目的：通用型CAR-T细胞作为一种预制化的免疫治疗手段，相比传统的自体CAR-T细胞疗法，具有显著的优势。它无需依赖患者自体细胞的提取与扩增，能够快速、方便地为患者提供治疗，从而大幅缩短治疗周期。然而，尽管通用CAR-T细胞在应用上具备了便捷性，其面临的免疫排斥问题仍然不容忽视。其中，移植物抗宿主病（GVHD）和机体免疫耐受（HVGR）是限制其临床疗效的重要因素。当前的策略，如通过删除T细胞受体（TCR）、人类白细胞抗原I类（HLA-I）和II类（HLA-II），虽能有效预防由同种异体T细胞引起的排斥反应，但由于自然杀伤细胞（NK细胞）依赖HLA-I进行自我识别，通用型CAR-T疗法仍然面临着NK细胞对CAR-T细胞免疫排斥的难题。为了解决这一问题，本研究探讨了通过过表达类凝集素转录体1（LLT1），一种已知的NK细胞抑制配体，来改善免疫排斥反应的可能性。本研究将LLT1的过表达引入敲降了TRAC（T细胞受体α恒定区），CD38和HLA-I/II表面抗原的通用型靶向CD38的CAR-T细胞中, 抑制异基因NK细胞与通用型CAR-T细胞的免疫排斥。

内容：（1）CD38通用CAR-T细胞的构建与基因编辑优化；（2）LLT1过表达维持CAR-T细胞的Naive/干细胞表型；（3）LLT1过表达增强CAR-T细胞的细胞毒性。（4）LLT1过表达增强CAR-T细胞的Naive/干细胞转录特征；（5）LLT1过表达赋予CAR-T细胞对异体NK细胞杀伤的抗性；（6）LLT1过表达增强CAR-T细胞对异体PBMCs杀伤的抗性；（7）LLT1过表达的CAR-T细胞在人源化T-ALL模型中的强效抗肿瘤活性。

方法：（1）通过CRISPR/Cas9技术敲除CD38基因，构建了CD38敲除的CAR-T细胞，解决了CAR-T细胞因CD38表达导致的自我杀伤问题。设计sgRNA靶向CD38、TRAC、B2M和CIITA基因，通过电转染实现多基因敲除，构建通用型CAR-T细胞；（2）利用慢病毒载体将LLT1基因与CD38 CAR共表达，生成4KO-LLT1 CAR-T细胞；（3）通过记忆表型、细胞毒性、增殖水平、细胞因子分泌和体内实验等多项功能学检测，评估了UCAR-T细胞的基本特性；（4）通过RNA测序技术探讨了LLT1分子在UCAR-T细胞中的作用机制，重点分析了其对细胞干性维持和细胞因子水平上升的相关信号通路的调控；（5）通过体外实验，分析了UCAR-T细胞与异体NK细胞的相互作用，并评估了LLT1分子对异体NK细胞杀伤的抗性。（6）通过体外同种异体淋巴细胞反应模型，对UCAR-T细胞和异基因外周血单核细胞的交互作用进行了研究，并评估了LLT1分子对免疫排斥反应的抑制作用；（7）在体内实验中，进一步通过人源化小鼠模型，评估了UCAR-T细胞的抗白血病效果及其在治疗血液肿瘤中的长期耐受性和持久性。

成果：（1）本研究通过基因编辑技术构建了敲除CD38、T细胞受体α恒定区、β-2微球蛋白（B2M）和II类主要组织相容性复合体转录激活因子（CIITA）的靶向CD38的通用型CAR-T细胞，并实现了类凝集素转录体1（LLT1）的过表达；（2）通过敲除B2M和CIITA，CD38靶向通用型CAR-T细胞表现出更高的增殖能力和IL-2分泌水平；（3）LLT1过表达的CAR-T细胞显示出更强的干性记忆表型标志物CD62L表达，并显著增强了其抗肿瘤活性；（4）转录组分析显示，LLT1的过表达不仅促进了与干性相关的标志物（如SELL、BCL6、TCF7和CD27）的上调，还导致了IL-10、IL-4、IL-5等免疫因子的水平升高，表明LLT1可以增强CAR-T细胞的抗肿瘤活性和维持CAR-T细胞的免疫记忆；（5）体外实验分析UCAR-T细胞与异体NK细胞共培养时的存活率与功能发挥，结果表明，LLT1的表达抑制了异体NK细胞对HLA^KOCAR-T的杀伤，保证了UCAR-T细胞抗肿瘤有效性；（6）体外实验分析UCAR-T细胞与异体PBMC细胞共培养时对异体免疫细胞的耐受性，LLT1过表达联合TCR和HLA基因敲低可显著降低CAR-T细胞的免疫原性，增强其对异源T细胞介导的溶解的抗性；（7）体内实验结果显示，LLT1过表达的CAR-T细胞抗白血病治疗中表现出显著的疗效，并且其在宿主免疫系统中的持久性得到有效提升，表明LLT1的引入可以有效增强UCAR-T细胞在体内的长期抗肿瘤功能。

结论：在本研究中，我们发现了在UCAR-T细胞表面过表达类凝集素转录体1（LLT1）这一NK细胞抑制性配体，能够有效防止UCAR-T遭受同种异体免疫排斥反应。这种LLT1过表达的通用型CAR-T细胞，具有较强的抗肿瘤活性和干性记忆性，在体外能有效杀伤ALL等多种细胞系，并能降低人源化ALL模型小鼠的体内肿瘤负荷并延长小鼠生存期。本研究为通用型CAR-T细胞的临床应用提供了新的技术路径和理论依据，尤其是在减少免疫排斥和提高疗效方面具有重要意义。

Objectives: Universal CAR-T cells provide significant advantages over traditional autologous CAR-T cell therapy as a preformed immunotherapy approach. Unlike autologous methods, universal CAR-T cells eliminate the need for patient-specific cell extraction and expansion, enabling faster and more convenient treatment delivery and shortening the treatment cycle. However, despite the convenience of universal CAR-T cells in application, the problem of immune rejection still cannot be ignored. However, despite their convenience, universal CAR-T cells face persistent challenges with immune rejection, particularly graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR), which remain major barriers to their clinical efficacy. Current strategies, such as deleting the T-cell receptor (TCR), human leukocyte antigen class I (HLA-I), and class II (HLA-II), effectively prevent rejection mediated by allogeneic T cells. However, since natural killer (NK) cells rely on HLA-I for self-recognition, this approach still leaves universal CAR-T cells vulnerable to NK cell rejection. To address this issue, this study investigates the potential of overexpressing lectin-like transcript factor 1 (LLT1), a known NK cell inhibitory ligand, to mitigate immune rejection. Specifically, LLT1 overexpression was introduced into universal anti-CD38 CAR-T cells with knocked-down T cell receptor alpha constant region (TRAC), CD38, and HLA-I/II surface antigens to prevent rejection by allogeneic NK cells.

Contents: (1) Construction and gene editing optimization of CD38 universal CAR-T cells. (2) LLT1 overexpression maintains the naive/stem cell phenotype of CAR-T cells. (3) LLT1 overexpression enhances the cytotoxicity of CAR-T cells in vitro and in vivo. (4) LLT1 overexpression enhances the naive/stem cell transcriptional characteristics of CAR-T cells. (5) LLT1 overexpression confers resistance to allogeneic NK cell killing in CAR-T cells. (6) LLT1 overexpression enhances resistance to allogeneic peripheral blood mononuclear cell (PBMC) killing in CAR-T cells. (7) LLT1-overexpressing CAR-T cells exhibit potent anti-tumor activity in a humanized T-ALL model.

Methods: (1) CD38, TCR, HLA-I, and HLA-II genes were knocked down in T cells using the CRISPR/Cas9 system, followed by lentiviral transduction. (2) To create CD38-targeting universal CAR-T cells, T cells were transduced with lentiviruses that encoded the LLT1 and CD38 CAR designs. (3) The basic characteristics of universal CAR-T (UCAR-T) cells were evaluated through multiple functional assays, including memory phenotype, cytotoxicity, proliferation, cytokine secretion, and in vivo experiments. (4) RNA sequencing was used to explore the mechanism of LLT1 in UCAR-T cells, focusing on its regulation of stemness maintenance and cytokine-related signaling pathways. (5) In vitro experiments analyzed the interaction between UCAR-T cells and allogeneic NK cells, evaluating the resistance conferred by LLT1 to NK cell killing. (6) An in vitro allogeneic lymphocyte reaction model was used to analyze the interaction between UCAR-T cells and allogeneic PBMCs, assessing the inhibitory effect of LLT1 on immune rejection. (7) In vivo experiments evaluated the anti-leukemia efficacy, long-term tolerance, and durability of UCAR-T cells in a humanized mouse model.

Results: (1) This study successfully constructed universal anti-CD38 CAR-T cells with knocked-out CD38, TRAC, β-2-microglobulin (B2M), and class II major histocompatibility complex transactivator (CIITA), alongside LLT1 overexpression. (2) B2M and CIITA knockout enhanced the proliferation capacity and IL-2 secretion levels of CD38-targeting universal CAR-T cells. (3) LLT1-overexpressing CAR-T cells exhibited higher expression of the naive/stem cell memory phenotype marker CD62L and demonstrated stronger anti-tumor activity. (4) Transcriptome analysis revealed that LLT1 overexpression upregulated stemness-related markers (e.g., SELL, BCL6, TCF7, CD27) and increased levels of immune factors (e.g., IL-10, IL-4, IL-5), indicating enhanced anti-tumor activity and immune memory maintenance. (5) In vitro co-culture experiments showed that LLT1 expression protected HLA^KO CAR-T cells from allogeneic NK cell killing, ensuring their anti-tumor efficacy. (6) Co-culture with allogeneic PBMCs demonstrated that LLT1 overexpression combined with TCR and HLA knockdown significantly reduced CAR-T cell immunogenicity and enhanced resistance to allogeneic T cell-mediated cytolysis. (7) In vivo experiments confirmed that LLT1-overexpressing CAR-T cells exhibited potent anti-leukemia activity and improved persistence in the host immune system, highlighting their long-term anti-tumor potential.

Conclusions: This study demonstrates that overexpressing lectin-like transcript factor 1 (LLT1), an NK cell inhibitory ligand, on the surface of UCAR-T cells effectively prevents allogeneic immune rejection. These LLT1-overexpressing universal CAR-T cells exhibit robust anti-tumor activity and stemness memory, effectively killing ALL cell lines in vitro, reducing tumor burden in humanized ALL model mice, and prolonging survival. This research provides novel technical approaches and theoretical foundations for the clinical application of universal CAR-T cells, particularly in reducing immune rejection and enhancing therapeutic efficacy.