BackgroundThe 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.MethodsWe 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.ResultsGenetic 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.ConclusionAccording 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.