Key Points Increased immune suppressors and PD-1 abrogates effector responses in CML patients at diagnosis. Enhanced net effector immune responses and decreased PD-1 and immune suppressors may promote sustained deep molecular response in CML.

Summary There is currently no biomarker that reliably predicts treatment‐free remission (TFR) in chronic myeloid leukaemia (CML). We characterised effector and suppressor immune responses at the time of tyrosine kinase inhibitor (TKI) cessation in patients from the CML8 and CML10 clinical studies. Natural killer (NK) cells with increased expression of activating NK receptors were higher in patients who achieved TFR. There was no difference in the proportion of CD4+ or CD8+ T cells. Furthermore, we found that FoxP3+ regulatory T cells (T reg) and monocytic myeloid‐derived suppressor cells (Mo‐MDSCs) were concomitantly decreased in TFR patients, suggesting that the effector and suppressor arms of the immune system work in concert to mediate TFR. A discovery cohort (CML10) was used to generate a predictive model, using logistic regression. Patients classified into the high‐risk group were more likely to relapse when compared with the low‐risk group (HR 7·4, 95% CI 2·9‐19·1). The model was successfully validated on the independent CML8 cohort (HR 8·3, 95% CI 2·2–31·3). Effective prediction of TFR success may be obtained with an effector‐suppressor score, calculated using absolute NK cell, T reg, and Mo‐MDSC counts, at TKI cessation, reflecting the contribution of both immune suppressors and effectors in the immunobiology underlying successful TFR.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.