Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by ...single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.
The ability to study cancer-immune cell communication across the whole tumor section without tissue dissociation is needed, especially for cancer immunotherapy development, which requires ...understanding of molecular mechanisms and discovery of more druggable targets. In this work, we assembled and evaluated an integrated experimental framework and analytical process to enable genome-wide scale discovery of ligand-receptors potentially used for cellular crosstalks, followed by targeted validation. We assessed the complementarity of four different technologies: single-cell RNA sequencing and Spatial transcriptomic (measuring over >20,000 genes), RNA
In Situ
Hybridization (RNAscope, measuring 4-12 genes) and Opal Polaris multiplex protein staining (4-9 proteins). To utilize the multimodal data, we implemented existing methods and also developed STRISH (Spatial TRanscriptomic
In Situ
Hybridization), a computational method that can automatically scan across the whole tissue section for local expression of gene (e.g. RNAscope data) and/or protein markers (e.g. Polaris data) to recapitulate an interaction landscape across the whole tissue. We evaluated the approach to discover and validate cell-cell interaction
in situ
through in-depth analysis of two types of cancer, basal cell carcinoma and squamous cell carcinoma, which account for over 70% of cancer cases. We showed that inference of cell-cell interactions using scRNA-seq data can misdetect or detect false positive interactions. Spatial transcriptomics still suffers from misdetecting lowly expressed ligand-receptor interactions, but reduces false discovery. RNAscope and Polaris are sensitive methods for defining the location of potential ligand receptor interactions, and the STRISH program can determine the probability that local gene co-expression reflects true cell-cell interaction. We expect that the approach described here will be widely applied to discover and validate ligand receptor interaction in different types of solid cancer tumors.
IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed ...tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis.
Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s) in ...immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice), and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i) the literature findings and ii) the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines.
Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is ...limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan.
We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with (
= 24) or without (
= 9) COVID-19 from nasal immune cells in children with (
= 14) or without (
= 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort (
= 42), comparing obese and nonobese subjects.
Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of
and
in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes.
These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.
We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or ...target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (
R
2
=
0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.
An anode-supported tubular solid oxide fuel cell (SOFC) with a thin electrolyte film for reduced temperature operation was fabricated by a wet cofire process. Instead of the traditional extrusion ...method, the anode substrate was formed by a tape-cast method. This allows design the substrate with some advantageous configurations that can improve the performance of SOFCs. Sc2O3 and Y2O3-stabilized zirconia electrolyte films were fabricated on the substrate by a modified slurry dip-coating method and then cofired. In order to use (La0.6Sr0.4)CoO3 cathode, a thin (CeO2)0.9(GdO1.5)0.1 interlayer was also fabricated by the slurry dip-coating method. The fabricated tubular cell generated electricity successfully between 600 and 850DGC. Power density of ca. 170 and 220 mW/cm2 was achieved at 600 and 700DGC, respectively, however, the ohmic resistance was larger than expected. Because (CeO2)0.9(GdO1.5)0.1 interlayer was used, ohmic resistance could be high due to the formation of a layer of (Zr,Ce)O2 solid solution at the zirconia/ceria interface. The gaseous diffusion resistance governed the performance between 700 and 850DGC. Better performances of the anode-supported tubular SOFCs can be achieved by further optimizations for the zirconia/ceria interface, control of both porosity and thickness of the anode substrate, as well as improvement of the (La0.6Sr0.4)CoO3 cathode.
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