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Growing Mya-1 T Cells for Adoptive Immunotherapy

Description

CD4+CD25+ FOXP3+ cells are recognized as the most reliable regulatory T cell subset. However, the intracellular nature of the FOXP3 transcription factor limits its use for the isolation or selection of viable regulatory T cells for adoptive immunotherapy. Nuclear localization of FOXP3 has been more strongly associated with induced regulatory T cell (Treg) function than increased expression of FOXP3 alone. Several different cell culture methods and T cell activation techniques can induce increased expression of FOXP3 in a variety of T cell models, but Rapamycin (an mTOR inhibitor) was recently shown to differentially induce nuclear localization of FOXP3 when compared with IL-10 and TGFβ. Feline Tregs have been well characterized and share many of the phenotypic and functional characteristics of murine and human Tregs. We cultured feline Mya-1 T cells in conditions that would differentially promote effector or regulatory phenotypes and correlated nuclear localization of FOXP3 with other quantitative morphologic features using imaging flow cytometry. We compared the morphologic features of cells with high intra-nuclear concentrations of FOXP3 cultured without IL-2, with IL-2, and with IL-2 and Rapamycin before and after non-specific antigenic stimulation with Concanavalin-A. This analysis may help identify a population of pure regulatory T cells that would be more likely to maintain regulatory function following in-vitro expansion and activation. Furthermore, the feline T cell model could help elucidate important differences between murine and human Treg cells that would further translational efforts in adoptive immunotherapy. Now, we ask if nuclear localization of FOXP3 could be used to identify other morphologic differences between activated effector and regulatory T cells using a feline T cell line.

ContributorsRojas, Arturo Nikolas (Author) / Sweazea, Karen (Thesis director) / Mexas, Angela (Committee member) / Murphy, Andrea (Committee member) / School of Nutrition and Health Promotion (Contributor) / Barrett, The Honors College (Contributor)

Created2018-05

P2RX7 promotes a pro-memory signature in effector CD8+ T cells dependent on Zeb2 negative regulation

Description

Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). The extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. High (P2RX7hi) P2RX7-expressing early effector cells show survival, memory and pluripotency genes. Conversely, many terminal effector (TE) and apoptosis genes are upregulated in low (P2RX7lo) P2RX7-expressing cells. Among these genes is the zinc-finger transcriptional repressor Zeb2, which promotes TE differentiation at the expense of the memory cell pool. Given that Zeb2 was higher in P2RX7lo early effector cells, we postulated that Zeb2 ablation would allow P2RX7-deficient CD8+ T cells to skew towards memory subsets. To test this, we used RNP-based CRISPR-Cas9 to knockout Zeb2 in wild type or P2RX7-deficient P14 cells. At the memory timepoint, Zeb2 ablation led to a rescue of the ability of P2RX7-deficient cells to differentiate into the CD62L+ Tcm and CD69hiCD103hi Trm subsets, as well as increase the population of each. Our data suggest that P2RX7 imprints a pro-memory signature that is, to some extent, dependent on the negative regulation of Zeb2.

ContributorsVan Dijk, Sarah (Author) / Holechek, Susan (Thesis director) / Borges da Silvs, Henrique (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor)

Created2021-12