Since there is a remarkable difference in susceptibility to peroral infection with Toxoplasma gondii among inbred strains of mice, we performed studies to examine the mechanism(s) of this difference in susceptibility. After peroral infection with the ME49 strain of T. gondii, C57BL/6 (B6) mice all died whereas BALB/c mice all survived. At day 7 of infection (when B6 mice began dying), massive necrosis of the villi and mucosal cells in the ilea were observed in B6 but not in BALB/c mice. To analyze the role of T cells in resistance against death and development of necrosis in the ilea after infection, studies were performed using athymic nude and euthymic control B6 and BALB/c mice. Athymic B6 mice all died after infection, but surprisingly, they survived significantly longer than control B6 mice, indicating that T cells predispose to early death in these mice. Necrosis in the ilea was observed in control B6 but not in athymic B6 mice; however, significantly less numbers of tachyzoites were observed in the ilea of the former than the latter mice. These results indicate that necrosis in the ilea of the B6 mice was not due to destruction of tissue by tachyzoites but was mediated by T cells. This deleterious effect of T cells appears to contribute to early death in these mice. In contrast, T cells conferred resistance against death in BALB/c mice but did not cause necrosis in their ilea. To analyze the T cell subset(s) that induces necrosis of the ilea in B6 mice, we examined histological changes of the small intestines after infection of mutant mice deficient in different T cell subsets (with the same H-2b haplotype as B6 mice). Mice deficient in alpha/beta or CD4+ T cells did not develop necrosis in the ilea, whereas wild-type control mice and mice deficient in gamma/delta or CD8+ T cells did, suggesting that the cells that induce necrosis in the ilea after infection are CD4+ alpha/beta T cells. Since interferon (IFN)-gamma has been shown to be critical for survival of BALB/c mice after infection with T. gondii, we examined the role of this cytokine in resistance/susceptibility of infected B6 mice. Treatment of B6 mice with anti-IFN-gamma monoclonal antibody shortly before they developed illness prolonged time to death and prevented necrosis in the ilea in these mice. These results indicate that IFN-gamma mediates necrosis in the ilea of B6 mice after infection. This CD4+ T cell-dependent, IFN-gamma-mediated necrosis of the small intestines appears to be a mechanism that underlies the genetic susceptibility of B6 mice to peroral infection with T. gondii, whereas the same cytokine plays a critical role in the resistance of genetically resistant BALB/c mice.

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.

The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm [del(9p)], unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The alpha- and beta 1-interferon genes have been assigned to this chromosome region (9p21-22). We now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5'-methylthioadenosine phosphorylase (EC 2.4.2.28), an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them.

We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. V alpha 24+V beta 11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-gamma production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, alpha-galactosylceramide (alpha-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.

Glucocorticoids are widely used to treat patients with autoimmune diseases such as systemic lupus erythematosus (SLE). However, regimens used to treat many such conditions cannot maintain disease control in the majority of SLE patients and more aggressive approaches such as high-dose methylprednisolone pulse therapy are used to provide transient reductions in disease activity. The primary anti-inflammatory mechanism of glucocorticoids is thought to be NF-kappaB inhibition. Recognition of self nucleic acids by toll-like receptors TLR7 and TLR9 on B cells and plasmacytoid dendritic cells (PDCs) is an important step in the pathogenesis of SLE, promoting anti-nuclear antibodies and the production of type I interferon (IFN), both correlated with the severity of disease. Following their activation by self-nucleic acid-associated immune complexes, PDCs migrate to the tissues. We demonstrate, in vitro and in vivo, that stimulation of PDCs through TLR7 and 9 can account for the reduced activity of glucocorticoids to inhibit the IFN pathway in SLE patients and in two lupus-prone mouse strains. The triggering of PDCs through TLR7 and 9 by nucleic acid-containing immune complexes or by synthetic ligands activates the NF-kappaB pathway essential for PDC survival. Glucocorticoids do not affect NF-kappaB activation in PDCs, preventing glucocorticoid induction of PDC death and the consequent reduction of systemic IFN-alpha levels. These findings unveil a new role for self nucleic acid recognition by TLRs and indicate that inhibitors of TLR7 and 9 signalling could prove to be effective corticosteroid-sparing drugs.

Interferons (IFNs) alpha/beta (type I) and gamma (type II) bind to distinct cell surface receptors, inducing transcription of overlapping sets of genes by intracellular pathways that have recently attracted much attention. Previous studies using cell lines selected for their inability to respond to IFN-alpha (ref. 4) have shown that the protein kinase Tyk2 plays a central role in the IFN alpha/beta response. Here we report the isolation of the cell line gamma 1A, selected for its inability to express IFN-gamma-inducible cell-surface markers, that is deficient in all aspects of the IFN-gamma response tested, but responds normally to IFNs alpha and beta. The mutant cells can be complemented by the expression of another member of the JAK family of protein tyrosine kinases, JAK2 (refs 6-9). Unlike IFNs alpha and beta, IFN-gamma induces rapid tyrosine phosphorylation of JAK2 in wild-type cells, and JAK2 immunoprecipitates from these cells show tyrosine kinase activity. These responses are absent in gamma 1A cells. JAK2 is therefore required for the response to IFN-gamma but not to IFNs alpha and beta.

OBJECTIVE

To compare tear cytokine and chemokine concentrations in asymptomatic control and Dysfunctional Tear syndrome (DTS) patients and determine the correlations between tear inflammatory mediators and clinical severity.

METHODS

Prospective observational cohort study.

METHODS

Concentrations of epidermal growth factor (EGF), interleukin (IL)-1 alpha (1alpha), 1 beta (1beta), 6, 10, 12, and 13, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and chemokines: IL-8 (CXC); macrophage inflammatory protein-1 alpha (MIP-1alpha) (CCL3); and regulated upon activation, normal T-cell expressed and secreted (RANTES CCL5) were measured by a multiplex immunobead assay in an asymptomatic control group and DTS patients with and without meibomian gland disease (MGD). Spearman correlations between tear cytokines and severity of irritation symptoms and ocular surface signs were calculated.

RESULTS

Tear concentrations of IL-6, IL-8 and TNF-alpha were significantly higher in DTS with and without MGD and EGF was significantly reduced in the DTS without MGD group compared with the control group. MIP-1alpha was greater in entire DTS and DTS without MGD groups than the control group and RANTES was greater in DTS with MGD than the control and DTS without MGD groups. IL-12 was significantly higher in the DTS with MGD than the DTS without MGD subgroup. Significant correlations were observed between IL-6 and irritation symptoms and between a number of cytokines and chemokines and clinical parameters.

CONCLUSIONS

As predicted, patients with DTS have higher levels of inflammatory mediators in their tears that show correlation with clinical disease parameters. Furthermore, different tear cytokine/chemokine profiles were observed in DTS patients with and without MGD groups.

The efficient induction of interferons alpha and beta (IFN-alpha/beta) in virus-infected cells is central to the antiviral response of a host and is regulated mainly at the level of gene transcription. Once produced, IFN-alpha/beta transmit signals to the cell interior via a specific receptor complex to induce an antiviral response. Recently, the auto-amplification mechanism of the IFN-alpha/beta system that follows viral infection has been identified. This mechanism is mediated by transcription factors of the IFN regulatory factor family and, in fact, may have evolved to render the system more robust in antiviral responses.

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.

Human interleukin (IL) 1 receptor-associated kinase 4 (IRAK-4) deficiency is a recently discovered primary immunodeficiency that impairs Toll/IL-1R immunity, except for the Toll-like receptor (TLR) 3- and TLR4-interferon (IFN)-alpha/beta pathways. The clinical and immunological phenotype remains largely unknown. We diagnosed up to 28 patients with IRAK-4 deficiency, tested blood TLR responses for individual leukocyte subsets, and TLR responses for multiple cytokines. The patients' peripheral blood mononuclear cells (PBMCs) did not induce the 11 non-IFN cytokines tested upon activation with TLR agonists other than the nonspecific TLR3 agonist poly(I:C). The patients' individual cell subsets from both myeloid (granulocytes, monocytes, monocyte-derived dendritic cells [MDDCs], myeloid DCs [MDCs], and plasmacytoid DCs) and lymphoid (B, T, and NK cells) lineages did not respond to the TLR agonists that stimulated control cells, with the exception of residual responses to poly(I:C) and lipopolysaccharide in MDCs and MDDCs. Most patients (22 out of 28; 79%) suffered from invasive pneumococcal disease, which was often recurrent (13 out of 22; 59%). Other infections were rare, with the exception of severe staphylococcal disease (9 out of 28; 32%). Almost half of the patients died (12 out of 28; 43%). No death and no invasive infection occurred in patients older than 8 and 14 yr, respectively. The IRAK-4-dependent TLRs and IL-1Rs are therefore vital for childhood immunity to pyogenic bacteria, particularly Streptococcus pneumoniae. Conversely, IRAK-4-dependent human TLRs appear to play a redundant role in protective immunity to most infections, at most limited to childhood immunity to some pyogenic bacteria.

Interferons (IFNs) are a heterogeneous family of cytokines that exhibits multiple biological activities. Upon viral infection, expression of type I IFNs (i.e., IFN-alpha and IFN-beta) is induced in a variety of differentiated cells but not in cells of embryonal origin. IRF-1 and IRF-2, which bind to the same cis-elements within the promoters of type I IFN and IFN-inducible MHC class I genes, were identified previously. Here we demonstrate that the expression of both IRF and IFN genes is developmentally regulated in mouse EC cells; these genes become functional only after cell differentiation. Furthermore, cDNA-directed IRF-1 produced in undifferentiated but not differentiated EC cells efficiently activates the transfected IFN-alpha and IFN-beta and endogenous IFN-alpha genes, whereas IRF-2 represses the IRF-1 effects. These findings emphasize the dual function of the IRF-responsive cis-elements as positive and negative regulators, since they can be occupied by transcriptionally active or inactive IRF molecules. This type of regulatory mechanism might operate in other cytokine systems.

Increasing evidence supports involvement of inflammation and immunity in atherogenesis. We report here that CD40 ligand (CD40L), an immunoregulatory signaling molecule heretofore considered largely restricted to recently activated CD4+ T lymphocytes, is expressed by human vascular endothelial cells (EC), smooth muscle cells (SMC), and human macrophages in vitro, and is coexpressed with its receptor CD40 on all three cells types in human atherosclerotic lesions in situ. Cultured human vascular EC, SMC, and human macrophages all constitutively expressed CD40L mRNA as well as protein. Stimulation with interleukin 1beta, tumor necrosis factor alpha, or interferon gamma increased surface levels and de novo synthesis of CD40L on all three cell types. CD40L expressed on EC, SMC, and macrophages exhibited biological activity, as it induced BB cells. Human vascular SMC also constitutively expressed CD40, the receptor for CD40L, and through CD40 signaling, human recombinant CD40L induced expression of proinflammatory cytokines in these cells, identifying SMC as a target for CD40L. Human atherosclerotic lesions (n = 8) showed expression of immunoreactive CD40L on EC, SMC, and macrophages, while normal arterial tissues (n = 5) contained no CD40L. In atheroma CD40L+ cells often also expressed CD40. These observations establish human vascular EC, SMC, and human macrophages as a novel source of CD40L, and point to T cell-independent CD40 signaling, and a broader function of this pathway in regulation of nonimmune cells, as illustrated here by potential autocrine and paracrine activation during atherogenesis.

The cytokine profile produced by peripheral blood mononuclear cells (PBMC) in response to leishmania antigens and the ability of interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) to modulate the immune response were evaluated in 21 mucosal leishmaniasis patients. Patients with mucosal disease exhibited increased gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) secretion and decreased IL-10 secretion compared to patients with classical cutaneous leishmaniasis. CD4(+) Th1 cells were the main source of IFN-gamma and TNF-alpha production in mucosal leishmaniasis patients. Evaluation of cytokine gene expression in PBMC of these patients showed that there was strong up-regulation of IFN-gamma transcripts upon stimulation with leishmania antigen, in contrast to the baseline levels of IL-10 mRNA. IL-10 suppressed IFN-gamma production by 48% in cell cultures from cutaneous leishmaniasis patients and by 86% in cell cultures from healthy subjects stimulated with purified protein derivative, whereas in similar conditions IL-10 suppressed IFN-gamma production by 19% in cell cultures from mucosal leishmaniasis patients stimulated with leishmania antigen. TGF-beta suppressed IFN-gamma levels to a greater extent in healthy subjects than in mucosal leishmaniasis and cutaneous leishmaniasis patients. These data indicate that a poorly modulated T-cell response in mucosal leishmaniasis patients leads to production of high levels of proinflammatory cytokines, such as IFN-gamma and TNF-alpha, as well as a decreased ability of IL-10 and TGF-beta to modulate this response. These abnormalities may be the basis for the pathological findings observed in this disease.

Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

The production of cytokines during aging, except interleukin (IL)-2, has been neglected in humans. We measured the in vitro production of IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and IL-1 beta by peripheral mononuclear cells from selected healthy young (mean age 26.8 years) and aged (mean age 80.2 years) subjects. Significant increases of IL-6, TNF-alpha and IL-1 beta levels were found in mitogen-stimulated cultures from aged donors, occurring at 24 to 72 h after stimulation. No significant differences were observed for IFN-gamma production. Proliferative capability of cells stimulated with PHA was not impaired in aged subjects. Since the amounts of all cytokines studied were similar in unstimulated cultures from young and aged subjects, and also serum levels of TNF-alpha did not differ, these data indicate that the cellular machinery for the production of these cytokines is well preserved in aging, and also that cells from old people are able to up-regulate their production in response to appropriate stimuli. The increases in cytokine synthesis were not dependent on changes in the number of monocytes, nor were they related to the significant rise of CD45RO+, and the concomitant decrease of CD45RA+, occurring in both CD4+ and CD8+ lymphocytes from aged subjects. The increased production of pro-inflammatory cytokines by stimulated mononuclear cells of healthy aged subjects may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis and dementia.

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-alpha and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-alpha production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-alpha production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1-positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-alpha from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1-positive endosomes accompanied by the loss of IFN-alpha production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-alpha and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-alpha response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs.

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-alpha and IFN-beta), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Several bacterial pathogens of medical importance are able to persist and replicate inside host mononuclear phagocytes. Protective immunity depends on specific T lymphocytes that induce granulomatous lesions at the sites of bacterial multiplication. Listeria monocytogenes is an intracellular pathogen that replicates inside mononuclear phagocytes and hepatocytes of mice. Invasion from the phagosomal compartment into the cytoplasmic compartment is the principal mechanism of intracellular survival. Early in infection, resistance against L. monocytogenes is mediated by polymorphonuclear phagocytes which destroy infected liver cells, followed by natural killer cells which activate macrophages by means of interferon-gamma (refs 6, 7). A specific immune response by T cells then develops which leads to sterile eradication of the microbes. T cells are also responsible for the highly effective protection in vaccinated mice against secondary infections. Although the role of alpha beta T cells has been demonstrated in these immune responses, that of gamma delta T cells is unclear. Here we use mice that selectively lack either alpha beta or gamma delta T cells as a result of targeted germ-line mutations in their T-cell receptor genes to investigate the relative roles of these T-cell populations during experimental infection with L. monocytogenes. We find that in primary listeriosis, either alpha beta or gamma delta T cells are sufficient for early protection. Resistance to secondary infection is mediated mainly by alpha beta T cells but also involves gamma delta T cells. Thus alpha beta T-cell-deficient mice can be rendered partially resistant by vaccination, and gamma delta T cells are shown to be responsible for this protective effect. In infected gamma delta T-cell-deficient mice we noticed the appearance of unusual liver lesions, indicating that gamma delta T cells have a unique regulatory role in this bacterial infection.

The cytokine transforming growth factor (TGF)-beta, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-beta receptor (TGF-betaR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-beta-sensitive CCL64 cells mediated by recombinant TGF-betabetabeta-containing glioma cell supernatant at an EC(50) of 0.1 mumol/L. SD-208 blocks autocrine and paracrine TGF-beta signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-beta reporter assays and strongly inhibits constitutive and TGF-beta-evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-beta-releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon gamma and tumor necrosis factor alpha by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-beta or of TGF-beta-containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-beta-induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-beta receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-beta activity.

OBJECTIVE

To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS).

METHODS

B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS.

RESULTS

B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha.

CONCLUSIONS

We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-alpha production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-alpha and IL-6 production and NF-kappaB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.

The current status of T-cell subset involvement in viral immunity is summarized for experimental studies in mice. The immunobiology of the normal host response is discussed, with particular reference to lymphocytic choriomeningitis (LCM) and influenza. The general impression is that CD8+ cytotoxic T lymphocytes, CD4+ TH1 cells, gamma interferon, and IL-2 are of major importance, with these different components of the immune system interacting to promote an optimal response. However, experiments with a variety of virus systems indicate that there is considerable plasticity, at least in young, adult mice. Other mechanisms often compensate if a key lymphocyte subset is absent throughout the development of the immune response. Influenza-infected mice depleted of either CD4+ or CD8+ T cells clear virus and recover, though the latter may not be true for the elimination of LCM virus. Emerging information on the involvement of gamma delta T cells in viral pneumonia is summarized, but there is as yet no understanding of the biological significance (if any) of these lymphocytes in viral immunity. The point is made that alpha beta T-cell memory to viruses is long-lived, and the need for antigen persistence to maintain such memory is questioned.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.