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Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

ABSTRACT: Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR.

SUBMITTER: Eckhardt J

PROVIDER: S-EPMC3636694 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

Eckhardt Jenny J Ostalecki Christian C Kuczera Katarzyna K Schuler Gerold G Pommer Ansgar J AJ Lechmann Matthias M

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20121116 2

Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this st ...[more]

PMID: 23160665

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Project description:BackgroundBarrett's esophagus (BE) is caused by gastroesophageal reflux with consecutive mucosal inflammation, predisposing patients to the development of esophageal adenocarcinoma (EAC). We investigated changes in T cell-related mucosal combinatorial molecular protein patterns in both diseases using the novel Multi-Epitope-Ligand-Cartography, a unique robotic whole-cell imaging technology that simultaneously visualizes dozens of proteins in structurally intact tissues and correlates cellular localization of proteins with function.ResultsBiopsies were taken during endoscopy from BE, EAC, and normal control tissue, and proteomic microscopy was performed on 32 different epitopes. When the significance level was set to p < 0.0005 and the search depth to five antibody combinations, controls and BE can be differentiated by 63, controls and EAC by 3222, and BE from EAC by 1521 distinct protein combinations.For example, the number of activated apoptotic naïve and memory T cells was significantly increased only in BE, whereas the number of activated apoptotic helper and regulatory T cells was significantly elevated in BE and EAC. In contrast, the number of activated apoptotic cytotoxic T cells was significantly elevated only in EAC. Confirming different pathways in BE and EAC, the number of T lymphocytes with p53 expression and downregulation of bcl2 expression (CD3+p53+Bcl2-NfkB-) was significantly increased in EAC compared to BE and controls. Interestingly, the number of precursor T cells (CD7+) was significantly elevated only in EAC. These cells lack Bax and caspase-8, suggesting impaired apoptosis in the early stages of T cell differentiation.ConclusionProteomic analysis showed for the first time that proteins, which are critically involved in the mucosal immune system of the esophagus, are distinctly expressed in BE and EAC, whereas others are comparably altered in both diseases, suggesting that many pathogenic events might be shared by both diseases. Topological proteomic analysis, therefore, helps us to understand the different pathogenic events in the underlying disease pathways.

Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation.

Project description:Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single ("classical") or multiple ("complex") anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the "multi-epitope-targeting" approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as "multi-epitope-targeting" agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases.

Dataset Information

Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

Publications

Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

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