Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

NF-κB c-Rel Is Crucial for the Regulatory T Cell Immune Checkpoint in Cancer.

Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.

Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

An NF-κB Transcription-Factor-Dependent Lineage-Specific Transcriptional Program Promotes Regulatory T Cell Identity and Function.

Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.

A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling.

We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor-alpha, exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon-beta is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like receptors induce both classes and others for an optimal response to microbial pathogens.

Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling.

BACKGROUND: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. RESULTS: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5' v1 and 3' v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. CONCLUSION: Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

How Do Liquid-Junction Potentials and Medium Polarity at Electrode Surfaces Affect Electrochemical Analyses for Charge-Transfer Systems?

The importance of electrochemical analysis for charge-transfer science cannot be overstated. Interfaces in electrochemical cells present certain challenges in the interpretation and the utility of the analysis. This publication focuses on: (1) the medium polarity that redox species experience at the electrode surfaces that is smaller than the polarity in the bulk media and (2) the liquid-junction potentials from interfacing electrolyte solutions of different organic solvents, namely, dichloromethane, benzonitrile, and acetonitrile. Electron-donor-acceptor pairs of aromatics with similar structures (i.e., 1-naphthylamine and 1-nitronaphthalene, 10-methylphenothiazine and 9-nitroanthracene, and 1-aminopyrene and 1-nitropyrene) serve as redox analytes for this study. Using the difference between the reduction potentials of the oxidized donors and the acceptors eliminates the effects of the liquid junctions on the analysis of charge-transfer thermodynamics. This analysis also offers a means for evaluating the medium polarity that the redox species experience at the surface of the working electrode and the effects of the liquid junctions on the measured reduction potentials. While the liquid-junction potentials between the dichloromethane and acetonitrile solutions amount to about 90 mV, for the benzonitrile-acetonitrile junctions, the potentials are only about 30 mV. The presented methods for analyzing the measured electrochemical characteristics of donors and acceptors illustrate a means for improved evaluation of the thermodynamics of charge-transfer systems.

Liver Metastasis Modulate Responses of Suppressive Macrophages and Exhausted T Cells to Immunotherapy Revealed by Single Cell Sequencing.

Liver metastasis is associated with immunotherapy resistance, although the underlying mechanisms remain incompletely understood. By applying single cell RNA-sequencing to a concurrent subcutaneous and liver tumor murine model to recapitulate liver metastases, it is identified that subsets within tumor-infiltrating exhausted CD8+ T (Tex) cells and immunosuppressive tumor-associated macrophages (TAMs) display opposite responses to concurrent liver tumors and anti-PD-1 treatment, suggesting a complex immune regulating network. Both angiogenic and interferon-reactive TAMs show increased frequencies in implanted liver tumors, and anti-PD-1 treatment further elevates the frequencies of angiogenic TAMs. Such TAMs frequencies negatively correlate with the proportions of cytotoxic T cell subsets. Further, expression of interferon-stimulated genes in TAMs is dramatically reduced under effective anti-PD-1 treatment, while such tendencies are diminished in mice with implanted liver tumors. Therefore, the study indicates that liver metastases could increase immunosuppressive TAMs frequencies and inhibit Tex responses to PD-1 blockade, resulting in compromised systemic antitumor immunity and limited immunotherapy efficacy.

Metformin selectively dampens the acute inflammatory response through an AMPK-dependent mechanism.

Metformin is a first-line drug in the treatment of type-2 diabetes mellitus (T2DM). In addition to its antigluconeogenic and insulin-sensitizing properties, metformin has emerged as a potent inhibitor of the chronic inflammatory response of macrophages. In particular, metformin treatment has been shown to reduce expression of interleukin (IL-) 1ß during long-term exposure to the pro-inflammatory stimulus lipopolysaccharide (LPS) through a reduction in reactive oxygen species (ROS), which decreases the levels of the hypoxia-inducible factor (HIF) 1-α, and through enhanced expression of IL-10. However, the effect of metformin on the acute inflammatory response, before significant levels of ROS accumulate in the cell, has not been explored. Here, we show that metformin alters the acute inflammatory response through its activation of AMP-activated protein kinase (AMPK), but independently of HIF1-α and IL-10, in primary macrophages and two macrophage-like cell lines. Thus, metformin changes the acute and the chronic inflammatory response through fundamentally distinct mechanisms. Furthermore, RNA-seq analysis reveals that metformin pretreatment affects the levels of a large yet selective subset of inflammatory genes, dampening the response to short-term LPS exposure and affecting a wide range of pathways and biological functions. Taken together, these findings reveal an unexpected complexity in the anti-inflammatory properties of this widely used drug.

STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets.

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/ß 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.

Toll-like Receptor (TLR)-induced Rasgef1b expression in macrophages is regulated by NF-κB through its proximal promoter.

Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.

Regulation of the NF-κB-Mediated Transcription of Inflammatory Genes.

The NF-κB family of transcription factors plays a central role in the inducible expression of inflammatory genes during the immune response, and the proper regulation of these genes is a critical factor in the maintenance of immune homeostasis. The chromatin environment at stimulus-responsive NF-κB sites is a major determinant in transcription factor binding, and dynamic alteration of the chromatin state to facilitate transcription factor binding is a key regulatory mechanism. NF-κB is in turn able to influence the chromatin state through a variety of mechanisms, including the recruitment of chromatin modifying co-activator complexes such as p300, the competitive eviction of negative chromatin modifications, and the recruitment of components of the general transcriptional machinery. Frequently, the selective interaction with these co-activators is dependent on specific post-translational modification of NF-κB subunits. Finally, the mechanisms of inducible NF-κB activity in different immune cell types seem to be largely conserved. The diversity of cell-specific NF-κB-mediated transcriptional programs is established at the chromatin level during cell differentiation by lineage-defining transcription factors. These factors generate and maintain a cell-specific chromatin landscape that is accessible to NF-κB, thus restricting the inducible transcriptional response to a cell-appropriate output.

Toward an understanding of the gene-specific and global logic of inducible gene transcription.

Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in a coordinated fashion. In the vertebrate immune system, the highly selective response to an environmental stimulus, often an invading microorganism, plays an especially important role in regulating the activities of, and interactions among, the many cell types involved in innate and adaptive immunity. It is now widely appreciated that the selective response to a stimulus requires the concerted action of signal transduction pathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.