Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control.

Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (<10,000 HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia (P < 0.0001) and are positively correlated with proviral HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/ß-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor (IL27RA) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC.IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins, and the blood is a key conduit for transporting such factors. Investigating the communication factors promoting effective immune responses and having potentially antiviral functions against HIV using a novel focused omics approach ("communicome") has the potential to significantly improve our knowledge of effective host immunity and accelerate the HIV cure agenda. Including 140 subjects with variable viral loads and measuring the plasma levels of >600 soluble proteins, our data highlight the importance of Th17 cells and Wnt/ß-catenin signaling in HIV control and especially identify the IL-27/IL-27 receptor subunit alpha (IL-27RA) axis as a predictor of plasma viral load and proviral copy number in the peripheral blood. These data may provide important guidance to therapeutic approaches in the HIV cure agenda.

Neuroprotective natural antibodies to assemblies of amyloidogenic peptides decrease with normal aging and advancing Alzheimer's disease.

A number of distinct beta-amyloid (Abeta) variants or multimers have been implicated in Alzheimer's disease (AD), and antibodies recognizing such peptides are in clinical trials. Humans have natural Abeta-specific antibodies, but their diversity, abundance, and function in the general population remain largely unknown. Here, we demonstrate with peptide microarrays the presence of natural antibodies against known toxic Abeta and amyloidogenic non-Abeta species in plasma samples and cerebrospinal fluid of AD patients and healthy controls aged 21-89 years. Antibody reactivity was most prominent against oligomeric assemblies of Abeta and pyroglutamate or oxidized residues, and IgGs specific for oligomeric preparations of Abeta1-42 in particular declined with age and advancing AD. Most individuals showed unexpected antibody reactivities against peptides unique to autosomal dominant forms of dementia (mutant Abeta, ABri, ADan) and IgGs isolated from plasma of AD patients or healthy controls protected primary neurons from Abeta toxicity. Aged vervets showed similar patterns of plasma IgG antibodies against amyloid peptides, and after immunization with Abeta the monkeys developed high titers not only against Abeta peptides but also against ABri and ADan peptides. Our findings support the concept of conformation-specific, cross-reactive antibodies that may protect against amyloidogenic toxic peptides. If a therapeutic benefit of Abeta antibodies can be confirmed in AD patients, stimulating the production of such neuroprotective antibodies or passively administering them to the elderly population may provide a preventive measure toward AD.

Novel role of human CD4 molecule identified in neurodegeneration.

The human CD4 molecule (hCD4) is expressed on T lymphocytes and macrophages and acts as a key component of the cellular receptor for HIV. At baseline, hCD4 transgenic mice expressed hCD4 on microglia, the resident mononuclear phagocytes of the brain, and showed no neuronal damage. Activation of brain microglia by peripheral immune challenges elicited neurodegeneration in hCD4 mice but not in nontransgenic controls. In post-mortem brain tissues from AIDS patients with opportunistic infections, but without typical HIV encephalitis, hCD4 expression correlated with neurodegeneration. We conclude that hCD4 may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system.

TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.

Abnormal accumulation of the amyloid-beta peptide (Abeta) in the brain appears crucial to pathogenesis in all forms of Alzheimer disease (AD), but the underlying mechanisms in the sporadic forms of AD remain unknown. Transforming growth factor beta1 (TGF-beta1), a key regulator of the brain's responses to injury and inflammation, has been implicated in Abeta deposition in vivo. Here we demonstrate that a modest increase in astroglial TGF-beta1 production in aged transgenic mice expressing the human beta-amyloid precursor protein (hAPP) results in a three-fold reduction in the number of parenchymal amyloid plaques, a 50% reduction in the overall Abeta load in the hippocampus and neocortex, and a decrease in the number of dystrophic neurites. In mice expressing hAPP and TGF-beta1, Abeta accumulated substantially in cerebral blood vessels, but not in parenchymal plaques. In human cases of AD, Abeta immunoreactivity associated with parenchymal plaques was inversely correlated with Abeta in blood vessels and cortical TGF-beta1 mRNA levels. The reduction of parenchymal plaques in hAPP/TGF-beta1 mice was associated with a strong activation of microglia and an increase in inflammatory mediators. Recombinant TGF-beta1 stimulated Abeta clearance in microglial cell cultures. These results demonstrate that TGF-beta1 is an important modifier of amyloid deposition in vivo and indicate that TGF-beta1 might promote microglial processes that inhibit the accumulation of Abeta in the brain parenchyma.

Dysregulation of signal transduction pathways as a potential mechanism of nervous system alterations in HIV-1 gp120 transgenic mice and humans with HIV-1 encephalitis.

HIV-1 associated central nervous system (CNS) disease involves neuronal damage and prominent reactive astrocytosis, the latter characterized by strong upregulation of the glial fibrillary acidic protein (GFAP) in astrocytes. Similar alterations are found in transgenic mice expressing the HIV-1 envelope protein gp120 in the CNS. Because alterations of astrocyte functions could contribute to neuronal impairment, we compared brains of gp120 transgenic mice and gp120-transfected C6 astrocytoma cells with controls and found that gp120 induced a prominent elevation of steady state GFAP mRNA levels, primarily due to transcript stabilization. Increased levels of GFAP mRNA were also found in nontransfected C6 cells exposed to recombinant gp120. Exposure of C6 cells or primary mouse astrocytes to soluble gp120 led to activation of PKC as indicated by redistribution and increase in PKC immunoreactivity at the single cell level. gp120 effects were diminished by inhibitors of protein kinase C (PKC) but not inhibitors of protein kinase A. PKC activity was upmodulated in gp120-transfected C6 cells and in the CNS of gp120 transgenic mice. Further, brain tissue from patients with HIV-1 encephalitis and from gp120 transgenic mice showed increased PKC immunoreactivity. Taken together, these results indicate that gp120-induced increases in PKC activity may contribute to the gliosis seen in gp120 transgenic mice as well as in HIV-1-infected humans and raise the question of whether dysregulation of signal transduction pathways represents a general mechanism of HIV-associated pathogenesis.

Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/- mice: isoform-specific effects on neurodegeneration.

Apolipoprotein (apo) E isoforms are key determinants of susceptibility to Alzheimer's disease. The apoE4 isoform is the major known genetic risk factor for this disease and is also associated with poor outcome after acute head trauma or stroke. To test the hypothesis that apoE3, but not apoE4, protects against age-related and excitotoxin-induced neurodegeneration, we analyzed apoE knockout (Apoe-/-) mice expressing similar levels of human apoE3 or apoE4 in the brain under control of the neuron-specific enolase promoter. Neuronal apoE expression was widespread in the brains of these mice. Kainic acid-challenged wild-type or Apoe-/- mice had a significant loss of synaptophysin-positive presynaptic terminals and microtubule-associated protein 2-positive neuronal dendrites in the neocortex and hippocampus, and a disruption of neurofilament-positive axons in the hippocampus. Expression of apoE3, but not of apoE4, protected against this excitotoxin-induced neuronal damage. ApoE3, but not apoE4, also protected against the age-dependent neurodegeneration seen in Apoe-/- mice. These differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of Alzheimer's disease and to the poor outcome after head trauma and stroke associated with apoE4 in humans.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

A cell surface ELISA for the screening of monoclonal antibodies to antigens on viable cells in suspension.

To simplify the screening of monoclonal antibodies to different human T cell surface molecules a live cell enzyme-linked immunosorbent assay (cell ELISA) has been established and optimized. The assay was performed in 96-well plates. By using living human T lymphocytes in suspension surface modification by fixation or insolubilization of the cells was avoided. Several parameters influencing sensitivity and specificity were studied. About 150 ng/ml of mouse monoclonal antibodies to cell surface antigens could be detected when using 5 x 10(4) cells per well and a 1/1000 dilution of the anti-mouse IgG-alkaline phosphatase conjugate. This sensitivity permitted the primary screening of cell specific antibodies from hybridoma supernatants. The same detection limit was obtained in flow cytometric analysis. If required, the sensitivity of the cell ELISA could be increased using higher cell numbers and conjugate concentration. When analysing different cell lines with selected antibodies the cell ELISA was found to be as sensitive and specific as the fluorescence assay. The assay was applied to the screening of supernatants from hybridomas developed against human T helper cell clones and the detection of V beta specificities of T cell clones.

Astroglial overproduction of TGF-beta 1 enhances inflammatory central nervous system disease in transgenic mice .

Cerebral expression of the injury response cytokine transforming growth factor-beta 1 (TGF-beta 1) has been found to be increased in several neurological diseases but it remains unclear whether its function is primarily beneficial or detrimental. Here we show that transgenic (tg) mice that overexpress bioactive (TGF-beta 1 in the central nervous system (CNS) and show no overt phenotype in the unmanipulated state, are more susceptible to the immune-mediated CNS disease experimental autoimmune encephalomyelitis (EAE). TGF-beta 1 tg mice with EAE showed an earlier onset of clinical symptoms, more severe disease and increased mononuclear cell infiltration in their spinal cords compared with non-tg littermate controls with EAE. Whereas previous observations indicated that increased peripheral levels of TGF-beta 1 can suppress EAE, our findings demonstrate that local expression of TGF-beta 1 within the CNS parenchyma can enhance immune cell infiltration and intensify the CNS impairment resulting from peripherally triggered autoimmune responses.

Dominant negative effects of apolipoprotein E4 revealed in transgenic models of neurodegenerative disease.

Apolipoprotein E fulfills fundamental functions in lipid transport and neural tissue repair after injury.(6,8) Its three most common isoforms (E2, E3, and E4) are critical determinants of diverse human diseases, including major cardiovascular and neurodegenerative disorders.(8,14) Apolipoprotein E4 is associated with an increased risk for Alzheimer's disease(3,5) and poor clinical outcome after head injury or stroke.(11,16) The precise role of apolipoprotein E4 in these conditions remains unknown. To characterize the effects of human apolipoprotein E isoforms in vivo, we analysed transgenic Apoe knockout mice that express apolipoprotein E3 or E4 or both in the brain. Hemizygous and homozygous apolipoprotein E3 mice were protected against age-related and excitotoxin-induced neurodegeneration, whereas apolipoprotein E4 mice were not. Apolipoprotein E3/E4 bigenic mice were as susceptible to neurodegeneration as apolipoprotein E4 singly-transgenic mice. At eight months of age neurodegeneration was more severe in homozygous than in hemizygous apolipoprotein E4 mice consistent with a dose effect. Thus, apolipoprotein E4 is not only less neuroprotective than apolipoprotein E3 but also acts as a dominant negative factor that interferes with the beneficial function of apolipoprotein E3. The inhibition of this apolipoprotein E4 activity may be critical for the prevention and treatment of neurodegeneration in APOE varepsilon4 carriers.

Functional role of TGF beta in Alzheimer's disease microvascular injury: lessons from transgenic mice.

Recent studies have implicated pro- and anti-inflammatory cytokines as integral to Alzheimer's disease (AD) pathogenesis. Among them, transforming growth factor-beta (TGF-beta) is emerging as an important factor in regulating inflammatory responses. This multifunctional cytokine might be centrally involved in several aspects of AD pathogenesis by regulating beta-amyloid precursor protein synthesis and processing, plaque formation, astroglial and microglial response and neuronal cell death. Among all of these potential roles, studies in transgenic and infusion animal models have shown that TGF-beta may primarily contribute to AD pathogenesis by influencing A beta production and deposition, which in turn might result in damage to the brain microvasculature. The lessons learned from these models are of great interest not only for understanding of the role of TGF-beta in AD, but also for future treatments where testing of anti-inflammatory agents such as ibuprofen and an amyloid vaccine hold great promise. In this regard, further elucidation of the signal pathways by which TGF-beta exerts its effect in AD might lead to specific targets for further therapeutic intervention.

Alzheimer's disease-like cerebrovascular pathology in transforming growth factor-beta 1 transgenic mice and functional metabolic correlates.

Alzheimer's disease (AD) is frequently associated with cerebrovascular changes, including perivascular astrocytosis, amyloid deposition, and microvascular degeneration, but it is not known whether these pathological changes contribute to functional deficits in AD. To characterize the temporal relationship between amyloid deposition, cerebrovascular abnormalities, and potential functional changes, we studied transgenic mice that express transforming growth factor-beta 1 (TGF-beta 1) at low levels in astrocytes. TGF-beta 1 induced a prominent perivascular astrocytosis, followed by the accumulation of basement membrane proteins in microvessels, thickening of capillary basement membranes, and later, around 6 months of age, deposition of amyloid in cerebral blood vessels. At 9 months of age, various AD-like degenerative alterations were observed in endothelial cells and pericytes. Associated with these morphological changes were changes in regional cerebral glucose utilization. Preliminary results showed that TGF-beta 1 mice had significantly decreased glucose utilization in the mammillary bodies, structures involved in mnemonic and learning processes. Glucose utilization tended to be decreased in several other brain regions as well; however, in the inferior colliculus, it was markedly higher in TGF-beta 1 mice than in controls. We conclude that chronic overproduction of TGF-beta 1 triggers a pathogenic cascade leading to AD-like cerebrovascular amyloidosis, microvascular degeneration, and local alterations in brain metabolic activity. Similar mechanisms may be involved in AD pathogenesis.

Induction of matrix metalloproteinase-2 in human immunodeficiency virus-1 glycoprotein 120 transgenic mouse brains.

Human immunodeficiency virus (HIV)-1 can invade the brain and cause degeneration of the central nervous system, resulting in a host of cognitive and motor impairments. HIV-1 glycoprotein 120 (gp120), has been implicated in the neurodegenerative effects of HIV infection. Here, gp120's neurotoxic potential is demonstrated in both transgenic mice and cultured cells. We observed that gp120 causes an induction of matrix metalloproteinase (MMP)-2 activity and protein in transgenic mouse brains and in transfected C6 cells. We propose that induced MMP-2 may contribute to a neurodegenerative environment by degrading extracellular matrix (ECM) fibronectin and type IV collagen.

T cells as antigen-presenting cells.

Human T cells express major histocompatibility complex (MHC) class II antigens and adhesion molecules characteristic of antigen-presenting cells (APCs), and recent in vitro and in vivo evidence supports an antigen-presenting function for T cells. In this guise, T cells provide downregulatory signals for the immune response by inducing anergy in T cells that have already been activated and cytotoxicity in resting T cells. Here, Werner Pichler and Tony Wyss-Coray suggest that this may represent an important negative mechanism for T-cell homeostasis.

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide.

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57-68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8+ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s,r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.

Peptide-induced T cell clones: specificity, MHC restriction, proliferation and cytokine pattern as a function of different stimulations.

CD4+ T cell clones were generated to tetanus toxin or to two tetanus toxin-derived peptides p2 (AA 830-834) and p30 (AA 947-976). 11 of the 24 p30-specific clones reacted to shorter p30 subunits (p301 or p302), and only 14 of the p2 or p30-specific clones reacted with TT presented by EBV-transformed B cell lines (B-LCL). The p30-specific clones were HLA-DP4 restricted. In contrast to autologous B cell lines, the majority of allogeneic, but HLA-DP4-positive cell lines failed to present p30 to the specific clones. We concluded that T cell clones are highly specific and that both, small alterations of the peptide length as well as discrete differences of the HLA-molecule may abrogate recognition of the peptide HLA complex by T cells. Moreover, use of peptides as stimulators of T cells may recruit and activate T cells which fail the "original" peptide, derived from normal antigen processing. Clones could usually be maintained in culture for 4-6 months, but with the help of freezing and thawing some clones are now available for over 2 years and still specific. Comparison of different autologous antigen-presenting cells, namely B-LCL and activated MHC class II-positive T cells revealed that not all clones were able to mount a proliferative response to peptide presentation by T cells, while all clones proliferated to B cells as APC. If stimulated with peptide and B-LCL, the clone proliferating to T cells as APC (so-called T responder clones) secreted a broad spectrum of cytokines (Th0-like) and were easier to maintain in culture. In contrast, clones which were unable to proliferate to peptide presentation, so-called T-nonresponder clones, showed a more restricted cytokine pattern and elevated or very low IL4/IFN gamma ratio upon antigen specific stimulation. However, all clones secreted at least small amounts of IL2, IL4, IFN gamma and TNF alpha, if stimulated by PMA and ionomycin. Thus, both chemical and antigen-specific stimulations should be considered if T cell clones are classified as Th1 or Th2, whereby those clones, which secrete a limited cytokine pattern after antigen stimulation only, might be named Th1 or Th2 like clones, while clones which even after PMA/ionomycin do not secrete all cytokines, might represent "real" Th1 or Th2 clones.

Comparative analysis of surface antigens in cultured human outer root sheath cells and epidermal keratinocytes: persistence of low expression of class I MHC antigens in outer root sheath cells in vitro.

In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, outer root sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular outer root sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.

Improved sensitization of antigen-presenting cells with transferrin-bound peptides: advantages in competition for antigen presentation.

T cells recognize peptides in association with major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). To sensitize APC for antigen presentation in vitro and in vivo, high concentrations of synthetic peptides can be added from the outside and bind to the MHC molecules, thereby mimicking naturally processed peptides. In this report we investigated whether the transferrin (Tf) molecule could be used as a carrier to introduce antigenic peptides into the antigen presentation pathway of APC. We coupled to Tf various MHC class II DR1 restricted peptides and compared the sensitization of DR1+ APC by the Tf-bound or by the soluble peptide, using peptide-specific T cell clones (TCC). The presentation of the Tf-bound peptides was MHC restricted and could be blocked by the fixation of the APC with glutaraldehyde or by the addition of an excess of Tf. Tf-bound peptides were more efficiently presented than soluble peptides, since smaller concentrations were required to sensitize APC. Moreover, they could compete with a soluble peptide for MHC restricted presentation with a very high efficiency if compared to soluble competing peptides. Tf peptide conjugates could even compete with the presentation of a native antigen like tetanus toxoid. Peptides bound to the transferrin molecule might be useful for immunization strategies, as the relevant bound peptides are efficiently presented to peptide-specific TCC.