The role of cell cycle regulation in maintaining the size of the thymic microenvironment

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A variety of genetic studies have provided evidence demonstrating that regulating TEC proliferation is a key component of the cellular mechanisms that maintain the postnatal thymus. The first such study was the analysis of a transgenic mouse line expressing Cyclin D1 from a Keratin 5 promoter (K5.CyclinD1) (111). These mice showed dramatic and continuous thymus growth, resulting in an outsized thymus that did not undergo aging-associated involution, and eventually caused the death of the animal. Analysis of TEC subsets using subset-specific markers showed that this hyperplastic thymus had a relatively normal organization, and displayed no characteristics of thymoma or thymic lymphoma. Analysis of thymocyte differentiation, including assaying selection using TCR transgenic models, showed no defects in T cell development, only increased overall numbers. Interestingly, no thymus phenotypes were observed in mice with constitutive activation of CDK4 (112, 113), which could be explained by low levels of CDK4 in TECs and/or by CDK4-independent functions of Cyclin D1 in these cells. These data indicate that over-expressing Cyclin D1 in TECs results in increased capacity for normal thymocyte differentiation, presumably through an increased availability of microenvironmental niches.

A number of transgenic and knockout strains of mice with alterations in the cell cycle regulatory machinery have further been shown to have thymic phenotypes. For instance, transgenic mice overexpressing E2F2 (but not E2F1) in the thymus led to thymic carcinomas (114, 115), suggesting a role for E2F2 in the expansion of thymic epithelial cells (TECs) (114), while a mild decrease in thymus size was observed in E2f1;E2f2 double knockout animals (116). TEC hyperplastic phenotypes have been observed in transgenic mice expressing viral oncoproteins such as E7 and SV40 LT that can inactivate members of the retinoblastoma (RB) family (117-120), consistent with a role for RB family members in the thymic epithelium. Finally, the analysis of mice with mutations in p16 family members and p21 family members, the two families of small cell cycle inhibitors controlling the kinase activity of Cyclin/CDK complexes suggest specific roles for p18^Ink4c and p27^Kip1 in suppressing the expansion of TECs (121) (122). p27 in particular is expressed at higher levels in the developing thymus (123) and low levels of p27 correlate with poor prognosis in patients with thymoma (124, 125). Loss of p27 function in mice increases thymocyte cellularity as a direct consequence of an expanded TEC compartment that maintains proper organization and function (122). This phenotype is similar to that of over-expression of CyclinD1 in TECs, although perhaps not as severe.

While collectively these data strongly support a role for the extended RB pathway in the expansion of the thymic epithelium, major issues need to be addressed before the role of cell cycle regulation in TECs is understood. Many of these experiments did not specifically manipulate these genes in TECs, and non-cell autonomous effects due to crosstalk cannot be ruled out. In addition, none of these studies explored a role for this pathway at different stages over the entire lifespan. Thus, the stage-specific roles of the RB pathway in the formation, growth, maintenance, and/or involution of the thymus remain to be determined, in particular whether they play a role in aging-associated involution. Furthermore, the extra- and intra-cellular signals converging on members of the RB pathway to normally regulate TEC proliferation and differentiation are unknown. Finally, the molecular and cellular mechanisms by which the RB pathway may regulate TEC expansion and function, in particular the balance between regulating proliferation and differentiation, and their relationship to other known regulators of TEC differentiation such as Foxn1 and the NFKB family, represent intriguing future areas of investigation.