Phenotypic hallmarks of the thymic microenvironment during involution
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.
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