Analysis of the human thymic perivascular space during aging
Kristina G. Flores, Jie Li, Gregory D. Sempowski, Barton F. Haynes, Laura P. Hale
Kristina G. Flores, Jie Li, Gregory D. Sempowski, Barton F. Haynes, Laura P. Hale
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Analysis of the human thymic perivascular space during aging

Abstract

The perivascular space (PVS) of human thymus increases in volume during aging as thymopoiesis declines. Understanding the composition of the PVS is therefore vital to understanding mechanisms of thymic atrophy. We have analyzed 87 normal and 31 myasthenia gravis (MG) thymus tissues from patients ranging in age from newborn to 78 years, using immunohistologic and molecular assays. We confirmed that although thymic epithelial space (TES) volume decreases progressively with age, thymopoiesis with active T-cell receptor gene rearrangement continued normally within the TES into late life. Hematopoietic cells present in the adult PVS include T cells, B cells, and monocytes. Eosinophils are prominent in PVS of infants 2 years of age or younger. In the normal adult and the MG thymus, the PVS includes mature single-positive (CD1a– and CD4+ or CD8+) T lymphocytes that express CD45RO, and contains clusters of T cells expressing the TIA-1 cytotoxic granule antigen, suggesting a peripheral origin. PBMCs bind in vitro to MECA-79+ high endothelial venules present in the PVS, suggesting a mechanism for the recruitment of peripheral cells to thymic PVS. Therefore, in both normal subjects and MG patients, thymic PVS may be a compartment of the peripheral immune system that is not directly involved in thymopoiesis.

Authors

Kristina G. Flores, Jie Li, Gregory D. Sempowski, Barton F. Haynes, Laura P. Hale

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LM-PCR detects ongoing TCR gene rearrangement in pediatric and adult thy...
LM-PCR detects ongoing TCR gene rearrangement in pediatric and adult thymocytes. (a) LM-PCR detects free signal ends generated by dsDNA breaks 3′ and 5′ of the Dβ2.1 TCR gene segment, corresponding to D-J and V-DJ rearrangements, respectively. (b) Specific LM-PCR products obtained from thymocytes from 2 normal individuals less than 6 months old, indicating ongoing V-DJ (lanes 1 and 3; 409 bp) and D-J (lanes 9 and 11; 492 bp) rearrangement. Controls with non–linker-ligated DNA amplified with primers 3 and 5 (lane 17) or 2 and 4 (lane 18) demonstrate the appropriately sized germline bands (868 and 956 bp, respectively). Lanes using mock-ligated DNA (lanes 2, 4, 10, and 12), DNA lacking the TCR loci (bacterial DNA ± linker ligation; lanes 5, 6, 13, and 14), linker alone (lanes 7 and 15), and PCR blanks (lanes 8 and 16) are negative. The higher molecular weight bands seen in lanes 1 and 11 probably represent dsDNA breaks corresponding to additional (nonproductive) rearrangements in cells with a rearranged Dβ2.1 locus. However, this remains to be formally demonstrated using probes and primers specific for sequences unique to these downstream regions. (c) LM-PCR signals generated from thymocytes obtained from a 24-year-old male. Eight-fold dilutions of DNA (decreasing concentration left to right) were linker ligated and subjected to LM-PCR as described. Signals corresponding to both D-J and V-DJ rearrangements were detected in 5 of 8 samples (donor age and gender: 24 M, 29 F, 27 F, 41 F, 42 F). Only D-J signals were detected in 3 samples (donor age and gender: 28 F, 34 F, 46 M). All tissues tested had immunohistologic evidence for thymopoiesis, with at least small foci of CD1a+, mib-1+ lymphocytes within a loose network of thymic epithelial cells. Template control reactions using primers 2 and 4 amplified the appropriately sized germline band.