Assistant Professor Univ. of Calgary Cumming Sch. of Med., Alberta, Canada
Disclosure(s):
Jongbok Lee, PhD: No financial relationships to disclose
Introduction/Rationale: 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.
