同种异体双阴性T细胞在CD8+ T细胞中诱导针对急性髓系白血病的天然样细胞毒性功能

Background: Acute myeloid leukemia (AML) is characterized by malignant cells of myeloid origin and poor long-term survival due to relapse following conventional therapies. CD3 + CD4 - CD8 - double-negative T cells (DNTs) are a rare T-cell subset that can kill AML cells without causing graft-vs-host disease in preclinical models. A prior phase I trial demonstrated the feasibility, safety, and potential efficacy of healthy donor-derived allogeneic DNT therapy among patients with relapsed AML. However, the interaction between allogeneic DNTs and host immune cells remains unclear. Purpose: This study aims to investigate how allogeneic DNTs invigorate CD8 + T cells to better target AML. Methods: CD8 + T cells were collected from patient samples or healthy peripheral blood mononuclear cells after incubation with DNTs or various activation factors (anti-CD3/CD28 beads, AML cells, or AML cells with DNTs), respectively, followed by CD8 positive selection. Proteomic and single-cell RNA-sequencing (scRNA-seq) techniques were performed to identify unique immune pathways in DNT-activated CD8 + (CD8 DA ) T cells to kill AML cells. Findings of CD8 DA T cells were verified through cytotoxic co-culture assays, immunophenotyping, genetic knockouts (KOs), and inhibition assays. Results: CD8 + T cells from AML patient samples co-cultured with DNTs were highly activated and exhibited potent cytotoxicity against autologous AML blasts, compared to those cultured in the absence of DNTs. Mass spectrometry identified a unique proteomic profile in CD8 DA T cells, which were enriched for innate immune response markers relative to the other forms of activation. Interestingly, a higher CD8 DA T-cell signature score in patients' CD8 + T cells correlated with a response to anti-CTLA4 and decitabine treatment. Furthermore, CD8 DA T cells can target MHC KO AML cells, while anti-DNAM-1 antibodies reduce CD8 DA T-cell potency, indicating an innate-like immune mechanism. ScRNA-seq conducted on co-cultures of CD8 + T cells with AML, in the presence of DNTs or anti-CD3/CD28 beads, revealed an enrichment of ferroptosis-related genes in AML cells in the group cultured with DNTs. Using various cell-death inhibitors, only ferroptosis inhibitor Liproxstatin-1 significantly reduced the cytotoxic function of CD8 DA T cells. Clinically, lower expression of GPX4 or AIFM2 (FSP1), negative regulators of ferroptosis, resulted in better survival among two AML cohorts. Conclusion: DNTs stimulate an innate-like ability in CD8 + T cells to kill AML in an MHC-independent, DNAM-1-dependent manner through ferroptosis, yielding a potential clinical benefit for patients with AML.