Abstract
Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection.
Highlights
As the first T cells to be produced by the fetal murine thymus, Dendritic epidermal T cells (DETC) provide almost unique insight into the development of non-MHC/CD1-restricted T cells
Skint-1 mRNA is readily detectable in thymic medullary epithelial cells [17, 31], albeit at different levels in different strains of mice, it has not proved possible to reliably detect Skint-1 protein at the surface of cells, formally casting doubt on whether it mediates its effects on DETC progenitors via cell surface interactions
In fetal thymic organ culture (FTOC) supplemented with anti-Skint-1-DV mAb, there was a marked inhibition of maturation, and likewise the number of T cell receptor (TCR)␥␦ϩ events was reduced, consistent with antiSkint-1 inhibiting Skint1-dependent maturation and selective expansion of DETC progenitors (Fig. 1, right panel)
Summary
Antibody Generation—Several monoclonal antibodies specific for Skint-1 DV were generated from rat serum following immunization with recombinant Skint-1 DV, and purified over a Pierce Sulfolink column, before concentration with Millipore Amicon-30 protein concentrator columns. Transfected cells were lysed in buffer (ice-cold 1% Nonidet P-40, 20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, containing protease inhibitors) and the lysates were analyzed by Western blotting performed using standard protocols. Reaggregate Thymic Organ Culture—Single cell suspensions were prepared from E14 to E15 fetal thymi by digestion with collagenase-dispase as described previously [19], and were spin-infected for 45 min with ϫ20 concentrated retrovirus from LinXE cells transfected with Skint-1 constructs in MSCVderived bicistronic vectors. A final wash without Triton was followed by resuspension of the protein pellet into 8 M urea solubilization buffer. The seven cycles were followed by a final structure calculation where the 20 conformers with the lowest CYANA target function were chosen as representative. The structural coordinates for Skint-1 DV have been deposited in the RCSB Protein Data Bank under accession code 2N4I
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