-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

1587 Immunological Footprints of Pediatric Acute Myeloid Leukemia in Local Bone Marrow T Cells

Program: Oral and Poster Abstracts
Session: 617. Acute Myeloid Leukemias: Biomarkers, Molecular Markers and Minimal Residual Disease in Diagnosis and Prognosis: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Diseases, immune mechanism, immunology, Myeloid Malignancies, Biological Processes, multi-systemic interactions
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Paula Rothämel1*, Semjon Willier, MD2*, Jonas Wilhelm1*, Fiona AG Becker-Dettling1*, Theresa Kaeuferle1*, Franziska Blaeschke1, Meino Rohlfs1*, Irene Schmid1*, Vera Binder1*, Christoph Klein1,3, Thomas Walzthoeni4,5*, Matthias Heinig5,6,7* and Tobias Feuchtinger, MD2,8,9

1Department of Pediatrics, Dr. von Hauner Children’s Hospital, LMU University Hospital, LMU Munich, Munich, Germany
2Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children’s Hospital, University Hospital, LMU Munich, Munich, Germany
3Gene Center, LMU Munich, Munich, Germany
4Core Facility Genomics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich-Neuherberg, Germany
5Institute of Computational Biology (ICB), Helmholtz Center Munich, German Research Center for Environmental Health, Oberschleißheim, Germany
6Department of Computer Science, TUM School of Computation, Information and Technology, Technical University of Munich, Garching, Germany
7German Centre for Cardiovascular Research (DZHK), Munich Heart Association, Partner Site Munich, Berlin, Germany
8German Center for Translational Cancer Research (DKTK), Partner Site Munich, Munich, Germany
9German Center for Infection Research (DZIF), Munich, Germany

Introduction: Despite notable advancements, childhood acute myeloid leukemia (AML) remains associated with one of the poorest prognoses among pediatric malignancies. Conventional chemotherapies carry significant toxicity, underscoring the urgent need for novel e.g. immunotherapy-based approaches. Despite AML being previously considered non-immunogenic due to its low mutational burden, the complex interplay between leukemic blasts and T cells within the bone marrow in pediatric patients remains largely unexplored. This study investigates the AML-induced footprint on local bone marrow T cells (bmT cells) representing tumor-infiltrating lymphocytes, with the aim to reveal functional points of action for future immunotherapy.

Methods: Cryopreserved bone marrow samples from both pediatric AML patients (n=29) and age-matched healthy bone marrow donors (HD, n=9) were analyzed. Multicolor flow cytometry quantified surface expression of inhibitory signaling and activation receptors, as well as T cell differentiation stages. RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) were performed of sorted CD8+ bmT cells. Bulk RNA and ATAC-Seq data was visualized by principal component analysis (PCA). Another PCA was performed on the combined dataset of RNA- and ATAC-Seq. Differential analysis using DESeq2 (fitType “parametric”, testType “Wald”) identified 135 differentially expressed genes in CD8+ bmT cells between HD and AML patients based on fold change (≥1/≤-1) and statistical criteria (s-value<0.05).

Results: Based on CD45RO, CD62L and CD95 surface expression, late differentiation stages of bmT cells were enriched in AML. Namely, the frequency of naïve bmT cells was reduced (33.9% vs. 57.5%, mean values, p=0.0018), while the frequency of effector memory bmT cells was increased in AML patients (21.5% vs. 9.4%, p=0.0010). This could be validated by RNA-Seq of sorted CD8+ bmT cells which documented higher expression of cytotoxic effector T cell genes such as Perforin, Granulysin and Granzyme B. In addition, our analysis also revealed higher expression of functional relevant genes associated with T cell cytotoxity (ADGRG1), effector function (BATF/TPX2) or exhaustion (SLAMF7) in AML patients. Next, surface expression of receptors for inhibitory signaling and activation was analyzed. Several of these receptors have also been described as immune checkpoints (ICP). Analyzing differential expression between HD and AML patients showed pronounced upregulation of inhibitory markers (TIM-3: 7.1% vs. 1.7%, p<0.0001, CD39: 7.7% vs. 2.0%, p=0.0103, LAG-3: 8.0% vs. 2.9%; CTLA-4: 3.9% vs. 1.6%, p=0.0024; PD-1: 34.8% vs. 21.3%, p=0.0016). Since CD8+ bmT cells showed the most promising effects in the flow cytometry analysis, this subpopulation of bmT cells was further characterized by RNA- and ATAC-Seq. Here, clustering of HD and AML patients’ CD8+ bmT cells was observed in PCA. Interestingly, PCA also revealed differential clustering of CD8+ bmT cells from AML primary and relapse samples. Confirming the observed differences in phenotype of bmT cells in AML patients, we identified distinct bmT cell populations specifically induced in AML patients compared to HD, based on phenotype and differential ICP expression patterns. Through tSNE analysis, we discovered a CD8+ effector memory T cell population with a TIM-3+PD-1+2B4+ phenotype, comprising 2.3% of all bmT cells in AML samples compared to only 0.03% in HD.

Conclusion: This study reveals significant alterations in the activation and inhibition potential of bmT cells in pediatric AML compared to HD, as confirmed by comprehensive analysis of the epigenome, transcriptome and surfaceome. We confirm the observed differences through the identification of a T cell population with a typically exhausted phenotype specific for AML patients when compared to HD, implying persistent antigen exposure and potential direct or indirect interactions with leukemic blasts. Although prospective clinical trials will have to confirm functional relevance in patient cohorts, this study provides crucial insights into the immune landscape of pediatric AML, underscoring the potential for targeted immunotherapeutic interventions.

Disclosures: Feuchtinger: Servier: Research Funding; Miltenyi Biotec: Research Funding.

*signifies non-member of ASH