，可在健康供体CAR T细胞中重现内源性CLL介导的T细胞缺陷。与CLL细胞接触无法充分激活CAR T细胞功能，但不会造成不可逆的损害。这种状态可以通过强抗原刺激或IL2来挽救，并不是由于免疫抑制所致。相反，这种激活缺陷归因于CLL细胞上低水平的共刺激分子，并且外源性共刺激能增强CAR T细胞的激活。接下来，我们评估了来自同一患者不同环境中的CLL细胞的刺激表型。淋巴结（LN）来源的CLL细胞具有较强的共刺激表型，并促进更好的CAR T细胞去颗粒化和细胞因子产生，而匹配的外周血CLL细胞则不然。最后，在体外经CD40L激活的CLL细胞获得了类似于LN来源的肿瘤的共刺激表型，并刺激了更强的CAR T细胞增殖、细胞因子产生和细胞毒性。这些数据共同表明，不充分的激活是CLL CAR T细胞反应不良的驱动因素。CLL细胞的共刺激表型驱动不同的CAR T细胞反应，并且可以通过改善共刺激信号来增强。©2022年作者；由美国癌症研究协会出版。

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling.CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.© 2022 The Authors; Published by the American Association for Cancer Research.