Fibroblast dependency during early thymocyte development maps to the CD25⁺ CD44⁺ stage and involves interactions with fibroblast matrix molecules

We have investigated the role of specific components of the thymic stroma during development of CD4^-8^- T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25⁺44⁺ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II⁺ thymic epithelial cells and fibroblasts, their direct descendants, CD25⁺44^- precursors, develop to the CD4⁺8⁺ stage in the presence of MHC class II⁺ thymic epithelial cells alone. Thus, CD25⁺44⁺ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to α4 integrin and CD44 interfere with the efficiency with which CD4⁺8⁺ cells are generated from CD25⁺44⁺ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.