Functional characterisation of two receptor interaction determinants in human thymic stromal lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is a pro-inflammatory cytokine with important pathological roles in Asthma bronchiale, malignant tumours and other diseases. The heterodimeric human TSLP receptor (hTSLPR) consists of the TSLP-binding subunit (TSLPRα) and the IL-7Rα-subunit. We studied the properties of hTSLP variants with mutations in their bipartite interaction interface towards IL-7Rα. One mutant (T46D/K101D) showed only mild impairment in receptor affinity but a massive reduction in biological activity. To facilitate the future development of hTSLP mutants with drug properties, we have devised a eukaryontic cytokine display assay with activity read-out and intrinsic genotype-phenotype coupling.

A recombinant antibody fragment directed to the thymic stromal lymphopoietin receptor (CRLF2) efficiently targets pediatric Philadelphia chromosome-like acute lymphoblastic leukemia.

Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.

A versatile platform for activity determination of cytokines and growth factors based on the human TSLP (thymic stromal lymphopoietin) receptor.

Cytokines and growth factors are signaling proteins involved in communication processes between cells. They are involved in the control of numerous essential physiological processes such as cell proliferation, gene transcription and differentiation; therefore being in the focus of basic and applied research. Many of them are also of relevance for human diseases. When observed as potential targets for pharmacological intervention and objects of structure/function studies, it is important to measure their biological activities, optionally along with potential inhibitors, in a convenient and rational manner. Such tests are frequently laborious to set up and their establishment is complicated by the necessity to employ problematic cell types and sophisticated assays. Here we present a robust and modular activity assay system which can be adapted to virtually all ligands that signal through dimerization of membrane receptors from different families. The technique rests on fusing ligand-binding domains of specific receptors to the transmembrane and intracellular components of the thymic stromal lymphopoietin (TSLP) receptor which translates signals into readily quantifiable luciferase expression in reporter cells. We show that the activation of various hematopoietic cytokine receptors, of receptor tyrosine kinases as well as of receptors bearing serine/threonine kinase domains by their respective ligands was faithfully reflected both upon transient and stable introduction of hybrid receptor and reporter gene constructs into the murine pro-B cell line Ba/F3. Moreover, we demonstrate the suitability of this platform for the functional characterization of cytokine/growth factor receptor inhibitors.

Characterization of SKAP/kinastrin isoforms: the N-terminus defines tissue specificity and Pontin binding.

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis, apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms, however, previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions, this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR, and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse, SKAP1 expression is detectable in testis at 4 weeks postnatally, when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore, we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1, our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells, including studies on male fertility.

Blockade of thymic stromal lymphopoietin (TSLP) receptor inhibits TSLP-driven proliferation and signalling in lymphoblasts from a subset of B-precursor ALL patients.

PURPOSE: The cytokine thymic stromal lymphopoietin (TSLP) and its receptor TSLPR are involved in intercellular communication in the course of allergic inflammation and have recently been implicated in the development of various malignancies including B cell precursor acute lymphoblastic leukemia (BCP-ALL). We studied TSLPR expression, TSLP-induced signal transduction and its antibody-mediated inhibition in long-term cultures of primary cells derived from B-precursor ALL patients. METHODS: TSLPR expression was determined by flow cytometry and Western blot analysis, cell proliferation, signal transduction via the JAK/STAT pathway was analysed by Western blot detection of STAT tyrosine phosphorylation and by measuring TSLP-dependent activation of a STAT-specific reporter gene construct. For inhibition studies a recently introduced antagonistic antibody to the TSLPRα-subunit was used. RESULTS: TSLPR surface expression was observed in leukemic lymphoblasts from two out of ten patients with BCP-ALL. Upon TSLP stimulation, the cells with the highest TSLPR expression level showed enhanced proliferation and JAK/STAT-mediated gene regulation in a dose-dependent manner. By employment of an inhibitory antibody to the TSLPR, both TSLP-triggered cell proliferation and STAT transcription factor activation were specifically inhibited. CONCLUSIONS: These results suggest that blockade of the TSLPR might be a therapeutic option for a subset of BCP-ALL patients.

Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range.

Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function.

Thymic stromal lymphopoietin (TSLP) is an interleukin-(IL)-7-like cytokine with emerging pathological importance for the development of atopic diseases such as allergic asthma bronchiale. The TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor, shares the IL-7R alpha-subunit with the IL-7 receptor system. The specific TSLPR alpha-chain shows similarities with the gammac receptor chain, but has some unusual features within the receptor family in both its ligand-binding and cytoplasmic domain. The murine TSLPR signals via the signal transducers and activators of transcription STAT5 and STAT3, but is unique among cytokine receptors in that it activates STATs without the involvement of Janus (JAK) tyrosine kinases, but instead utilizes the Src type kinase Tec. Here, we show by Western blotting and reporter gene experiments in combination with the application of a specific JAK inhibitor that the human TSLP receptor, in contrast, requires the function of JAK1 and JAK2 for STAT activation. Moreover, we demonstrate that the human TSLPR mediates gene regulation not only through STAT5 and STAT3 but has also the potential to mediate transcription via STAT1. Our work should help to understand more thoroughly how TSLP triggers inflammatory responses in the course of atopic diseases.