Deconvolution of the Response to Bacillus Calmette–Guérin Reveals NF-κB-Induced Cytokines As Autocrine Mediators of Innate Immunity

Bacillus Calmette–Guérin (BCG) is used as a vaccine and diagnostic test for tuberculosis, as well as immunotherapy in the treatment of bladder cancer. While clinically useful, the response to mycobacterial stimulation is complex and the induced protein signature remains poorly defined. We characterized the cell types directly engaged by BCG, as well as the induced cytokine loops that transmit signal(s) to bystander cells. Standardized whole-blood stimulations and mechanistic studies on single and purified cell populations identified distinct patterns of activation in monocytes as compared to neutrophils and invariant lymphocyte populations. Deconvoluting the role of Toll-like receptor 2/4 and Dectin-1/2 in the inflammatory response to BCG, we revealed Dectin-1/2 as dominant in neutrophils as compared to monocytes, which equally engaged both pathways. Furthermore, we quantified the role of NF-κB and NADPH/reactive oxygen species (ROS)-dependent cytokines, which triggered a JAK1/2-dependent amplification loop and accounted for 40–50% of the induced response to BCG. In sum, this study provides new insight into the molecular and cellular pathways involved in the response to BCG, establishing the basis for a new generation of immunodiagnostic tools

Deconvolution of the Response to Bacillus Calmette–Guérin Reveals NF-κB-Induced Cytokines As Autocrine Mediators of Innate Immunity

International audienceBacillus Calmette-Guérin (BCG) is used as a vaccine and diagnostic test for tuberculosis, as well as immunotherapy in the treatment of bladder cancer. While clinically useful, the response to mycobacterial stimulation is complex and the induced protein signature remains poorly defined. We characterized the cell types directly engaged by BCG, as well as the induced cytokine loops that transmit signal(s) to bystander cells. Standardized whole-blood stimulations and mechanistic studies on single and purified cell populations identified distinct patterns of activation in monocytes as compared to neutrophils and invariant lymphocyte populations. Deconvoluting the role of Toll-like receptor 2/4 and Dectin-1/2 in the inflammatory response to BCG, we revealed Dectin-1/2 as dominant in neutrophils as compared to monocytes, which equally engaged both pathways. Furthermore, we quantified the role of NF-κB and NADPH/reactive oxygen species (ROS)-dependent cytokines, which triggered a JAK1/2-dependent amplification loop and accounted for 40-50% of the induced response to BCG. In sum, this study provides new insight into the molecular and cellular pathways involved in the response to BCG, establishing the basis for a new generation of immunodiagnostic tools

Single-Cell and Bulk RNA-Sequencing Reveal Differences in Monocyte Susceptibility to Influenza A Virus Infection Between Africans and Europeans

International audienceThere is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use single-cell RNA-sequencing to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. We show widespread inter-individual variability in the percentage of IAV-infected monocytes. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3 , MX1 and OAS3 . Upon IAV challenge, we find that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. We also uncover a stronger resistance of CD16 + monocytes to IAV infection, together with CD16 + -specific mRNA expression of IL6 and TNF in response to IAV. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16 + monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Based on these data, we hypothesize that higher basal monocyte activation, driven by environmental factors and/or weak-effect genetic variants, underlies the lower cellular susceptibility to IAV infection of individuals of African ancestry relative to those of European ancestry. Further studies are now required to investigate how such cellular differences in IAV susceptibility translate into population differences in clinical outcomes and susceptibility to severe influenza

Molecular Analyte Profiling of the Early Events and Tissue Conditioning Following Intravesical Bacillus Calmette-Guerin Therapy in Patients With Superficial Bladder Cancer

International audiencePURPOSE:We characterized the innate immune response to intravesical bacillus Calmette-Guerin therapy using a systems approach based on proteomic and cytometric screens.MATERIALS AND METHODS:Blood and urine were collected from patients receiving intravesical bacillus Calmette-Guerin therapy before, and 2 and 4 hours after bacillus Calmette-Guerin treatment, at the first and third instillation. Proteomic and cytometry based screens were performed.RESULTS:Molecular analyte profiling revealed a prime/boost pattern to the innate response to intravesical bacillus Calmette-Guerin. We identified 36 statistically significant changes in the proteins induced during the third instillation compared to the initial treatment. These analytes were classified into 3 categories of 1) plasma proteins that leaked into the urine, 2) cytokines/chemokines produced locally during the first hours of inflammation and 3) other innate molecules that modulate the bladder microenvironment. To characterize the marked increase in the inflammatory response after multiple treatments we evaluated the cells present in the urine and again a prime/boost response was revealed. For the locally produced analytes it was possible to define the cell source(s) and, thus, provide a first generation map of what occurs during the initial phase of bacillus Calmette-Guerin therapy.CONCLUSIONS:This study provides in vivo information concerning the ability of bacillus Calmette-Guerin to sensitize the tissue microenvironment to enhance innate responses and establishes a framework for improving vaccination strategies while decreasing adverse events

Bacillus Calmette-Guérin strain differences have an impact on clinical outcome in bladder cancer immunotherapy

BACKGROUND Whether the commonly used bacillus Calmette-Guérin (BCG) strains Connaught and Tice confer different treatment responses in non-muscle-invasive bladder cancer (NMIBC) is unknown. OBJECTIVES To compare clinical efficacy, immunogenicity, and genetics of BCG Connaught and Tice. DESIGN, SETTING, AND PARTICIPANTS A prospective randomized single-institution trial with treatment of 142 high-risk NMIBC patients with BCG Connaught or Tice. INTERVENTION Patients were randomized to receive six instillations of BCG Connaught or Tice. For experimental studies, BCG strains were compared in C57Bl/6 mice. Bladders and lymphoid tissues were analyzed by cytometry and the latter cultivated to detect live BCG. BCG genomic DNA was sequenced and compared with reference genomes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Recurrence-free survival was the primary end point of the clinical study. The Kaplan-Meier estimator was used for estimating survival and time-to-event end points. Nonparametric tests served for the analysis of the in vivo results. RESULTS AND LIMITATIONS Treatment with BCG Connaught conferred significantly greater 5-yr recurrence-free survival compared with treatment with BCG Tice (p=0.0108). Comparable numbers of patients experienced BCG therapy-related side effects in each treatment group (p=0.09). In mice, BCG Connaught induced stronger T-helper cell 1-biased responses, greater priming of BCG-specific CD8(+) T cells, and more robust T-cell recruitment to the bladder than BCG Tice. Genome sequencing of the BCG strains revealed candidate genes potentially involved in the differential clinical responses. CONCLUSIONS BCG strain may have an impact on treatment outcome in NMIBC immunotherapy. PATIENT SUMMARY We compared the efficacy of two commonly used bacillus Calmette-Guérin (BCG) strains for the treatment of NMIBC and found that treatment with BCG Connaught prevented recurrences more efficiently than BCG Tice. Comparison of the immunogenicity of the two strains in mice indicated superior immunogenicity of BCG Connaught. We also identified genetic differences that may explain the differential efficacy of the Connaught and Tice BCG strains. TRIAL REGISTRATION NCT00003779

Dissecting human population variation in single-cell responses to SARS-CoV-2

International audienceHumans display substantial interindividual clinical variability after SARS-CoV-2 infection 1–3 , the genetic and immunological basis of which has begun to be deciphered 4 . However, the extent and drivers of population differences in immune responses to SARS-CoV-2 remain unclear. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells—from 222 healthy donors of diverse ancestries—that were stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces weaker, but more heterogeneous, interferon-stimulated gene activity compared with influenza A virus, and a unique pro-inflammatory signature in myeloid cells. Transcriptional responses to viruses display marked population differences, primarily driven by changes in cell abundance including increased lymphoid differentiation associated with latent cytomegalovirus infection. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell composition on population disparities in immune responses, with genetic variants exerting a strong effect on specific loci. Furthermore, we show that natural selection has increased population differences in immune responses, particularly for variants associated with SARS-CoV-2 response in East Asians, and document the cellular and molecular mechanisms by which Neanderthal introgression has altered immune functions, such as the response of myeloid cells to viruses. Finally, colocalization and transcriptome-wide association analyses reveal an overlap between the genetic basis of immune responses to SARS-CoV-2 and COVID-19 severity, providing insights into the factors contributing to current disparities in COVID-19 risk

Environmental and genetic drivers of population differences in SARS-CoV-2 immune responses

The RNA sequencing data generated and analyzed in this study have been deposited in the Institut Pasteur data repository, OWEY, which can be accessed via the following link: https://doi.org/XXXX. The genome-wide genotyping data generated or used in this study have been deposited in OWEY and can be accessed at the following URL: https://doi.org/XXXX. Data access and use is restricted to academic research related to the variability of the human immune response.Humans display vast clinical variability upon SARS-CoV-2 infection 1–3 , partly due to genetic and immunological factors 4 . However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk

Unravelling the determinants of human health in French Polynesia: the MATAEA project

BackgroundFrench Polynesia is a French overseas collectivity in the Southeast Pacific, comprising 75 inhabited islands across five archipelagoes. The human settlement of the region corresponds to the last massive migration of humans to empty territories, but its timeline is still debated. Despite their recent population history and geographical isolation, inhabitants of French Polynesia experience health issues similar to those of continental countries. Modern lifestyles and increased longevity have led to a rise in non-communicable diseases (NCDs) such as obesity, diabetes, hypertension, and cardiovascular diseases. Likewise, international trade and people mobility have caused the emergence of communicable diseases (CDs) including mosquito-borne and respiratory diseases. Additionally, chronic pathologies including acute rheumatic fever, liver diseases, and ciguatera, are highly prevalent in French Polynesia. However, data on such diseases are scarce and not representative of the geographic fragmentation of the population. ObjectivesThe MATAEA project aims to estimate the prevalence of several NCDs and CDs in the population of the five archipelagoes, and identify associated risk factors. Moreover, genetic analyses will contribute to determinate the sequence and timings of the peopling history of French Polynesia, and identify causal links between past genetic adaptation to island environments, and present-day susceptibility to certain diseases. MethodsThis cross-sectional survey is based on the random selection of 2,100 adults aged 18-69 years and residing on 18 islands from the five archipelagoes. Each participant answered a questionnaire on a wide range of topics (including demographic characteristics, lifestyle habits and medical history), underwent physical measurements (height, weight, waist circumference, arterial pressure, and skin pigmentation), and provided biological samples (blood, saliva, and stool) for biological, genetic and microbiological analyses. ConclusionFor the first time in French Polynesia, the MATAEA project allows to collect a wide range of data to explore the existence of indicators and/or risk factors for multiple pathologies of public health concern. The results will help health authorities to adapt actions and preventive measures aimed at reducing the incidence of NCDs and CDs. Moreover, the new genomic data generated in this study, combined with anthropological data, will increase our understanding of the peopling history of French Polynesia

TRIM5 is an innate immune sensor for the retrovirus capsid lattice

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice