Biology subjects

Jakobsen, M. R.

Publications and source records attributed to Jakobsen, M. R..

5 recordsLinked to original sources

bioRxiv2025

Using high-dimensional immunophenotyping for mapping immune cell composition in rapid cryopreserved liquid biopsies

Liquid biopsies, such as a blood sample, are non-invasive and simple ways to monitor biomarkers in clinical settings. For cancer immunotherapy settings a major clinical goal is to identify host immunological biomarkers that can be used to monitor progression of disease, treatment efficacy, or even adverse effects. Here, the profiling and validation of blood circulating immune cells may allow continuously survey on how the immune system alters during disease progress and treatment. However, such practice will require proper sampling and development of a robust high-dimensional phenotypic analysis. Here, we designed a 37-marker spectral flow cytometry assay with the purpose of conducting high-dimensional immunophenotyping as monitoring the immune status in patients. The assay was developed to profile all major immune cell compartments, their differentiation as well as activation markers, including numerous checkpoint markers. Importantly, we validated this assay in context of how liquid biopsies should be processed to ensure minimal effect of cryopreservation compared to immunophenotyping of a freshly acquired blood sample. Our work highlights important considerations for which phenotypic alternations can be introduced with different cryopreservation methods. Essentially, we identified a fast and simple processing pipeline where cryopreservation of immune cells showed minimal changes in the immunophenotyping profile. This approach can certainly be implemented in clinical settings and will help diagnostic clinical sites with a strong and validated tool applicable for monitoring individual patients immunological status.

immunology↗

bioRxiv2023

Biofilms and core pathogens shape the tumour microenvironment and immune phenotype in colorectal cancer

ObjectiveGrowing evidence links bacterial dysbiosis with colorectal cancer (CRC) carcinogenesis, characterized by an increased presence of core pathogens such as Bacteroides fragilis and Fusobacterium nucleatum. Here, we characterized the in situ biogeography and transcriptional interactions between bacteria and the host in mucosal colon biopsies. DesignThe influence of CRC core pathogens and biofilms on the tumour microenvironment (TME) was investigated in biopsies from patients with and without CRC (paired normal tissue and healthy tissue biopsies) using fluorescence in situ hybridization and dual-RNA sequencing. ResultsTissue-invasive, mixed-species biofilms enriched for B. fragilis and F. nucleatum were observed in CRC tissue, especially in right-sided tumours. Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. CRC samples with high bacterial activity demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors. In contrast, the gene expression profiles of CRC samples with low bacterial activity resembled healthy tissue samples. Moreover, immune cell profiling showed that B. fragilis and F. nucleatum modulated the TME and correlated with increased infiltration of neutrophils and CD4+ T-cells. Overall, bacterial activity was critical for the immune phenotype and correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells. ConclusionBiofilms and core pathogens shape the TME and immune phenotype in CRC. Our results support that Fusobacterium spp. may provide a future therapeutic target to reduce biofilms and the inflammatory response in the TME while highlighting the importance of widening the scope of bacterial pathogenesis in CRC beyond core pathogens.

cancer biology↗

bioRxiv2023

T-cell derived extracellular vesicles prime macrophages for improved STING based cancer immunotherapy

A key phenomenon in cancer is the establishment of a highly immunosuppressive tumor microenvironment (TME). Despite advances in immunotherapy, where the purpose is to induce tumor recognition and hence hereof tumor eradication, the majority of patients applicable for such treatment still fail to respond. It has been suggested that high immunological activity in the tumor is essential for achieving effective response to immunotherapy, which therefore have led to exploration of strategies that triggers inflammatory pathways. Here activation of the stimulator of interferon genes (STING) signaling pathway has been considered an attractive target, as it is a potent trigger of pro-inflammatory cytokines and type I and III interferons. However, immunotherapy combined with targeted STING agonists has not yielded sustained clinical remission in humans. This suggest a need for exploring novel adjuvants to improve the innate immunological efficacy. Here, we demonstrate that extracellular vesicles (EVs), derived from activated CD4+ T cells (T-EVs), sensitizes macrophages to elevate STING activation, mediated by IFN{gamma} carried on the T-EVs. Our work support that T-EVs can disrupt the immune suppressive environment in the tumor by reprogramming macrophages to a pro-inflammatory phenotype, and priming them for a robust immune response towards STING activation.

immunology↗

bioRxiv2021

Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here we show that in COVID-19 patients, circulating plasmacytoid dendritic cells (pDCs) decline early after symptom onset and this correlated with COVID-19 disease severity. This transient depletion coincides with decreased expression of antiviral type I IFN and the systemic inflammatory cytokines CXCL10 and IL-6. Importantly, COVID-19 disease severity correlated with decreased pDC frequency in peripheral blood. Using an in vitro stem cell-based human pDC model, we demonstrate that pDCs directly sense SARS-CoV-2 and in response produce multiple antiviral (IFN and IFN{lambda}1) and inflammatory (IL-6, IL-8, CXCL10) cytokines. This immune response is sufficient to protect epithelial cells from de novo SARS-CoV-2 infection. Targeted deletion of specific sensing pathways identified TLR7-MyD88 signaling as being crucial for production of the antiviral IFNs, whereas TLR2 is responsible for the inflammatory IL-6 response. Surprisingly, we found that SARS-CoV-2 engages the neuropilin-1 receptor on pDCs to mitigate the antiviral IFNs but not the IL-6 response. These results demonstrate distinct sensing pathways used by pDCs to elicit antiviral vs. immunopathological responses to SARS-CoV-2 and suggest that targeting neuropilin-1 on pDCs may be clinically relevant for mounting TLR7-mediated antiviral protection. One Sentence SummarypDCs sense SARS-CoV-2 and elicit antiviral protection of lung epithelial cells through TLR7, while recognition of TLR2 elicits an IL-6 inflammatory response associated with immunopathology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/469755v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@fe64daorg.highwire.dtl.DTLVardef@18f4278org.highwire.dtl.DTLVardef@54de50org.highwire.dtl.DTLVardef@1cf67cb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO SARS-CoV-2 sensing by plasmacytoid dendritic cells. SARS-CoV-2 is internalized by pDCs via a yet unknown endocytic mechanism. The intracellular TLR7 sensor detects viral RNA and induces a signaling cascade involving MyD88-IRAK4-TRAF6 (1) to induce CXCL10 and, via IRF7 phosphorylation and translocation, inducing type I and III Interferons (2). Once secreted, type I and III IFNs initiate autocrine and paracrine signals that induce the expression of IFN stimulated genes (ISGs), thereby facilitating an antiviral response that can protect the cell against infection. However, SARS-CoV-2, has the intrinsic property to facilitate CD304 signaling, potentially by interfering with IRF7 nuclear translocation, thereby inhibiting type I IFN production and thus reducing the antiviral response generated by the pDC (4). Furthermore, the SARS-CoV-2 envelope (E) glycoprotein is sensed by the extracellular TLR2/6 heterodimer and this facilitates production of the inflammatory IL-6 cytokine (5). Illustration was created with BioRender.com C_FIG

immunology↗

bioRxiv2018

Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

Combination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

bioengineering↗