Abstract 163: Allogeneic double-negative T cells induce an innate-like cytotoxic function in CD8+T cells against acute myeloid leukemia.

Abstract 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+ (CD8DA) T cells to kill AML cells. Findings of CD8DA 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 CD8DA T cells, which were enriched for innate immune response markers relative to the other forms of activation. Interestingly, a higher CD8DA T-cell signature score in patients’ CD8+ T cells correlated with a response to anti-CTLA4 and decitabine treatment. Furthermore, CD8DA T cells can target MHCKO AML cells, while anti-DNAM-1 antibodies reduce CD8DA 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 CD8DA 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. Citation Format: Leanne Palichuk, Enoch Tin, Pauline Douglas, Ruzena Filandrova, David Schriemer, Juan Arteaga, Michele Nawata, Sandeep Kaur, Sonia Cerquozzi, Michelle Geddes, Lynn Savoie, Jongbok Lee. Allogeneic double-negative T cells induce an innate-like cytotoxic function in CD8+T cells against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 163.