Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

psupertime: supervised pseudotime analysis for time-series single-cell RNA-seq data.

MOTIVATION: Improvements in single-cell RNA-seq technologies mean that studies measuring multiple experimental conditions, such as time series, have become more common. At present, few computational methods exist to infer time series-specific transcriptome changes, and such studies have therefore typically used unsupervised pseudotime methods. While these methods identify cell subpopulations and the transitions between them, they are not appropriate for identifying the genes that vary coherently along the time series. In addition, the orderings they estimate are based only on the major sources of variation in the data, which may not correspond to the processes related to the time labels. RESULTS: We introduce psupertime, a supervised pseudotime approach based on a regression model, which explicitly uses time-series labels as input. It identifies genes that vary coherently along a time series, in addition to pseudotime values for individual cells, and a classifier that can be used to estimate labels for new data with unknown or differing labels. We show that psupertime outperforms benchmark classifiers in terms of identifying time-varying genes and provides better individual cell orderings than popular unsupervised pseudotime techniques. psupertime is applicable to any single-cell RNA-seq dataset with sequential labels (e.g. principally time series but also drug dosage and disease progression), derived from either experimental design and provides a fast, interpretable tool for targeted identification of genes varying along with specific biological processes. AVAILABILITY AND IMPLEMENTATION: R package available at github.com/wmacnair/psupertime and code for results reproduction at github.com/wmacnair/psupplementary. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.

Simulation-based inference of differentiation trajectories from RNA velocity fields.

We report Cytopath, a method for trajectory inference that takes advantage of transcriptional activity information from the RNA velocity of single cells to perform trajectory inference. Cytopath performs this task by defining a Markov chain model, simulating an ensemble of possible differentiation trajectories, and constructing a consensus trajectory. We show that Cytopath can recapitulate the topological and molecular characteristics of the differentiation process under study. In our analysis, we include differentiation trajectories with varying bifurcated, circular, convergent, and mixed topologies studied in single-snapshot as well as time-series single-cell RNA sequencing experiments. We demonstrate the capability to reconstruct differentiation trajectories, assess the association of RNA velocity-based pseudotime with actually elapsed process time, and identify drawbacks in current state-of-the art trajectory inference approaches.

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Educational status-related disparities in awareness, treatment and control of cardiovascular risk factors in India.

OBJECTIVE: To determine association of socioeconomic status, defined by educational status (ES), with awareness, treatment and control of cardiovascular risk factors. METHODS: We performed an epidemiological study at 11 cities in India using cluster sampling. 6198 subjects (3426 men, 2772 women, response 62%, age 48±10 years) were evaluated for sociodemographic, lifestyle, anthropometric and biochemical factors. ES was categorised according to years of schooling into low (≤10 years), medium (11-15 years) and high (>15 years). Risk factors were diagnosed according to current guidelines. Awareness, treatment and control status were determined for hypertension, diabetes and hypercholesterolaemia. For smoking/tobacco use, quit rate was determined. Descriptive statistics are reported. RESULTS: Age-adjusted and sex-adjusted prevalence (%) of various risk factors in low, medium and high ES subjects was hypertension 31.8, 29.5 and 34.1, diabetes 14.5, 15.3 and 14.3, hypercholesterolaemia 24.0, 23.9 and 27.3, and smoking/tobacco use 24.3, 14.4 and 19.0. Significantly increasing trends with low, medium and high ES were observed for hypertension awareness (30.7, 37.8, 47.0), treatment (24.3, 29.2, 35.5) and control (7.8, 11.6, 15.5); diabetes awareness (47.2, 51.5, 56.4), treatment (38.3, 41.3, 46.0) and control (18.3, 15.3, 22.8); hypercholesterolaemia awareness (8.9, 22.4, 18.4), treatment (4.1, 6.2, 7.9) and control (2.8, 3.2, 6.9), as well as for smoking/tobacco quit rates (1.6, 2.8, 5.5) (χ(2) for trend, p<0.05). CONCLUSIONS: Low ES subjects in India have lower awareness, treatment and control of hypertension, diabetes and hypercholesterolaemia and smoking quit rates.