Skint-1 is a highly specific, unique selecting component for epidermal T cells.

αß T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.

Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes.

Although local regulation of T-cell responses by epithelial cells is increasingly viewed as important, few molecules mediating such regulation have been identified. Skint1, a recently identified member of the Ig-supergene family expressed by thymic epithelial cells and keratinocytes, specifies the murine epidermal intraepithelial lymphocyte (IEL) repertoire. Investigating whether Skint1-related molecules might regulate IEL in other compartments, this study focuses on buytrophilin-like 1 (Btnl1), which is conspicuously similar to Skint1 and primarily restricted to small intestinal epithelium. Btnl1 protein is mostly cytoplasmic, but surface expression can be induced, and in vivo Btnl1 can be detected adjacent to the IEL. In a newly developed culture system, enforced epithelial cell expression of Btnl1 attenuated the cells' response to activated IEL, as evidenced by suppression of IL-6 and other inflammatory mediators. These findings offer a unique perspective on emerging genetic data that Btnl genes may comprise novel and important local regulators of gut inflammation.

Identifying and antagonizing the interactions between layilin and glycosylated collagens.

Layilin is a small type I transmembrane receptor thought to bridge extracellular ligands with the cytoskeleton through its intracellular interactions with the scaffolding protein talin. Recent bulk- and single-cell RNA sequencing experiments have repeatedly found layilin to be highly upregulated in key T cell sub-populations in multiple disease states, suggesting its importance to the adaptive immune response. Despite this prevalence, little is known about layilin's precise role in mediating extracellular interactions or how these interactions can be modulated in disease states. Here we take advantage of layilin's dependence on calcium ions to discover its interactions with highly glycosylated type II, IV, V, and VI collagens. Toward exploring layilin's role in disease, we exploited the Ca2+ dependence in a differential phage display strategy to engineer species cross-reactive antibodies that block this interaction.

Skint1, the prototype of a newly identified immunoglobulin superfamily gene cluster, positively selects epidermal gammadelta T cells.

B cells, alphabeta T cells and gammadelta T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. alphabeta T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules. The molecules that select gammadelta T cells are unknown. Vgamma5+Vdelta1+ cells comprise 90% of mouse epidermal gammadelta T cells. By mapping and genetic complementation using a strain showing loss of Vgamma5+Vdelta1+ cells due to a failure of thymic selection, we show that this defect is caused by mutation in Skint1, a newly identified gene expressed in thymus and skin that encodes a protein with immunoglobulin-like and transmembrane domains. Skint1 is the prototypic member of a rapidly evolving family of at least 11 genes in mouse, with greatest similarity to the butyrophilin genes. These findings define a new family of proteins mediating key epithelial-immune interactions.

Phosphatidylinositol 3-kinase improves the efficiency of positive selection.

We have generated transgenic mice expressing the amino-terminal fragment of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110ABD) in thymocytes. Expression of p110ABD results in constitutive activation of PI3K and in significant increases in the numbers of mature, single-positive thymocytes. We previously reported that the increase in mature cells was in part due to a defect in thymic emigration. In this study we identify another component to this phenotype. Expression of p110ABD results in an enhancement of positive selection, without alterations in thymocyte lifespan or negative selection. Since PI3K can affect activation of Btk, which in turn potentiates calcium fluxes, during B cell development, our results suggest that PI3K could play a role in the regulation of Itk kinases in T cells, and that both cell types share a common signaling network to modulate calcium responses downstream of their antigen receptor.

Selection of the cutaneous intraepithelial gammadelta+ T cell repertoire by a thymic stromal determinant.

Intraepithelial lymphocytes constitute a group of T cells that express mainly monospecific or oligoclonal T cell receptors (TCRs). Like adaptive TCR alphabeta+ T cells, intraepithelial lymphocytes, a subset enriched in TCR gammadelta+ T cells, are proposed to be positively selected by thymically expressed self agonists, yet no direct evidence for this exists at present. Mouse dendritic epidermal T cells are prototypic intraepithelial lymphocytes, displaying an almost monoclonal TCR gammadelta+ repertoire. Here we describe an FVB substrain of mice in which this repertoire was uniquely depleted, resulting in cutaneous pathology. This phenotype was due to failure of dendritic epidermal T cell progenitors to mature because of a heritable defect in a dominant gene used by the thymic stroma to 'educate' the natural, skin-associated intraepithelial lymphocyte repertoire to be of physiological use.

Phosphatidylinositol 3-kinase regulates thymic exit.

To understand the role of PI3K during T cell development, we generated transgenic mice expressing the N terminus of the PI3K catalytic subunit (p110(ABD); ABD, adaptor binding domain) in thymocytes. Expression of p110(ABD) activates endogenous p110 and results in the accumulation of mature single-positive CD3(high)heat-stable Ag(low) thymocytes. This is mostly due to a defect in emigration of those cells, as shown by the delayed appearance of peripheral T cells in neonatal transgenic mice and by competitive adoptive transfer experiments. Although the mechanisms underlying these effects of PI3K are not yet clear, our results show an important role for PI3K activity in the regulation of mature thymocyte exit to the periphery.