LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells
Abstract Background The benefit of universal CAR-T cells over autologous CAR-T cell therapy is that they are a treatment that is ready to use. However, the prevention of graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR) remains challenging. Deleting class I of human leukocyte an...
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2025-01-01
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| Series: | Journal of Experimental & Clinical Cancer Research |
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| Online Access: | https://doi.org/10.1186/s13046-025-03273-2 |
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| author | Shuxian Zhu Shiyu Zuo Chuo Li Xingjie You Erlie Jiang Xiaoming Feng Yuechen Luo |
| author_facet | Shuxian Zhu Shiyu Zuo Chuo Li Xingjie You Erlie Jiang Xiaoming Feng Yuechen Luo |
| author_sort | Shuxian Zhu |
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| description | Abstract Background The benefit of universal CAR-T cells over autologous CAR-T cell therapy is that they are a treatment that is ready to use. However, the prevention of graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR) remains challenging. Deleting class I of human leukocyte antigen (HLA-I) and class II of human leukocyte antigen (HLA-II) can prevent rejection by allogeneic T cells; however, natural killer (NK) cell rejection due to the loss of self-recognition remains unresolved. This study tested whether the overexpression of Lectin-like transcript 1 (LLT1), an NK cell inhibitory ligand, in T cell receptor (TCR) and HLA-I/II disrupted universal CD38-targeting CAR-T cells could prevent rejection by allogeneic NK cells. Methods We generated CD38-targeting universal CAR-T cells by transducing T cells with lentiviruses encoding the CD38 CAR and LLT1 constructs. T cells were subjected to CD38, TCR, HLA-I, and HLA-II gene knockdown using CRISPR/Cas9, followed by lentiviral transduction. We performed cytotoxicity, proliferation, and cytokine assays to evaluate the functionality of universal chimeric antigen receptor-T cell (UCAR-T) cells and conducted in vitro and in vivo assays, including allogeneic responses and RNA sequencing, to assess their resistance to allogeneic T and NK cells, anti-leukemia efficacy, and persistence in treating hematologic malignancies. Results Genetic editing of CD38 universal CAR-T cells, including CD38, T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), and class II major histocompatibility complex transactivator (CIITA) knockdowns, was successfully achieved. In vitro, LLT1 overexpression boosted CAR-T cell proliferation and antitumor activity, leading to a transcriptional signature characterized by elevated stemness-related markers (SELL, BCL6, TCF7, and CD27) and increased levels of IL-10 and other cytokines. It also effectively mitigates rejection by allogeneic NK and T cells. In a humanized T-cell acute lymphoblastic leukemia (T-ALL) model, CD38 allogeneic universal CAR-T cells demonstrated superior survival rates and tumor clearance with reduced inflammatory responses. Conclusion According to these results, LLT1 overexpression enhances UCAR-T cell activity and prevents allogeneic rejection, providing essential insights for the development of universal CAR-T cell therapy. |
| format | Article |
| id | doaj-art-29493353804a47b49f35285d3f7f2c4d |
| institution | Kabale University |
| issn | 1756-9966 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Experimental & Clinical Cancer Research |
| spelling | doaj-art-29493353804a47b49f35285d3f7f2c4d2025-01-26T12:57:56ZengBMCJournal of Experimental & Clinical Cancer Research1756-99662025-01-0144112210.1186/s13046-025-03273-2LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cellsShuxian Zhu0Shiyu Zuo1Chuo Li2Xingjie You3Erlie Jiang4Xiaoming Feng5Yuechen Luo6State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeGeriatric Medical Center, Division of Geriatric Gastroenterology, The First Affiliated Hospital of Kunming Medical UniversityState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeState Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical CollegeAbstract Background The benefit of universal CAR-T cells over autologous CAR-T cell therapy is that they are a treatment that is ready to use. However, the prevention of graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR) remains challenging. Deleting class I of human leukocyte antigen (HLA-I) and class II of human leukocyte antigen (HLA-II) can prevent rejection by allogeneic T cells; however, natural killer (NK) cell rejection due to the loss of self-recognition remains unresolved. This study tested whether the overexpression of Lectin-like transcript 1 (LLT1), an NK cell inhibitory ligand, in T cell receptor (TCR) and HLA-I/II disrupted universal CD38-targeting CAR-T cells could prevent rejection by allogeneic NK cells. Methods We generated CD38-targeting universal CAR-T cells by transducing T cells with lentiviruses encoding the CD38 CAR and LLT1 constructs. T cells were subjected to CD38, TCR, HLA-I, and HLA-II gene knockdown using CRISPR/Cas9, followed by lentiviral transduction. We performed cytotoxicity, proliferation, and cytokine assays to evaluate the functionality of universal chimeric antigen receptor-T cell (UCAR-T) cells and conducted in vitro and in vivo assays, including allogeneic responses and RNA sequencing, to assess their resistance to allogeneic T and NK cells, anti-leukemia efficacy, and persistence in treating hematologic malignancies. Results Genetic editing of CD38 universal CAR-T cells, including CD38, T cell receptor alpha constant (TRAC), beta-2-microglobulin (B2M), and class II major histocompatibility complex transactivator (CIITA) knockdowns, was successfully achieved. In vitro, LLT1 overexpression boosted CAR-T cell proliferation and antitumor activity, leading to a transcriptional signature characterized by elevated stemness-related markers (SELL, BCL6, TCF7, and CD27) and increased levels of IL-10 and other cytokines. It also effectively mitigates rejection by allogeneic NK and T cells. In a humanized T-cell acute lymphoblastic leukemia (T-ALL) model, CD38 allogeneic universal CAR-T cells demonstrated superior survival rates and tumor clearance with reduced inflammatory responses. Conclusion According to these results, LLT1 overexpression enhances UCAR-T cell activity and prevents allogeneic rejection, providing essential insights for the development of universal CAR-T cell therapy.https://doi.org/10.1186/s13046-025-03273-2Universal CAR-T CellsNK Cell InhibitionAllogeneic RejectionT Cell Stemness |
| spellingShingle | Shuxian Zhu Shiyu Zuo Chuo Li Xingjie You Erlie Jiang Xiaoming Feng Yuechen Luo LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells Journal of Experimental & Clinical Cancer Research Universal CAR-T Cells NK Cell Inhibition Allogeneic Rejection T Cell Stemness |
| title | LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells |
| title_full | LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells |
| title_fullStr | LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells |
| title_full_unstemmed | LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells |
| title_short | LLT1 overexpression renders allogeneic-NK resistance and facilitates the generation of enhanced universal CAR-T cells |
| title_sort | llt1 overexpression renders allogeneic nk resistance and facilitates the generation of enhanced universal car t cells |
| topic | Universal CAR-T Cells NK Cell Inhibition Allogeneic Rejection T Cell Stemness |
| url | https://doi.org/10.1186/s13046-025-03273-2 |
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