Double-Negative T Cells Induce Ferroptosis-Dependent, MHC-Independent Cytotoxicity of CD8+ T Cells Against AML 2256323

Abstract

Abstract Introduction Acute myeloid leukemia (AML) is a blood cancer originating from myeloid cells in the bone marrow with poor prognosis. CD3+CD4-CD8-double negative T cells (DNTs) is a rare subset of immune cells that have shown strong anti-AML properties. Our previous work demonstrated that allogeneic DNTs from healthy donors effectively targeted AML in vitro and in vivo without off-tumor toxicity in preclinical models and early phase clinical trials. However, the mechanisms by which DNTs influence host immune cells’ anti-leukemic activity remain unclear. Methods We cultured patient or healthy donor CD8+ T cells with autologous AML blasts or AML cell lines and characterized their immunophenotype and anti-leukemic activity. We performed proteomic analysis comparing CD8+ T cells activated by DNT+AML, AML alone, or CD3/CD28 beads, and differentially expressed proteins and pathways were identified and validated using functional assays. We conducted blocking experiments targeting ferroptosis and generated MHC-I knockout AML cells to test whether DNT-activated CD8+ T cells can kill AML cells in an MHC-independent manner. Results CD8+ T cells co-cultured with DNTs mediated cytotoxicity toward AML cells. Proteomic profiling revealed that CD8+ T cells activated by DNT+AML upregulated innate immune response and ferroptosis-pathways. Blocking ferroptosis markedly inhibited their killing capacity, indicating that CD8+ T cell cytotoxicity depends on ferroptosis. Notably, these CD8+ T cells could kill MHC-I—deficient AML cells, suggesting an antigen-independent mechanism. Conclusion These findings show that, beyond direct cytotoxicity, DNTs promote a non-conventional anti-leukemic activity of CD8+ T cells through ferroptosis-dependent mechanisms. This synergy between DNTs and CD8+ T cells represents a novel immune axis that could be leveraged to enhance adoptive cell therapy efficacy against AML. Funding Source Alberta Cancer Foundation, Kids Cancer Care, CIHR Project Grant, Cancer Research Society Operating Grant Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)