Single cell RNA sequencing to uncover intestinal cell-type specific cis-eQTL driving inherited predisposition to IBD

IBD is characterized by a chronic idiopathic inflammation of the gastrointestinal (GI) tract and consist of two main forms: ulcerative colitis and Crohn’s disease. The importance of genetic susceptibility has been well established through Genome Wide Association Studies (GWAS), which have identified over 200 risk loci for IBD. However, the « true causative » genes in these loci have been identified for only few on the basis of independently associated coding variants. Fine-mapping studies suggested that most risk variants cause “cis”-eQTL in disease relevant cell types, but recent post-GWAS studies could not find matching cis-eQTLs for the majority of risk loci (137/200). This indicates that the relevant cell types were either not present amongst the analyzed cell populations or under-represented. In this study, we performed cis-eQTL analysis with single cell RNA-seq of human gut biopsies to uncover the truly relevant cell types with higher resolution and unbiased approach. Biopsies were collected from three GI locations (ileum, transverse colon, rectum) from the same individuals. Cell suspensions were prepared, tagged by location and cell fraction using TotalseqB hashtag antibodies for multiplexing and processed to the 10X Genomics Chromium. Data were analyzed using Cellranger and Seurat to identify the cell clusters and marker genes. In total, 50 individuals’ biopsies data were integrated. Simultaneously, genotype was analyzed with Infinium OmniExpress-24v1 chip from 1 ml blood and imputed. Both scRNA-seq data and imputed genotypes were input to qtltools v1.3.1 for cis-eQTL anlaysis. Analysis are actually ongoing and will certainly generate new set of cell-based eQTL and determine whether some of these drive inherited predisposition to IBD by comparing the corresponding expression association patterns with disease association patterns using methods developed in our laboratory

Kinetics and persistence of the cellular and humoral immune responses to BNT162b2 mRNA vaccine in SARS-CoV-2-naive and -experienced subjects

Background: Understanding and measuring the individual level of immune protection and its persistence at both humoral and cellular levels after SARS-CoV-2 vaccination is mandatory for the management of the vaccination booster campaign. Our prospective study was designed to assess the immunogenicity of the BNT162b2 mRNA vaccine in triggering the humoral and the cellular immune response in healthcare workers up to 6 months after two doses vaccination. Methods: This prospective study enrolled 208 healthcare workers from the Liège University Hospital (CHU) of Liège in Belgium. All participants received two doses of BioNTech/Pfizer COVID-19 vaccine (BNT162b2). Fifty participants were SARS-CoV-2 experienced (self-reported SARS-CoV-2 infection) and 158 were naïve (no reported SARS-CoV-2 infection) before the vaccination. Blood sampling was performed at the day of the first (T0) and second (T1) vaccine doses administration, then at 2 weeks (T2), 4 weeks (T3) and 6 months (T4) after the 1st vaccine dose administration. A total of 1024 blood samples were collected. All samples were tested for the presence of anti-Spike antibodies using DiaSorin LIAISON SARS-CoV-2 TrimericS IgG assay. Neutralizing antibodies against the SARS-CoV-2 Wuhan-like variant strain were quantified in all samples using a Vero E6 cell-based neutralization-based assay. Cell-mediated immune response was evaluated at T4 on 80 participants by measuring the secretion of IFN- on peripheral blood lymphocytes using the QuantiFERON Human IFN- SARS-CoV-2, Qiagen. All participants were monitored on weekly-basis for the novo SARS-COV-2 infection for 4 weeks after the 1st vaccine dose administration. We analyzed separately the naïve and experienced participants. Findings: We found that anti-spike antibodies and neutralization capacity levels were significantly higher in SARS-CoV-2 experienced healthcare workers (HCWs) compared to naïve HCWs at all time points analyzed. Cellular immune response was similar in the two groups six months following 2nd dose of the vaccine. Reassuringly, most participants had a detectable cellular immune response to SARS-CoV-2 six months after vaccination. Besides the impact of SARS-CoV-2 infection history on immune response to BNT162b2 mRNA vaccine, we observed a significant negative correlation between age and persistence of humoral response. Cellular immune response was, however, not significantly correlated to age, although a trend towards a negative impact of age was observed. Conclusions: Our data strengthen previous findings demonstrating that immunization through vaccination combined with natural infection is better than 2 vaccine doses immunization or natural infection alone. It may have implications for personalizing mRNA vaccination regimens used to prevent severe COVID-19 and reduce the impact of the pandemic on the healthcare system. More specifically, it may help prioritizing vaccination, including for the deployment of booster doses

Three-dimensional cultured Liver-on-a-Chip with mature hepatocyte-like cells derived from human pluripotent stem cells

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Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

Abstractrag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.</jats:p

Characterization of phenotypic and transcriptional differences in human pluripotent stem cells under 2D and 3D culture conditions

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine