**3.2 Mechanisms of thymic stromal cell-mediated structural thymic atrophy**

In light of the aforementioned evidence of age-related TEC defects and the decline in total TEC numbers in the aged, atrophied thymus, we now move to discuss the underlying mechanisms of these alterations. Many studies have been conducted to identify factors involved in the cellular and molecular aspects of TEC aging (cytokines, transcription factors, microRNAs, sex steroids, etc.). The single most predominant factor currently accepted as significantly contributing to this phenomenon is the TEC autonomous transcription factor FoxN1. This idea was based on the athymic nude mouse phenotype [67, 68]. FoxN1 is expressed mainly in epithelial cells of the thymus and skin to regulate epithelial cell differentiation in these organs [67]. It is thereby responsible for thymic organogenesis and subsequent T cell development in the thymus [16], as well as hair follicle development in the skin [69, 70]. Many past and current studies utilize nude mice, which exhibit a null mutation in FoxN1 resulting in the lack of hair and the thymus, which explains the lack of T cells in these mice [71, 72].

FoxN1 is noted to be reduced in expression in the age-related atrophied thymus and has even been described as one of the first markers of the onset of thymic involution [73, 74]. The question is whether this reduced FoxN1 expression is due to TEC aging, which results in a decline in many TEC-associated genes, or if primary FoxN1 decline with aging induces a TEC defect that then results in age-related thymic involution. This cause-and-effect relationship had been substantially debated prior to the generation of a conditional knock-out (cKO) FoxN1 mouse model [75]. In this model, the murine FoxN1 gene is *loxP*-floxed and the uCreERT is introduced through crossbreeding [76]. In this model, the tamoxifen (TM)-inducible ubiquitous Cre-recombinase (uCreERT ) transgene has a low level of spontaneous activation, even without TM induction [77, 78], causing gradual excision of the FoxN1*flox/flox* gene over time. This results in progressive loss of FoxN1 with age and thymic involution that is positively correlated with reduced FoxN1 levels [79]. Supplying exogenous FoxN1, such as via plasmid [79] or transgene [80, 81], into the aged thymus greatly reduces thymic atrophy and improves function. Additionally, the use of FoxN1 reporter mice has enabled further elucidation of the timeline and kinetics of thymic atrophy with age [82]. For example, one group recently published a study demonstrating that the reduction in FoxN1 initiates the onset of thymic involution, beginning predominantly in the cTEC compartment [82]. Therefore, a decline in FoxN1 expression with aging causally induces flaws in TEC homeostasis, thereby resulting in age-related thymic atrophy, as opposed to the notion that age-induced thymic atrophy causes FoxN1 decline in the thymus.
