This Core will support the Projects with both standard MLR monitoring and flow cytometry, and advanced technology including immune cell subset assays by CyTOF analysis, and T cell clonal repertoire assays by high throughput sequencing of TCR genes from appropriate T cell populations, and mRNA expression in DC populations by RNAseq. The Core will support both preclinical models in Projects 2-3 by analyzing mouse cells, and the clinical study in Project 1 by analyzing human cells. In the case of CyTOF, staining of cells for up to 40 targets simultaneously using heavy metal conjugated mAbs and analysis by spectroscopy is expected to lead to the identification of new subsets of adaptive and innate immune cells that will provide additional insights into cells that contribute to tolerance in the preclinical and clinical studies. In the case of high throughput sequencing, alloreactive T cell clones will be identified and purified from pretransplant MLR cultures with recipient responder cells and donor stimulator cells. Clonal frequencies will be determined in recipients before and after tolerance induction to search for evidence of clonal deletion. In addition, the breadth of the T cell clonal repertoire will be examined to study long term immune reconstitution.
The Scientific Core will utilize both standard methods and new techniques to support all Projects. A new method that enables rapid sequencing of T cell receptor genes will be used to analyze T cell recognition of antigens in donor transplants. In addition, a CyTOF instrument will be used to analyze immune cells with improved precision such that new subsets of these cells can be identified that contribute to tolerance of transplants.
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