SEARCH · bioRxiv Science

Results for “immunology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources

bioRxiv2020

Expansion of SARS-CoV-2-specific Antibody-secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific antibodies are typically a major predictor of protective immunity, yet B cell and antibody responses during COVID-19 are not fully understood. Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) concentrations were higher in serum of patients who were hospitalized for longer, supporting the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and provides tools to study immune responses to SARS-CoV-2 infection and vaccination.

immunology↗

bioRxiv2020

High-throughput Screening with Pluripotent Stem Cells Identifies CUDC-907 as an Effective Compound for Restoring the Proinflammatory Phenotype of Nakajo-Nishimura Syndrome

Nakajo-Nishimura syndrome (NNS) is an autoinflammatory disorder caused by a homozygous mutations in PSMB8 gene. The administration of systemic corticosteroids is partially effective, but continuous treatment causes severe side effects. We previously established a pluripotent stem cell (PSC)-derived NNS disease model that reproduces several inflammatory phenotypes including the overproduction of monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein-10 (IP-10). Here we performed high-throughput compound screening (HTS) using this PSC-derived NNS model to find potential therapeutic candidates and identified CUDC-907 as an effective inhibitor of the release of MCP-1 and IP-10. CUDC-907 did not induce cell death within therapeutic concentrations and was also effective on primary patient cells. Further analysis indicated that the inhibitory effect was post-transcriptional. These findings suggest that HTS with PSC-derived disease models is useful for finding drug candidates for autoinflammatory diseases. Significance statementIn this study, we identified a histone deacetylase inhibitor CUDC-907 as a potential effective compound for ameliorating overproduction of inflammatory chemokines in an autoinflammatory disease named Nakajo-Nishimura syndrome. We performed high-throughput screening using pluripotent stem cell-derived monocytic cell lines. Our data prove the validity of screening system as a versatile platform for seeking candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cells.

immunology↗

bioRxiv2020

Unveiling the inter individual variability of human fibroblasts innate immune response using robust cell-based protocols

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our "Add-only" protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

immunology↗

bioRxiv2020

Infinity Flow: High-throughput single-cell quantification of 100s of proteins using conventional flow cytometry and machine learning

Modern immunologic research increasingly requires high-dimensional analyses in order to understand the complex milieu of cell-types that comprise the tissue microenvironments of disease. To achieve this, we developed Infinity Flow combining hundreds of overlapping flow cytometry panels using machine learning to enable the simultaneous analysis of the co-expression patterns of 100s of surface-expressed proteins across millions of individual cells. In this study, we demonstrate that this approach allows the comprehensive analysis of the cellular constituency of the steady-state murine lung and to identify novel cellular heterogeneity in the lungs of melanoma metastasis bearing mice. We show that by using supervised machine learning, Infinity Flow enhances the accuracy and depth of clustering or dimensionality reduction algorithms. Infinity Flow is a highly scalable, low-cost and accessible solution to single cell proteomics in complex tissues.

immunology↗

bioRxiv2020

Divergent SARS-CoV-2-specific T and B cell responses in severe but not mild COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding both the immunological processes providing specific immunity and potential immunopathology underlying the pathogenesis of this disease may provide valuable insights for potential therapeutic interventions. Here, we quantified SARS-CoV-2 specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. The observed disparate T and B cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

immunology↗

bioRxiv2020

Generation and utilization of a HEK-293T murine GM-CSF expressing cell line

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GMCSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF ([~]180ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture alveolar macrophages without an altering inflammatory activation cascade. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

immunology↗

bioRxiv2020

ADA2 is a lysosomal DNase regulating the type-I interferon response

Deficiency of adenosine deaminase 2 (DADA2) is a severe, congenital syndrome, which manifests with hematologic, immunologic and inflammatory pathologies. DADA2 is caused by biallelic mutations in ADA2, but the function of ADA2, and the mechanistic link between ADA2 deficiency and the severe inflammatory phenotype remains unclear. Here, we show that monocyte-derived proteomes from DADA2 patients are highly enriched in interferon response proteins. Using immunohistochemistry and detailed glycan analysis we demonstrate that ADA2 is post-translationally modified for sorting to the lysosomes. At acidic, lysosomal pH, ADA2 acts as a novel DNase that degrades cGAS/Sting-activating ligands. Furthermore, we define a clear structure-function relationship for this acidic DNase activity. Deletion of ADA2 increased the production of cGAMP and type I interferons upon exposure to dsDNA, which was reverted by ADA2 overexpression or deletion of STING. Our results identify a new level of control in the nucleic acid sensing machinery and provide a mechanistic explanation for the pathophysiology of autoinflammation in DADA2. One Sentence SummaryADA2 is a lysosomal nuclease controlling nucleic acid sensing and type I interferon production.

immunology↗

bioRxiv2020

Weight and organ specific immune cell profiling of Sleeve Gastrectomy

Sleeve gastrectomy (SG) has profound, immediate weight-loss independent effects on obesity related diabetes (T2D). Our prior studies have shown that immunologic remodeling may play a part in this metabolic improvement. However, to date, little is known about how the major immune cell populations change following SG. Using mass cytometry with time of flight analysis (CyTOF) we aimed to broadly explore the organ-specific immune cell repertoire induced by SG. Surgery was performed on obese, insulin resistant and lean mice in order to understand surgery-specific phenotypes. We identified a shift within the splenic B cell compartment with a reduction in follicular and an increase in innate-like B cell subsets in SG animals. There was a concomitant increase in multiple circulating immunoglobulin classes. Further, SG animals had a conserved increase in splenic neutrophils and a tendency toward M2 macrophage polarization. Others have shown that these, weight-loss independent, surgery-specific changes are linked to improved glucose metabolism and thus, may be a major contributor to post SG physiology. Characterizing the complex immune milieu following SG is an important step toward understanding the physiology of SG and the potential therapies therein.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsSG(Sleeve Gastrectomy)(T2D)Type 2 Diabetes(TNF-α)tumor necrosis factor alpha(VAT)visceral adipose tissuePOD(post-operative Day)GLP-1(glucagon like peptide 1)OGTT(oral glucose tolerance test)ITT(insulin tolerance test)DIO(diet induced obesity)CyTOF(cytometry with time of flight analysis)ARG1(arginase 1)TGR5(Takeda g-protein bile acid receptor)RYGB(Roux-en-Y gastric bypass)View Full Text

immunology↗

bioRxiv2020

A three dimensional human immune-tumor cell model reveals the importance of isotypes in antibody-based immunotherapy

Monoclonal antibodies (mAb) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates in the patients, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a novel pre-clinical human immune – three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer culture, of human CD20+ B-cell lymphoma. We show that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, mediating strong infiltration of monocytic effector cells into 3D spheroids. Hence, the human immune-3D tumor model is an attractive ex vivo system to help filter out mAbs for best efficacy in cancer immunotherapy.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

SARS-CoV-2 reactive T cells in uninfected individuals are likely expanded by beta-coronaviruses

The current pandemic is caused by the SARS-CoV-2 virus and large progress in understanding the pathology of the virus has been made since its emergence in late 2019. Several reports indicate short lasting immunity against endemic coronaviruses, which contrasts repeated reports that biobanked venous blood contains SARS-CoV-2 reactive T cells even before the outbreak in Wuhan. This suggests there exists a preformed T cell memory in individuals not exposed to the pandemic virus. Given the similarity of SARS-CoV-2 to other members of the Coronaviridae family, the endemic coronaviruses appear likely candidates to generate this T cell memory. However, given the apparent poor immunological memory created by the endemic coronaviruses, other immunity against other common pathogens might offer an alternative explanation. Here, we utilize a combination of epitope prediction and similarity to common human pathogens to identify potential sources of the SARS-CoV-2 T cell memory. We find that no common human virus, other than beta-coronaviruses, can explain the pre-existing SARS-CoV-2 reactive T cells in uninfected individuals. Our study suggests OC43 and HKU1 are the most likely pathogens giving rise to SARS-CoV-2 preformed immunity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Modelling the interplay between the CD4+/CD8+ T-cell ratio and the expression of MHC-I in tumours

Describing the anti-tumour immune response as a series of cellular kinetic reactions from known immunological mechanisms, we create a mathematical model that shows the CD4+/CD8+ T-cell ratio, T-cell infiltration and the expression of MHC-I to be interacting factors in tumour elimination. Methods from dynamical systems theory and non-equilibrium statistical mechanics are used to model the T-cell dependent anti-tumour immune response. Our model predicts a critical level of MHC-I expression which determines whether or not the tumour escapes the immune response. This critical level of MHC-I depends on the helper/cytotoxic T-cell ratio. However, our model also suggests that the immune system is robust against small changes in this ratio. We also find that T-cell infiltration and the specificity of the intra-tumour TCR repertoire will affect the critical MHC-I expression. Our work suggests that the functional form of the time evolution of MHC-I expression may explain the qualitative behaviour of tumour growth seen in patients.Mathematics Subject Classification (2010) MSC 37C25 · MSC 82C99 · MSC 37N25 · MSC 92B99Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Discriminating Mild from Critical COVID-19 by Innate and Adaptive Immune Single-cell Profiling of Bronchoalveolar Lavages

How innate and adaptive lung immune responses to SARS-CoV-2 synchronize during COVID-19 pneumonitis and regulate disease severity is poorly established. To address this, we applied single-cell profiling to bronchoalveolar lavages from 44 patients with mild or critical COVID-19 versus non-COVID-19 pneumonia as control. Viral RNA-tracking delineated the infection phenotype to epithelial cells, but positioned mainly neutrophils at the forefront of viral clearance activity during COVID-19. In mild disease, neutrophils could execute their antiviral function in an immunologically controlled fashion, regulated by fully-differentiated T-helper-17 (TH17)-cells, as well as T-helper-1 (TH1)-cells, CD8+ resident-memory (TRM) and partially-exhausted (TEX) T-cells with good effector functions. This was paralleled by orderly phagocytic disposal of dead/stressed cells by fully-differentiated macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, hence facilitating lung tissue repair. In critical disease, CD4+ TH1- and CD8+ TEX-cells were characterized by inflammation-associated stress and metabolic exhaustion, while CD4+ TH17- and CD8+ TRM-cells failed to differentiate. Consequently, T-cell effector function was largely impaired thereby possibly facilitating excessive neutrophil-based inflammation. This was accompanied by impaired monocyte-to-macrophage differentiation, with monocytes exhibiting an ATP-purinergic signalling-inflammasome footprint, thereby enabling COVID-19 associated fibrosis and worsening disease severity. Our work represents a major resource for understanding the lung-localised immunity and inflammation landscape during COVID-19.

immunology↗

bioRxiv2020

Single-cell analysis of mosquito hemocytes identifies signatures of immune cell sub-types and cell differentiation

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

immunology↗

bioRxiv2020

The antibody response to the glycan α-Gal correlates with COVID-19 disease symptoms

The coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. The characterization of the immunological mechanisms involved in disease symptomatology and protective response is important to advance in disease control and prevention. Humans evolved by losing the capacity to synthesize the glycan Gal1-3Gal{beta}1-(3)4GlcNAc-R (-Gal), which resulted in the development of a protective response against pathogenic viruses and other microorganisms containing this modification on membrane proteins mediated by anti--Gal IgM/IgG antibodies produced in response to bacterial microbiota. In addition to anti--Gal antibody-mediated pathogen opsonization, this glycan induces various immune mechanisms that have shown protection in animal models against infectious diseases without inflammatory responses. In this study, we hypothesized that the immune response to -Gal may contribute to the control of COVID-19. To address this hypothesis, we characterized the antibody response to -Gal in patients at different stages of COVID-19 and in comparison with healthy control individuals. The results showed that while the inflammatory response and the anti-SARS-CoV-2 (Spike) IgG antibody titers increased, reduction in anti--Gal IgE, IgM and IgG antibody titers and alteration of anti--Gal antibody isotype composition correlated with COVID-19 severity. The results suggested that the inhibition of the -Gal-induced immune response may translate into more aggressive viremia and severe disease inflammatory symptoms. These results support the proposal of developing interventions such as probiotics based on commensal bacteria with -Gal epitopes to modify the microbiota and increase the -Gal-induced protective immune response and reduce the severity of COVID-19.

immunology↗

bioRxiv2020

Temporally integrated single cell RNA sequencing analysis of controlled and natural primary human DENV-1 infections

Controlled dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To bridge this knowledge gap, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 controlled human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both controlled and natural DENV infection resulted in overlapping patterns of inflammatory gene upregulation, natural DENV infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by controlled and natural primary DENV infection are similar, but that natural DENV infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state

immunology↗

bioRxiv2020

Delineation of a molecularly distinct terminally differentiated memory CD8 T cell population

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated circulating memory cells can be further divided into effector memory (TO_SCPLOWEMC_SCPLOW) and central memory (TO_SCPLOWCMC_SCPLOW) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was simultaneously terminally-fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally-differentiated effector memory CD8 T cell population (terminal-TO_SCPLOWEMC_SCPLOW) was conflated within the conventional TO_SCPLOWEMC_SCPLOW population, prompting redefinition of the classical characteristics of TO_SCPLOWEMC_SCPLOW cells. Murine terminal-TO_SCPLOWEMC_SCPLOW were transcriptionally, functionally, and developmentally unique compared to TO_SCPLOWEMC_SCPLOW cells. Through mass cytometry and single-cell RNAseq analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-TO_SCPLOWEMC_SCPLOW population of CD8 T cells that was transcriptionally distinct from TO_SCPLOWEMC_SCPLOW and TO_SCPLOWCMC_SCPLOW. A key finding of this study was that parsing of terminal-TO_SCPLOWEMC_SCPLOW from conventionally defined TO_SCPLOWEMC_SCPLOW challenges classical characteristics of TO_SCPLOWEMC_SCPLOW biology, including enhanced presence in lymphoid tissues, robust IL-2 production and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-TO_SCPLOWEMC_SCPLOW and clarification of TO_SCPLOWEMC_SCPLOW biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

immunology↗

bioRxiv2020

Host transcriptional response to TB preventive therapy differentiates two sub-groups of IGRA-positive individuals

We investigated the longitudinal whole blood transcriptional profile responses to tuberculosis preventive therapy of 18 IGRA-positive tuberculosis contacts and IGRA-negative, tuberculosis-unexposed healthy controls. Longitudinal unsupervised clustering analysis with a subset of 474 most variable genes in antigen-stimulated blood separated the IGRA+ participants into two distinct subgroups, one of which clustered with the IGRA-negative controls. 117 probes were significantly differentially expressed over time between the two cluster groups, many of them associated with immunological pathways important in mycobacterial control. We contend that the differential host RNA response reflects lack of M.tuberculosis (Mtb) viability in the group that clustered with the IGRA-unexposed healthy controls, and Mtb viability in the group (1/3 of IGRA-positives) that clustered away. Gene expression patterns in the blood of IGRA+ individuals emerging during the course of PT, which reflect Mtb viability, could have major implications in the identification of risk of progression, treatment stratification and biomarker development.

immunology↗

bioRxiv2020

Human thymopoiesis selects unconventional CD8+ α/β T cells that respond to multiple viruses.

T cell receptors (TCRs) are formed by stochastic gene rearrangements, theoretically generating >1019 sequences. They are selected during thymopoiesis, which releases a repertoire of about 108 unique TCRs per individual. How evolution shaped a process that produces TCRs that can effectively handle a countless and evolving set of infectious agents is a central question of immunology. The paradigm is that a diverse enough repertoire of TCRs should always provide a proper, though rare, specificity for any given need. Expansion of such rare T cells would provide enough fighters for an effective immune response and enough antigen-experienced cells for memory. We show here that human thymopoiesis releases a large population of CD8+ T cells harboring /{beta} paired TCRs that (i) have high generation probabilities and (ii) a preferential usage of some V and J genes, (iii) are shared between individuals and (iv) can each recognize and be activated by multiple unrelated viral peptides, notably from EBV, CMV and influenza. These polyspecific T cells may represent a first line of defense that is mobilized in response to infections before a more specific response subsequently ensures viral elimination. Our results support an evolutionary selection of polyspecific /{beta} TCRs for broad antiviral responses and heterologous immunity.

immunology↗