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bioRxiv2020

Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines

Lymphoblastoid Cell Lines (LCLs) are generated by transforming primary B cells with Epstein-Barr Virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, proliferation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

immunology↗

bioRxiv2020

BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1{+/-}0.2 and 1.24{+/-}0.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development,1, 2 and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.

immunology↗

bioRxiv2020

Tumor-responsive, multifunctional CAR-NK cells cooperate with impaired autophagy to infiltrate and target glioblastoma

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking have rendered glioblastoma (GBM) highly resistant to therapy. As a result, GBM immunotherapies have failed to demonstrate sustained clinical improvements in patient overall survival (OS). To overcome these obstacles, here we describe a novel, sophisticated combinatorial platform for GBM: the first multifunctional immunotherapy based on genetically-engineered, human NK cells bearing multiple anti-tumor functions, including local tumor responsiveness, that addresses key drivers of GBM resistance to therapy: antigen escape, poor immune cell homing, and immunometabolic reprogramming of immune responses. We engineered dual-specific CAR-NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally-released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site specific activity in the tissue and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells, but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising new NK cell-based combinatorial strategy that can target multiple clinically-recognized mechanisms of GBM progression simultaneously.

immunology↗

bioRxiv2020

Identification, Characterization, and Targeting of a Rare and Temporal Dendritic Cell State that Facilitates Adaptive Immune Responses

The heterogeneity of innate immune cells facilitates efficient antigen presentation and immune activation in the presence of pathogens via cooperativity of various cell subsets and cell states but also obscures the contribution of individual antigen presenting cells (APCs) to overall immune response.1 It has been hypothesized that a small number of APCs, which are more sensitive to the initial pathogen stimulus, are responsible for coordinating neighboring APCs in an effort to share the metabolic strain associated with heightened pathogen sensitivity.2 In this study, we have identified a temporally-controlled state of dendritic cells (DCs) that demonstrate greater sensitivity to toll-like-receptor (TLR) agonists and secrete the majority of paracrine activating cytokines (TNF, IL-6...ect). We were able to isolate this distinct population of DCs preferentially phagocytosed the majority of fluorescently labeled, TLR agonist conjugated microparticles (MPs).3 We call this population First Responder cells (FRs) due to their ability to first uptake the MPs and activate neighboring APCs via paracrine signaling. We show that FRs exist in this state for <3 hours, cycle through this state on a <24-hour timescale and show a distinct mRNA profile. Furthermore, FRs are necessary for generation of adaptive responses both in vitro and in vivo. We also show that we can improve both IgG titers and CD8 responses in vivo by targeting two highly upregulated receptors on FR cells, DAP12 and PRG2. Given the significance of FR involvement in APC activation, this study has broad immunological value because it offers a critical first evaluation of a new APC cell state but also has important translational value for improving vaccine efficacy via FR targeting.

immunology↗

bioRxiv2020

Neutralizing antibodies from early cases of SARS-CoV-2 infection offer cross-protection against the SARS-CoV-2 D614G variant.

The emergence of a SARS-CoV-2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross-neutralize against the G614 variant. In this report, profiling of the anti-SARS-CoV-2 humoral immunity reveals similar neutralization profiles against both S protein variants, albeit waning neutralizing antibody capacity at the later phase of infection. These findings provide further insights towards the validity of current immune-based interventions. IMPORTANCERandom mutations in the viral genome is a naturally occurring event that may lead to enhanced viral fitness and immunological resistance, while heavily impacting the validity of licensed therapeutics. A single point mutation from aspartic acid (D) to glycine (G) at position 614 of the SARS-CoV-2 spike (S) protein, termed D614G, has garnered global attention due to the observed increase in transmissibility and infection rate. Given that a majority of the developing antibody-mediated therapies and serological assays are based on the S antigen of the original Wuhan reference sequence, it is crucial to determine if humoral immunity acquired from the original SARS-CoV-2 isolate is able to induce cross-detection and cross-protection against the novel prevailing D614G variant.

immunology↗

bioRxiv2020

Intercellular telomere transfer extends T cell lifespan

The common view is that T-lymphocytes activate telomerase, a DNA polymerase that extends telomeres at chromosome ends, to delay senescence. We show that independently of telomerase, T cells elongate telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs). Upon contact with T cells, APCs degraded shelterin to donate telomeres, which were cleaved by TZAP, and then transferred in extracellular vesicles (EVs) at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled them to fuse with T cell chromosomal ends causing an average lengthening of [~]3000 base pairs. Thus, we identify a previously unknown telomere transfer program that supports T cell lifespan.

immunology↗

bioRxiv2020

SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets

Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.

immunology↗

bioRxiv2020

Tropomyosin in mugwort cross-reacts to house dust mite, eliciting non-Th2 response in allergic rhinitis patients sensitized to house dust mite

BackgroundMugwort and house dust mite (HDM) are two of the most common inhalant allergens in Asia; however, whether or not mugwort affects polysensitized HDM+ allergic rhinitis (AR) patients has not been elucidated. MethodsOverall, 15884 AR outpatients were assessed for clinical status. Amino acid sequences of mugwort were determined by mass spectrometry. Afterward, cross-reactivity between mugwort tropomyosin and Dermatophagoides pteronyssinus 10 (Der p10) was analysed by ELISA inhibition and basophils activation experiments. To compare immunologic responses eliciting by two different tropomyosins, peripheral blood mononuclear cells (PBMCs) of HDM-monosensitized patients were stimulated by mugwort, HDM, Der p10 and synthetic peptides representing mugwort tropomyosin respectively. ResultsPolysensitized HDM+AR patients were mainly sensitized to cat and mugwort, and the positive rate of monosensitized HDM+AR out-clinic patients was increased during the mugwort pollen season. Mugwort tropomyosin protein had similar structural domains to HDM tropomyosin, Der p10. ELISA inhibition experiment showed synthetic mugwort tropomyosin peptide inhibited IgE binding to Der p10; mugwort tropomyosin peptide activated basophils which were primed by HDM-specific IgE. Unlike HDM and Derp 10, mugwort and mugwort tropomyosin mainly induced IFN-{gamma} and IL-17, release in PBMCs of monosensitized HDM+AR patients, but not IL-5. ConclusionsPan-allergen tropomyosin is a major protein accounting for the cross-reactivity between mugwort and HDM, which reminds HDM+ patients to reduce mugwort exposure in mugwort pollen season in virtue of the tropomyosin induced mild inflammation.

immunology↗

bioRxiv2020

Early induction of SARS-CoV-2 specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients

Virus-specific humoral and cellular immunity act synergistically to protect the host from viral infection. We interrogated the dynamic changes of virological and immunological parameters in 12 patients with symptomatic acute SARS-CoV-2 infection from disease onset to convalescence or death. We quantified SARS-CoV-2 viral RNA in the respiratory tract in parallel with antibodies and circulating T cells specific for various structural (NP, M, ORF3a and spike) and non-structural proteins (ORF7/8, NSP7 and NSP13). We observed that while rapid induction and quantity of humoral responses were associated with increased disease severity, an early induction of SARS-CoV-2 specific T cells was present in patients with mild disease and accelerated viral clearance. These findings provide further support for a protective role of SARS-CoV-2 specific T cells over antibodies during SARS-CoV-2 infection with important implications in vaccine design and immune-monitoring.

immunology↗

bioRxiv2020

The Theory and Practice of the viral dose in neutralization assay: insights on SARS-CoV-2 "doublethink" effect.

Due to the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for reliable high-throughput serological assays in order to evaluate the immunological responses against SARS-COV-2 virus and to enable population screening, as well as vaccines and drugs efficacy testing. Several serological assays for SARS-CoV-2 are now becoming available in the market. However, it has also become extremely important to have well-established assays with desirable high sensitivity and specificity. To date, the micro-neutralization (MN) assay, is currently considered the gold-standard being capable of evaluating and detecting, functional neutralizing antibodies (nAbs). Several protocols exist for microneutralization assays which vary in several steps of the protocol: cell seeding conditions, number of cells seeded, virus amount used in the infection step, virus-serum-cells incubation period etc. These potential differences account for a high degree of variability and inconsistency of the results and using a harmonized protocol for the micro-neutralization assay could potentially solve this. Given this situation, the main aim of our study was to carry out SARS-CoV-2 wild type virus MN assay in order to investigate which optimal tissue culture infective dose 50 (TCID50) infective dose in use is the most adequate choice for implementation in terms of reproducibility, standardization possibilities and comparability of results. Therefore, we assessed the MN by using two different viral infective doses: a standard dose of 100 TCID50/well and a lower dose of 25 TCID50/well. The results obtained, yielded by MN on using the lower infective dose (25 TCID50), were in line with those obtained with the standard infective dose; in some cases, however, we detected a titre that was one or two dilution steps higher, which maintained all negative samples negative. This suggesting that the lower dose can potentially have a positive impact on the detection and estimation of neutralizing antibodies present in a given sample, showing higher sensitivity but similar specificity and therefore, it would require a more accurate assessment and cross-laboratories standardisation especially when MN is employed as serological assay of choice for pre-clinical and clinical studies.

immunology↗

bioRxiv2020

The central THαβ immunity associated cytokine: IL-10 has a strong anti-tumor ability toward established cancer models in vivo and toward cancer cells in vitro

Cancer immunotherapy is a promising new approach for cancer treatment. We propose to use a new host immunological pathway: TH{beta} immunity for cancer treatment. TH{beta} immunity is a natural host immunity which can be used IFN-/{beta} and its major effectors are IL-10 and IL-15. Thus, we here used these cytokines for treatment both in a mice breast cancer 4T1 model and in a neuroblastoma NXS2 model. 4T1 cells and NXS2 cells were inoculated subcutaneously to wild type BALB/c female mice and AJ mice, respectively. Both Cytokines and an antibody treatment with variable dosages were given. Our results show that IL-10 and IL-15 treatments have significant effects on not only tumor volume shrinkage and but also on mice survival. However, IFN-{gamma} even worsens the tumor volume and mice survival. Antibodies plus IL-10 is no better than IL-10 alone for cancer immunotherapy. This can be due to GD2 antigen expression on immune cells. Moreover, an anti-GD2 antibody could harm immune cells themselves. We found that IL-10 has direct toxicity on tumor cells in vitro. Our results conclude that using TH{beta} immunity is a good strategy for cancer immunotherapy.

immunology↗

bioRxiv2020

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands

The microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota can modulate reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific pathogen-free (SPF) and germ-free (GF) mice. Using SPF and GF mice intranasally exposed to lipopolysaccharide (LPS), a component of Gram-negative bacteria, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs. Plain Language summaryMicrobiota represents an important partner of immunologic system at the interface between immune cells and epithelium. It is well known, notably in the gut, that the microbiota contributes in shaping efficient and safe defenses. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. In this study, we postulate that endogenous microbiota could dampen an excessive reactivity of pulmonary tissue to external stimuli. Thus, we sought to study the innate immune reaction switched on by viral or bacterial ligands in respiratory tract cells coming from mice with or without microbiota (germ-free condition, GF). Altogether, our results show a higher inflammatory reaction in GF condition. This study represents a step forward to better establish the link between the microbiota and the reactivity of the lung tissue. Not only these data demonstrate that the microbiota educates the pulmonary innate immune system, but also contributes the emerging concept of using respiratory commensal bacteria as potential next-generation probiotics to prevent susceptibility to respiratory diseases.

immunology↗

bioRxiv2020

Efficacy and immunogenicity of different BCG doses in BALB/c and CB6F1 mice when challenged with H37Rv or Beijing HN878

In this study, 2 strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guerin (BCG) Danish doses from 3x105 to 30 CFU/mouse, followed by either immunogenicity evaluation or aerosol infection with Mycobacterium tuberculosis (a laboratory strain H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3x105 - 3000 CFU (BALB/c) and 3x105-300 CFU (CB6F1). Whereas, both strains of mice when infected with H37Rv, significant protection was seen in BCG doses at 3x105 - 300 CFU. Immunological evaluation revealed interesting results; i) both strains of mice demonstrated a significant increase in the frequencies of BCG-specific IFN{gamma}+ IL2+ TNF+ CD4 T cells in the BCG doses at 3x105 - 3000 CFU (BALB/c) and 3x105 - 300 CFU (CB6F1); ii) secretion of IL2 and IFN{gamma} were correlated with the bacterial burden in the lungs of HN878 infected CB6F1 mice. The study demonstrated a BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight index) is protective in both strains of mice and the use of a virulent clinical isolate in testing new tuberculosis vaccine/advancing research is recommended.

immunology↗

bioRxiv2020

Bifidobacteria-mediated immune system imprinting early in life

Immune-microbe interactions early in life influence an individuals risk of developing allergies, asthma and some autoimmune disorders. Breastfeeding helps guide the development of healthy immune-microbe relationships, in part by providing nutrients to specialized microbes that in turn benefit the host and its developing immune system. Such bacteria having co-evolved with humans are associated with reduced risks of immune mediated diseases but are increasingly rare in modern societies. Here we map an immunological sequence of events, triggered by microbial colonization that distinguish children with different gut bacterial composition. Lack of bifidobacterial species is associated with elevated markers of intestinal inflammation and immune dysregulation and in a randomized trial of breastfed infants, the infant-adapted Bifidobacterium infantis EVC001 silenced intestinal Th2 and Th17 immune responses, while inducing IFN{beta}, and its metabolites skew T-cell polarization in vitro, from Th2 towards Th1, suggesting a healthier immune imprinting during the first critical months of life. HIGHLIGHTSAn ordered sequence of immune changes after birth, driven by microbial interactions Low gut Bifidobacterium abundance is associated with markers of intestinal inflammation Feeding B. infantis EVC001 silenced intestinal Th2 and Th17 but upregulates IFN{beta} B. infantis EVC001 metabolites and/or enteric cytokines skew naive T-cell polarization towards Th1 and away from Th2

immunology↗

bioRxiv2020

IL-12 signaling promotes TET2-mediated DNA demethylation during CD8 T cell effector differentiation

CD8 T cell memory differentiation endows T cells with an ability to rapidly induce effector functions upon pathogen re-encounter. While it is well established that substantial epigenetic remodeling occurs during the effector stage of the immune response, the signaling events that imprint CD8 T cells with these stable epigenetic programs are not well-defined. To gain insight into the signaling determinants of effector-associated epigenetic programming among CD8 T cells, we explored the role of IL-12 in the imprinting of IFNg expression during human CD8 T cell priming. We observed that TCR-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promotor. However, TCR stimulation in the presence of the inflammatory cytokine, IL-12, resulted in significant and stable demethylation of the IFNg locus that was commensurate with an increase in IFNg expression. We further show that IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent passive demethylation in an ex vivo human CAR T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector epigenetic programming in CD8 T cells during the primary immune response and serve as proof of concept that signal 3 cytokines can be used to guide the induction of epigenetically regulated traits among T cells used for adoptive immunotherapies.

immunology↗

bioRxiv2020

Binding site localization on non-homogeneous cell surfaces using topological image averaging

Antibody binding to cell surface proteins plays a crucial role in immunity and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. In the present work, we have developed a method for imaging-based localization of binding sites on cellular surface proteins. The cellular morphology typically varies, and antibodies often bind in a non-homogenous manner, making traditional particle-averaging strategies challenging for accurate native antibody localization. Nanometer-scale resolution is achieved through localization in one dimension, namely the distance from a bound ligand to a reference surface, by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology.

immunology↗

bioRxiv2020

Poised cell circuits in human skin are activated in disease

The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence SummarySingle cell atlas of human skin reveals cell circuits which are quantitatively and qualitatively reconfigured in inflammatory skin disease.

immunology↗

bioRxiv2020

Platypus: an open-access software for integrating lymphocyte single-cell immune repertoires with transcriptomes

High-throughput single-cell sequencing (scSeq) technologies are revolutionizing the ability to molecularly profile B and T lymphocytes by offering the opportunity to simultaneously obtain information on adaptive immune receptor repertoires (VDJ repertoires) and transcriptomes. An integrated quantification of immune repertoire parameters such as germline gene usage, clonal expansion, somatic hypermutation and transcriptional states opens up new possibilities for the high-resolution analysis of lymphocytes and the inference of antigen-specificity. While multiple tools now exist to investigate gene expression profiles from scSeq of transcriptomes, there is a lack of software dedicated to single-cell immune repertoires. Here, we present Platypus, an open-source software platform providing a user-friendly interface to investigate B cell receptor (BCR) and T cell receptor (TCR) repertoires from single-cell sequencing experiments. Platypus provides a framework to automate and ease the analysis of single-cell immune repertoires while also incorporating transcriptional information involving unsupervised clustering, gene expression, and gene ontology. To showcase the capabilities of Platypus, we use it to analyze and visualize single-cell immune repertoires and transcriptomes from B and T cells from convalescent COVID-19 patients, revealing unique insight into the repertoire features and transcriptional profiles of clonally expanded lymphocytes. Platypus will expedite progress by increasing accessibility to the broader immunology community by facilitating the analysis of single-cell immune repertoire and transcriptome sequencing.

immunology↗