**5. Trends in rejuvenation of age-related thymic atrophy**

Rejuvenation of aged thymic function is one of the strategies to reduce inflammaging because it can reduce self-reactive Tcon cell release and potentially readjust tTreg cell function so that the adaptive immune aspects of inflammaging may be ameliorated. Several strategies to rejuvenate the atrophied thymus have been reported, including: (1) TEC stem cell-based strategies, including utilization of human embryonic/pluripotent stem cells [110–112], FoxN1eGFP/+ knock-in epithelial cells [113], young TEC-based [114] or inducible TEC-based [115] strategies; (2) cytokine-to-TEC based therapy, such as keratinocyte growth factor (KGF) [116, 117] and IL-22 [118–120]; (3) genetically-based methods (enhancement of exogenous FoxN1 expression with FoxN1 cDNA plasmid and FoxN1 transgene) [79–81], and (4) epigenetically-based methods (via exosomes extracted from young healthy serum) [121].

As to the genetic rejuvenation strategy via exogenous FoxN1, intrathymic injection of plasmid vectors carrying FoxN1-cDNA into middle-aged and aged mice was able to partially rescue thymic atrophy and function. The investigators observed increased thymic size and thymocyte number in the treated group compared to mice receiving empty vector [79]. Another group utilized an inducible FoxN1 overexpression reporter gene system, and it was demonstrated that *in vivo* upregulation of FoxN1 expression in middle-aged and aged mice resulted in increased thymic size and thymocyte numbers as well as increased numbers of early thymic progenitor cells [81]. Additionally, the ratio of mTECs to cTECs, which is normally declined, was restored to normal levels [81].

As to cell-based therapy, this has also been investigated as a potential source of thymic rejuvenation via the use of exogenous TECs from newborn thymi. The investigators, after observing that circulating factors alone (via a heterochronic parabiosis model, in which young and aged mice are surgically joined resulting in mutual influence of blood-borne factors [122–130]) did not rejuvenate the aged thymus, utilized a model of direct transplantation of TECs from newborn mice intrathymically into middle-aged recipients [114]. This group observed renewed growth of the thymus as well as enhanced T cell generation [114].

Other groups are investigating the use of reprogrammed mouse embryonic fibroblasts (MEF), as sources of exogenous FoxN1, as a means of generating *de novo* ectopic thymus. One such group generated induced TECs (iTECs) from MEF cells by initiating FoxN1 expression that converted MEF cells into epithelial-like cells *in vitro* [115]. Then, these iTECs, after some testing, were re-aggregated and grafted under the kidney capsule of syngenic adult mice to evaluate the ability of these iTECs to develop into a functional thymus-like organ. Interestingly, the grafts were seeded by host T cell progenitors and reflected thymocyte distributions associated

with the normal thymus at endpoint (4 weeks after engraftment). Additionally, typical thymus microstructure was observed in these grafts [115].

The overarching conclusions taken from these cytokine, cellular, genetic, or epigenetically-based rejuvenation strategies are that FoxN1 expression is a key target for rejuvenating TECs, resulting in a more functional thymus able to produce normal T cells. However, we need to recognize that any rejuvenation therapy has its pitfalls. For example, intrathymic injection of newborn TECs can rejuvenate middle-aged thymus [114], but the source of newborn TECs is limited and may not be ideal as a translational therapy. Additionally, generation of an ectopic *de novo* thymus under the kidney capsule [115] can generate naïve T cells, but this does not remedy the increased self-reactive T cells released by the original atrophied thymus remaining in the host. Also, the use of cytokines may help revitalize the thymus, but as a systemic therapy could present various detrimental side-effects. Therefore, further studies to develop practical and effective therapies are necessary.
