Phenotypically immature thymocytes are present in adult thymus tissues meeting immunohistologic criteria for thymopoiesis. Lymphocytes from a 78-year-old male thymus were analyzed by flow cytometry using combinations of fluorescently labeled mAb’s. The majority of lymphocytes present were CD3⁺ (a), with 82% CD4⁺, CD8⁺ double-positive (b). 3% of cells were reactive with CD19 and CD20 mAb’s (c), and were consequently identified as B lymphocytes. Of the CD4^hi cells, more than 90% were CD45RO⁺ (d), 97% were CD38⁺ (e), and 97% were CD45RA^– (f), consistent with an immature phenotype. Similar results were seen with gating on CD8⁺ lymphocytes. Very few mature T cells were present in this sample, consistent with the observed lack of lymphocytes infiltrating the PVS on immunohistochemical sections. Although the majority of cells present in this thymus were immature thymocytes, the absolute numbers of thymocytes obtained for analysis were less than 1% of those obtained per gram of tissue from pediatric thymus.

Phenotypes of cells present in the thymic PVS. (a) H&E staining, in addition to cytokeratin immunoperoxidase staining (brown), demonstrates that eosinophils are present outside the TE network and thus within the PVS in thymus tissues expressing IL-5 mRNA. The majority of cells shown in this field are eosinophils, with both mature and immature morphologies represented. Arrows denote representative eosinophils. (b) Cytokeratin (brown) and CD20 (red) double staining shows that CD20⁺ cells are present within both the thymic medulla (M) and the PVS (*), but are rare within the cortex (C). (c) Cytokeratin (brown) and TIA-1 (red) double staining identifies TIA-1⁺ cells within the PVS.

Thymic PVS increases with age in normal individuals and in patients with MG. The percent thymic PVS determined from H&E–stained sections is shown as a function of age quintile. (a) Data for normal individuals are expressed as mean ± SD for the indicated number of cases. Quintile 1: 7 ± 2%, n = 18; quintile 2: 12 ± 7%, n = 19; quintile 3: 37 ± 19%, n = 10; quintile 4: 55 ± 18%, n = 21; quintile 5: 82 ± 16%, n = 19. (b) Data for patients with MG are expressed as mean ± SD for the indicated number of cases. Quintile 2: 23 ± 13%, n = 2; quintile 3: 61 ± 34%, n = 8; quintile 4: 83 ± 14%, n = 13; quintile 5: 96 ± 2%, n = 8. (c–h) Cytokeratin immunoperoxidase staining (brown color) outlines TES, with an H&E counterstain. Letters denote representative regions of thymic cortex (C), medulla (M), and PVS (P). (c) Quintile 1. (d) Quintile 2. (e) Quintile 3. (f) Quintile 4. (g) Quintile 5. (h) Thymus with follicular hyperplasia from a 20-year-old female with MG. Cytokeratin and H&E staining shows that primary and secondary follicles are located outside the cytokeratin network, within the PVS. ×25 (original magnification).

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.

Phenotypes of cells present in the thymic PVS. Thymus from a 42-year-old female with MG is shown in order to allow examination of relatively large areas of PVS within a single field. TES active in thymopoiesis is highlighted using a C to indicate active cortex, with an arrow pointing to the medulla. The cytokeratin stain (b) also demonstrates inactive TES (arrowheads in a and b) surrounded by PVS (P). The following immunoperoxidase stains demonstrate phenotypes of cells present in both TES and PVS: (c) CD3, T cells; (d) CD20, B cells; (e) CD1a, immature thymocytes; (f) mib-1, proliferating cells (note the positive reaction of both CD1a and mib-1 mAb’s [brown] with thymocytes in cortex [C] but not in medulla [arrow]); (g) CD8, immature thymocytes and mature CTLs; (h) CD45RO, immature thymocytes and mature memory T cells. (i) MECA-79 immunostaining highlights HEV in the thymic PVS (P). Inset depicts a MECA-79 ⁺ HEV at higher magnification.