Dengue-2 structural proteins associate with human proteins to produce a coagulation and innate immune response biased interactome.

BACKGROUND: Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. METHODS: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. RESULTS: Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. CONCLUSIONS: Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.

Preservation of self: an overview of E3 ubiquitin ligases and T cell tolerance.

Until recently ubiquitination of a protein was thought to simply serve the mundane task of targeting a protein for proteasomal degradation. Accumulating evidence over the past decade has demonstrated the importance of ubiquitination in non-degradative functions including regulating cellular signaling, that highlight its role in human disease and thus potential development of novel therapeutics. Much has been written about ubiquitination in the immune system, in this review we will outline our current knowledge of ubiquitination with respect to T cell tolerance. Specifically, we will provide on overview of E3 ubiquitin ligases and their role in various states of CD4+ T cell tolerance: central and peripheral.

Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis.

Although systemic administration of neutralizing anti-TNF antibodies has been used successfully in treating rheumatoid arthritis, there is a potential for side effects. We transduced a collagen reactive T-cell hybridoma with tissue-specific homing properties to assess therapeutic effects of local delivery to inflamed joints of anti-TNF single-chain antibodies (scFv) by adoptive cellular gene therapy. Cell culture medium conditioned with 1 x 10(6) scFv producer cells/ml had TNF neutralizing capacity in vitro equivalent to 50 ng/ml anti-TNF monoclonal antibody. Adding a kappa chain constant domain to the basic scFv (construct TN3-Ckappa) gave increased in vitro stability and in vivo therapeutic effect. TN3-Ckappa blocked development of collagen-induced arthritis in DBA/1LacJ mice for >60 days. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection. No significant variations in cell proliferation or cytokine secretion were found in splenocytes or peripheral lymphocytes. IL-6 expression was blocked in the diseased paws of mice in the scFv treatment groups compared to controls. In conclusion, we have shown that local expression of an anti-inflammatory agent blocks disease development without causing demonstrable systemic immune function changes. This is encouraging for the potential development of safe adoptive cellular therapies to treat autoimmunity.

A complicated relationship: fulfilling the interactive needs of the T lymphocyte and the dendritic cell.

T cells recognize antigenic peptides displayed on the surface of MHC-bearing antigen-presenting cells (APCs), and with sufficient costimulation become activated. However, the ability of an APC (even bearing the correct peptide) to initiate and fulfill the requirements for T cell activation is not easily achieved. Naive T cells use multiple copies of a single receptor to survey the vast array of peptides presented on an APC, and require multiple receptor engagements to initiate T cell activation. Dendritic cells (DCs) are specialized cells with optimal capabilities for priming naive CD4+ T cells. Activation occurs, after initial antigen recognition by T cells, followed by a rapid dialogue between the T cells and the DCs. The resulting changes in both the cytoskeleton and the expression or regulation of cell-surface molecules on both cell types act to further strengthen engagement. In this report, we review the fundamentals of CD4+ T helper cell : DC interactions and discuss recent data concerning the molecular characteristics of this engagement.

Bioluminescence imaging of lymphocyte trafficking in vivo.

Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

CD4(+)CD25(+) T cells facilitate the induction of T cell anergy.

T cell anergy is characterized by the inability of the T cell to produce IL-2 and proliferate. It is reversible by the addition of exogenous IL-2. A similar state of unresponsiveness is observed when the proliferative response of murine CD4(+)CD25(-) T cells is suppressed in vitro by coactivated CD4(+)CD25(+) T cells. We have developed a suppression system that uses beads coated with anti-CD3 and anti-CD28 Abs as surrogate APCs to study the interaction of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in vitro. CD4(+)CD25(+) T cell-induced suppression, in this model, was not abrogated by blocking the B7-CTLA-4 pathway. When the CD4(+)CD25(-) T cells were separated from the CD4(+)CD25(+) suppressor cells after 24 h of coactivation by the Ab-coated beads, the CD4(+)CD25(-) T cells were unable to proliferate or to produce IL-2 upon restimulation. The induction of this anergic phenotype in the CD4(+)CD25(-) T cells correlated with the up-regulated expression of the gene related to anergy in lymphocytes (GRAIL), a novel anergy-related gene that acts as a negative regulator of IL-2 transcription. This system constitutes a novel mechanism of anergy induction in the presence of costimulation.

Gene therapy in autoimmune disease.

Recent work on gene therapies for autoimmune disease has continued to provide insight into the pathogenesis of autoimmunity. Reliable, effective and targeted gene therapy applications have been achieved by using transduced dendritic cells and antigen-specific T cells as delivery vehicles. Bioluminescence imaging has been implemented to visualize cell trafficking and homing in vivo. As a first step into human gene therapy, a phase I clinical trial for assessing the feasibility and safety of gene transfer has been completed in a group of rheumatoid arthritis patients.

Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit.

CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of "regulatory" cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-gamma production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

Immunization with DNA encoding an immunodominant peptide of insulin prevents diabetes in NOD mice.

DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer and has more recently been used to prevent autoimmune disease. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T-cell-mediated destruction of the insulin-secreting beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In this study we demonstrate that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin (residues 9-23 of the B chain) protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B chain peptide show significantly lower disease incidence and delayed onset of disease when compared to controls. Protection appears to be mediated by insulin B (9-23)-specific down-regulation of IFN-gamma. Our results confirm that DNA vaccination has a protective effect on autoimmunity, the understanding of which will reveal new insights into the immune system and open doors for novel therapies.

Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis.

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

T cell receptor (TCR)-mediated repertoire selection and loss of TCR vbeta diversity during the initiation of a CD4(+) T cell response in vivo.

We recently described a novel way to isolate populations of antigen-reactive CD4(+) T cells with a wide range of reactivity to a specific antigen, using immunization with a fixed dose of nominal antigen and FACS((R)) sorting by CD4(high) expression. Phenotypic, FACS((R)), functional, antibody inhibition, and major histocompatibility complex-peptide tetramer analyses, as well as T cell receptor Vbeta sequence analyses, of the antigen-specific CD4(high) T cell populations demonstrated that a diverse sperm whale myoglobin 110-121-reactive CD4(+) T cell repertoire was activated at the beginning (day 3 after immunization) of the immune response. Within 6 d of immunization, lower affinity clones were lost from the responding population, leaving an expanded population of oligoclonal, intermediate affinity (and residual high affinity) T cells. This T cell subset persisted for at least 4 wk after immunization and dominated the secondary immune response. These data provide evidence that CD4(+) T cell repertoire selection occurs early in the immune response in vivo and suggest that persistence and expansion of a population of oligoclonal, intermediate affinity T cells is involved in CD4(+) T cell memory.

Polyarginine enters cells more efficiently than other polycationic homopolymers.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.

Rapid and efficient vascular transport of arginine polymers inhibits myointimal hyperplasia.

BACKGROUND: We recently discovered that short polymers of arginine efficiently translocate across the cytoplasmic membrane independent of the basic amino acid transporter. We evaluated the kinetics and biological effects of heptamers of L-arginine and D-arginine (L-R7 and D-R7, respectively) in vascular cells. We assessed the effects of these peptides on the NO synthesis pathway and vascular cell proliferation. METHODS AND RESULTS: Human umbilical vein endothelial cell and rabbit vascular segments were incubated in medium containing biotin-labeled L-R7 or D-R7. Both polymers rapidly translocated through the vessel wall and into the vascular cells in a dose- and time-dependent fashion. At a dose of 10 micromol/L for 30 minutes, 100% of the endothelial cells showed evidence of cytoplasmic and nuclear localization of the peptides. To evaluate the biological effects of the polymer translocation on myointimal formation, rabbit jugular vein segments were incubated with polymers (10 micromol/L, 30 minutes) or vehicle before arterial interposition grafting. Planimetric measurement 28 days after surgery revealed that L-R7 and D-R7 substantially reduced myointimal formation compared with the control condition (intima/media ratio: control 1. 50.5, L-R7 0.40.2, and D-R7 0.80.2; P:<0.05). Furthermore, basal nitrate and nitrite production from L-R7-treated grafts was significantly higher than that from both control and D-R7-treated veins. Studies in vitro of cultured vascular smooth muscle cells revealed that both polymers also exhibit an NO-independent inhibition of vascular smooth muscle cell proliferation. CONCLUSIONS: Short polymers of arginine have the unique ability of vascular cell translocation, and they also have direct biological effects. These attributes are potentially useful in treating myointimal hyperplasia.

A novel transcription factor, T-bet, directs Th1 lineage commitment.

Naive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNgamma. T-bet expression correlates with IFNgamma expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNgamma gene and induces endogenous IFNgamma production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNgamma and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs.

Gene therapy for autoimmune disease.

Autoantigen-specific CD4(+) T lymphocytes have been implicated in the pathogenesis of autoimmune diseases. Tissue-specific homing properties of autoantigen-specific CD4(+) T cells suggested that these cells might be ideal vehicles for delivery of retroviral-encoded regulatory proteins in a site-specific manner as a therapy for autoimmune diseases. Application of retroviral transduction of autoantigen-reactive CD4(+) T cells in gene therapy of autoimmunity must include systems capable of targeting these rare populations of antigen-activated T cells. Studies discussed below suggest that retroviral transduction of autoantigen-specific murine CD4(+) T cells may provide a method to target and isolate nontransformed autoantigen-specific murine CD4(+) T cells and provide a rational approach to gene therapy in animal models of autoimmunity.

Identification and characterization of the antigen-specific subpopulation of alloreactive CD4+ T cells in vitro and in vivo.

We report the identification and characterization of the small subpopulation of alloantigen-specific T cells in vitro and in vivo. This subpopulation of T cells was distinguished by up-regulation of cell surface CD4 expression. These CD4high T cells were alloantigen specific in proliferation assays in vitro, and they expressed memory/activation markers, including CD44high and CD69high. Further studies demonstrated that these allospecific CD4high cells were also present (< or = 1% of CD4+ T cells) in vivo in BALB/c (H-2d) recipients of C57BL/6 (H-2b) skin allografts. CD4high T cells isolated from regional draining lymph nodes in these skin graft recipients reacted in a donor-specific fashion to C57BL/6 splenocyte stimulator cells in mixed lymphocyte culture. Adoptive transfer of CD4high, but not CD4normal T cells, just before skin engraftment in CD4 knockout mice, reconstituted rejection. The discovery that a small subpopulation of CD4high lymph node cells contained all of the alloantigen-specific T cells may allow study of tissue-specificity and subsequent alloantigen identification in transplantation.

Ligand-independent down-regulation of IFN-gamma receptor 1 following TCR engagement.

Activated T lymphocytes modulate the level of many molecules on their cell surface, including cytokine receptors. This regulation of cytokine receptor expression affects the ability of T cells to respond to cytokines and thus influences the outcome of an immune response. The receptor for IFN-gamma, a proinflammatory cytokine, consists of two copies of a ligand binding chain (IFN-gammaR1) as well as two copies of a second chain (IFN-gammaR2) required for signal transduction. The expression of IFN-gammaR2 is down-regulated at the mRNA level on CD4+ T cells when they differentiate into the Th1, but not the Th2, phenotype. This down-regulation has been demonstrated to depend on the ligand, IFN-gamma, which is produced by Th1 but not Th2 T cells. The regulation of the cell-surface expression of IFN-gamma receptors during primary T cell activation has not been reported. Naive and differentiated T lymphocytes express IFN-gammaR1 at the mRNA level and as a cell-surface protein. In this study, we present evidence that cell-surface expression of IFN-gammaR1 is transiently down-regulated on the surface of naive CD4+ T cells shortly after TCR engagement. Furthermore, this down-regulation is not mediated by the ligand, IFN-gamma, but results from TCR engagement and can be inhibited by cyclosporin A.

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.