Discovery of T Cell Antigens by High-Throughput Screening of Synthetic Minigene Libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery

Mesenchymal cell survival in airway and interstitial pulmonary fibrosis

Fibrotic reactions in the airways of the lung or the pulmonary interstitium are a common pathologic outcome after exposure to a wide variety of toxic agents, including metals, particles or fibers. The survival of mesenchymal cells (fibroblasts and myofibroblasts) is a key factor in determining whether a fibroproliferative response that occurs after toxic injury to the lung will ultimately resolve or progress to a pathologic state. Several polypeptide growth factors, including members of the platelet-derived growth factor (PDGF) family and the epidermal growth factor (EGF) family, are prosurvival factors that stimulate a replicative and migratory mesenchymal cell phenotype during the early stages of lung fibrogenesis. This replicative phenotype can progress to a matrix synthetic phenotype in the presence of transforming growth factor-β1 (TGF-β1). The resolution of a fibrotic response requires growth arrest and apoptosis of mesenchymal cells, whereas progressive chronic fibrosis has been associated with mesenchymal cell resistance to apoptosis. Mesenchymal cell survival or apoptosis is further influenced by cytokines secreted during Th1 inflammation (e.g., IFN-γ) or Th2 inflammation (e.g., IL-13) that modulate the expression of growth factor activity through the STAT family of transcription factors. Understanding the mechanisms that regulate the survival or death of mesenchymal cells is central to ultimately developing therapeutic strategies for lung fibrosis

NSAID induced perforated peptic ulcer in a pediatric sickle cell patient

Peptic ulcer disease is a relatively rare entity in the pediatric population. Given the trend toward multimodal pain control for pain crises in Sickle Cell Disease patients, they are at an increased risk of developing complications secondary to peptic ulcer disease. We discuss a case of a Sickle Cell Disease patient on multimodal therapy that presented with a perforated peptic ulcer requiring emergent surgery. While multimodal therapy helps ease the dependency on narcotic pain medication, it does present other potential problems like potential bleeding or perforation. For those that can be categorized in this select group of patients, routine surveillance with esophagogastroduodenoscopy should be considered for those at highest risk to prevent devastating complications

Lessons Learned during Dengue Outbreaks in the United States, 2001–2011

Public health authorities should involve the clinical and laboratory community and engage the local community in vector control and case reporting

Screening and pool deconvolution ELISPOTs.

<p><b>Screening: a</b>) and <b>b</b>) are bar graphs of primary screening results from two newly diagnosed T1D subjects. Panel a) is subject ND2, panel b) is subject ND3. Each bar represents the IFNγ spot number for an individual cultured ELISPOT well stimulated with a single minigene pool. Red line indicates 5× standard deviations of wells stimulated with mock transfected autologous B cells. Red circles indicate three wells that scored positive in both screens. <b>Deconvolution c</b>) and <b>d</b>): cultured IFNγ ELISPOT assays testing ND2-derived CD8+ T cell responses against individual minigenes from <b>c</b>) pool 85 and <b>d</b>) pool 308. Individual minigenes were amplified using minigene specific primers. Subsequent PCR reactions added T7 and common sequences, and full length individual minigenes were rebuilt and tested as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029949#pone-0029949-g001" target="_blank">Fig. 1</a>. Targeted minigenes encode 33 residue peptides displayed in red. <b>Peptide epitope mapping e</b>) and <b>f</b>): Direct IFNγ ELISPOT assays testing overlapping 15 residue peptides from GLIPR1 and EpCAM minigenes targeted in b) and c). Peptides were tested in triplicate using a direct 24 hour IFNγ ELISPOT assay with 1×10⁵ CD8+ T cells/well. Purple residues indicate non-antigen derived sequences encoded by minigene flanking sequences. Red residues indicate 9 residue peptide epitope identified in subsequent epitope mapping experiments.</p

Responses to GLIPR1 4–12 and EpCAM 140–148 incases and controls, and HLA restriction studies.

<p>Spot number and minigene source for provisionally positive minigene pools. Each score represents a single cultured ELISPOT well stimulated with minigenes derived from the indicated genes. Minigenes in each pool are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029949#pone.0029949.s002" target="_blank">Table S2</a>. Positive scores exceeded 5 standard deviations of mean scores of wells stimulated with mock transfected autologous B cells. Pool number 9, 103 and 106 scored positive in both subjects.</p

Overview of synthetic minigene screening.

<p><b>a</b>) Libraries are synthesized on programmable microarrays, cleaved from the chip surface and provided as a single mixture of antisense oligonucleotide templates. <b>b</b>) Initial 96 well PCR reactions utilize individual sense, pool specific primers (green, purple, orange arrows) in combination with a common primer (red arrows) to amplify specific pools of antisense templates (multicolor regions) from the mixed oligo library. Synthesis of a complement for the common primer is dependent upon synthesis of the sense strand primed by a single, unique pool-specific primer in each well. This subdivides the library into ordered arrays of minigene pools, with 10 defined minigenes/well. <b>c</b>) A second PCR reaction sews a stop codon and a human beta-globin 3′ UTR (purple+gold boxes) onto each minigene using the common primer domain as an overlap. PCR is driven by the sense pool specific primer, and an antisense primer extending from a 130 base oligo dT tail through the 3′ end of the UTR. Inclusion of an oligo dT tail on the antisense strand encodes a polyA template on the end of each mature minigene. This template allows synthesis of poly-adenylated mRNA during <i>in vitro</i> transcription. <b>d</b>) Arrays of minigene pools are purified and subject to <i>in vitro</i> transcription in the presence of a cap analogue, producing an array of defined, fully translatable mRNA pools. <b>e</b>) IVT products are transfected into autologous CD40L expanded B cells for use as antigen presenting cells. Transfected APCs are used as stimulators and targets for <i>in vitro</i> stimulations and IFNγ ELISPOT assays.</p

Responses to GLIPR1 4–12 and EpCAM 140–148 in cases and controls, and HLA restriction studies.

<p>Responses to <b>a</b>) GLIPR1 (4–12) <b>b</b>) and EpCAM (140–148) in T1D, at risk and control subjects. In both panels, PBMCs from the indicated patient groups were stimulated with GLIPR1 or EpCAM peptides (filled bars) vs. control peptide (open bars) overnight. Anti-CD3/CD28 stimulation was used as a positive control and all responses to antibody stimulation were too numerous to count (data not shown). The graphs display the average raw number of spots from triplicate wells +/− the standard error. The (*) indicates for a given patient that the experimental peptide responses were significantly higher (FDR <0.05) than the response to the control peptide. The number of patients who significantly responded to the each experimental peptide is indicated by the fraction in the graph. PBMC from subject ND2 used for discovery of each epitope were not available and are not included in these graphs. <b>c</b>) HLA-A*0201 binding assay for GLIPR1 epitope TLATIAWMV on T2 cells. 1×10⁶ T2 cells were incubated with 20 µg/ml of the indicated peptide in media for 4 hours at 37°C, stained for HLA-A*0201 and evaluated by flow cytomety. <b>d</b>). Presentation of GLIPR1 eptiope by HLA A*0201. T2 cells were loaded with 10 µg/ml GLIPR1 epitope TLATIAWMV or control epitope from pyruvate dehydrogenase for two hours at room temperature, washed and mixed with purified CD8+ T cells from one responding subject (T1D #2) in a direct IFNγ assay and was found to be statistically significant compared to the control peptide (FDR<0.05).</p