SUBTRACTIVE ISOLATION OF PHAGE-DISPLAYED SINGLE-CHAIN ANTIBODIES TO THYMIC STROMAL CELLS BY USING INTACT THYMIC FRAGMENTS

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of su ch microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we h ave employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intac t, mildly fixed thymic fragments as target tissue and lymphocytes as a bsorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4- 4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium spec ific, TB4-28 detects an epitope expressed on both epithelial- and mese nchymal-derived stromal cells. TB4-4 reacts with all cortical epitheli al cells and with other endoderm-derived epithelia, but this reagent l eaves the majority of medullary epithelial cells unstained. In contras t, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cel ls. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection o f evolutionary conserved epitopes expressed on subsets of thymic strom al cells.