Search

Through scRNA-seq and ATAC-seq, Pisu et al. characterize specific lung macrophage subsets that either restrict or promote M. tuberculosis growth. Comparable macrophage populations are present in the human airways, and cells show evidence of epigenetic imprinting., In this study, we detail a novel approach that combines bacterial fitness fluorescent reporter strains with scRNA-seq to simultaneously acquire the host transcriptome, surface marker expression, and bacterial phenotype for each infected cell. This approach facilitates the dissection of the functional heterogeneity of M. tuberculosis–infected alveolar (AMs) and interstitial macrophages (IMs) in vivo. We identify clusters of pro-inflammatory AMs associated with stressed bacteria, in addition to three different populations of IMs with heterogeneous bacterial phenotypes. Finally, we show that the main macrophage populations in the lung are epigenetically constrained in their response to infection, while inter-species comparison reveals that most AMs subsets are conserved between mice and humans. This conceptual approach is readily transferable to other infectious disease agents with the potential for an increased understanding of the roles that different host cell populations play during the course of an infection., Graphical Abstract

Antibody-mediated long-lasting protection from harmful pathogens depends on collaboration of immune cells within immunological niches. Stoler-Barak et al. introduce an approach that enables the visualization of all the germinal center niches and activated B cells within intact lymph nodes., Germinal centers (GCs) are sites wherein B cells proliferate and mutate their immunoglobulins in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). Here, we mapped the location of single B cells in the context of intact lymph nodes (LNs) throughout the GC response, and examined the role of BCR affinity in dictating their position. Imaging of entire GC structures and proximal single cells by light-sheet fluorescence microscopy revealed that individual B cells that previously expressed AID are located within the LN cortex, in an area close to the GC LZ. Using in situ photoactivation, we demonstrated that B cells migrate from the LZ toward the GC outskirts, while DZ B cells are confined to the GC. B cells expressing very-low-affinity BCRs formed GCs but were unable to efficiently disperse within the follicles. Our findings reveal that BCR affinity regulates B cell positioning during the GC response.

HIV-1 cure research seeks to decrease or eliminate the latent reservoir. The evaluation of such curative strategies requires accurate measures of the reservoir. Gaebler et al. describe a combined multicolor qPCR and next-generation sequencing method that enables the sensitive and specific characterization of the HIV-1 latent reservoir., HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.

B cells edited by CRISPR/Cas9 to express anti–HIV-1 antibodies participate in humoral immune reactions after immunization and secrete neutralizing serum titers of anti-HIV antibodies in vivo., A small number of HIV-1–infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1–3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization., Graphical Abstract

This study uses single-cell RNA sequencing to investigate the transcriptional heterogeneity of all cell types known to comprise the acutely injured spinal cord in mice. The authors investigate both cell subtype heterogeneity and potential signaling interactions between myeloid, vascular, and macroglia cells., The wound healing process that occurs after spinal cord injury is critical for maintaining tissue homeostasis and limiting tissue damage, but eventually results in a scar-like environment that is not conducive to regeneration and repair. A better understanding of this dichotomy is critical to developing effective therapeutics that target the appropriate pathobiology, but a major challenge has been the large cellular heterogeneity that results in immensely complex cellular interactions. In this study, we used single-cell RNA sequencing to assess virtually all cell types that comprise the mouse spinal cord injury site. In addition to discovering novel subpopulations, we used expression values of receptor–ligand pairs to identify signaling pathways that are predicted to regulate specific cellular interactions during angiogenesis, gliosis, and fibrosis. Our dataset is a valuable resource that provides novel mechanistic insight into the pathobiology of not only spinal cord injury but also other traumatic disorders of the CNS.

Single cell RNAseq analyses were performed on T cells from MC38 and CT26 tumor models. PCA-based sub-clustering and STARTRAC analyses revealed fine-tuned T cell subsets and dynamic in tumor. CCR8 was identified as a novel target on Tregs to synergize with anti–PD-1 for cancer immunotherapy., Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/β sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti–PD-1 treatment in MC38 and B16F10 tumor models., Graphical Abstract

Veglia et al. characterize the heterogeneity of polymorphonuclear neutrophils (PMNs) in the tumor microenvironment, spleen, and peripheral blood. The identification of neutrophil populations with potent immune suppressive activity opens selective targeting opportunities., In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

Zhuang et al. identify SARS-CoV-2–specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. SARS-CoV-2–specific T cells are protective and cross-reactive in vivo, and are shaped by type I IFN signaling., Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2–specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2–infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2–specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2–infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.

McCann et al. describe the development and characterization of a new mouse model for studying HIV-specific T cell responses and testing various immunotherapeutic strategies, which they validate by demonstrating enhanced therapeutic effects of autologous HIV-specific T cells augmented with cytokine-loaded nanogels., HIV-specific CD8+ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4+ T cells from HIV+ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8+ T cell response have a profound impact on the emergence and persistence of escape mutations. This “participant-derived xenograft” model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.

By using genomic and transcriptomic sequencing technologies, Ye et al. identify AID-dependent and/or polymerase η-associated mechanisms of mutagenesis in human germinal center–derived/related B cell lymphomas and link aberrant immunoglobulin gene diversification processes to different disease subtypes., Both somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase (AID). Dysregulation of these processes has been linked to B cell lymphomagenesis. Here we performed an in-depth analysis of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) genomes. We characterized seven genomic mutational signatures, including two B cell tumor-specific signatures, one of which is novel and associated with aberrant SHM. We further identified two major mutational signatures (K1 and K2) of clustered mutations (kataegis) resulting from the activities of AID or error-prone DNA polymerase η, respectively. K1 was associated with the immunoglobulin (Ig) switch region mutations/translocations and the ABC subtype of DLBCL, whereas K2 was related to the Ig variable region mutations and the GCB subtype of DLBCL and FL. Similar patterns were also observed in chronic lymphocytic leukemia subtypes. Thus, alterations associated with aberrant CSR and SHM activities can be linked to distinct developmental paths for different subtypes of B cell lymphomas., Graphical Abstract

Mast cells (MCs) comprise two lineages that differ in tissue localization and ontologic origin. Derakhshan et al. define the transcriptomes of these lineages during airway inflammation, identifying distinct programs associated with each lineage and a role for TGF-β signaling in inducible MC development., Murine mast cells (MCs) contain two lineages: inducible bone marrow–derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by β7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. β7High MCs expand and mature during lung inflammation as part of a TGF-β–inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow–derived MCs (BMMCs) identified a requirement for SCF in this TGF-β–mediated development and found that epithelial cells directly elicit TGF-β–dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development.

Diagnosis of EBV-driven T/NK-cell lymphoproliferative disorders and chronic active EBV diseases is often difficult. Fournier et al. report a flow-FISH cytometry assay allowing rapid identification of EBV-infected cells in blood and accurate diagnoses. It represents a powerful tool to study pathophysiological mechanisms of EBV-LPD., Epstein-Barr virus (EBV) preferentially infects epithelial cells and B lymphocytes and sometimes T and NK lymphocytes. Persistence of EBV-infected cells results in severe lymphoproliferative disorders (LPDs). Diagnosis of EBV-driven T or NK cell LPD and chronic active EBV diseases (CAEBV) is difficult, often requiring biopsies. Herein, we report a flow-FISH cytometry assay that detects cells expressing EBV-encoded small RNAs (EBERs), allowing rapid identification of EBV-infected cells among PBMCs. EBV-infected B, T, and/or NK cells were detectable in various LPD conditions. Diagnosis of CAEBV in 22 patients of Caucasian and African origins was established. All exhibited circulating EBV-infected T and/or NK cells, highlighting that CAEBV is not restricted to native American and Asian populations. Proportions of EBV-infected cells correlated with blood EBV loads. We showed that EBV-infected T cells had an effector memory activated phenotype, whereas EBV-infected B cells expressed plasma cell differentiation markers. Thus, this method achieves accurate and unambiguous diagnoses of different forms of EBV-driven LPD and represents a powerful tool to study their pathophysiological mechanisms.

The emergence of SARS-CoV-2 has created a need for robust assays of plasma and monoclonal antibody neutralization potency. Pseudotyped HIV-1– and vesicular stomatitis virus–based reporter viruses and a replication-competent vesicular stomatitis virus/SARS-CoV-2 chimera represent useful tools to assess neutralizing antibodies., The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2–specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies., Graphical Abstract

Highly efficient gene editing in primary myeloid cells is made possible by optimized non-viral delivery of CRISPR-Cas9. This enables single and multiplexed gene knockout in monocytes, macrophages, and dendritic cells to advance innate immunity research and cell therapy., Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity., Graphical Abstract

By single-cell RNA sequencing, Zakharov et al. examine the cellular contents of pancreatic islets at three developmental stages in autoimmune diabetes. From diabetes inception, islets show marked cellular heterogeneity, which progresses with time and includes T cells, macrophages, and dendritic cells., Tissue-specific autoimmune diseases are driven by activation of diverse immune cells in the target organs. However, the molecular signatures of immune cell populations over time in an autoimmune process remain poorly defined. Using single-cell RNA sequencing, we performed an unbiased examination of diverse islet-infiltrating cells during autoimmune diabetes in the nonobese diabetic mouse. The data revealed a landscape of transcriptional heterogeneity across the lymphoid and myeloid compartments. Memory CD4 and cytotoxic CD8 T cells appeared early in islets, accompanied by regulatory cells with distinct phenotypes. Surprisingly, we observed a dramatic remodeling in the islet microenvironment, in which the resident macrophages underwent a stepwise activation program. This process resulted in polarization of the macrophage subpopulations into a terminal proinflammatory state. This study provides a single-cell atlas defining the staging of autoimmune diabetes and reveals that diabetic autoimmunity is driven by transcriptionally distinct cell populations specialized in divergent biological functions.

Single-cell transcriptome analysis of epithelial cells from human ileum, colon, and rectum reveals different nutrient-absorption preferences in the small and large intestine, providing a rich resource for further characterization of human intestine cell constitution and functions., The intestine plays an important role in nutrient digestion and absorption, microbe defense, and hormone secretion. Although major cell types have been identified in the mouse intestinal epithelium, cell type–specific markers and functional assignments are largely unavailable for human intestine. Here, our single-cell RNA-seq analyses of 14,537 epithelial cells from human ileum, colon, and rectum reveal different nutrient absorption preferences in the small and large intestine, suggest the existence of Paneth-like cells in the large intestine, and identify potential new marker genes for human transient-amplifying cells and goblet cells. We have validated some of these insights by quantitative PCR, immunofluorescence, and functional analyses. Furthermore, we show both common and differential features of the cellular landscapes between the human and mouse ilea. Therefore, our data provide the basis for detailed characterization of human intestine cell constitution and functions, which would be helpful for a better understanding of human intestine disorders, such as inflammatory bowel disease and intestinal tumorigenesis., Graphical Abstract

This work demonstrates a novel strategy that enables the identification of HCMV-derived T cell epitopes by mass spectrometry. It provides a panel of novel T cell epitopes and presents evidence for their involvement in physiologic immune control of HCMV infections., In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identifying the repertoire of T cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. Here, we present a novel approach that employs HCMV deletion mutant viruses lacking HLA class I immunoevasins and allows direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. We identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. Multiple HCMV-directed specificities in the memory T cell pool of single individuals indicate that physiologic anti-HCMV T cell responses are directed against a broad range of antigens. Thus, the unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T cell specificities in seropositive individuals.

Ghaedi et al. identify IL-18R+IL-33R− ILC progenitors, which differentiate into multiple ILC lineages, in the lung of neonatal and adult RORα lineage tracer mice. ILC2s in neonatal mouse lungs are divided into distinct cytokine and amphiregulin-producing effector ILC2s., Lung group 2 innate lymphoid cells (ILC2s) drive allergic inflammation and promote tissue repair. ILC2 development is dependent on the transcription factor retinoic acid receptor–related orphan receptor (RORα), which is also expressed in common ILC progenitors. To elucidate the developmental pathways of lung ILC2s, we generated RORα lineage tracer mice and performed single-cell RNA sequencing, flow cytometry, and functional analyses. In adult mouse lungs, we found an IL-18Rα+ST2− population different from conventional IL-18Rα−ST2+ ILC2s. The former was GATA-3intTcf7EGFP+Kit+, produced few cytokines, and differentiated into multiple ILC lineages in vivo and in vitro. In neonatal mouse lungs, three ILC populations were identified, namely an ILC progenitor population similar to that in adult lungs and two distinct effector ILC2 subsets that differentially produced type 2 cytokines and amphiregulin. Lung ILC progenitors might actively contribute to ILC-poiesis in neonatal and inflamed adult lungs. In addition, neonatal lung ILC2s include distinct proinflammatory and tissue-repairing subsets.