In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes

We previously established three mouse cell lines (Aire⁺ TEC1, Aire⁺ TEC2 and Aire⁺ DC) from the medullary thymic epithelial cells (mTECs) and dendritic cells (mDCs). These cells constitutively expressed ''autoimmune regulator (Aire) gene'' and they exhibited various features of self antigen-presenting cells (self-APCs) present in the thymic medullary region. Here, we confirmed our previous observation that Aire⁺ thymic epithelial cells adhere to fresh thymocytes and kill them by inducing apoptosis, thus potentially reproducing in vitro some aspects of the negative selection of T cells in vivo. In this system, a single Aire⁺ cell appeared able to kill ,30 thymocytes within 24 hrs. Moreover, we observed that ectopic expression of peripheral tissue-specific antigens (TSAs), and expression of several surface markers involved in mTEC development, increased as Aire⁺ cell density increases toward confluency. Thus, these Aire⁺ cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state. Surprisingly, an in vitro co-culture system consisting of Aire⁺ cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4⁺ killer and CD4^- rescuer) that may determine the fate (dead or alive) of the differentiating Aire⁺ mTECs. Thus, our in vitro coculture system appears to mimic a part of ''in vivo thymic crosstalk''.