Double-negative T cells utilize a TNFα-JAK1-ICAM-1 cytotoxic axis against acute myeloid leukemia

• Upon engaging DNT-susceptible AML, DNTs secrete TNFα which sensitizes AML to DNT killing, including DNT-resistant AML

• TNFα noncanonically signals through JAK1 to upregulate ICAM-1 on AML for increased DNT anti-leukemic function

Allogeneic double-negative T cells (DNTs) are a rare T cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-vs-host disease in an allogeneic setting. A phase I clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we utilized a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a TNFα-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, LFA-1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy.