COMPREHENSIVE CHARACTERIZATION OF THE BONE MARROW MICROENVIRONMENT IDENTIFIES A DISTINCT IMMUNE SIGNATURE ASSOCIATED WITH TUMOR BURDEN IN ACUTE MYELOID LEUKEMIA

Background Acute myeloid leukemia (AML) is a heterogeneous and aggressive disease characterized by the uncontrolled clonal expansion of hematopoietic progenitor cells. The role of the immune system in AML progression and treatment response is under investigation, but a comprehensive phenotypic characterization of the immune microenvironment in the AML bone marrow (BM) niche is still lacking. Aims This study aimed to identify an immune BM signature related to tumor burden in AML patients. Methods The BM immunological profile of AML patients was analyzed by assessing the composition of selected key immune cell populations (CD4, CD8, regulatory T cells, B cells, regulatory B (B regs) cells, natural killer (NK) cells, monocytes, dendritic cells and myeloid-derived suppressor cells (MDSC)), along with their respective subtypes. Notably, the expression of a large array of specific immune checkpoints was also evaluated. For this purpose, BM samples from 35 AML patients (pts) at diagnosis and 6 healthy donors (HD) were analyzed using multiparametric flow cytometry. Additionally, AML cells were quantified and characterized based on CD34, CD117, CD33, CD13, HLA-DR expression, and of selected leukemia- associated aberrant lineage markers (CD7, CD56, CD25). To assess changes in the cell population composition of the AML BM niche and the expression of immune checkpoints after therapy, we analyzed 6 pts 1 month after diagnosis. All six patients were refractory to chemo/immunotherapy. Results At diagnosis our results show increased proportions of naı̈ve T cells in AML patients. Of note, an increased expression of immune checkpoints was observed in AML patients compared to HD: LAG3 in NK cells, TIM1 in naı̈ve B cells and CD95 in regulatory B cells. Focusing on the myeloid compartment, AML MDSCs displayed an enrichment of a specific immune checkpoint (LOX1). Interestingly, a significant positive correlation was observed between AML cell percentages and the proportion of LOX1+ MDSCs and PD1+ total B cells. Moreover, we identified a positive correlation between CD56+ AML cells and CD95+ B cells, as well as between CD25+ AML cells and both PD1+ NK and PD1+ B cells. After therapy, we observed a trend toward reduction of proportion of naı̈ve T cells and the expression of LAG3 and LOX1 in NK and MDSC, respectively. Interestingly, a significant positive correlation was observed between AML cell percentages and the proportion of CD160+ and CD244+ CD4+ T cells, as well as between AML cells and TIM3+ CD8+ early effector T cells. Summary/Conclusion Overall these data, although requiring validation in larger AML patient cohorts, highlight that there is a sequestration of naı̈ve T cell in the BM, where the tumor burden is directly related to the expression of selected immune checkpoints in T cells, NK cells, and MDSCs. In the context of refractory disease, T cell exhaustion, evidenced by the upregulation of CD160, CD244, and TIM3, may contribute to leukemia persistence. Importantly, the relationship between the expression of specific leukemia-associated aberrant lineage markers and the immunosuppressive microenvironment may help to identify a comprehensive signature with prognostic potential in AML.