转化性/难治性T细胞恶性肿瘤患者的治疗选择有限。由于自体T细胞制品可能存在爆炸性污染以及针对T系列抗原的CAR-T细胞产生内源性杀伤，因此在T细胞恶性肿瘤中使用嵌合抗原受体(chimeric antigen receptor, CAR)-T细胞疗法具有挑战性。最近，外源性双阴性T细胞(double-negative T cells, DNTs)已被证明可作为即用型细胞免疫治疗的安全选择，并且可以进行CAR基因转导。在本研究中，我们探讨了外源性DNTs对T细胞恶性肿瘤的抗肿瘤活性，以及利用抗CD4-CAR (CAR4)-DNTs作为T细胞恶性肿瘤的细胞免疫治疗的潜力。我们通过流式细胞仪细胞毒性测定和异种移植模型研究了DNTs和CAR4-DNTs对T细胞急性淋巴细胞白血病（T-ALL）和外周T细胞淋巴瘤（PTCL）的抗肿瘤活性。我们通过转膜实验和阻断实验研究了其作用机制。 外源性DNTs能够诱导体外T-ALL和PTCL的内源性抗肿瘤细胞毒性，但在体内需要使用高剂量的DNTs才能达到治疗效果。将DNTs转导第三代CAR4显著增强了DNTs对T细胞恶性肿瘤的效力。CAR4-DNTs相对于空载体转导的DNTs来说，在体内和体外对CD4+ T-ALL和PTCL具有更优的细胞毒性。CAR4-DNTs能够消除体外的T-ALL和PTCL细胞系以及原发性T-ALL干细胞。CAR4-DNTs能够有效渗透到肿瘤组织中，延缓肿瘤进展，并延长T-ALL和PTCL的裸鼠异种移植模型的存活期。此外，使用PI3Kδ抑制剂伊地那麦促进了CAR4-DNTs的记忆表型，增强了它们在体内的持久性和抗白血病细胞效力。在DNT介导和CAR4-DNT介导的T-ALL和PTCL消灭过程中，LFA-1、NKG2D以及穿孔素/颗粒酶B释放途径起到了重要作用。 这些结果表明，CAR4-DNTs能够有效靶向T-ALL和PTCL，并为外源性CAR4-DNTs作为T细胞恶性肿瘤的细胞免疫治疗提供了支持。© 作者（或其雇主）2023。经CC BY-NC授权许可复用。商业再利用被禁止。请参阅权利和许可。BMJ出版。

Patients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous T-cell products and fratricide of CAR-T cells targeting T-lineage antigens. Recently, allogeneic double-negative T cells (DNTs) have been shown to be safe as an off-the-shelf adoptive cell therapy and to be amendable for CAR transduction. Here, we explore the antitumor activity of allogeneic DNTs against T-cell malignancies and the potential of using anti-CD4-CAR (CAR4)-DNTs as adoptive cell therapy for T-cell malignancies.Healthy donor-derived allogeneic DNTs were ex vivo expanded with or without CAR4 transduction. The antitumor activity of DNTs and CAR4-DNTs against T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) were examined using flow cytometry-based cytotoxicity assays and xenograft models. Mechanisms of action were investigated using transwell assays and blocking assays.Allogeneic DNTs induced endogenous antitumor cytotoxicity against T-ALL and PTCL in vitro, but high doses of DNTs were required to attain therapeutic effects in vivo. The potency of DNTs against T-cell malignancies was significantly enhanced by transducing DNTs with a third-generation CAR4. CAR4-DNTs were manufactured without fratricide and showed superior cytotoxicity against CD4+ T-ALL and PTCL in vitro and in vivo relative to empty-vector transduced-DNTs. CAR4-DNTs eliminated T-ALL and PTCL cell lines and primary T-ALL blasts in vitro. CAR4-DNTs effectively infiltrated tumors, delayed tumor progression, and prolonged the survival of T-ALL and PTCL xenografts. Further, pretreatment of CAR4-DNTs with PI3Kδ inhibitor idelalisib promoted memory phenotype of CAR4-DNTs and enhanced their persistence and antileukemic efficacy in vivo. Mechanistically, LFA-1, NKG2D, and perforin/granzyme B degranulation pathways were involved in the DNT-mediated and CAR4-DNT-mediated killing of T-ALL and PTCL.These results demonstrate that CAR4-DNTs can effectively target T-ALL and PTCL and support allogeneic CAR4-DNTs as adoptive cell therapy for T-cell malignancies.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.