In this paper, we have extensively characterized the thymic PVS of adults and provided data suggesting a peripheral origin for PVS lymphocytes. Our identification of MECA-79+ HEVs that are capable of binding PBMCs suggests a mechanism for egress of peripheral lymphocytes into the PVS.
Our data on the relative proportions of the TES and PVS in normal thymus are consistent with those of Steinmann (2
), who used silver staining to identify PVS containing reticular fibers associated with extracellular matrix deposition. The PVS can also be identified by immunostains for fibronectin and laminin (3
), and by mAb TE7 (11
). In this study, we delineated the PVS from the TES (cortex and medulla) using reactivity of the epithelial component of the TES with cytokeratin mAb’s as a marker. We have determined the epithelial content of aging thymus, including the development of immunohistologic assays to evaluate the extent of ongoing thymopoiesis in archival FFPE thymus tissues.
We also report a definitive molecular assay for ongoing thymopoiesis in the human thymus, using the LM-PCR assay to detect dsDNA breaks that occur specifically during TCR gene rearrangement. Previous studies have used LM-PCR to investigate V-D-J recombination in mouse thymocytes (14
). Recent studies have measured the levels of TCR excised circles (TRECs) in blood lymphocytes to estimate levels of thymopoiesis (30
). However, because TCR excised circles are long-lived in peripheral T cells, and are lost only by dilution during cellular proliferation, a direct correlation between TREC levels and ongoing thymopoiesis cannot be made with this assay. The LM-PCR assay provides a snapshot of ongoing TCR gene rearrangement at the time of tissue harvest, and this provides a definitive measure of thymopoiesis in cases where fresh thymus tissue is available.
Our studies of lymphocytes in the TES demonstrated that the presence of mature CTLs, as indicated by expression of the TIA-1 granule antigen, is rare within the TES. These results fail to directly support the hypothesis of Hartwig and Steinmann (6
) that such cells are responsible for induction of atrophy in aging thymus. We did find that TIA-1+
CTLs are often present in the PVS of both normal and MG adult thymus tissues (Figure c). Because cells in the PVS are separated from the TES by a basement membrane (3
), the anatomic localization of thymic CTLs make it less likely that these CTLs induce atrophy of the TES through direct effects on thymic epithelial cells. However, CTLs in the PVS could secrete cytokines that may have direct or indirect effects on the process of thymic atrophy.
We also studied the PVS of MG human thymus tissues in parallel with normal thymus tissue. As reported previously, we found that histologic changes that occur in normal human thymus with aging also occur in MG thymus. The follicular hyperplasia observed in some of the MG thymus tissues is histologically similar to (although quantitatively greater than) changes occurring in normal thymus during aging. Therefore, both normal and MG thymus can be used to illustrate the changes that occur in the PVS and TES of the human thymus with aging.
We report several other novel observations. First, although eosinophils have previously been identified within thymus tissue of infants (33
), their location within the PVS and their decreased prevalence with age has never been described. Our data showed that thymus from children 2 years of age or younger potentially contains sufficient IL-5 to support eosinophil differentiation, consistent with the hypothesis that eosinophils differentiate in situ within the PVS of pediatric thymus early in life. However, because IL-5 has been shown to also be chemotactic for eosinophils (35
), increased eosinophil migration into the PVS of IL-5–expressing pediatric thymus tissues cannot be ruled out.
Although several studies have suggested that lymphocytes present within the PVS may be of peripheral origin (see below), our study is the first to demonstrate that these PVS lymphocytes are immunophenotypically consistent with peripheral mature cells. Although many surface markers expressed on PVS lymphocytes are also expressed on lymphocytes present in the thymic medulla, the presence of large numbers of CD1a–, CD45RO+, CD38–/low T cells, combined with the presence of TIA-1+–activated CTLs within the PVS, demonstrated that at least some PVS T cells are not new virgin T cells exiting from the thymic medulla, and therefore most likely migrated from the periphery. The presence of B cells located clearly within the thymic medulla (Figure b) raises the question of whether those cells arose in the medulla, in contrast to PVS B cells that may come from the periphery. The contribution of the thymus to the process of B-cell development has not been studied adequately, and the potential contributions of thymic B cells to the peripheral immune B-cell repertoire remain unclear.
The question of whether T cells in the thymic PVS originate within the thymus or migrate from the periphery has been previously addressed in animal models. Savino et al. (29
) described the existence of giant thymic PVS in the nonobese diabetic mouse. Cells within the PVS of these animals were resistant to sublethal irradiation with x-rays and hydrocortisone treatment, and were concluded to be mature lymphocytes, consistent with our more extensive phenotypic characterization of PVS cells in human thymus.
Finally, the identification and localization of HEVs within the PVS of human thymus is important. The existence of HEVs within the thymus has previously been reported in preleukemic mice (37
), although those authors did not recognize the existence of the PVS and described these HEVs as occurring within the thymic medulla. “Occasional, predominately medullary MECA-79+
HEV[s]” were reported previously in postnatal human thymus (38
); however, the significance was not discussed and anatomic location was similarly imprecise. The expression of MECA-79 (a ligand for L-selectin) on the HEVs in the PVS suggests a mechanism by which peripheral lymphocytes could access the thymic PVS, similar to mechanisms described in peripheral lymph nodes and sites of inflammation (38
). Our studies using in vitro lymphocyte binding assays demonstrate that peripheral blood lymphocytes can bind to thymic HEVs and thus potentially migrate into the thymic PVS. The function of these PVS lymphocytes in relation to the process of thymic involution has not been established, although temporally, the largest infiltrate volumes are seen in the age ranges where more rapid involution occurs. The mechanisms regulating HEV development in the PVS are still unknown.
Taken together, our studies suggest that the thymic PVS is a compartment of the peripheral immune system that is not directly involved in thymopoiesis. Understanding the relationships between the trafficking of peripheral lymphocytes to thymic PVS should lead to insights into mechanisms of thymic atrophy and control of postnatal thymic function.