Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

BHLHE40 Regulates Myeloid Cell Polarization through IL-10-Dependent and -Independent Mechanisms.

Better understanding of the host responses to Mycobacterium tuberculosis infections is required to prevent tuberculosis and develop new therapeutic interventions. The host transcription factor BHLHE40 is essential for controlling M. tuberculosis infection, in part by repressing Il10 expression, where excess IL-10 contributes to the early susceptibility of Bhlhe40-/- mice to M. tuberculosis infection. Deletion of Bhlhe40 in lung macrophages and dendritic cells is sufficient to increase the susceptibility of mice to M. tuberculosis infection, but how BHLHE40 impacts macrophage and dendritic cell responses to M. tuberculosis is unknown. In this study, we report that BHLHE40 is required in myeloid cells exposed to GM-CSF, an abundant cytokine in the lung, to promote the expression of genes associated with a proinflammatory state and better control of M. tuberculosis infection. Loss of Bhlhe40 expression in murine bone marrow-derived myeloid cells cultured in the presence of GM-CSF results in lower levels of proinflammatory associated signaling molecules IL-1ß, IL-6, IL-12, TNF-α, inducible NO synthase, IL-2, KC, and RANTES, as well as higher levels of the anti-inflammatory-associated molecules MCP-1 and IL-10 following exposure to heat-killed M. tuberculosis. Deletion of Il10 in Bhlhe40-/- myeloid cells restored some, but not all, proinflammatory signals, demonstrating that BHLHE40 promotes proinflammatory responses via both IL-10-dependent and -independent mechanisms. In addition, we show that macrophages and neutrophils within the lungs of M. tuberculosis-infected Bhlhe40-/- mice exhibit defects in inducible NO synthase production compared with infected wild-type mice, supporting that BHLHE40 promotes proinflammatory responses in innate immune cells, which may contribute to the essential role for BHLHE40 during M. tuberculosis infection in vivo.

Selective Glycan Labeling of Mannose-Containing Glycolipids in Mycobacteria.

Mycobacterium tuberculosis (Mtb) is one of history's most successful human pathogens. By subverting typical immune responses, Mtb can persist within a host until conditions become favorable for growth and proliferation. Virulence factors that enable mycobacteria to modulate host immune systems include a suite of mannose-containing glycolipids: phosphatidylinositol mannosides, lipomannan, and lipoarabinomannan (LAM). Despite their importance, tools for their covalent capture, modification, and imaging are limited. Here, we describe a chemical biology strategy to detect and visualize these glycans. Our approach, biosynthetic incorporation, is to synthesize a lipid-glycan precursor that can be incorporated at a late-stage step in glycolipid biosynthesis. We previously demonstrated selective mycobacterial arabinan modification by biosynthetic incorporation using an exogenous donor. This report reveals that biosynthetic labeling is general and selective: it allows for cell surface mannose-containing glycolipid modification without nonspecific labeling of mannosylated glycoproteins. Specifically, we employed azido-(Z,Z)-farnesyl phosphoryl-ß-d-mannose probes and took advantage of the strain-promoted azide-alkyne cycloaddition to label and directly visualize the localization and dynamics of mycobacterial mannose-containing glycolipids. Our studies highlight the generality and utility of biosynthetic incorporation as the probe structure directs the selective labeling of distinct glycans. The disclosed agents allowed for direct tracking of the target immunomodulatory glycolipid dynamics in cellulo. We anticipate that these probes will facilitate investigating the diverse biological roles of these glycans.

Biosynthetic Glycan Labeling.

Glycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of d-arabinofuranose (d-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of d-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.

IL-1ß Induces the Rapid Secretion of the Antimicrobial Protein IL-26 from Th17 Cells.

Th17 cells play a critical role in the adaptive immune response against extracellular bacteria, and the possible mechanisms by which they can protect against infection are of particular interest. In this study, we describe, to our knowledge, a novel IL-1ß dependent pathway for secretion of the antimicrobial peptide IL-26 from human Th17 cells that is independent of and more rapid than classical TCR activation. We find that IL-26 is secreted 3 hours after treating PBMCs with Mycobacterium leprae as compared with 48 hours for IFN-γ and IL-17A. IL-1ß was required for microbial ligand induction of IL-26 and was sufficient to stimulate IL-26 release from Th17 cells. Only IL-1RI+ Th17 cells responded to IL-1ß, inducing an NF-κB-regulated transcriptome. Finally, supernatants from IL-1ß-treated memory T cells killed Escherichia coli in an IL-26-dependent manner. These results identify a mechanism by which human IL-1RI+ "antimicrobial Th17 cells" can be rapidly activated by IL-1ß as part of the innate immune response to produce IL-26 to kill extracellular bacteria.

Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput.

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.

The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.

SirT1 is required in the male germ cell for differentiation and fecundity in mice.

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

Variability in tuberculosis granuloma T cell responses exists, but a balance of pro- and anti-inflammatory cytokines is associated with sterilization.

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few "multi-functional" T cells were observed. However, granulomas were found to be "multi-functional" with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.

RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device.

Studies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000-8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10-20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays.

Engineered bromodomains to explore the acetylproteome.

MS-based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti-acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine-containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot-to-lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.

Computer aided manual validation of mass spectrometry-based proteomic data.

Advances in mass spectrometry-based proteomic technologies have increased the speed of analysis and the depth provided by a single analysis. Computational tools to evaluate the accuracy of peptide identifications from these high-throughput analyses have not kept pace with technological advances; currently the most common quality evaluation methods are based on statistical analysis of the likelihood of false positive identifications in large-scale data sets. While helpful, these calculations do not consider the accuracy of each identification, thus creating a precarious situation for biologists relying on the data to inform experimental design. Manual validation is the gold standard approach to confirm accuracy of database identifications, but is extremely time-intensive. To palliate the increasing time required to manually validate large proteomic datasets, we provide computer aided manual validation software (CAMV) to expedite the process. Relevant spectra are collected, catalogued, and pre-labeled, allowing users to efficiently judge the quality of each identification and summarize applicable quantitative information. CAMV significantly reduces the burden associated with manual validation and will hopefully encourage broader adoption of manual validation in mass spectrometry-based proteomics.

Molecular characterization of EGFR and EGFRvIII signaling networks in human glioblastoma tumor xenografts.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with a mean survival of 15 months with the current standard of care. Genetic profiling efforts have identified the amplification, overexpression, and mutation of the wild-type (wt) epidermal growth factor receptor tyrosine kinase (EGFR) in ≈ 50% of GBM patients. The genetic aberration of wtEGFR is frequently accompanied by the overexpression of a mutant EGFR known as EGFR variant III (EGFRvIII, de2-7EGFR, ΔEGFR), which is expressed in 30% of GBM tumors. The molecular mechanisms of tumorigenesis driven by EGFRvIII overexpression in human tumors have not been fully elucidated. To identify specific therapeutic targets for EGFRvIII driven tumors, it is important to gather a broad understanding of EGFRvIII specific signaling. Here, we have characterized signaling through the quantitative analysis of protein expression and tyrosine phosphorylation across a panel of glioblastoma tumor xenografts established from patient surgical specimens expressing wtEGFR or overexpressing wtEGFR (wtEGFR+) or EGFRvIII (EGFRvIII+). S100A10 (p11), major vault protein, guanylate-binding protein 1(GBP1), and carbonic anhydrase III (CAIII) were identified to have significantly increased expression in EGFRvIII expressing xenograft tumors relative to wtEGFR xenograft tumors. Increased expression of these four individual proteins was found to be correlated with poor survival in patients with GBM; the combination of these four proteins represents a prognostic signature for poor survival in gliomas. Integration of protein expression and phosphorylation data has uncovered significant heterogeneity among the various tumors and has highlighted several novel pathways, related to EGFR trafficking, activated in glioblastoma. The pathways and proteins identified in these tumor xenografts represent potential therapeutic targets for this disease.

Proximity labeling defines the phagosome lumen proteome of murine and primary human macrophages.

Proteomic analyses of the phagosome has significantly improved our understanding of the proteins which contribute to critical phagosome functions such as apoptotic cell clearance and microbial killing. However, previous methods of isolating phagosomes for proteomic analysis have relied on cell fractionation with some intrinsic limitations. Here, we present an alternative and modular proximity-labeling based strategy for mass spectrometry proteomic analysis of the phagosome lumen, termed PhagoID. We optimize proximity labeling in the phagosome and apply PhagoID to immortalized murine macrophages as well as primary human macrophages. Analysis of proteins detected by PhagoID in murine macrophages demonstrate that PhagoID corroborates previous proteomic studies, but also nominates novel proteins with unexpected residence at the phagosome for further study. A direct comparison between the proteins detected by PhagoID between mouse and human macrophages further reveals that human macrophage phagosomes have an increased abundance of proteins involved in the oxidative burst and antigen presentation. Our study develops and benchmarks a new approach to measure the protein composition of the phagosome and validates a subset of these findings, ultimately using PhagoID to grant further insight into the core constituent proteins and species differences at the phagosome lumen.

PathMHC: a workflow to selectively target pathogen-derived MHC peptides in discovery immunopeptidomics experiments for vaccine target identification.

Vaccine-elicited T cell responses can contribute to immune protection against emerging infectious disease risks such as antimicrobials-resistant (AMR) microbial pathogens and viruses with pandemic potential, but rapidly identifying appropriate targets for T cell priming vaccines remains challenging. Mass spectrometry (MS) analysis of peptides presented on major histocompatibility complexes (MHCs) can identify potential targets for protective T cell responses in a proteome-wide manner. However, pathogen-derived peptides are outnumbered by self peptides in the MHC repertoire and may be missed in untargeted MS analyses. Here we present a novel approach, termed PathMHC, that uses computational analysis of untargeted MS data followed by targeted MS to discover novel pathogen-derived MHC peptides more efficiently than untargeted methods alone. We applied this workflow to identify MHC peptides derived from multiple microbes, including potential vaccine targets presented on MHC-I by human dendritic cells infected with Mycobacterium tuberculosis . PathMHC will facilitate antigen discovery campaigns for vaccine development.

Protease shaving of Mycobacterium tuberculosis facilitates vaccine antigen discovery and delivery of novel cargoes to the Mtb surface.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of infectious disease death and lacks a vaccine capable of protecting adults from pulmonary TB. Studies have shown that Mtb uses a variety of mechanisms to evade host immunity. Secreted Mtb proteins such as Type VII secretion system substrates have been characterized for their ability to modulate anti-Mtb immunity; however, studies of other pathogens such as Salmonella Typhi and Staphylococcus aureus have revealed that outer membrane proteins can also interact with the innate and adaptive immune system. The Mtb outer membrane proteome has received relatively less attention due to limited techniques available to interrogate this compartment. We filled this gap by deploying protease shaving and quantitative mass spectrometry to identify Mtb outer membrane proteins which serve as nodes in the Mtb-host interaction network. These analyses revealed several novel Mtb proteins on the Mtb surface largely derived from the PE/PPE class of Mtb proteins, including PPE18, a component of a leading Mtb vaccine candidate. We next exploited the localization of PPE18 to decorate the Mtb surface with heterologous proteins and deliver these surface-engineered Mtb to the phagosome. Together, these studies reveal potential novel targets for new Mtb vaccines as well as facilitate new approaches to study difficult to study cellular compartments during infection.

Validation and quantification of peptide antigens presented on MHCs using SureQuant.

Vaccines and immunotherapies that target peptide-major histocompatibility complexes (peptide-MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing vaccines and immunotherapies to target peptide-MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as 'SureQuant') can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition. SureQuant MHC has three main use cases: (i) conclusive confirmation of the identities of putative MHC peptides via comparison with an internal synthetic stable isotope labeled (SIL) peptide standard; (ii) accurate relative quantification by using pre-formed heavy isotope-labeled peptide-MHC complexes (hipMHCs) containing SIL peptides as internal controls for technical variation; and (iii) absolute quantification of each target peptide by using different amounts of hipMHCs loaded with synthetic peptides containing one, two or three SIL amino acids to provide an internal standard curve. Absolute quantification can help determine whether the abundance of a peptide-MHC is sufficient for certain therapeutic modalities. SureQuant MHC therefore provides unique advantages for immunologists seeking to confidently validate antigenic targets and understand the dynamics of the MHC repertoire. After synthetic standards are ordered (3-4 weeks), this protocol can be carried out in 3-4 days and is suitable for individuals with mass spectrometry experience who are comfortable with customizing instrument methods.

CD8+ T cell priming that is required for curative intratumorally anchored anti-4-1BB immunotherapy is constrained by Tregs.

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical success has been limited by on-target, off-tumor activity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of a ɑ4-1BB antibody fused to the collagen-binding protein LAIR. While combination treatment with an antitumor antibody (TA99) shows only modest efficacy, simultaneous depletion of CD4+ T cells boosts cure rates to over 90% of mice. Mechanistically, this synergy depends on ɑCD4 eliminating tumor draining lymph node regulatory T cells, resulting in priming and activation of CD8+ T cells which then infiltrate the tumor microenvironment. The cytotoxic program of these newly primed CD8+ T cells is then supported by the combined effect of TA99 and ɑ4-1BB-LAIR. The combination of TA99 and ɑ4-1BB-LAIR with a clinically approved ɑCTLA-4 antibody known for enhancing T cell priming results in equivalent cure rates, which validates the mechanistic principle, while the addition of ɑCTLA-4 also generates robust immunological memory against secondary tumor rechallenge. Thus, our study establishes the proof of principle for a clinically translatable cancer immunotherapy.

Fc-engineered antibodies promote neutrophil-dependent control of Mycobacterium tuberculosis.

Mounting evidence indicates that antibodies can contribute towards control of tuberculosis (TB). However, the underlying mechanisms of humoral immune protection and whether antibodies can be exploited in therapeutic strategies to combat TB are relatively understudied. Here we engineered the receptor-binding Fc (fragment crystallizable) region of an antibody recognizing the Mycobacterium tuberculosis (Mtb) capsule, to define antibody Fc-mediated mechanism(s) of Mtb restriction. We generated 52 Fc variants that either promote or inhibit specific antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole-blood model of infection. While there is likely no singular Fc profile that universally drives control of Mtb, here we found that several Fc-engineered antibodies drove Mtb restriction in a neutrophil-dependent manner. Single-cell RNA sequencing analysis showed that a restrictive Fc-engineered antibody promoted neutrophil survival and expression of cell-intrinsic antimicrobial programs. These data show the potential of Fc-engineered antibodies as therapeutics able to harness the protective functions of neutrophils to promote control of TB.

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.