Analysis of thymic stromal cell populations using flow cytometry

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Abstract

The complexity of the lymphostromal interplay that is essential to αβT-cell development is reflected by the heterogeneity of both lymphocytes and thymic stromal cells. While panels of monoclonal antibodies have described many of the cellular components of these microenvironments, the means to quantify stromal cell subsets using flow cytometry remains poorly defined. This study refines and compares various stromal cell isolation procedures and determines the effects of various digestion enzymes on important surface molecules. Three- and four-color flow cytometry is used to correlate established and novel stromal cell markers to define thymic fibroblasts, epithelium and a unique subset of thymic endothelium that express MHC class II. This work provides a basis for the purification of thymic stromal cells for further phenotypic, functional and genetic analysis.

Introduction

The thymus provides a unique microenvironment that efficiently generates αβT-lymphocytes capable of responding to foreign peptide in the context of self-MHC. This microenvironment is established through the interplay of developing thymocytes with a non-lymphocytic component broadly termed the thymic stroma. The latter consists of a phenotypically diverse group of cells, including epithelium, endothelium, reticular fibroblasts, macrophages, dendritic cells and neuroendocrine cells (Boyd et al., 1993). These cells collectively provide cell surface molecules, cytokines and extracellular matrix elements that are essential for various stages of T-cell development (Anderson et al., 1996). While the various subsets of thymocytes in the thymus have been studied in depth, relatively little is known about the stromal cell types that influence their development.

Earlier ultrastructural studies initially demonstrated the diversity of thymic epithelial cells based on morphology and electron lucency, identifying six discrete subsets (types 1–6) (van de Wijngaert et al., 1984). Further investigations into thymic stromal cells (TSC) have involved immunohistological analysis using panels of monoclonal antibodies (mAbs) specific for stromal antigens (for example, van Vliet et al., 1984, de Maagd et al., 1985, Lobach et al., 1985, Godfrey et al., 1988). Such work has defined stromal cells (particularly the epithelial cells) into discrete subsets on the basis of morphology and the expression of intracellular and cell surface markers (Kampinga et al., 1989). However, the coexpression of these various markers and population kinetics of the stromal cell subsets have been difficult to evaluate by histological studies alone. A previous study attempting to resolve some of these issues using flow cytometric analysis of thymic stromal cells found extensive overlap between the distributions of determinants thought to be exclusive in their expression (Izon et al., 1994). Here, we describe the usage of multicolor flow cytometry to distinguish the various thymic stromal populations using mAbs to surface antigens.

The stromal cell yields from various enzymatic digestion methods are compared and the epitope sensitivities of important markers determined. As found previously, flow cytometry reveals a more extensive distribution of some stromal antigens than would have been predicted from immunohistology, highlighting the greater sensitivity of the former technique. However, exclusion of certain contaminating cell types has enabled clearer correlation of TSC reactive mAbs on stromal cell subsets. FACS purification of these stromal subsets will hopefully aid in the determination of the precise functional roles each of these cell types have in T-cell development. Furthermore, genetic and antigenic analysis of individual populations may lead to a better understanding of the molecules that mediate these functions.

Section snippets

Animals

CBA×C57BL/6 F1 mice at 3 days or 4 weeks of age were used as a source of thymic stromal cells. Mice were bred and maintained by the Monash University Central Animal Services.

Thymic stromal cell isolation by collagenase digestion

The thymic stromal cell isolation procedure used was based on that described by us previously (Shortman et al., 1989). Two to three thymi were dissected from freshly killed mice and trimmed of fat and connective tissue. Small cuts into the capsules were made with a pair of fine scissors and the thymi were gently agitated in

Analysis of thymic stromal cells isolated by collagenase digestion

To determine the enrichment and numbers of thymic stromal cells that could be obtained using collagenase digestion, cells isolated from a series of incubations were analyzed for CD45 expression, which detects all cells derived from haematopoietic stem cells (HSCs) (reviewed by Thomas, 1989). This enabled discrimination of CD45 epithelium, endothelium, fibroblasts and neuroendocrine cells from all other cells in the thymus.

Table 1 shows that incubation of thymic fragments in a series of three

Conclusion

This study refines isolation protocols for thymic stromal cells and characterizes the major subsets using a panel of surface reactive mAbs. Collagenase digestion of thymic fragments from 4-week-old and neonatal mice enriched CD45 negative and positive stromal cells appropriate for immunofluorescent labeling and flow cytometry. However, collagenase digestion did not dissociate all CD45 stromal cells, leaving up to 20% remaining as aggregates. Incubation with trypsin ensures maximal recovery but

Acknowledgements

The authors wish to thank Dr. A Farr for kindly providing mAbs. This work was supported by grants from the Australian NH&MRC.

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