NF-κB2-deficient mice have impaired T and B cell responses. We found, however, that in these mice there was severe infiltration of lymphocytes into multiple organs and increased activity of autoantibodies to peripheral tissue antigens in a manner similar to that of autoimmune regulator-deficient (Aire-deficient) mice. We further demonstrated that NF-κB2 was required for thymic Aire gene transcriptional regulation. The Nfkb2 -/-thymus had distinct cortical and medullar structures, but reduced Aire and target gene expression of peripheral tissue antigens. Engraftment of Nfkb2 -/-thymic stroma to nude mice recapitulated the autoimmune phenotype of the native Nfkb2 -/-mice, confirming a key defect in central tolerance. Lymphotoxin β receptor (LTβR) ligation-induced Aire gene expression was also largely abolished in the absence of NF-κB2. Thus NF-κB2 downstream of LTβR plays an important role in the regulation of central tolerance in an Aire-dependent manner.

The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment because Relb and Aire deficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions. Relb ͉ T cell differentiation ͉ thymic medulla

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

BackgroundThe identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.Methodology/Principal FindingsFirst, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×105 genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶104–1∶105 and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶103–1∶104 and were protected from hepatitis, but two out of the five rabbits showed virus shedding.Conclusions/SignificanceRabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.

Classical swine fever virus (CSFV), which causes typical clinical characteristics in piglets, including hemorrhagic syndrome and immunosuppression, is linked to hepatitis C and dengue virus. Oxidative stress and a reduced mitochondrial transmembrane potential are disturbed in CSFV-infected cells. The balance of mitochondrial dynamics is essential for cellular homeostasis. In this study, we offer the first evidence that CSFV induces mitochondrial fission and mitophagy to inhibit host cell apoptosis for persistent infection. The formation of mitophagosomes and decline in mitochondrial mass relevant to mitophagy were detected in CSFV-infected cells. CSFV infection increased the expression and mitochondrial translocation of Pink and Parkin. Upon activation of the PINK1 and Parkin pathways, Mitofusin 2 (MFN2), a mitochondrial fusion mediator, was ubiquitinated and degraded in CSFV-infected cells. Mitophagosomes and mitophagolysosomes induced by CSFV were, respectively, observed by the colocalization of LC3-associated mitochondria with Parkin or lysosomes. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of mitophagosomes to lysosomes. Mitochondrial fission caused by CSFV infection was further determined by mitochondrial fragmentation and Drp1 translocation into mitochondria using a confocal microscope. The preservation of mitochondrial proteins, upregulated apoptotic signals and decline of viral replication resulting from the silencing of Drp1 and Parkin in CSFV-infected cells suggested that CSFV induced mitochondrial fission and mitophagy to enhance cell survival and viral persistence. Our data for mitochondrial fission and selective mitophagy in CSFV-infected cells reveal a unique view of the pathogenesis of CSFV infection and provide new avenues for the development of antiviral strategies.

Mycobacterium tuberculosis PtpA is a secreted effector protein that dephosphorylates several proteins in the host cell cytoplasm, such as p-JNK, p-p38, and p-VPS33B, leading to suppression of host innate immunity. Here we show that, in addition, PtpA enters the nucleus of host cells and regulates the expression of host genes, some of which are known to be involved in host innate immunity or in cell proliferation and migration (such as GADD45A). PtpA can bind directly to the promoter region of GADD45A in vitro. Both phosphatase activity and DNA-binding ability of PtpA are important in suppressing host innate immune responses. Furthermore, PtpA-expressing Mycobacterium bovis BCG promotes proliferation and migration of human lung adenoma A549 cells in vitro and in a mouse xenograft model. Further research is needed to test whether mycobacteria, via PtpA, might affect cell proliferation or migration in humans.

Lymph node (LN) hypertrophy, the increased cellularity of LNs, is the major indication of the initiation and expansion of the immune response against infection, vaccination, cancer or autoimmunity. The mechanisms underlying LN hypertrophy remain poorly defined. Here, we demonstrate that LIGHT (TNFSF14) is a novel factor essential for LN hypertrophy after CFA immunization. Mechanistically, LIGHT is required for the influx of lymphocytes into but not egress out of LNs. In addition, LIGHT is required for DC migration from the skin to draining LNs. Compared with WT mice, LIGHT−/− mice express lower levels of chemokines in skin and addressins in LN vascular endothelial cells after CFA immunization. We unexpectedly observed that LIGHT from radioresistant rather than radiosensitive cells, likely Langerhans cells, is required for LN hypertrophy. Importantly, antigen-specific T cell responses were impaired in DLN of LIGHT−/− mice, suggesting the importance of LIGHT regulation of LN hypertrophy in the generation of an adaptive immune response. Collectively, our data reveal a novel cellular and molecular mechanism for the regulation of LN hypertrophy and its potential impact on the generation of an optimal adaptive immune response.

Lymph nodes (LNs) maintain active homeostasis at steady state. However, in response to changes in the local environment, such as local infection, cancer, vaccination, and autoimmune disease, dramatic remodeling of LN occurs. This remodeling includes changes in size, lymph and blood flow, immune cell trafficking and cellularity, lymphatic and blood vessel growth and activation, as well as microarchitecture. Therefore, inflammatory conditions often lead to enlarged nodes; after local inflammation resolves, LNs actively regress in size and return to steady state. Remodeling of lymphatic vessels (LVs) and blood vessels (BVs) during both the expansion and regression phases are key steps in controlling LN size as well as function. The cells, membrane-associated molecules, and soluble cytokines that are essential for LV and BV homeostasis as well as dynamic changes in the expansion and regression phases have not been well defined. Understanding the underlying cellular and molecular mechanisms behind LN remodeling would help us to better control undesired immune responses (e.g. inflammation and autoimmune diseases) or promote desired responses (e.g. antitumor immunity and vaccination). In this review, we focus on how the closely related tumor necrosis factor (TNF) members: LIGHT (TNFSF14), lymphotoxin-αβ, and TNF-α contribute to the remodeling of LNs at various stages of inflammation.