Analysis of the human thymic perivascular space during aging (original) (raw)
Thymic PVS volume increases with age in normal subjects and in MG patients. Although grossly detectable thymic atrophy may not occur until puberty, analysis of thymic PVS in normal thymus tissues ranging in age from 0 to 78 years (Table 1) clearly demonstrates that a statistically significant increase in the PVS has occurred by 2–10 years of age (age quintile 2; P < 0.01 vs. age quintile 1 [0–1 year]) (Figure 1a). The percent PVS increases progressively with age in normal individuals (Figure 1a), in agreement with previous studies by Steinmann (2). The increase in PVS volume is accompanied by accumulation of lymphocytes within the PVS (Figure 1, c–h), particularly in quintiles 2–4.
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).
The contribution of the TES and the PVS to the overall composition of MG thymus during aging was determined and compared with normal thymus. The percent PVS is increased in MG thymuses compared with age-matched normal thymuses (quintile 2, P < 0.07; quintile 3, P < 0.08; quintile 4, P < 0.0006; quintile 5, P < 0.018) (Figure 1b). A portion of this increase is due to follicular hyperplasia present in a subset of MG thymus tissue (Table 1). Cytokeratin staining (Figure 1h) clearly demonstrates that the primary and secondary follicles present in MG thymus with follicular hyperplasia occur in the PVS, not in the TES, similar to what is observed in normal aging thymus (5). Therefore, the thymic follicular hyperplasia seen in the PVS of MG thymus is an exaggerated state of the perivascular infiltration with lymphocytes that normally occurs in aging thymus.
The adipocyte and lymphocyte content of the PVS similarly changes with age. Adipocytes are rare in quintile 1, and increase progressively in quintiles 2–5, as shown in Figure 1. The lymphocyte content of the PVS increases rapidly in quintiles 2 and 3; a significant fraction of PVS area in these age ranges contains lymphocytes (Figure 1, d and e), as reported previously by Steinmann (2). The lymphocyte content of the PVS begins to decrease in quintile 4, with a corresponding increase in the fraction of PVS composed of adipose tissue in quintiles 4 and 5, relative to earlier quintiles (Figure 1, f and g). The PVS of most tissue samples in quintile 5 contains predominately adipocytes, with few lymphocytes (Figure 1g).
Immunohistologic identification of cells undergoing thymopoiesis in the TES. The TES in both normal and MG thymus decreases with age as PVS increases (Figure 1, a and b). To study age-related changes in thymopoiesis, we have determined the phenotype of lymphocytes present in the TES of normal and MG thymus tissues. CD1a is expressed on immature CD4+, CD8+ (double-positive) cortical thymocytes that have successfully completed rearrangement of the TCR β-chain gene locus (19), but it is not expressed on medullary thymocytes or mature T cells (20). We found that CD1a immunoreactivity in infant thymus (age 0–1 year) was limited to thymocytes within the thymic cortex (data not shown). Some dendritic cells located in the thymic medulla also reacted with CD1a mAb; however, these cells were clearly distinguished from immature thymocytes by their location, larger size, and multiple dendritic processes. Rare CD1a+ cells with dendritic cell morphology were also present in the PVS. The majority of the CD1a+ cortical thymocytes also reacted with mib-1 mAb specific for the Ki-67 nuclear proliferation antigen, with less frequent mib-1+ cells present in the thymic medulla and in the PVS (not shown). CD1a+, mib-1+ cortical thymocytes also reacted strongly with antibodies specific for CD3, CD4, CD8, CD38, and CD45RO (not shown), as expected for immature thymocytes (20, 21). Only rare isolated lymphocytes within the TES were reactive with TIA-1 mAb, specific for granules present in activated CTLs. Cytokeratin staining revealed a loose network of thymic epithelial (TE) cells within the cortex in association with large numbers of CD1a+, mib-1+ thymocytes, with a somewhat denser pattern of TE cells in the medulla (not shown). The majority of TE cells in both cortex and medulla were surrounded by multiple thymocytes. Taken together, these studies suggest that thymus tissues that are active in the process of thymopoiesis can be identified immunohistologically in FFPE tissues by the presence of immature CD1a+, mib-1+ thymocytes in a loose network of TE cells that allows thymocyte–TE cell interactions.
Of the 118 normal and MG thymus tissues examined, 116 contained at least some regions meeting our immunohistologic criteria for thymopoiesis. However, the absolute numbers of immature CD1a+, mib-1+ thymocytes undergoing thymopoiesis decreased progressively as the percent TES decreased with age and in MG. Thymocytes derived from a 78-year-old male thymus shown by immunohistochemistry to contain CD1a+, mib-1+ thymocytes displayed an immature phenotype. These thymocytes were CD4+ and CD8+, expressed CD45RO and CD38, and were negative for CD95, CD62L, and CD45RA (Figure 2). This thymus was highly atrophic with few PVS lymphocytes present. Although the absolute numbers of immature thymocytes were greatly decreased, the relative proportions were similar to those in pediatric thymus.
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 CD4hi 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.
LM-PCR detects ongoing TCR gene rearrangement in pediatric and adult human thymocytes. To provide molecular confirmation of thymopoiesis, we used an LM-PCR assay (22, 14) to identify dsDNA breaks indicative of D-J or V-DJ TCR gene rearrangement in thymocytes derived from pediatric and adult thymus tissues. This assay detects dsDNA breaks that are transiently generated at recombination signal sequences by the V-D-J recombinase complex during the process of TCR gene rearrangement (Figure 3a). The presence of LM-PCR signals in a given tissue is thus indicative of ongoing thymopoiesis.
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.
LM-PCR analysis detected DNA breaks associated with both D-J and V-DJ rearrangement of the TCRB gene in 14 of 14 pediatric thymocyte samples tested (2 representative samples are shown in Figure 3b, lanes 1, 3, 9, and 11). This assay is specific for ongoing human TCRB gene rearrangement: no LM-PCR signals are detected in DNA from human spleen or tonsil, mouse thymocytes (data not shown), bacteria (Figure 3b, lanes 5, 6, 13, and 14), or mock linker-ligated thymocytes (Figure 3b, lanes 2, 4, 10, and 12).
LM-PCR was used to determine whether the phenotypically immature thymocytes present in older adult thymuses were undergoing thymopoiesis. LM-PCR analysis detected dsDNA breaks corresponding to both D-J and V-DJ TCR gene rearrangement in most thymocyte DNA samples derived from normal adult and MG thymus tissues that showed immunohistologic evidence of thymopoiesis (Figure 3c). LM-PCR signals corresponding to D-J TCR gene rearrangement were detected in 8 of 8 adult thymus tissues tested, with V-DJ signals detected in 5 of 8 samples.
Eosinophils are prominent in the PVS of pediatric thymus. The PVS of infant thymus (quintile 1; 0–1 year) contains few lymphocytes. The majority of the cells present in the PVS of infants are spindle-shaped, consistent with fibroblasts or preadipocytes. However, large clusters of eosinophils are also frequently found in the PVS of infant thymus (Figure 4a). Many of these eosinophils have the bilobed nucleus characteristic of mature eosinophils; also present are cells containing similar eosinophilic granules and large single-lobed or indented nuclei with chromatin patterns that are consistent with the eosinophilic myelocyte and eosinophilic metamyelocyte stages of eosinophil differentiation. Clusters of eosinophils are smaller and less abundant in older children (quintile 2; 2–10 years). Eosinophils are rare to nondetectable in older thymus tissues from age quintiles 3–5 (11 years or older).
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.
The eosinophils present within the PVS of pediatric thymus could have differentiated there in situ or could have developed inside the TES or outside the thymus, and then migrated into the PVS. To investigate the ability of the thymus to support the differentiation of eosinophils, we analyzed thymic IL-5 mRNA production as a function of age. In humans, IL-5 is highly specific for stimulating the production, activation, and survival of eosinophils (23). In mice, the expression of IL-5 is sufficient to induce the full pathway of eosinophil differentiation (24). Therefore, we analyzed the expression of IL-5 in 44 normal human thymus tissues by RNase protection assay. We found that IL-5 was readily detectable in most normal thymus tissues from patients 2 years of age or younger (IL-5 = 1.13 ± 0.28 % of GAPDH signal, range 0–3.28, n = 14). IL-5 mRNA was undetectable in thymus derived from patients 3 years of age or older (n = 30). The level of IL-5 mRNA correlates with the observed incidence of eosinophilia within the thymic PVS in patients 3 years of age or younger. In particular, thymus tissues with high IL-5 content had moderate to large clusters of eosinophils present in the PVS (Figure 4a). Conversely, thymic tissues with undetectable IL-5 mRNA levels did not demonstrate eosinophilia of the thymic PVS. Taken together with the observation that some of the eosinophils present in the PVS exhibit nuclear morphologies characteristic of immature eosinophils, these data suggest that the eosinophils present in the thymic PVS of infants could differentiate in the PVS in response to thymic IL-5 production.
PVS lymphocytes have a phenotype consistent with peripheral lymphocytes. As described above, T lymphocytes present within the PVS are not positive for both CD1a and mib-1 and are therefore most likely mature T lymphocytes. Mature T lymphocytes present in the PVS may be newly generated mature virgin T cells emigrating from the thymus or T cells recirculating from the periphery. No phenotypic markers currently exist to specifically detect recent thymic emigrants in humans. Therefore, we determined the immunoreactivity of PVS lymphocytes with a panel of antibodies directed at lymphocyte differentiation and activation markers, using single, double, and triple immunohistochemical and immunofluorescence assays to locate the PVS (by lack of cytokeratin reactivity; Figure 5, a and b) and to identify appropriate markers on lymphocytes. Results are summarized in Table 2.
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.
Immunoreactivity of lymphocytes present in the thymic compartments
When large numbers of lymphocytes are present in the PVS of normal thymus (usually in age quintiles 3 and 4), the infiltrate consists of both T cells and B cells (Figure 5, c and d). T cells in the PVS are CD1a– and most are also mib-1– (Figure 5, e and f). Triple immunostains, combining a cytokeratin immunoperoxidase stain to view the TES with CD4-FITC and CD8-PC5 fluorescent antibodies, demonstrated that PVS T cells are single-positive, expressing either CD4 or CD8, but not both (not shown; see also Figure 5g). Most PVS T cells express the CD45RO surface antigen (Figure 5h) rather than the CD45RA antigen typically expressed on peripheral virgin T cells (not shown). Although large numbers of CD45RA+ lymphocytes are present both in the thymic medulla and in the PVS, the majority of these cells are B cells (Figure 4b). The cell types present in the PVS of 50 normal adult and 31 age-matched MG thymus tissues are similar, with further expansion of the PVS in MG thymus with follicular hyperplasia by infiltrates of primarily B lymphocytes, often with prominent germinal centers. (Figure 1h).
We observed that a subset of the CD8+ T cells present in the PVS also express the TIA-1 antigen characteristic of activated CTLs (Figure 4c), indicating that these cells are not recent thymic emigrants, but are mature CTLs from the periphery (25). We also found differences in CD38 expression between T cells in the TES and the PVS. Although cortical and medullary thymocytes express relatively high reactivity with CD38 mAb (21) (Figure 2e), most T cells present in the PVS do not have high reactivity with CD38. However, germinal center B cells are moderately to strongly reactive with CD38 mAb’s (Table 2). Using cytokeratin immunoperoxidase staining to locate the TES, combined with CD3-FITC and CD38-PE mAb’s, we confirmed that virtually all CD3+ T cells within the TES are also strongly positive for CD38 mAb (data not shown). Conversely, the majority of CD3+ cells in the PVS are not strongly reactive with CD38 mAb. Less than 5% of PVS CD3+ cells reacted strongly with CD38 mAb, and these cells were frequently located within or adjacent to B-cell follicles with germinal centers, consistent with an activated T-cell phenotype. The relative lack of CD38 immunostaining of most PVS T cells is also consistent with a peripheral origin for most PVS lymphocytes, because most peripheral blood lymphocytes are also CD38– (21).
PBMCs bind to MECA-79+ HEVs in the PVS of adult normal and MG thymus. We observed that HEVs could frequently be identified in normal thymus PVS when large numbers of lymphocytes were present in the PVS. Similarly, thymus from adult patients with MG that had large amounts of PVS lymphoid infiltrates also contained HEVs within the PVS. Peripheral lymph node HEVs have previously been shown to be immunoreactive with MECA-79 mAb, which recognizes a ligand for L-selectin (13). We found that the HEVs present in the PVS of adult normal and MG thymus react strongly with MECA-79 mAb (Figure 5i), similarly to HEVs in lymph nodes that have been shown to support lymphocyte recirculation through a mechanism that is dependent on MECA-79 and L-selectin (26). Thymic PVS HEVs did not react with mAb MECA-367 (not shown), which is specific for a mucosal addressin previously shown to direct migration of lymphocytes to Peyer’s patches and mesenteric lymph nodes (12, 27, 28).
To determine whether the thymic PVS HEVs can potentially support the emigration of peripheral lymphocytes, we performed in vitro assays of PBMC binding to tissue sections, as described previously (17, 18). We found that PBMCs bound specifically to thymic PVS HEVs, with 14 ± 3 lymphocytes (mean ± SD) bound per HEV in the 4 thymus tissues tested. These studies demonstrate that PBMCs bind well to the MECA-79+ thymic HEVs, which can thus potentially direct the migration of peripheral lymphocytes into the thymic PVS. Additional studies will be needed to determine the precise mechanisms for lymphocyte emigration into the PVS.