Phenotypic hallmarks of the thymic microenvironment during involution

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There are several hallmarks of aging-associated involution that are easily assayed by histological approaches and more quantitative flow cytometric techniques. Key classically defined morphological changes are cortical thinning, disintegration of the CMJ, and significant reduction in size. These characteristics are all easily identified using hematoxylin and eosin stained paraffin sections (Figure 2A) or staining of frozen sections with compartment-specific markers (Figure 2B, C). Histological (A-C) and thymocyte cellularity (D) analysis throughout postnatal life in the mouse indicates that there is a close correlation between thymocyte numbers and stromal organization. After a very rapid logarithmic increase in thymus size and cellularity in the first postnatal week, thymus size and cellularity level off at around 3 weeks postnatal, with maximum size and cortical thickness at about 4 weeks. we observe the first clear drop in T cell numbers and thymus size between 6 and 7 weeks, cortical thinning is observed at 2 months, and by 3 months we observe the first signs of disorganization at the CMJ (regarded as an early hallmark of the onset of involution). After 3 months, a significant decrease occurs in the frequency of the subset of medullary TECs defined by binding high levels of the lectin, Ulex europaeus agglutinin-1 (UEA-1). By 6 months the thymus is already dramatically smaller, and CMJ degeneration is clearly progressing. In addition to depletion of the TEC compartment, accumulation of adipocytes in the perivascular space is another characteristic feature of the aging thymus (19-21). Although the precise origin of intrathymic adipocytes remains unknown, recent evidence suggests that adipocytes may arise via epithelial to mesenchymal transition (22). Involution becomes progressively more severe at subsequent ages, ultimately resulting in a near total breakdown of compartmental organization.

Alterations in composition of the thymus stromal compartment have been quantified by flow cytometric analysis of stromal cell subsets during thymus involution. The results have shown changes in the frequency of TECs relative to mesenchymal cells, a reduced ratio of mTECs to cTECs, reduced numbers of both major TEC subsets, specific reductions in the levels of MHC Class II expression and decline in the numbers of UEA-1+ cells. However, it is important to recognize that morphological changes in TECs with aging can result in changes in the ease of isolation of specific subsets. Therefore, specific changes in the relative frequencies of different TEC subsets must also be validated by independent methods, such as immunostaining in tissue sections. Technical difficulties in isolating thymus stromal cells and their cellular morphology make it difficult to accurately quantify changes in specific subsets during involution. New approaches to evaluating changes in the thymic stromal composition and organization are needed to move the field forward.