Deciphering the regulatory landscapte of fetal and adult γδ T-cell development at single-cell resolution

γδ T cells with distinct properties develop in the embryonic and adult thymus and have been identified as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases, and tissue homeostasis. Despite their potential value for immunotherapy, differentiation of γδ T cells in the thymus is incompletely understood. Here, we establish a high‐resolution map of γδ T‐cell differentiation from the fetal and adult thymus using single‐cell RNA sequencing. We reveal novel sub‐types of immature and mature γδ T cells and identify an unpolarized thymic population which is expanded in the blood and lymph nodes. Our detailed comparative analysis reveals remarkable similarities between the gene networks active during fetal and adult γδ T‐cell differentiation. By performing a combined single‐cell analysis of Sox13, Maf, and Rorc knockout mice, we demonstrate sequential activation of these factors during IL ‐17‐producing γδ T‐cell (γδT17) differentiation. These findings substantially expand our understanding of γδ T‐cell ontogeny in fetal and adult life. Our experimental and computational strategy provides a blueprint for comparing immune cell differentiation across developmental stages

KAT Ligation for Rapid and Facile Covalent Attachment of Biomolecules to Surfaces

The efficient and bioorthogonal chemical ligation reaction between potassium acyltrifluoroborates (KATs) and hydroxylamines (HAs) was used for the surface functionalization of a self-assembled monolayer (SAM) with biomolecules. An alkane thioether molecule with one terminal KAT group (S-KAT) was synthesized and adsorbed onto a gold surface, placing a KAT group on the top of the monolayer (KAT-SAM). As an initial test case, an aqueous solution of a hydroxylamine (HA) derivative of poly(ethylene glycol) (PEG) (HA-PEG) was added to this KAT-SAM at room temperature to perform the surface KAT ligation. Quartz crystal microbalance with dissipation (QCM-D) monitoring confirmed the rapid attachment of the PEG moiety onto the SAM. By surface characterization methods such as contact angle and ellipsometry, the attachment of PEG layer was confirmed, and covalent amide-bond formation was established by X-ray photoelectron spectroscopy (XPS). In a proof-of-concept study, the applicability of this surface KAT ligation for the attachment of biomolecules to surfaces was tested using a model protein, green fluorescent protein (GFP). A GFP was chemically modified with an HA linker to synthesize HA-GFP and added to the KAT-SAM under aqueous dilute conditions. A rapid attachment of the GFP on the surface was observed in real time by QCM-D. Despite the fact that such biomolecules have a variety of unprotected functional groups within their structures, the surface KAT ligation proceeded rapidly in a chemoselective manner. Our results demonstrate the versatility of the KAT ligation for the covalent attachment of a variety of water-soluble molecules onto SAM surfaces under dilute and biocompatible conditions to form stable, natural amide bonds

Validation of the Medical Research Council and a newly developed prognostic index in patients with malignant glioma: How useful are prognostic indices in routine clinical practice?

Although different prognostic indices for malignant gliomas have been developed, their validity outside of clinical trials has not been widely tested. The aim of this study was to determine whether the Medical Research Council (MRC) brain tumour prognostic index was able to stratify patients for survival managed in routine practice, and secondly to compare the results with our newly developed prognostic score which included tumour grade and only 3 prognostic groups. The MRC and the new prognostic index were calculated for a group of 119 adult patients with malignant glioma managed by surgical resection/biopsy and post-operative radiotherapy. For the MRC and new score, 6 and 3 prognostic groups were defined, respectively. For all patients median survival was 11 (2-66) months. The overall survival rate at 12 and 24 months were 43% and 18%, respectively. The MRC median and two-year survival rates were 14 months and 26% for a score of 1-10, 14 months and 27% for a score of 11-15, 13 months and 22% for a score of 16-20, 8 months and 10% for a score of 21-25, 8 months and 0% for those scoring 26-33. There was only one patient in the 34-38 group. For the new prognostic index, median and two-year survival rates were respectively 16 and 26%; 12 and 23%; 8 and 7% for the good, intermediate and poor prognostic groups. Both indices were significant factors for survival in univariate analysis (MRC index, p = 0.0089, new index p = 0.0002), but not in multivariate analysis. Both the MRC and our newly devised prognostic score were able to separate patients into good and poor prognostic groups, which may aid in treatment decisions, although there was less differentiation between the MRC groups especially over the first year. Both scores use routinely available factors. However, inclusion of tumour grade in the new score may be an advantage over the MRC index

Single-cell RNA-sequencing identifies the developmental trajectory of C-Myc-dependent NK1.1^-T-bet⁺

Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4−CD8αβ−TCRαβ+NK1.1− IEL precursors (NK1.1− IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1− IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a high-resolution molecular framework for thymic IEL development of NK1.1− IELPs and deepen our understanding of this still elusive cell type