Investigating Mechanisms of Immune Suppression Secondary to an Inflammatory Microenvironment

The immune system plays a dynamic role in cancer progression. The theory of immunoediting suggests that the relationship between the tumor cell and the immune cell is one that is in flux: initially highly active and responsive to tumor antigen to one that has escaped immune responsiveness. Once the tumor has formed and effectively “escaped”, there are multiple mechanisms that work against a conventional immune response. The tumor cell clones that have escaped the elimination phase are those that are less immunogenic. These clones downregulate MHC, have increased apoptosis through DAMP-driven mechanisms, and suppressive cell phenotypes are driven through cytokine-driven cues and innate feed-forward mechanisms. This project aims to understand three different mechanisms of tumor-induced immune suppression: the first, a model of lung adenocarcinoma, exploring innate NK subversion by repression of the NKG2D-MICA/B axis; the second, a model of mammary carcinoma used as a basis to explore differences in NK trafficking and exclusion from the tumor microenvironment due to upregulation of inhibitory signaling mediators; the third, a mechanism of T cell suppression induced by excessive production of danger associated molecular pattern (DAMP) S100A9 in Myelodysplastic Syndromes (MDS). Lung Cancers of all subtypes are the leading cancer death in both men and women in the United States. While progress has been made in developing immunotherapies, we chose to focus on basic mechanisms of immunosuppression in order to identify ways to reinvigorate tumor-immune responsiveness. Micro-RNAs (miRs) function via RNA interference and are implicated in cancer development and progression due to their effects on genes involving the cell cycle, apoptosis, and metabolism. Our group has previously shown that Transforming Growth Factor-beta (TGFβ) inhibits NK cell function by means of inducing miR-183 and subsequent repression of DAP12, thus halting NK cell signaling. Here, we found that NKG2 ligand MICA/B which is normally upregulated in situations of stress or damage is suppressed in the presence of high levels of miR-183. This miR, employed by the suppressive TGFβ, renders NK cells dysfunctional by depleting MICA/B from the tumor cell itself so that the NK cell cannot recognize it through activating receptor NKG2D. This is a profound mechanism that hits at one of the earliest effector mechanisms in the tumor microenvironment. Micro-RNA 155 is another oncomir that is expressed in NK cells and other leukocytes, where it is upregulated by inflammatory stimuli in the tumor microenvironment. SH2-containing inositol polyphosphate 5-phosphatase (SHIP-1) negatively regulates interferon gamma production in NK cells and regulates the actin cytoskeleton, having diverse effects on NK cell motility. SHIP-1 is a defined target of miR-155, and thus SHIP-1 is decreased in the context of excessive miR-155 expression. This miR is thus important to NK cell function, as we showed in our work that in the absence of this miR, there exists a dysfunctional NK cell that cannot traffic or produce proper amounts of cytokine in response to normal stimuli. Here, we found that miR-155 regulates chemotaxis and NK tumor infiltration downstream of SHIP-1 using a mouse model of mammary carcinoma. This is a basic science mechanism that teases apart the complexity of the relationship between this miR and leukocyte trafficking into the tumor bed, identifying another mechanism of immune subversion in this case mediated by a lack of trafficking present in the inflammatory tumor microenvironment, and thus has applicability to many tumor types. MDS are a disease driven by excessive inflammation secondary to peripheral blood cytopenias and hypercellular bone marrow. This disease is predominately driven by an excess of myeloid derived suppressor cells (MDSC) and suppressive cytokines that dampen conventional mechanisms of immunity. The role of T cells in the disease is incompletely understood. Here, we report on a newly identified mechanism of T cell suppression in the disease mediated by DAMP S100A9 through the Pattern Recognition Receptor (PRR) Receptor for Advanced Glycation Endproducts (RAGE). Rather than inducing a potent immune response as DAMP/PRR signaling should, we uniquely found that T cells are inhibited in their function when exposed to exogenous DAMP signaling. T cell proliferation is almost completely shut down, and interferon gamma production is halted. While many PRRs are present on adaptive cells, RAGE was found to be exclusive to CD4+hi T cells, whereby S100A9 signals through. These T cells did not express any lineage specificity, and produced normal amounts of cytokine when activated with anti-CD3/28. Treatment with S100A9 was able to induce RAGE expression on these T cells, indicating a positive feed forward loop. This RAGE expression was dynamic and decreased rapidly after T cell receptor (TCR) engagement, indicating that this RAGE/S100A9 signaling response may act as a pre-activation checkpoint for the T cell prior to responding to antigen. We found that RAGE+ T cells are significantly increased in the bone marrow of patients with MDS, and thus this may represent a way to capitalize on a lack of T cell responsiveness in a disease with typically limited treatment options. The work presented here furthers our understanding of tumor-immune biology from both a basic science approach and more translational approaches. The questions asked throughout this work attempt to unpack the complexity of the relationship between the immune cell and the cancer cell in a push-pull that, given future efforts for translation, can improve outcomes and in the case of the RAGE/S100A9 axis, be translated into new therapies