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Previously, we detailed the characterization of thymic shared Ag-1, a unique marker of immature thymocytes and isolated thymic stromal cells, defined by the mAb MTS 35. In this study, the functional relevance of this molecule to thymopoiesis was investigated by the addition of purified MTS 35 to fetal thymus organ culture. It down-regulated thymic shared Ag-1 expression and dramatically reduced thymocyte cell yield through inhibition of alpha beta-TcR+ T cell differentiation, post CD3-CD4-CD8- triple negative thymocytes. These effects were specific for the mAb MTS 35, because controls, which include both isotype-matched and other lymphostromal mAb, showed no similar effects. These results demonstrate that thymic shared Ag-1 is a functionally important marker of early thymocyte differentiation, particularly with regard to the alpha beta-TcR lineage.

In a previous study, we raised a mAb (MTS 35) reacting with a plasma membrane Ag expressed on both cortical thymocytes and a subset of thymic medullary epithelial cells. In view of the shared expression of this molecule, we have defined it as thymic shared Ag-1 (TSA-1). Considering its selective reactivity with cortical, but not medullary thymocytes, the relevance of TSA-1 as a marker of immature T cells was investigated in detail in this study, using multicolor flow cytometric analysis. TSA-1 was found on all immature thymocyte subsets (CD3-4-8-, CD3-4+8-, CD3-4-8+, CD3-4+8+, CD3low4+8+). Conversely, CD3high4+8- and CD3high4-8+ thymocytes, early thymic migrants and peripheral T cells were TSA-1-. More refined gating and analysis of the transitional CD3intermediate/high4+8+ thymocytes, proposed candidates for negative selection, demonstrated that approximately one half were TSA-1-. In fact, there was a directly inverse relationship between TSA-1 and CD3 expression on thymocytes. In the periphery, TSA-1 was detected on B lymphocytes. TSA-1 is PI-linked and has a molecular mass of 17 kDa nonreduced, or 12 to 13 kDa reduced. Through cross-correlation analysis, this molecule was distinct from H-2K, PNA-R, CD5, CD11a/18, Thy-1, HSA, Ly6A/E, Ly6C, ThB, CD25, CD44. Hence TSA-1 appears to be a unique marker which exquisitely separates mature from immature thymocytes.

Both cyclophosphamide (Cy) and testosterone propionate (TP) treatments ablate B cells in chickens. Essential bursal microenvironmental elements, however, are altered or lost following TP treatment, while bursae from Cy-injected birds can be reconstituted with donor precursors. These two models can thus be utilized to distinguish which bursal stromal molecules are functionally most important in the specific microenvironment of this organ. Monoclonal antibodies (mAb) reactive with non-lymphoid components of the chicken bursa of Fabricius have been used to examine bursal sections from birds treated with Cy or TP. Molecules have been identified on the epithelial buds and follicle-associated epithelium (FAE) that are enhanced following Cy treatment (MUI-52 and 58) and are absent in TP-treated birds. The expression of these molecules may correspond to the ability of Cy-treated but not TP-treated bursae to attract lymphoid precursors. Molecules have also been identified on cells in the subepithelial mesenchymal layer (MUI-63, 65 and 75). These cells interact with the surface epithelium (sEp) prior to epithelial bud formation, an interaction which appears to be TP sensitive. Additionally, two potentially important molecules have been identified in the bursal medulla (MUI-54 and 72) which may have an interactive role with developing B lymphocytes.