Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.

A molecular single-cell lung atlas of lethal COVID-19.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection1,2, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1ß and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1+ pathological fibroblasts3 contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

A Modified Injector and Sample Acquisition Protocol Can Improve Data Quality and Reduce Inter-Instrument Variability of the Helios Mass Cytometer.

Mass cytometry is a powerful tool for high-dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigm's third-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing, we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median and higher coefficients of variation in many antibody-associated signal intensities. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multisite study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces interinstrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multicenter studies. © 2019 International Society for Advancement of Cytometry.

Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant's near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to ß-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium.

Plasticity of intragraft alloreactive T cell clones in human gut correlates with transplant outcomes.

The site of transition between tissue-resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity, and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single-cell level after human intestinal transplantation. Host-versus-graft (HvG)-reactive T cells were mainly distributed to TRM, effector T (Teff)/TRM, and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to Teff/TRM clusters in association with rejection. By integrating pre- and post-transplantation (Tx) mixed lymphocyte reaction-determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in the human gut, providing novel insight into TRM biology.

Anti-PD-1 immunotherapy with androgen deprivation therapy induces robust immune infiltration in metastatic castration-sensitive prostate cancer.

When compared to other malignancies, the tumor microenvironment (TME) of primary and castration-resistant prostate cancer (CRPC) is relatively devoid of immune infiltrates. While androgen deprivation therapy (ADT) induces a complex immune infiltrate in localized prostate cancer, the composition of the TME in metastatic castration-sensitive prostate cancer (mCSPC), and the effects of ADT and other treatments in this context are poorly understood. Here, we perform a comprehensive single-cell RNA sequencing (scRNA-seq) profiling of metastatic sites from patients participating in a phase 2 clinical trial (NCT03951831) that evaluated standard-of-care chemo-hormonal therapy combined with anti-PD-1 immunotherapy. We perform a longitudinal, protein activity-based analysis of TME subpopulations, revealing immune subpopulations conserved across multiple metastatic sites. We also observe dynamic changes in these immune subpopulations in response to treatment and a correlation with clinical outcomes. Our study uncovers a therapy-resistant, transcriptionally distinct tumor subpopulation that expands in cell number in treatment-refractory patients.

Neoadjuvant chemoradiation alters the immune microenvironment in pancreatic ductal adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a grim prognosis despite complete surgical resection and intense systemic therapies. While immunotherapies have been beneficial with many different types of solid tumors, they have almost uniformly failed in the treatment of PDAC. Understanding how therapies affect the tumor immune microenvironment (TIME) can provide insights for the development of strategies to treat PDAC. We used quantitative multiplexed immunofluorescence (qmIF) quantitative spatial analysis (qSA), and immunogenomic (IG) analysis to analyze formalin-fixed paraffin embedded (FFPE) primary tumor specimens from 44 patients with PDAC including 18 treated with neoadjuvant chemoradiation (CRT) and 26 patients receiving no treatment (NT) and compared them with tissues from 40 treatment-naïve melanoma patients. We find that relative to NT tumors, CD3+ T cell infiltration was increased in CRT treated tumors (p = .0006), including increases in CD3+CD8+ cytotoxic T cells (CTLs, p = .0079), CD3+CD4+FOXP3- T helper cells (Th, p = .0010), and CD3+CD4+FOXP3+ regulatory T cells (Tregs, p = .0089) with no difference in CD68+ macrophages. IG analysis from micro-dissected tissues indicated overexpression of genes involved in antigen presentation, T cell activation, and inflammation in CRT treated tumors. Among treated patients, a higher ratio of Tregs to total T cells was associated with shorter survival time (p = .0121). Despite comparable levels of infiltrating T cells in CRT PDACs to melanoma, PDACs displayed distinct spatial profiles with less T cell clustering as defined by nearest neighbor analysis (p < .001). These findings demonstrate that, while CRT can achieve high T cell densities in PDAC compared to melanoma, phenotype and spatial organization of T cells may limit benefit of T cell infiltration in this immunotherapy-resistant tumor.

Blocking IL1 Beta Promotes Tumor Regression and Remodeling of the Myeloid Compartment in a Renal Cell Carcinoma Model: Multidimensional Analyses.

PURPOSE: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1ß may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types. EXPERIMENTAL DESIGN: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1ß plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model. RESULTS: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1ß reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1ß plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs. CONCLUSIONS: Single-cell RNA-seq analyses show that IL1ß blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell-independent mechanisms. IL1ß is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

Potent Neutralizing Antibodies Directed to Multiple Epitopes on SARS-CoV-2 Spike.

The SARS-CoV-2 pandemic rages on with devasting consequences on human lives and the global economy 1,2 . The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this novel coronavirus. Here we report the isolation of 61 SARS-CoV-2-neutralizing monoclonal antibodies from 5 infected patients hospitalized with severe disease. Among these are 19 antibodies that potently neutralized the authentic SARS-CoV-2 in vitro , 9 of which exhibited exquisite potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng/mL. Epitope mapping showed this collection of 19 antibodies to be about equally divided between those directed to the receptor-binding domain (RBD) and those to the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that are overlapping with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody targeting RBD, a second targeting NTD, and a third bridging two separate RBDs revealed recognition of the closed, "all RBD-down" conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

Development of a Comprehensive Antibody Staining Database Using a Standardized Analytics Pipeline.

Large-scale immune monitoring experiments (such as clinical trials) are a promising direction for biomarker discovery and responder stratification in immunotherapy. Mass cytometry is one of the tools in the immune monitoring arsenal. We propose a standardized workflow for the acquisition and analysis of large-scale mass cytometry experiments. The workflow includes two-tiered barcoding, a broad lyophilized panel, and the incorporation of a fully automated, cloud-based analysis platform. We applied the workflow to a large antibody staining screen using the LEGENDScreen kit, resulting in single-cell data for 350 antibodies over 71 profiling subsets. The screen recapitulates many known trends in the immune system and reveals potential markers for delineating MAIT cells. Additionally, we examine the effect of fixation on staining intensity and identify several markers where fixation leads to either gain or loss of signal. The standardized workflow can be seamlessly integrated into existing trials. Finally, the antibody staining data set is available as an online resource for researchers who are designing mass cytometry experiments in suspension and tissue.

Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that Are Maintained by a Circulating Pool.

Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.

Average Overlap Frequency: A simple metric to evaluate staining quality and community identification in high dimensional mass cytometry experiments.

High dimensional cytometry now allows measurement of over 50 parameters in a single sample, and is typically visualized using sophisticated dimensionality-reducing methods and analyzed with automated clustering algorithms. While these tools facilitate the identification and presentation of key findings, it remains challenging to effectively monitor and report the staining quality of individual markers. We present the Average Overlap Frequency (AOF), a simple and efficient metric to evaluate and quantify the robustness of staining and clustering quality in high-dimensional data. We leverage the AOF to compare and determine the optimal storage conditions for stained whole blood samples prior to mass cytometry analysis. We also show that the AOF can be easily incorporated as part of automated analysis pipelines in large scale immune monitoring studies and used to flag and exclude samples with poor staining quality. We propose that the AOF may be incorporated as an essential quality control metric to better identify and report the underlying sample quality in all CyTOF and other high-dimensional cytometry experiments.

Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.

Silencing Mycobacterium smegmatis by using tetracycline repressors.

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as co-repressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems.