Burkhard Ludewig

Ribose 2′- O -methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5′ cap structures of higher eukaryote mRNAs have ribose 2′-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2′-O-methylation of mRNA remains elusive. Here we show that 2′-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2′-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2′-O-methylation of mRNA suggests that RNA modifications such as 2′-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Smooth Muscle Cells in Transplant Atherosclerotic Lesions Are Originated From Recipients, but Not Bone Marrow Progenitor Cells

Background—Smooth muscle cell (SMC) accumulation in the intima of vessels is a key event in the pathogenesis of transplant atherosclerosis. The traditional hypothesis that SMCs in the lesion are derived from the media of the donor vessel has been challenged by recent observations, but the cell origin is still not well established. Methods and Results—Here, we use a simplified model of artery allografts in transgenic mice to clearly identify the source of SMCs in transplant atherosclerosis. Aortic segments donated by BALB/c mice allografted to ROSA26 (C57B/6) mice expressing &bgr;-galactosidase (gal) in all tissues showed that neointimal cells derived exclusively from host cells. It was also demonstrated that SMCs of neointimal and atherosclerotic lesions in vessels allografted to mice expressing &bgr;-gal only in SMCs (SM-LacZ) or to apoE-deficient/SM-LacZ mice originated from the recipient, and not donor vessels. Interestingly, bone marrow transplantation of SM-LacZ &bgr;-gal–expressing cells into aortic allograft recipients revealed completely negative &bgr;-gal staining of neointimal and atherosclerotic lesions. However, a population of &bgr;-gal–positive cells in lesions of allografts was observed in chimeric mice with ROSA26 &bgr;-gal–expressing marrow cells. When bone marrow cells from both ROSA26 and SM-LacZ mice were cultured and stimulated with platelet-derived growth factor-BB, &agr;-actin and &bgr;-gal double-positive cells were found, suggesting that bone marrow cells have an ability to differentiate into SMCs. Conclusions—Thus, we provide strong evidence that SMCs of neointimal and atherosclerotic lesions in allografts are derived from the recipients and that non–bone marrow–derived progenitor cells are a possible source of SMCs in atherosclerotic lesions.

Form follows function: lymphoid tissue microarchitecture in antimicrobial immune defence

Secondary lymphoid organs (SLOs) are tissues that facilitate the induction of adaptive immune responses. These organs capture pathogens to limit their spread throughout the body, bring antigen-presenting cells into productive contact with their cognate lymphocytes and provide niches for the differentiation of immune effector cells. Therefore, the microanatomy of SLOs defines the ability of an organism to respond to pathogens. SLO microarchitecture is, at the same time, extremely adaptable to environmental changes. In this Review, we discuss recent insights into the function and plasticity of the SLO microenvironment with regards to antimicrobial immune defence.

Lack of Conventional Dendritic Cells Is Compatible with Normal Development and T Cell Homeostasis, but Causes Myeloid Proliferative Syndrome

Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c(hi) dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.

Induction, binding specificity and function of human ICOS

Recently, we have identified the inducible co‐stimulator (ICOS), an activation‐dependent, T cell‐specific cell surface molecule related to CD28 and CTLA‐4. Detailed analysis of human ICOS presented here shows that it is a 55‐60‐kDa homodimer with differently N‐glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12‐myristate 13‐acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up‐regulated early on all T cells, including the CD28– subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen‐presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7‐1 or B7‐2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross‐interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co‐induces a variety of cytokines including IL‐4, IL‐5, IL‐6, IFN‐γ, TNF‐α, GM‐CSF, but not IL‐2, and superinduces IL‐10. Furthermore, ICOS co‐stimulation prevents the apoptosis of pre‐activated T cells. The human ICOS gene maps to chromosome 2q33 – 34.

Aggravation of viral hepatitis by platelet-derived serotonin.

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8+ T cell–dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8+ T cell–dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8+ T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.

Coronavirus Non-Structural Protein 1 Is a Major Pathogenicity Factor: Implications for the Rational Design of Coronavirus Vaccines

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

Plasmacytoid dendritic cells control T-cell response to chronic viral infection

Infections with persistent viruses are a frequent cause of immunosuppression, autoimmune sequelae, and/or neoplastic disease. Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferon (IFN-I) and other cytokines in response to virus-derived nucleic acids. Persistent viruses often cause depletion or functional impairment of pDCs, but the role of pDCs in the control of these viruses remains unclear. We used conditional targeting of pDC-specific transcription factor E2-2 to generate mice that constitutively lack pDCs in peripheral lymphoid organs and tissues. The profound impact of pDC deficiency on innate antiviral responses was revealed by the failure to control acute infection with the cytopathic mouse hepatitis virus. Furthermore, pDC-deficient animals failed to clear lymphocytic choriomeningitis virus (LCMV) from hematopoietic organs during persistent LCMV infection. This failure was associated with reduced numbers and functionality of LCMV-specific CD4+ helper T cells and impaired antiviral CD8+ T-cell responses. Adoptive transfer of LCMV-specific T cells revealed that both CD4+ and CD8+ T cells required IFN-I for expansion, but only CD4+ T cells required the presence of pDCs. In contrast, mice with pDC-specific loss of MHC class II expression supported normal CD4+ T-cell response to LCMV. These data suggest that pDCs facilitate CD4+ helper T-cell responses to persistent viruses independently of direct antigen presentation. Thus pDCs provide an essential link between innate and adaptive immunity to chronic viral infection, likely through the secretion of IFN-I and other cytokines.

B cell homeostasis and follicle confines are governed by fibroblastic reticular cells

Fibroblastic reticular cells (FRCs) are known to inhabit T cell–rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.

In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity

Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC) transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore, important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.