In JoVE (2)
Articles by Maran L. Sprouse in JoVE
Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice Thomas Lee1, Ivan Shevchenko1, Maran L. Sprouse1, Maria Bettini1, Matthew L. Bettini1 1Department of Pediatrics, Baylor College of Medicine We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.
Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs Maran L. Sprouse1, Gabriele Blahnik2, Thomas Lee1, Natalie Tully1, Pinaki Benarjee3, Eddie A. James2, Maria J. Redondo4, Matthew L. Bettini1, Maria Bettini1 1 The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.
Other articles by Maran L. Sprouse on PubMed
Rapid Identification and Expression of Human TCRs in Retrogenic Mice Journal of Immunological Methods. Dec, 2016 | Pubmed ID: 27589924 Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector. This streamlined approach for generating human:mouse chimeric TCR vectors allows for rapid TCR expression in humanized-retrogenic (hu-Rg) mice through retroviral mediated stem cell gene transfer. Using widely available techniques and equipment, this method is easily adaptable by most laboratories. This is the first TCR identification protocol that is efficiently combined with subsequent in vivo TCR expression.
Ectopic Expression of Self-Antigen Drives Regulatory T Cell Development and Not Deletion of Autoimmune T Cells Journal of Immunology (Baltimore, Md. : 1950). Aug, 2017 | Pubmed ID: 28835461 Type 1 diabetes is a T cell-mediated autoimmune disease that is characterized by Ag-specific targeting and destruction of insulin-producing β cells. Although multiple studies have characterized the pathogenic potential of β cell-specific T cells, we have limited mechanistic insight into self-reactive autoimmune T cell development and their escape from negative selection in the thymus. In this study, we demonstrate that ectopic expression of insulin epitope B:9-23 (InsB9-23) by thymic APCs is insufficient to induce deletion of high- or low-affinity InsB9-23-reactive CD4(+) T cells; however, we observe an increase in the proportion and number of thymic and peripheral Foxp3(+) regulatory T cells. In contrast, the MHC stable insulin mimetope (InsB9-23 R22E) efficiently deletes insulin-specific T cells and prevents escape of high-affinity thymocytes. Collectively, these results suggest that Ag dose and peptide-MHC complex stability can lead to multiple fates of insulin-reactive CD4(+) T cell development and autoimmune disease outcome.