Comprehensive characterization of the bone marrow microenvironment identifies an exhausted immune signature associated with tumor burden in Acute Myeloid Leukemia

The bone marrow (BM) immune microenvironment of Acute myeloid leukemia (AML), an heterogeneous and aggressive malignancy, is incompletely characterized. We analyzed BM samples from 38 AML patients at diagnosis and 9 healthy donors (HD) using multiparametric flow cytometry to profile key immune populations (CD4, CD8, regulatory T cells (Tregs), B cells, regulatory B cells (Bregs), natural killer cells (NK), monocytes, dendritic cells (DC) and myeloid-derived suppressor cells (MDSC)), along with their respective subtypes. A broad panel of immune checkpoints was also evaluated. AML blasts were characterized by CD34, CD117, CD33, CD13, HLA-DR and of aberrant marker (CD7, CD56, CD25) expression. Four refractory patients were monitored after immuno- or chemotherapy (after 1 cycle n=4; after 2 cycles n=2). At diagnosis, AML patients showed increased CD4/CD8 naïve T cells and higher proportions of CD8 effector, intermediate effector T cells, and naïve Tregs compared to HD. Plasmacytoid DCs were also expanded. Conversely, Bregs and double-negative B cells were markedly reduced. Focusing on immune checkpoints, significant higher expression of TIM1, CD244 and TIGIT was observed in naïve B cells, T cells and Tregs, respectively. Blast burden correlated with increased expression of immune checkpoints: TIM3/LAG3/CTLA4/NKG2D on NK cells; TIM3/TIGIT on CD4 T cells; LAG3/BTLA/CTLA4 on CD8 T cells; CTLA4 on Tregs; and CD1D on B and Breg cells. Specific aberrant AML phenotypes also associated with immune exhaustion: CD56+ AML cells correlated with the expression of PD1/CD244/CD160/TIGIT on CD4 and CD244/LAG3 on CD8 T cells, LAG3 on Tregs, and TIGIT on B cells; CD7+ AML cells correlated with CD244/TIM3 on CD4 T cells and TIM3 on Tregs; CD25+ AML cells correlated with CTLA4/TIM3 on CD8 T cells. After therapy, refractory patients showed a reduction in naïve T cells and a significant decrease in Bregs and double-negative B cells compared to baseline, along with an increase in effector Tregs. In conclusion, AML is associated with sequestration of naïve T cells in the BM and with a tumor burden-associated upregulation of specific exhaustion-related immune checkpoints in T, B, and regulatory subsets. Refractory disease is marked by a shift toward a more immunosuppressive phenotype. Overall, this study, pending validation in larger cohorts, provides a deeper understanding of AML immune-pathogenesis and has the potential to identify novel targets for immunotherapeutic strategies.