The repertoire of Aire-regulated peripheral tissue antigens in single thymic medullary epithelial cells

Thymic medullary epithelial cells (MECs) express a wide range of peripheral tissue antigens (PTAs), many of which are dependent on the transcriptional regulator, AutoImmune Regulator or Aire. While Aire is known to be critically important for shaping a self-tolerant T-cell repertoire, its role in MEC maturation and function remains poorly defined. Two models currently exist to explain the phenomenon of PTA expression in MECs. The terminal differentiation model contends that the most mature MECs have the highest surface expression of CD80/MHC Class II molecules and express the most diverse array of PTAs. In contrast, the developmental model proposes that the most mature MECs express a restricted repertoire of PTAs as a consequence of differentiation programs that emulate those of extrathymic epithelial lineages. Implicit in this model is a requirement for the expression in MECs of the transcription factors necessary for the differentiation of other epithelial lineages. Using a sensitive and reproducible single-cell PCR assay, I report that Aire-expressing MECs are heterogeneous and express a diverse set of PTAs at variable frequencies and combinations at the single-cell level. Moreover, single Aire-expressing MECs tend to co-express genes within an Aire-regulated cluster, but in a stochastic manner. The stochastic expression of PTAs was associated with differential allelic expression, with some cells expressing PTAs monoallelically or biallelically in a probabilistic manner. Lastly, I use real-time quantitative RT-PCR to show that thymic expression of Ins2 does not depend on the expression of Pdx1 or cMaf , transcription factors known to be important for the development of the pancreas and for insulin transcription. These results argue strongly in favor of the terminal differentiation model, and refine it.