Regulating the regulators: Overcoming regulatory T cell suppression in cancer and identifying inhibitors of inflammation using RNA interference

The immune system has evolved to protect the host against the attack of foreign, potentially pathogenic, microorganisms. However, breakdowns in the regulatory mechanisms that control immune homeostasis precipitate the development of numerous diseases.;Regulatory T cells (Tregs) are a specialized subset of immune cells which prevent the over-activation of the immune response. Although Tregs are essential for preventing autoimmune diseases, tumor specific Tregs can inhibit the generation of protective anti-tumor immunity. The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) has been shown to be effective in overcoming immune tolerance and promoting tumor rejection. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of Treg suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated tumor cell clones that express GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance antitumor immunity and delay tumor growth in syngeneic BALB/c mice. Dissection of the the mechanisms of anti-tumor activity in vivo demonstrated that the localized expression of GITR-L specifically promotes the local accumulation of CD8+ T cells, which are essential for controlling tumor growth.;Toll like receptors (TLR) are important pathogen sensors in the innate immune response. Although activation of TLRs is critical for pathogen clearance, excessive activation can be deleterious and is associated with numerous chronic inflammatory diseases. In an attempt to identify novel genes involved in the regulation of TLR dependent proinflammatory responses, we developed a murine macrophage cell line stably transfected with a construct expressing diphtheria toxin A under the control of the TNF-alpha promoter. Thus, only cells which harbor shRNA sequences that inhibit proinflammatory signals will be positively selected. By utilizing an RNAi-based forward genetic approach, we identified unique shRNA sequences that inhibit genes necessary for LPS (TLR4) and Pam3Cys (TLR2) dependent TNF-alpha production. Importantly, the inhibition of proinflammatory cytokine production appears to be unique to MyD88 dependent signaling pathways as poly (I:C) (TLR3) dependent TNF-alpha production was not inhibited.