Single-cell sequencing is an emerging technology in the field of immunology and oncology that allows researchers to couple RNA quantification and other modalities, like immune cell receptor profiling at the level of an individual cell. A number of workflows and software packages have been created to process and analyze single-cell transcriptomic data. These packages allow users to take the vast dimensionality of the data generated in single-cell-based experiments and distill the data into novel insights. Unlike the transcriptomic field, there is a lack of options for software that allow for single-cell immune receptor profiling. Enabling users to easily combine mRNA and immune profiling, scRepertoire was built to process data derived from 10x Genomics Chromium Immune Profiling for both T-cell receptor (TCR) and immunoglobulin (Ig) enrichment workflows and subsequently interacts with a number of popular R packages for single-cell expression, such as Seurat. The scRepertoire R package and processed data are open source and available on GitHub and provides in-depth tutorials on the capability of the package.

CD8+ T cells are important effectors of adaptive immunity against pathogens, tumors, and self antigens. Here, we asked how human cognate antigen-responsive CD8+ T cells and their receptors could be identified in unselected single-cell gene expression data. Single-cell RNA sequencing and qPCR of dye-labeled antigen-specific cells identified large gene sets that were congruently up- or downregulated in virus-responsive CD8+ T cells under different antigen presentation conditions. Combined expression of TNFRSF9, XCL1, XCL2, and CRTAM was the most distinct marker of virus-responsive cells on a single-cell level. Using transcriptomic data, we developed a machine learning-based classifier that provides sensitive and specific detection of virus-responsive CD8+ T cells from unselected populations. Gene response profiles of CD8+ T cells specific for the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein differed markedly from virus-specific cells. These findings provide single-cell gene expression parameters for comprehensive identification of rare antigen-responsive cells and T cell receptors.

Hexahydrophthalic anhydride (HHPA) and methyltetrahydrophthalic anhydride (MTHPA) belong to the group of the acid anhydrides and, among other applications, are used in the production of epoxy resins. These substances are known as potent low-molecular allergens and induce predominantly type I allergies according to Coombs and Gell. We examined 110 employees exposed to HHPA and MTHPA. With all of them a RAST was carried out with the commercially available conjugates of phthalic anhydride (PA) and a skin prick test with 1% and 5% acetonic solutions of PA. In 109 of these sera a radio allergo sorbent test (RAST) was carried out with the not commercially available conjugates of HHPA and of MTHPA. With complaints connected with the workplace the working materials used (HHPA, MTHPA) were also checked by means of the skin prick test. With at least one positive immunological finding (in the RAST and/or skin prick test) in connection with complaints at the workplace, we performed a workplace-related inhalation test under experimental conditions. Specific IgE against acid anhydrides was detected in a total of 17 (15.4%) persons. In the challenge test, six (5.4%) sensitisations were shown to be clinically relevant. On inclusion of borderline positive findings with PA conjugates the RAST produced three false negative and one false positive finding compared with a RAST with HHPA and MTHPA conjugates. With the conjugates of trimellitic anhydride, in no case could specific IgE be detected. The skin prick test led, in comparison with the RAST, to three false positive and three false negative findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Aims/hypothesis The beta cell protein tetraspanin 7 is a target of autoantibodies in individuals with type 1 diabetes. The aim of this study was to identify autoantibody epitope-containing regions and key residues for autoantibody binding. Methods Autoantibody epitope regions were identified by immunoprecipitation of luciferase-tagged single or multiple tetraspanin 7 domains using tetraspanin 7 antibody-positive sera. Subsequently, amino acids (AAs) relevant for autoantibody binding were identified by single AA mutations. Results In tetraspanin 7 antibody-positive sera, antibody binding was most frequent to tetraspanin 7 proteins that contained the NH 2 -terminal cytoplasmic domain 1 (C1; up to 39%) or COOH-terminal C3 (up to 22%). Binding to C3 was more frequent when the domain was expressed along with the flanking transmembrane domain, suggesting that conformation is likely to be important. Binding to external domains was not observed. Single AA mutations of C3 identified residues Y246, E247 and R239 as critical for COOH-terminal binding of 9/10, 10/10 and 8/10 sera tested, respectively. Mutation of cysteines adjacent to the transmembrane domain at either residues C235 or C236 resulted in both decreased (8/178 and 15/178 individuals, respectively; >twofold decrease) and increased (30/178 and 13/178 individuals, respectively; >twofold increase) binding in participant sera vs wild-type protein. Conclusions/interpretationWe hypothesise that conformation and, potentially, modification of protein terminal ends of tetraspanin 7 may be important for autoantibody binding in type 1 diabetes.