Rapid quantification of naive alloreactive T cells by TNF-alpha production and correlation with allograft rejection in mice

Allograft transplantation requires chronic immunosuppression, but there is no effective strategy to evaluate the long-term maintenance of immunosuppression other than assessment of graft function. The ability to monitor naive alloreactive T cells would provide an alternative guide for drug therapy at early, preclinical stages of graft rejection and for evaluating tolerance-inducing protocols. To detect and quantify naive alloreactive T cells directly ex vivo, we used the unique ability of naive T cells to rapidly produce TNF-alpha but not IFN-gamma. Naive alloreactive T cells were identified by the production of TNF-alpha after a 5-hour in vitro stimulation with alloantigen and were distinguished from effector/memory alloreactive T cells by the inability to produce IFN-gamma. Moreover, naive alloreactive T cells were not detected in mice tolerized against specific alloantigens. The frequency of TNF-alpha-producing cells was predictive for rejection in an in vivo cytotoxicity assay and correlated with skin allograft rejection. Naive alloreactive T cells were also detected in humans, suggesting clinical relevance. We conclude that rapid production of TNF-alpha can be used to quantify naive alloreactive T cells, that it is abrogated after the induction of tolerance, and that it is a potential tool to predict allograft rejection

Gene Expression Profiling during Early Acute Febrile Stage of Dengue Infection Can Predict the Disease Outcome

Background: We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples. Methodology/Principal Findings: The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2-3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. Conclusions/Significance: Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection. © 2009 Nascimento et al

Idd Loci Synergize to Prolong Islet Allograft Survival Induced by Costimulation Blockade in NOD Mice

OBJECTIVE—NOD mice model human type 1 diabetes and are used to investigate tolerance induction protocols for islet transplantation in a setting of autoimmunity. However, costimulation blockade–based tolerance protocols have failed in prolonging islet allograft survival in NOD mice

The relationship of interacting immunological components in dengue pathogenesis

The World Health Organization (WHO) estimates that there are over 50 million cases of dengue fever reported annually and approximately 2.5 billion people are at risk. Mild dengue fever presents with headache, fever, rash, myalgia, osteogenic pain, and lethargy. Severe disease can manifest as dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF). Symptoms of DSS/DHF are leukopenia, low blood volume and pressure encephalitis, cold and sweaty skin, gastrointestinal bleeding, and spontaneous bleeding from gums and nose. Currently, there are no therapeutics available beyond supportive care and untreated complicated dengue fever can have a 50% mortality rate. According to WHO DSS/DHF is the leading cause of childhood mortality in some Asian countries. Dendritic cells are professional antigen presenting cells that are primary targets in a dengue infection. Dengue binds to Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN). DC-SIGN has a high affinity for ICAM3 which is expressed in activating T-cells. Previous studies have demonstrated an altered T-cell phenotype expressed in dengue infected patients that could be potentially mediated by dengue-infected DCs

Identification by Automated Screening of a Small Molecule that Selectively Eliminates Neural Stem Cells Derived from hESCs but Not Dopamine Neurons

BACKGROUND:We have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo. METHODOLOGY/PRINCIPAL FINDINGS:In this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug. CONCLUSIONS/SIGNIFICANCE:In conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. © 2013 Nascimento et al

Synergistic Interactions between the NS3hel and E Proteins Contribute to the Virulence of Dengue Virus Type 1

Dengue virus constitutes a significant public health problem in tropical regions of the world. Despite the high morbidity and mortality of this infection, no effective antiviral drugs or vaccines are available for the treatment or prevention of dengue infections. The profile of clinical signs associated with dengue infection has changed in recent years with an increase in the number of episodes displaying unusual signs. We use reverse genetics technology to engineer DENV-1 viruses with subsets of mutations previously identified in highly neurovirulent strains to provide insights into the molecular mechanisms underlying dengue neuropathogenesis. We found that single mutations affecting the E and NS3hel proteins, introduced in a different genetic context, had a synergistic effect increasing DENV replication capacity in human and mosquito derived cells in vitro. We also demonstrated correlations between the presence of these mutations and viral replication efficiency, viral loads, the induction of innate immune response genes and pathogenesis in a mouse model. These results should improve our understanding of the DENV-host cell interaction and contribute to the development of effective antiviral strategies

GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a “T cell–driven” methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens

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Altered cytokine responses of dengue-specific CD4+ T cells to heterologous serotypes

The interplay of different inflammatory cytokines induced during a dengue (DEN) virus infection plays a role in either protection or increased disease severity. We measured the frequencies and characterized the cytokine responses of DEN virus-specific memory CD4+ T cells in PBMC of six volunteers who received experimental live attenuated monovalent DEN vaccines. IFN-gamma and TNF-alpha responses to inactivated DEN Ags were detected in up to 0.54 and 1.17% of total circulating CD4+ T cells, respectively. Ags from the homologous serotype elicited the highest IFN-gamma response. The ratio of TNF-alpha- to IFN-gamma-producing CD4+ T cells was higher after stimulation with Ags from heterologous DEN serotypes. Peptide-specific CD4+ T cell frequencies of up to 0.089% were detected by direct staining using HLA class II tetramers. IFN-gamma and TNF-alpha responses to individual HLA class II-restricted peptide epitopes were detected in up to 0.05 and 0.27% of CD4+ T cells, respectively. Peptide sequences from the homologous serotype elicited a variety of cytokine response patterns. TNF-alpha- to IFN-gamma-positive CD4+ T cell ratios varied between peptides, but the ratio of the sum of responses was highest against heterologous serotypes. These results demonstrate epitope sequence-specific differences in T cell effector function. These patterns of effector responses may play a role in the immunopathogenesis of DEN hemorrhagic fever