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bioRxiv2017

Transplacental protection against asthma by maternal treatment with a bacterial-derived immunomodulatory agent

Studies in European and US farming populations have documented major reductions in asthma prevalence in offspring of mothers exposed to microbial breakdown products present in farm dusts and unprocessed foods. This was associated with enhancement of innate immune competence in the offspring. We sought to (i) identify a safe therapeutic that would reproduce these immunomodulatory effects in a murine model, (ii) elucidate underlying mechanism(s)-of-action, and (iii) develop a scientific rationale for progressing this approach to human trials. We demonstrate in mice that maternal treatment during pregnancy with the microbial-derived immunomodulator OM85, which has been used clinically in adults and children in Europe for >30 years for bolstering resistance to infection-associated airways inflammation, markedly reduces the susceptibility of the offspring of treated mothers to development of experimental atopic asthma. We identify bone marrow precursors of the dendritic cell populations responsible for airway mucosal immune surveillance as the primary targets for the asthma-protective effects of maternal OM85 treatment in the offspring.

immunology↗

bioRxiv2017

The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis infection.

Protection from infectious disease relies on two distinct mechanisms. \"Antimicrobial resistance\" directly inhibits pathogen growth, whereas \"infection tolerance\" controls tissue damage. A single immune-mediator can differentially contribute to these mechanisms in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase complex (Phox) produces anti-microbial superoxides and protects from tuberculosis in humans. However, Phox-deficient mice do not display the expected defect in resistance to M. tuberculosis leaving the role of this complex unclear. We re-examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was due to unregulated Caspase1 activation, IL-1{beta} production, and neutrophil influx into the lung. These studies demonstrate that Phox-derived superoxide protect against TB by promoting tolerance to persistent infection, and highlight a central role for Caspase1 in regulating TB disease progression.

immunology↗

bioRxiv2017

The Role of Conformational Dynamics in Abacavir-Induced Hypersensitivity Syndrome

Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavirs specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots. Further, the presence of abacavir has a direct impact on the dynamics and the conformational space available to peptides bound to HLA-B*57:01, likely influencing abacavir-induced immune self-reactivity. Our results support hypersensitivity models in which abacavir-binding alters the equilibrium proportions of neopeptide conformations in a manner favourable to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.

immunology↗

bioRxiv2017

Single-cell RNA-sequencing redefines blood cell type classification in mosquitoes

Mosquito blood cells are ancestral immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond the ambiguous morphological and functional criteria used for their classification. Here we combined the power of single-cell RNA-sequencing, imaging flow cytometry and single-molecule RNA hybridization to analyze blood cells of the malaria mosquito Anopheles gambiae. By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional machinery. Analyses of differentially expressed genes identified transcriptional signatures of two distinct cell types that challenge the current morphology-based classification of these cells. We further demonstrated an active transfer of a cellular marker between blood cells that confounds their identity. We propose that cell-to-cell exchange is broadly relevant for cell type classification and may account for the remarkable cellular diversity observed in nature.

immunology↗

bioRxiv2018

A temporally dynamic Foxp3 autoregulatory transcriptional circuit controls the effector Treg programme

Regulatory T cells (Treg) are negative regulators of the immune response. Whilst thymic Treg generation is well studied, it is not known whether and how Foxp3 transcription is induced and regulated in the periphery during immune responses. Here we use Foxp3 Timer of cell kinetics and activity (Tocky) mice, which report real-time Foxp3 gene transcription by measuring the spontaneous maturation of Fluorescent Timer protein from Blue to Red fluorescence, to identify the flux of Foxp3-to Foxp3+ T cells within the periphery and analyse the real-time activity of Foxp3 transcription. Using a murine model of skin allergy, we show that both the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased at inflamed sites. These persistent dynamics of Foxp3 transcription determine the effector Treg programme, and are dependent on a Foxp3 autoregulatory transcriptional circuit, as evidenced by analysis of T cells lacking functional Foxp3 protein. Such reactive and persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules including CTLA-4 and effector-Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof-of-principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high frequency Foxp3 expressors are depleted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T cell regulation, and establishes the Foxp3-Tocky system as a tool to investigate the mechanisms behind T cell immunotherapies.

immunology↗

bioRxiv2017

Macrophage resilience and control of intracellular bacteria requires sst1-dependent integration of c-Myc, stress response and type I IFN pathways

Host susceptibility to tuberculosis and several other intracellular bacteria is controlled by a mouse genetic locus, sst1. Necrotic inflammatory lesions, similar to human TB granulomas, are a hallmark of the sst1 susceptible phenotype. Our previous work established that increased disease severity in sst1 susceptible mice reflects dysfunctional macrophage effector or tolerance mechanisms, but the molecular mechanisms have been unclear.\n\nWe demonstrate that sst1-deficient macrophages develop aberrant, biphasic responses to TNF, characterized by super-induction of stress and type I interferon pathways after prolonged TNF stimulation. This late stage response was initiated by oxidative stress and Myc. It was driven via a JNK - IFN{beta} - PKR feed-forward circuit locking the susceptible macrophages in a state of escalating stress. Consequently, prolonged TNF stimulation of the susceptible macrophages reduced their resilience to subsequent infection with intracellular bacteria.\n\nThe data support a generalizable paradigm in host - pathogen interactions, where susceptibility emerges gradually within inflammatory tissue due to imbalanced macrophage responses to growth, differentiation, activation and stress stimuli prior to encountering pathogens. This explains how successful pathogens may locally bypass mechanisms of resistance in otherwise immunocompetent hosts and suggests novel therapeutic strategies.

immunology↗

bioRxiv2017

A primate specific loss of function polymorphism in TLR2 gene decreases inflammation and protects humans from organ dysfunction in Malaria

Polymorphisms of TLR genes could regulate and contribute functionally to innate immunity and inflammation. TLR2, a promiscous receptor recognizes Pathogen Associated Molecular Patterns from several microbes, bacterial, viral, protozoan and helminths. We demonstrate that monocytes of humans with deletion polymorphism in TLR2 gene (a 23 bp deletion in 5 UTR region) respond more vigorously in vitro to several TLR2 ligands in comparison to those with insertion allele. Lower primates such as Rhesus monkeys and Baboon display deletion genotype while insertion is found in higher primates viz., Orangutan, Chimpanzees and Gorilla. Enhanced inflammation is a hallmark of pathogenesis in human severe malaria leading to bad prognosis and odds ratio of patients prone to develop severe malaira such as multi organ failure with del/del genotype was found to be very high. Based on induction of inflammatory cytokines by normal human PBMCs in vitro and circulating cytokine levels in cohorts of patients with severe P. falciparum malaria, we propose that insertion of a 23bp sequence in 5UTR region of TLR2 gene could have led to moderated TLR2 induced inflammation thus offering survival advantage to higher primates by rendering them relatively refractory to multi-organ dysfunction in severe malaria.\n\nOne Sentence SummaryA 23bp deletion in TLR2 gene is associated with high inflammation and susceptibility to organ dysfunction in Plasmodium falciparum malaria.

immunology↗

bioRxiv2017

B1a B cells require autophagy for metabolic homeostasis and self-renewal

Specific metabolic programs are activated by immune cells to fulfil their functional roles, which include adaptations to their microenvironment. B1 B cells are tissue-resident, innate-like B cells. They have many distinct properties, such as the capacity to self-renew and the ability to rapidly respond to a limited repertoire of epitopes. The metabolic pathways that support these functions are unknown. We show that B1 B cells are bioenergetically more active than B2 B cells, with higher rates of glycolysis and oxidative phosphorylation, and depend on glycolysis. They acquire exogenous fatty acids, and store lipids in droplet form. Autophagy is differentially activated in B1a B cells, and deletion of the autophagy gene Atg7 leads to a selective loss of B1a B cells due to a failure of self-renewal. Autophagy-deficient B1a B cells downregulate critical metabolic genes and accumulate dysfunctional mitochondria. B1 B cells therefore, have evolved a distinct metabolism adapted to their residence and specific functional properties.

immunology↗

bioRxiv2017

Pharmacological Macrophage Attenuation by Imatinib Reverts Hepatic Steatosis and Inflammation in Obese Mice

AimsNon-alcoholic fatty liver disease (NAFLD) has become one of the most common liver diseases worldwide. As macrophages play a key role in NAFLD, therapies targeting macrophages have been postulated. Indeed, strategies depleting macrophages or blocking monocyte recruitment into the liver improve NAFLD, however, are not feasible in clinical practice. Our goal was to assess whether attenuation of macrophages can be achieved by imatinib, an anti-leukemia drug with known anti-inflammatory and anti-diabetic properties, and how this impacts NAFLD.\n\nMaterials and MethodsMurine macrophages were polarized in vitro to different activation states in the presence or absence of imatinib; mice on high fat diet orally treated with imatinib or vehicle; and human monocytes of diabetic patients and healthy controls treated with or without imatinib for translational application.\n\nResultsImatinib specifically attenuated pro-inflammatory murine macrophages in vitro and in vivo. In livers of obese mice, imatinib caused Kupffer cells to adopt an attenuated phenotype via modulation of the TNF-pathway. This immune-modulation resulted in markedly improved hepatic steatosis along with beneficial effects on liver function, lipids and systemic inflammation. The immune-dampening effect of imatinib also prevailed in human monocytes, indicating translational applicability.\n\nConclusionsImmune-modulation of myeloid cells as exemplified by imatinib may be a novel therapeutic strategy in patients with NAFLD.

immunology↗

bioRxiv2017

Caffeine downregulates inflammatory pathways involved in autoimmunity

ObjectivesCaffeine is a widely consumed pharmacologically active product. In the present study, we focused on characterizing immunomodulatory effects of caffeine on peripheral blood mononuclear cells (PMBCs).\n\nMethodsThe effect of caffeine on gene expression profiles was initially evaluated using RNA sequencing data. Validation experiments were performed to confirm the results and examine dose-dependent effects of caffeine on PBMCs from healthy subjects. Gene expression levels were measured by real-time quantitative PCR, and cytokine production was determined using a multiplex cytokine assay.\n\nResultsCaffeine at high doses showed a robust downregulatory effect of immune-related genes in PBMCs. Functional annotation analysis of downregulated genes revealed significant enrichment in cytokine activity and in genes related to several autoimmune diseases including lupus and rheumatoid arthritis. Dose-dependent validation experiments showed significant downregulation at the mRNA levels of key inflammatory genes including STAT1, TNF, and PPARG. TNF and PPARG were suppressed even with the lowest caffeine dose tested, which corresponds to the serum concentration of caffeine after administration of one cup of coffee. Cytokine levels of IL-8, MIP-1{beta}, IL-6, IFN-{gamma}, GM-CSF, TNF, IL-2, IL-4, MCP-1, and IL-10 were decreased significantly with caffeine treatment.\n\nConclusionOur findings indicate potential downregulatory effects of caffeine on key inflammatory genes and cytokines, which play important role in autoimmunity. Further studies exploring therapeutic or disease-modulating potential of caffeine in autoimmune diseases and exploring the mechanisms involved are warranted.

immunology↗

bioRxiv2018

Representativeness and robustness of TCR repertoire diversity assessment by high-throughput sequencing

High-throughput sequencing (HTS) has the potential to decipher the diversity of T cell repertoires and their dynamics during immune responses. Applied to T cell subsets such as T effector and T regulatory cells, it should help identify novel biomarkers of diseases. However, given the extreme diversity of TCR repertoires, understanding how the sequencing conditions, including cell numbers, biological and technical sampling and sequencing depth, impact the experimental outcome is critical to properly use of these data. Here we assessed the representativeness and robustness of TCR repertoire diversity assessment according to experimental conditions. By comparative analyses of experimental datasets and computer simulations, we found that (i) for small samples, the number of clonotypes recovered is often higher than the number of cells per sample, even after removing the singletons; (ii) high sequencing depth for small samples alters the clonotype distributions, which can be corrected by filtering the datasets using Shannon entropy as a threshold; (iii) a single sequencing run at high depth does not ensure a good coverage of the clonotype richness in highly polyclonal populations, which can be better covered using multiple sequencing. Altogether, our results warrant better understanding and awareness of the limitation of TCR diversity analyses by HTS and justify the development of novel computational tools for improved modelling of the highly complex nature of TCR repertoires.

immunology↗

bioRxiv2018

Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureus homolog of Cas9) and SpCas9 (S. pyogenes homolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigen-specific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

immunology↗

bioRxiv2018

BRD4 Deficiency Selectively Affects a Unique Developmental Subpopulation in Thymocytes

The bromodomain protein BRD4 is a driver in both inflammatory diseases and cancers. It has multiple functions, contributing to chromatin structure and transcription through its intrinsic HAT and kinase activities. Despite the wide-ranging characterization of BRD4, little is known about its in vivo function. In the present study, we have examined the role of BRD4 in T cell development by conditional deletion at various stages of thymocyte differentiation. We found that BRD4 is critical for normal T cell development. Surprisingly, BRD4 selectively regulates the progression of immature CD8 single positive (ISP) thymocytes into quiescent DP thymocytes. In striking contrast, BRD4 deletion does not affect the extensive proliferation associated with the differentiation of double negative (DN) into ISP cells. Nor does it affect the maturation of double positive (DP) into conventional CD4+ and CD8+ thymocytes. These studies lead to the unexpected conclusion that BRD4 selectively regulates preselection ISP thymocytes.\n\nOn-line SummaryImmature CD8 single-positive (ISP) thymocytes are identified as a molecularly-distinct thymocyte subpopulation, dependent on BRD4 for progression to the DP stage. DN and DP are BRD4-independent. These findings provide new insights into BRD4, a therapeutic target in inflammation and cancer.

immunology↗

bioRxiv2018

HCMV glycoprotein B subunit vaccine efficacy was mediated by non-neutralizing antibody effector functions

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to-date, demonstrating approximately 50% efficacy in preventing HCMV acquisition in phase II trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, though were poor mediators of NK cell activation. Altogether, these data suggest that non-neutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.\n\nSignificanceThe CDC estimates that every hour, a child is born in the United States with permanent neurologic disability resulting from human cytomegalovirus (HCMV) infection - more than is caused by Down syndrome, fetal alcohol syndrome, and neural tube defects combined. A maternal vaccine to block transmission of HCMV to the developing fetus is a necessary intervention to prevent these adverse outcomes. The gB/MF59 vaccine is the most successful tested clinically to-date, achieving 50% reduction in HCMV acquisition. This manuscript establishes the function and epitope specificity of the humoral response stimulated by this vaccine that may explain the partial vaccine efficacy. Understanding the mechanism of gB/MF59-elicited protective immune responses will guide rational design and evaluation of the next generation of HCMV vaccines.

immunology↗

bioRxiv2018

Type-2 CD8+ T lymphocytes responsive to PGD2 and LTE4 in severe eosinophilic asthma

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia - a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

immunology↗

bioRxiv2018

MAIT cells contribute to protection against lethal influenza infection in vivo

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post infection. Activation was modulated via cytokines independently of MR1. MAIT cell-deficient MR1-/- mice showed enhanced weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2-/-{gamma}C-/- mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.

immunology↗

bioRxiv2018

Highly efficient and versatile plasmid-based gene editing in primary T cells

Adoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

immunology↗

bioRxiv2018

Analysis of human immunoglobulin VDJ and DJ rearrangements shows N region synthesis by concatenation of cytosine-rich strands preferentially originating from trimmed germline gene segments

The formation of non-templated (N) regions during immunoglobulin gene rearrangement is a major contributor to antibody diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH-rearrangements and 8,728 unique human DJH-rearrangements containing exactly one identifiable D-gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3-overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3-ends of VH-, D-, and JH-gene segments indicating a dependency of the transferase mechanism upon the nuclease mechanism.

immunology↗