S100A9 Contributes to T Cell Dysfunction through Its Interaction with RAGE in MDS

We have previously reported that the overexpression of S100A9 drives the development of Myelodysplastic Syndrome (MDS) through expansion of Myeloid Derived Suppressor Cells (MDSC) and promotion of pyroptosis. Despite the identified role of S100A9's effects on MDSC, and hematopoietic stem and progenitor cells (HSPC), as well as their establishment of an immunosuppressive microenvironment, the effects of S100A9 on adaptive immunity in MDS progression are less clear. Here, we report for the first time the unidentified role of S100A9 on T cell function in MDS that may lead to impaired immunosurveillance in the disease. Danger Associated Molecular Pattern (DAMP) S100A9 is a known ligand for the Pattern Recognition Receptor (PRR) Receptor for Advanced Glycation Endproducts (RAGE). We investigated RAGE surface expression by flow cytometry on MDS bone marrow resident T cells vs those derived from healthy donor bone marrow. We found significantly (p=0.04) higher RAGE surface expression on T cells from MDS bone marrow, and this expression was restricted to the CD4 lineage. To ascertain the effects of S100A9 on RAGE+CD4+ T cell function, we performed flow cytometry in a time course experiment post-T cell activation. Without S100A9 treatment, cell surface RAGE expression was low in activated T cells from healthy donors, but extended treatment with recombinant human S100A9 resulted in increased RAGE expression, suggesting a positive feedback loop for this DAMP. Unlike activated T cells, T cells not exposed to activating conditions did not display upregulated RAGE expression after S100A9 treatment. This indicates that this may be a post-activation switch, acting as a checkpoint for the T cell in the context of excessive damage signaling by DAMP S100A9. In order to further characterize the functional consequences of RAGE engagement, we performed transcription factor staining paired with a cytometric bead array for secreted cytokines in activated T cells treated with S100A9. Tumor Necrosis Factor Alpha (TNFa), IL-10 and IL-6 were induced by S100A9, indicating perhaps some degree of polarization induced by this DAMP. Commercially available RAGE V-domain inhibitor FPS-ZM1 blunted this cytokine signaling, indicating a significant portion of this cytokine production is indeed mediated through RAGE. In addition, we performed lipophilic dye dilution assays to track the effects S100A9 has on T cell proliferation following activation. S100A9 significantly decreased proliferative response under normal stimulatory conditions. Similar inhibition was seen in T cells derived from PBMC, MDS, or healthy donor bone marrow resident T cells, suggesting that the consequences of RAGE engagement are not disease specific. To rule out apoptosis as a potential cause for this halt in proliferation, we stained the cells with Annexin V and Propidium Iodide. To further elucidate how S100A9 might be affecting T cell proliferation, we analyzed cell cycle profiles following activation and S100A9 treatment. T cells treated with S100A9 showed a repressed cell cycle prior to G2, compared to T cells activated without any S100A9 treatment, suggesting a possible G1/S arrest. The evidence obtained in our study suggests any role of T cell dysfunction mediated by RAGE in MDS may be directly linked to the increased levels of S100A9 in the bone marrow microenvironment. Our work represents a novel mechanism of T cell dysfunction that may lead to a lack of responsiveness in the context of a disease known to overexpress the RAGE ligand S100A9. Capitalizing on this novel checkpoint can potentially be used both as novel biomarker and as a therapeutic target in the future to restore T cell immunosurveillance to a functional state in MDS.