The immunosuppressive bone marrow microenvironment is an important contributor to the limited success of immunotherapy in acute myeloid leukemia (AML), but the cellular interactions underlying AML immune evasion are incompletely understood. We therefore generated a single-cell spatial transcriptomic and proteomic atlas using 148 bone biopsies from 113 individuals comprising pediatric and adult AML at diagnosis and non-leukemic controls. We observed an expansion of regulatory T cells (Tregs) in AML, with stronger colocalization between Tregs and macrophages compared to non-leukemic bone marrow. Distinct cellular neighborhoods were enriched for myeloid progenitor-like cells together with macrophages and T cells, which correlated with higher macrophage and T cell immune checkpoint expression. Moreover, these neighborhoods were associated with specific AML subtypes, especially KMT2A-rearranged and RUNX1::RUNX1T1 AML. These spatial patterns were validated by identification of malignant cells via in situ fusion detection in RUNX1::RUNX1T1 cases. Functional experiments revealed that macrophages and AML cells not only actively recruit Tregs, but also promote naïve T cell differentiation into Tregs. Spatially informed ligand-receptor analysis predicted the involvement of the Galectin-9 – CD44/TIM-3 axis in this immunosuppressive crosstalk, which was supported by in vitro inhibition of CD44 and/or TIM-3 preventing macrophage- and AML-induced Treg differentiation. Collectively, this comprehensive spatial map of the AML bone marrow identified tripartite crosstalk between AML, macrophages, and T cells mediated by the Galectin-9 – CD44/TIM-3 axis as a key component of the immunosuppressive microenvironment. Targeting Galectin-9 – CD44/TIM-3 interactions may be a promising strategy to overcome immune evasion and enhance immunotherapeutic success in AML. Highlights Spatial transcriptomic and proteomic atlas of pediatric and adult acute myeloid leukemia (AML) bone marrow Increased colocalization of macrophages and regulatory T cells (Tregs) in AML Macrophages and AML cells induce differentiation of naïve T cells to Tregs in vitro, which can be prevented by inhibition of CD44 or TIM-3
M. van der Meulen, Emma S. Pool, A. Perzolli et al.· bioRxiv· 0 citations
BACKGROUND
Relapse of acute myeloid leukemia (AML) after allogeneic stem cell transplantation (alloSCT) remains a major cause of treatment failure, with poor long-term survival. Donor lymphocyte infusion (DLI) can induce a graft-versus-leukemia effect but may also lead to graft-versus-host disease (GvHD) associated morbidity and mortality. In this study we evaluated the 5-year overall survival of a treatment strategy combining cytoreduction, dose-escalated DLI and interferon-α in patients with relapsed AML after T-cell depleted alloSCT.
METHODS
In this single-center, retrospective cohort study we analyzed 84 adult patients with relapsed AML after first alloSCT with in vitro T-cell depletion (TCD), and in a proportion of patients combined with in vivo TCD. The study was performed at Leiden University Medical Center between 2005 and 2020, with follow-up through January 2025. Patients with low-burden morphologic relapse (≤10% bone marrow blasts) received DLI without prior cytoreduction; patients with higher-burden morphologic relapse received cytoreductive therapy followed by DLI three weeks later. In the absence of GvHD three weeks after DLI, interferon-α was administered to augment the alloimmune response. Quality of life in long-term survivors was assessed exploratively.
RESULTS
Six patients received supportive care only, leaving 78 patients treated per protocol. Patients with low-burden morphologic relapse (n=8) received DLI without cytoreduction and achieved a 5-year OS of 60% (95% CI 22-98). In seven additional patients, higher-burden morphologic relapse was unexpectedly diagnosed at the time of a pre-scheduled prophylactic or preemptive DLI. Cytoreduction was deferred to await the effect of this DLI, yet none of these patients survived beyond 18 months. Among 63 patients with higher-burden morphologic relapse who received re-induction therapy with intent to proceed to DLI, 44 (70%) ultimately received DLI, of whom 25 (57%) developed GvHD. The 5-year OS for the re-induction cohort was 7% (95% CI 1-13). Of 44 patients who reached DLI, 24 (55%) achieved complete remission. In this subset, 2- and 5-year RFS were 35% and 19%, respectively, with 5-year cumulative incidences of relapse and non-relapse mortality of 25% (95% CI 12-45) and 42% (95% CI 25-51). Transplantation in first complete remission (HR 0.40, p=0.017) and relapse occurring more than 6 months after alloSCT (HR 0.44, p=0.036) were associated with improved OS. Long-term survivors reported good global mental health but persistent GvHD-related symptoms and impaired social and functional well-being.
CONCLUSION
A DLI-based strategy can induce durable remission in patients with low-burden morphologic AML relapse after TCD alloSCT, even without prior cytoreduction. In higher-burden morphologic relapse, however, effective cytoreduction followed by DLI and interferon-α is essential, yet long-term outcomes remain poor, constrained by GvHD-associated non-relapse mortality as well as recurrent leukemia. These results define the long-term boundaries of therapeutic DLI and provide a contemporary reference standard for novel post-transplant relapse interventions.
J. Bergsma, E. Argiro, K. Oosterink et al.· Transplantation and Cellular...· 0 citations
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