Mechanisms of single-stranded RNA and type I interferon induced suppression of adaptive immune responses

Abstract

Toll-like receptors (TLRs) belong to the pattern-recognition receptors (PRR) expressed mainly by cells of the innate immune system and recognize conserved structures derived from microbes. Stimulation of TLRs by their respective TLR ligands induces the production of proinflammatory cytokines, chemokines, and maturation of dendritic cells (DCs), which plays a crucial role in the induction of adaptive immunity. Strinkingly, we and others have shown that the systemic exposure to TLR ligands suppresses the generation of adaptive T cell immunity towards subsequent infections. Our group has recently observed that the systemic injection of single-stranded RNA (ssRNA), which is recognized by endosomal TLR7, inhibited antigen-specific cytotoxic T lymphocyte (CTL) responses in the spleen in a type I interferon (IFN) dependent manner. Whereas the immunostimulatory effects of TLR stimulation and type I IFNs are well described, it remains poorly understood how the suppression of adaptive immune responses is mediated by TLR ligands and type I IFN, respectively. In this thesis, the underlying mechanisms of ssRNA- and type I IFN-mediated inhibition of adaptive immunity were investigated. <br /> The results presented demonstrate that the systemic administration of ssRNA leads to the suppression of CTL responses against subsequent infections with viral and bacterial pathogens, which was attributed to an impaired expansion and activation of antigen-specific CD8 T cells. Furthermore, we detected that the proliferation and cytokine production of antigen-specific CD4 T cells was impeded, which was associated with a downregulation in MHC class II molecule expression on ssRNA-preactivated splenic DCs. These results clearly indicate that a systemic injection of ssRNA inhibited the generation of CD4 T cell help, which contributed to the suppression of antigen-specific CTL responses. This interpretation was supported by the observation that ssRNA-induced CTL suppression was circumvented in the presence of effector or memory antigen-specific CD4 T helper cells. The generation of T cell immunity was not impaired if ssRNA was targeted to the cytoplasm by complexation with the carrier in vivo-jetPEI, thereby avoiding the recognition by TLR7. TLR7-mediated recognition of systemic ssRNA stimulated the production of type I IFNs, which were essential in ssRNA-induced CTL suppression. We identified plasmacytoid DCs (pDCs) to respond immediately to ssRNA stimulation by releasing early type I IFNs. Based on our experiments in LysM-specific IFNAR1 deficient, STAT1 deficient, and IRF7 deficient mice, we assume that ssRNA-induced early type I IFNs are amplified via the JAK-STAT pathway in splenic macrophages. The resulting secretion of substantial amounts of IFNα affected splenic DCs, which were impaired in their ability to cross-prime antigen-specific CD8 T cells, thus contributing to the lack of antigen-specific cytotoxicity.<br /> A clinical relevance of RNA-mediated immune suppression was shown in a combinatorial anti-tumor therapy model, mimicking small-interfering RNA (siRNA) application and induction of tumor-specific CTL responses. We could demonstrate that, depending on the carrier used, systemic in vivo siRNA application interfered with an adenoviral-based melanoma vaccination due to the lack of a tumor-specific CTL response.<br /> Moreover, we revealed that CTL suppression was not restricted to the application of synthetic RNA oligoribonucleotides. Infection with an Influenza virus impeded the generation of the CTL response against a subsequent adenovirus infection, which was ascribed to the production of type I IFNs. This observation implies that type I IFN-triggering viruses in general might inhibit adaptive immune responses in a similar fashion as we observed with systemic ssRNA. <br /> Taken together, our results point out that ssRNA- and virus-induced type I IFNs cannot only stimulate, but also inhibit antigen-specific T cell responses in the spleen. Furthermore, we indicate that a therapeutic siRNA application in vivo, e.g. in tumor therapies, might lead to undesired off-target effects, such as immune suppression, due to the recognition of siRNA by nucleic acid receptors such as TLR7.