T cell precursor development depends upon poorly defined interactions between thymocyte precursors and stromal cells involving cell surface molecules, extracellular matrix (ECM) components and soluble growth factors. To determine whether presentation of soluble factors by ECM is involved in early T cell development, we analyzed expression of ECM components in individual thymic stromal subsets and investigated their ability to present ECM-associated IL-7, a factor known to be important during early thymocyte development. We show that MHC class II(+) thymic epithelium and fibroblasts - essential requirements for development of CD4(-)8(-)precursors - both show surface expression of ECM components such as fibronectin and heparan sulfate. Use of biotinylated IL-7 protein indicates that both cell types bind IL-7, while enzymatic disruption of specific ECM components indicates that IL-7 presentation by both cell types is dependent upon heparan sulfate. However, disruption of IL-7 presentation specifically on fibroblasts does not affect their ability to contribute to T cell development. Collectively, these data suggest that while ECM-mediated presentation of IL-7 may be a general function of thymic stromal cells during thymocyte development, heparan sulfate-mediated IL-7 presentation specifically by fibroblasts is not essential and that the specific requirement for fibroblasts in early development involves additional undefined interactions.

We describe a novel method of cell purification involving two stage immunomagnetic selection which permits isolation of cells based on a second cell surface marker without the need for removal of beads used in the first selection step. This approach takes advantage of the size differences between commercially available immunomagnetic beads and/or particles and their differing properties in terms of attraction to magnetic fields of various strengths. The first stage of separation involves positive selection of cells using the Miltenyi MiniMacs system, utilising 50 nm MicroBeads and a MiniMacs magnet. Cells obtained from this procedure—still rosetted with 50 nm MicroBeads—can then be subjected to further positive or negative selection using either streptavidin M280 or anti-rat M450 Dynabeads, without the need for prior bead removal, since the strength of the magnetic field of the Dynal separator is sufficient to attract the larger Dynabeads but not the MicroBeads. Here, we show that this system can be used to isolate a number of cell types including very rare target cell populations such as haemopoietic stem cells, using two different surface markers without perturbing subsequent functional capacity.

Thymocyte positive selection results in maturation to the single-positive stage, while negative selection results in death by apoptosis, Although kinetic analyses indicate only 3-5% of CD4(+)8(+) cells reach the single-positive stage, the balance of positive and negative selection and the nature and quantity of cells mediating maximal negative selection are uncertain. Here, using a system where the number and type of stromal cells and thymocytes can be controlled, we investigated the maturation of CD4(+)8(+) thymocytes in the presence or absence of thymic epithelium and dendritic cells (DC) from wild-type (wt) and H-2M(-/-) mice expressing different peptide arrays. We find that titration of wt DC into reaggregates of wt epithelium has a dramatic effect on the number of CD4(+) cells generated, with 1% DC causing a maximal 80% reduction, Moreover, while addition of 1% wt DC into cultures of H-2M(-/-) epithelium causes a 90% reduction in CD4(+) cells, no effect was observed when similar numbers of wt thymic epithelium were added. Collectively, these data provide the first accurate indication of the quantity and quality of stromal cells required for maximal negative selection in the thymus, demonstrate the importance of peptide diversity in T cell selection, and highlight a large degree of overlap between positive and negative selection events.