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Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCIIloCD80lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis., Author Summary T cells, an essential component of the immune system, generate protective immune responses against pathogenic organisms and cancer cells. T cells are produced in the thymus, which provides a unique microenvironment required for T cell development. Distinct subsets of thymic epithelial cells (TECs) in the outer cortex and inner medulla provide signals required for the survival and differentiation of immature T cells, referred to as thymocytes. Medullary TECs (mTECs) play a critical role in preventing autoimmunity because they have the unique ability to express peptides found in other organs throughout the body. Presentation of self-peptides to thymocytes causes deletion of cells that express high affinity self-reactive receptors. Numerous studies have established that a major signaling pathway mediated by NFκB family members is indispensable for mTEC development. However, whether other signaling pathways are also required has remained an open question. Here, we use gain- and loss-of-function genetic approaches to demonstrate that another pathway, mediated by Stat3 signaling, plays an important role in mTEC development and homeostasis. We show that constitutive Stat3 activation enhances the survival of immature mTECs and bypasses the requirement for thymocyte-derived signals in medullary region formation. In contrast, Stat3 depletion reduces mTEC cellularity and impairs medullary region formation.

The thymus maintains T cell receptor (TCR) repertoire diversity through perpetual release of self-MHC restricted naive T cells. However, thymus involution during the aging process reduces naïve T cell output, leading to defective immune responsiveness to newly encountered antigens. We have shown that age-related thymus involution is prevented in mice expressing a keratin 5 promoter-driven Cyclin D1 (K5.D1) transgene in thymic epithelial cells (TECs). Sustained T cell output from K5.D1 thymi prevents the age-associated decline of naïve T cells in the periphery. We find that K5.D1 recent thymic emigrants (RTEs) undergo typical phenotypic maturation. Competitive adoptive transfer studies with K5.D1 and nontransgenic RTEs indicate that K5.D1 RTEs are incorporated into the peripheral T cell pool comparably to nontransgenic controls. In addition, functional studies show that K5.D1 peripheral T cells are responsive to anti-CD3 and anti-CD28 stimulation in vitro. Collectively, these data show that preventing thymus involution not only sustains T cell output, but also provides functionally competent RTEs during aging.

Naïve T cells are continually exported from the thymus and maintain peripheral TCR repertoire diversity. As the thymus involutes with age, thymopoiesis and T cell output wane, reducing the number of recent thymic emigrants (RTEs). Although homeostatic proliferation of memory T cells sustains peripheral T cell numbers, the decrease in naïve T cells restricts immune responsiveness to new antigens. It has been proposed that RTE output would be sustained if thymus involution were prevented. To test this hypothesis, we analyzed RTEs in mice that do not undergo thymus involution due to a keratin 5 promoter-driven Cyclin D1 transgene (K5.D1) expressed in thymic epithelial cells. This transgene maintains functional thymic microenvironmental niches that support thymocyte progenitor maturation. We introduced a RAG2p-GFP transgene to discriminate RTEs from mature naïve (MN) T cells in K5.D1 mice. As previously reported, RTEs decline with age in RAG2p-GFP controls. In contrast, the number of RTEs increases with age in K5.D1;RAG2p-GFP mice. Despite a lower frequency of MN T cells in K5.D1 mice, the number of MN T cells is increased by ~4 fold in 5-6 month old K5.D1;RAG2p-GFP mice compared to RAG2p-GFP littermates. The increase in naïve T cells does not displace existing memory T cells, supporting the notion that naïve and memory T cells occupy distinct homeostatic niches. These data demonstrate that preventing thymus involution sustains the naïve T cell compartment during aging.