Deconstructing the Thymic Microenvironment Through Genesis to Senescence

Abstract

The thymus is essential for adaptive immunity, orchestrating the differentiation of hematopoietic progenitors into various T-cell lineages. Thymic epithelial cells (TECs) impart this unique function by mediating the major checkpoints in T-cell differentiation while also imposing stringent tolerance processes required to prevent autoimmunity. Achieving these feats requires extensive TEC specialization and the formation of distinct thymic microenvironments. These features change extensively throughout life, from the growth phases of the embryonic and perinatal thymus, into the steady-state adult, through responses to acute injury and regeneration and, finally, during age-related thymic involution. Here we review how hypothesis and technology have shaped the field's understanding of the thymic microenvironment. We focus on how the development of single-cell technologies has revealed a remarkably diverse cellular landscape shaped by progenitor cell differentiation, TEC proliferation, AIRE-mediated transcriptional processes, and the differentiation of thymic mimetic cell lineages.

Keywords: T‐cell differentiation; epithelium; single‐cell technologies; stromal cells; thymus; tolerance.

FIGURE 1

The thymic microenvironment during embryogenesis, neonatal and aged stages. The thymic microenvironment undergoes major changes through development, followed by a gradual decline during involution. aaTECs, age‐associated thymic epithelial cells; Cld3/4, claudin 3/4; CMJ, cortico‐medullary junction; DCs, dendritic cells; int. mTECs, intermediate mTECs; LSP, lymphoid seeding progenitors; NCCs, neural crest cells; SP, single positive T cells; term. mTECs, terminally differentiated mTECs.

FIGURE 2

Representative confocal images of Krt19 (A) and Krt10 (B) expression in the thymus of aged Foxn1^nTnG mice. The medullary region is identified by high tdTomato expression and relatively high TEC cell density, whereas the cortical region is characterized by low tdTomato expression and lower TEC density. The aaTEC1 region is distinguished by a very high density of TECs, with the dotted line marking its boundary. C: Cortex; M: Medulla; Scale bar: 50 μm. Thymic lobes were fixed in paraformaldehyde (PFA) and sectioned into 200 μm slices using a vibratome. The sections were blocked with 0.3% Triton X‐100 in PBS and subsequently stained with specific antibodies diluted in 0.1% Triton X‐100 in PBS. Following staining, the sections were cleared transparent using EasyIndex optical clearing solution (LifeCanvas). Imaging was performed using a Zeiss LSM 980 confocal microscope, with representative images presented as 10 μm projections.

FIGURE 3

Reduced number of mimetic cells in young and aged Foxn1 ^nTnG mice. (A) Representative contour plots identifying tuft, corneocyte, and microfold mimetic cells in thymi from 2‐month‐old or 18‐month‐old Foxn1 ^nTnG mice. Cells were gated on CD45⁻/EpCAM⁺/GFP⁺ cells. (B) Quantification of mimetic cell subsets, presented as the total number of cells (upper panels) and the proportion of GFP⁺ mTECs (lower panels). Data are from 2‐month‐old (n = 6) and 18‐month‐old (n = 8) Foxn1^nTnG mice. Statistics were generated with two‐tailed paired t test. ns = non‐significant, *p = 0.03, **p = 0.002, ***p = 0.0002.