Hepatitis B virus (HBV) infection isCurrently, only interferon (IFN)-␣ and nucleoside or nucleotide analogues have been shown to be effective in suppressing HBV replication and in inducing clinical remission of liver disease. 2 However, increasing evidence suggests that immune mediators such as Toll-like receptor (TLR) ligands could be used successfully as therapeutic agents in HBV 3 or other viral infections. [4][5][6] TLRs play a crucial role in early host defense by recognizing so-called pathogen-associated molecular patterns that are essential for the survival of the microorganism but are not present in eukaryotes. 7 To date, 13 mammalian TLRs have been identified (10 in humans and 13 in mice), each containing a unique extracellular domain and a conserved cytoplasmic Toll/interleukin (IL)-1 receptor domain. 8 After ligand binding, the cytoplasmic Toll/IL-1 receptor domain of TLRs associates with intracellular adapters and activates downstream signaling molecules, including the transcription factors nuclear factor B, interferon regulatory factor-1, interferon regulatory factor-7, and mitogen-activated protein kinases, which lead to the activation of type I IFNs, proinflammatory cytokines, or costimulatory molecules. 9 Five adapter proteins

Summary Little is known of how the Toll‐like receptor (TLR) system can modulate the function of non‐parenchymal liver cells (NPC) as a major component of the innate and adaptive immune system of the liver. To investigate the diversification of TLR signalling pathways in NPC, we isolated Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild‐type C57BL/6 mice and examined their responses to TLR1 to TLR9 agonists. The data show that KC respond to all TLR ligands by producing tumour necrosis factor‐α (TNF‐α) or interleukin‐6 (IL‐6), to TLR3 and TLR4 ligands only by producing interferon‐β (IFN‐β), to TLR1 and TLR8 ligands by significantly up‐regulating major histocompatibility complex (MHC) class II and costimulatory molecules, and to TLR1, ‐2, ‐4 and ‐6 ligands by inducing high levels of T‐cell proliferation and IFN‐γ production in the mixed lymphocyte reaction (MLR). Similarly, LSEC respond to TLR1 to ‐4, ‐6, ‐8 and ‐9 ligands by producing TNF‐α, to TLR3 and ‐4 ligands by producing IL‐6, and to TLR3 ligands by producing IFN‐β. Interestingly, despite significant up‐regulation of MHC class II and co‐stimulatory molecules in response to TLR8 ligands, LSEC stimulated by TLR1, ‐2 or ‐6 could stimulate allogeneic T cells as assessed by MLR. By contrast, myeloid dendritic cells, used as positive control for classical antigen‐presenting cells, respond to TLR1, ‐2, ‐4 and ‐9 ligands by both up‐regulation of CD40 and activation of allogeneic T cells. In conclusion, NPC display a restricted TLR‐mediated activation profile when compared with ‘classical’ antigen‐presenting cells which may, at least in part, explain their tolerogenic function in the liver.

The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n ¼ 46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk À/À T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK R29H mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

Homozygocity mappingWe searched for homozygous regions in the DNA samples of subjects IV-4 and IV-3 using GeneChip Human Mapping 250K NspI Array of Affymetrix. The chip allows genotyping of SNPs with an average distance of approximately 50 kb between the markers. Digestion with NspI, ligation of the adaptors, and amplification with generic primers that recognize the adaptor sequence were followed by fragmentation, end labeling, and hybridization to the chip in accordance with the manufacturer's instructions. Homozygous regions greater than 5.0 Mb were manually detected. 15 Mutation analysisMutation analysis was performed by direct sequencing of PCR fragments obtained after nested amplification of the exonic and flanking intronic region coding sequences of ITK 17 exons. Primers to amplify the genomic DNA samples were designed according to GenBank sequences. Direct cycle sequencing of all PCR fragments was performed with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and analyzed by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Analyzed sequences were compared with the cDNA and genomic DNA sequences in GenBank accession numbers NM_005546 (human ITK mRNA). Constructs and immunoblot analyses Results and Discussion Clinical phenotypeThe family tree and a clinical summary of the affected family are presented in Figure 1A and Table 1. Subject IV-5 presented at the age of 4.5 years with fever, lymphadenopathy and splenomegaly. Her past history was significant for recurrent febrile episodes which had started at four years of age. Lymph node biopsy revealed Hodgkin's lymphoma, CD30+ and EBV-LMP + and CD20-. She was treated with a regimen for advanced stage Hodgkin's disease with a good response. Four months off IL-2-inducible T-cell kinase deficiency haematologica | 2011; 96(3) 473 therapy she presented with fever, skin rash, lung disease, splenomegaly, trilineage cytopenia, hypertriglyceridemia, hypofibrinogenemia and hyperferritinemia. Bone marrow evaluation showed hemophagocytosis with Hodgkin cells positive for CD20, CD30 and EBV-LMP. Serum EBV PCR showed 125,850 copies/mL and positive EBNA IgG.Immunoglobulins were low (IgG 463 mg/dL, undetectable IgA and IgM). She was diagnosed with relapsed Hodgkin's lymphoma and hemophagocytic lymphohistiocytosis. Therapy with steroids, rituximab and chemotherapy (VP-16, vinorelbine and gemcitabine) was started but she developed further disease progression with respiratory failure and died. Subject IV-3 presented at five years of age with fever and lymphadenopathy. His past history included a profound sensorineural hearing defect, mild mental retardation and recurrent infections. Laboratory tests showed hypogammaglobulinemia (IgG 291 mg/dL, IgA and IgM below 42 and 32, respectively), positive anti-EBV VCA IgG, and negative anti-EBNA. EBV PCR was not carried out. A lymph node biopsy showed mixed cellularity Hodgkin's lymphoma with numerous Hodgkin's cells and few CD30 positive and CD20 negative Reed-Sternberg cells...

IntroductionDendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system, and they are pivotal in the initiation of immune responses against viruses. 1 However, a number of viruses are able to infect DCs, and several recent studies have investigated the effect of such infections on the biology of DCs. Although only very few studies report enhanced or unchanged functions of DCs after viral infection, most viruses seem to impair functional properties of DCs (for review, see Pollara et al 2 ). These data imply that viruses infect DCs as a strategy of immune escape.In most reports that demonstrate the impairment of DCs by viruses, the morphology, phenotype, viability, and the ability to secrete cytokines of infected DCs was found to be altered, suggesting that viruses might have a significant impact on the DC-mediated initiation of an immune response in an infected host. 2 In fact, there is 1 report that directly shows that measles virus infection of DCs can result in unstable DC-T-cell contacts and, as a consequence, in impaired T-cell activation. 3 Retroviruses, such as HIV, are also known to infect DCs and interfere with their maturation. 4 Maturation of DCs can easily be measured by analyzing the expression of costimulatory molecules such as CD40, CD80, and CD86, or maturation markers like the molecule CD83. 5 However, the biological consequences of retrovirus infection of DCs for antigen presentation to T cells has not been investigated in detail so far. The most important functional properties of DCs are T-cell engagement and subsequent activation, which is the critical step in inducing adaptive immunity after infection. Since the interaction of DCs and naive T cells requires physical cell-cell contact, we used a 3D collagen matrix model 6 to investigate the contact duration and kinetics of virus-infected DCs with naive T cells. Friend virus (FV) is a retroviral complex comprised of 2 components: a replicationcompetent helper virus called Friend murine leukemia virus (F-MuLV), which is nonpathogenic in adult mice; and a replicationdefective but pathogenic component called spleen focus-forming virus (SFFV). 7 Coinfection of cells by the 2 viruses allows SFFV to spread by being packaged into F-MuLV-encoded virus particles. FV infection of susceptible adult mice induces polyclonal proliferation of erythroid precursor cells, causing severe splenomegaly. This proliferation is caused by the binding of the SFFV envelope glycoprotein to the erythropoietin receptors of nucleated erythroid cells. 8 In susceptible mice, FV subsequently transforms erythroid precursor cells, leading to fully malignant erythroleukemias. 9 However, beside erythroid precursor cells, FV can also infect a variety of other cells types, including B cells, monocytes, and granulocytes. 10 In addition, FV induces a severe generalized immunosuppression during acute infection 9,10 ; however, it is unknown whether this is the result of a functional impairment of APCs due to virus infection. Susceptibility to both FV-i...