Abstract
Thymic Treg cell differentiation occurs via a two-step process. Step one generates Treg cell progenitors (TregP) and is driven by strong TCR interactions with antigens presented in the thymus. Step two is initiated by activation of STAT5 via IL-2, or IL-15, leading to mature Treg cells capable of emigrating from the thymus and mediating immune tolerance and homeostasis in peripheral tissues. Herein we describe an in vitro TregP cell differentiation assay that models the second, cytokine dependent, step of thymic Treg cell development. It can be utilized with relative ease to determine if a population of thymocytes represents a potential progenitor population for Treg cells as well as test how different cytokines or chemical inhibitors modulate the differentiation of known TregP cell populations into mature Treg cells.