S126: il-9 secreted by leukemia stem cells induces the epigenetic activation of bm-infiltrating cd4+ t cells in acute myeloid leukemia

Background: Acute myeloid leukemia (AML) is a clonal disease of hematopoietic stem and progenitor cells (HSPCs) that results in the expansion of undifferentiated progenitor cells. Disease development and progression is driven by so-called leukemia stem cells (LSCs). Similar to HSPCs, LSCs interact in the bone marrow (BM) microenvironment with different cell types including BM stromal cells, endothelial cells and also immune cells. However, LSCs efficiently avoid the elimination by the immune system through expressing immune-inhibitory molecules or downregulating crucial immunological recognition pathways. We recently documented that BM-infiltrating CD8+ T cells in AML patients have a silenced gene expression and are functionally impaired. Although CD4+ T cells are key players in regulation of the adaptive immune system, their functional role and related signaling in leukemia patients is poorly understood. Aims: This work aimed to characterize BM-infiltrating CD4+ T cells and investigate their interaction with LSCs and CD8+ T cells. Methods: We analyzed the transcriptome of well-defined populations of LSCs/HSCs and progenitors as well as matching immune cells (CD8+ or CD4+ T cell lymphocytes) from BM of 30 newly diagnosed AML patients (different risk categories) and seven controls. Next, numerous tens of thousands of predictive networks were modeled using an unbiased correlation analysis to identify potential linkages between genes expressed in the leukemia stem/progenitor fraction and matched immune cells in all AML risk groups and controls. The revealed immune-dependent dysregulated signaling pathways in CD4+ T cells and their possible connections with the other examined cell groups were functionally verified. Results: In contrast to BM infiltrating CD8+ T cells that show a silenced gene expression pattern, transcriptomic analysis of BM-infiltrating CD4+ T cells in AML revealed an activation of immune-related signaling pathways with skewing towards a TH1 polarization and a lack of TH9 immunophenotype. To study possible interactions of CD4+ T cells with pared AML LSCs, progenitors and CD8+ T cells in the BM, we conducted an unbiased comprehensive correlation network modeling. Within all mapped networks, a node was defined as a gene expressed in any of the studied cell populations, and a hub (high-degree correlated) was a gene in one cell that correlated significantly with more than 15 different genes in the other cell type. IL9 gene was identified as a potential hub in AML LSCs regulating BM-infiltrating CD4+ T cells. Functional validations revealed that IL-9 produced by AML LSCs could epigenetically activate CD4+ T cells in the bone marrow. IL-9R signaling in CD4+ T cells activated JAK/STAT signaling and increased histone methylation. IL-9 activated CD4+ T cells produced different cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-gamma. IFN-gamma secreted by activated CD4+ T cells significantly increased the colony forming capacity of AML LSCs. Blockage of JAK/STAT signaling or silencing of key regulatory genes for histone methylation (lysine methyltransferase 2A, KMT2A & lysine methyltransferase 2E, KMT2E) decreased the activation/proliferation of CD4+ T and diminished the production of TNF-α and IFN-gamma. Summary/Conclusion: Our findings indicated that AML LSCs activate BM-infiltrating CD4+ T lymphocytes by secreting IL-9. IL9R signaling in CD4+ T cells leads to JAK/STAT signaling and increased histone methylation resulting in effector cell differentiation and production of IFN-gamma which in turn expands LSCs and contributes to disease progression. Keywords: Hematopoietic stem and progenitor cells, Epigenetic, AML, CD4+ T cells