Expression of The αβ T-Cell Receptor Is Necessary for The Generation of The Thymic Medulla

The architecture of the thymus of mice that congenitally fail to express the αβ T-cell receptor (TCRαβ) has been examined by immunohistology. In these mice, a defined mutation was introduced into the TCRc gene by homologous recombination. By using antibodies specific for cortical or medullary epithelium and for major histocompatibility complex antigens, the network of cortical epithelium in these mice was shown to be essentially unaltered in comparison with that of normal mice. In contrast, the thymic medulla was considerably reduced in size. This analysis shows that expression of the αβ TCR but not the γδ TCR is obligatory for establishing the thymic medulla and suggests that the growth of medullary epithelial cells may require contact with TCRαβ-expressing cells.


INTRODUCTION
The thymic microenvironment is essential for the differentiation of T cells. Prothymocytes migrate from sites of haematopoiesis into the thymus and there undergo a series of differentiation and expansion events, during which they express either aft or , forms of the TCR (reviewed by von Boehmer, 1988). TCRafl-expressing thymocytes undergo negative selection to remove autoreactive TCRs and positive selection to generate MHC-restricted TCRs. Both epithelial and dendritic cells are mediators of the selection processes, with the epithelial cells being particularly important in positive selection (Benoist and Mathis, 1989;Vukmanovic et al., 1992).
The thymic lobules of normal mice are arranged into an outer cortex and an inner medulla. In contrast to the normal thymus, the network of medullary epithelial cells is poorly developed in scid mice (Shores et al., 1991;Surh et al., 1992). However, injection of mature T cells or transfer of normal bone marrow cells restores this network of epithelial cells (Shores et al., 1991;Surh et al., 1992). These observations suggest that mature T cells may be necessary for the development of the thymic medulla. *Corresponding author.
We have previously generated mice that fail to express the cfl TCR by disrupting the TCRc chain by homologous recombination (Philpott et al., 1992). In these mice, thymocytes arrest at the double-positive (CD4+CD8+) stage of development and fail to mature into single positive (CD4+ or CD8+ thymocytes. We also observed that the thymic medulla, characterized by its less dense cellularity in histological preparations, was undetectable morphologically. However, the basis for the disruption of the cellular architcture of the medulla was not elucidated. To determine whether this loss extended to the network of medullary epithelial cells or other cells in the microenvironment, thymi from these mice were analyzed using a series of monoclonal antibodies specific for thymic epithelial subsets, and major histocompatibility complex antigens. In the present study, we show that there is a drastic reduction of medulla epithelial cells, suggesting that contact with TCRafl cells are necessary for the maintenance of the thymic medulla.

Mice
Mice used in this study were congenitally deficient in mature c8 TCR (TCRc-/-) expressing cells as 176 D.B. PALMER et al. described by Philpott et al. (1992). The control mice were wild type (WT) littermates expressing normal levels of the c]/TCR. Immunohistochemistry Thymi were removed from 4-8-week-old mice, snap frozen in liquid nitrogen, and stored at -70 C. Fivemicron frozen sections were cut from the tissue, air dried, fixed in acetone, and stained using the indirect immunoperoxidase method at room temperature. Slides were incubated with primary antibody for 1 hour and after washing in Tris-buffered saline (TBS), pH 7.6, were incubated for a further hour with biotinylated secondary antibody diluted in TBS containing 2% normal mouse serum. After washing for 20 min, the sections were incubated with avidinperoxidase (Sigma, UK) and visualized by adding diaminobenzidine (DAB, Sigma) (10 mg/15 ml with 12 1 of H202). Slides were counterstained with haematoxylin, dehydrated, and mounted using LPX mountant (BDH, UK). Positively staining cells were identified by the presence of a brown reaction product.

Thymic Epithelial Staining
Generation of mAbs raised against thymic epithelium has revealed molecular heterogeneity within the thymus microenvironment (Ritter and Crispe, 1992). The mAbs NLDC-145 and 4F1 recognize cortical epithelium, and NLDC-145 also weakly reacts against dendritic cells (Kraal et al., 1986;Kanariou et al., 1989). In contrast, the mAbs ER-TR5 and IVC4 react specifically with medullary epithelium (van Vliet et al., 1984;Kanariou et al., 1989). Although both sets of anti-cortical and antimedullary epithelium antibodies were used, only staining with NLDC-145 and ER-TR5 are shown. Staining with either NLDC-145 or 4F1 on normal sections of thymus revealed a typical network of cortical epithelial cells, which are characterized by long thin processes separated by densely packed thymocytes (Fig. l a). In contrast, the network of medullary epithelial cells in a normal thymus, as revealed by the mAbs ER-TR5 and IVC4, possessed shorter processes that were surrounded by fewer lymphoid cells (Fig. lc).
The thymic cortex of TCRc-/mice showed no apparent differences in comparison with that of the normal cortex, using either NLDC-145 or 4F1 (Fig. lb). In contrast, the mAbs ER-TR5 and IVC4 showed that the thymic medulla was considerably reduced in size in the TCRa-/mice (Fig. l d).
Staining with the anti-keratin antibody revealed the total network of epithelial cells in the thymus and confirmed these findings in both the wild type and TCRc-/mice (Figs. le and lf). Macrophages were evenly distributed in the thymus of both control and TCRc-/mice (data not shown).

MHC Antigen Expression
On the normal thymus MHC class II distribution in the cortex revealed a reticular network of epithelial cells, whereas the medulla showed confluent staining of MHC class II molecules (Fig. 2a). This pattern of staining was in agreement with that shown by Rouse et al. (1979). In the TCRc mutant mice, class II expression of the cortical epithelial cells was unaltered. However, only a small area of dense medullary class II staining was evident, underscoring the small size of the medulla in these mice (Fig. 2b). MHC class I staining produced a similar picture, although the overall expression is weaker, of dense staining in the medulla that was almost absent in the thymus from TCRc-/mice (data not shown). DISCUSSION We have previously shown that the less dense cellularity characteristic of the thymic medulla was lacking in thymic sections from mice congenitally deficient in expression of the TCRc gene (Philpott et