Growing Mya-1 T Cells for Adoptive Immunotherapy

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.