Reduced Humoral and Cellular Immune Response to Primary COVID-19 mRNA Vaccination in Kidney Transplanted Children Aged 5-11 Years.

The situation of limited data concerning the response to COVID-19 mRNA vaccinations in immunocom-promised children hinders evidence-based recommendations. This prospective observational study investigated humoral and T cell responses after primary BNT162b2 vaccination in secondary immunocompromised and healthy children aged 5-11 years. Participants were categorized as: children after kidney transplantation (KTx, n = 9), proteinuric glomerulonephritis (GN, n = 4) and healthy children (controls, n = 8). Expression of activation-induced markers and cytokine secretion were determined to quantify the T cell response from PBMCs stimulated with peptide pools covering the spike glycoprotein of SARS-CoV-2 Wuhan Hu-1 and Omicron BA.5. Antibodies against SARS-CoV-2 spike receptor-binding domain were quantified in serum. Seroconversion was detected in 56% of KTx patients and in 100% of the GN patients and controls. Titer levels were significantly higher in GN patients and controls than in KTx patients. In Ktx patients, the humoral response increased after a third immunization. No differences in the frequency of antigen-specific CD4+ and CD8+ T cells between all groups were observed. T cells showed a predominant anti-viral capacity in their secreted cytokines; however, this capacity was reduced in KTx patients. This study provides missing evidence concerning the humoral and T cell response in immunocompromised children after COVID-19 vaccination.

Cu-doped TiO2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies.

Nanoparticle-mediated cancer immunotherapy holds great promise, but more efforts are needed to obtain nanoformulations that result in a full scale activation of innate and adaptive immune components that specifically target the tumors. We generated a series of copper-doped TiO2 nanoparticles in order to tune the kinetics and full extent of Cu2+ ion release from the remnant TiO2 nanocrystals. Fine-tuning nanoparticle properties resulted in a formulation of 33% Cu-doped TiO2 which enabled short-lived hyperactivation of dendritic cells and hereby promoted immunotherapy. The nanoparticles result in highly efficient activation of dendritic cells ex vivo, which upon transplantation in tumor bearing mice, exceeded the therapeutic outcomes obtained with classically stimulated dendritic cells. Efficacious but simple nanomaterials that can promote dendritic cancer cell vaccination strategies open up new avenues for improved immunotherapy and human health.

A genotype-phenotype screening system using conditionally immortalized immature dendritic cells.

Here, we describe a protocol for CRISPR/Cas9-mediated gene knockout in conditionally immortalized immature dendritic cells (DCs), which can be limitlessly expanded before differentiation. This facilitates the genetic screening of DC functions in vitro including assessment of phagocytosis, cytokine production, expression of co-stimulatory or co-inhibitory molecules, and antigen presentation, as well as evaluation of the capacity to elicit anticancer immune responses in vivo. Altogether, these approaches described in this protocol allow investigators to link the genotype of DCs to their phenotype. For complete details on the use and execution of this protocol, please refer to Le Naour et al. (2020).

Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions.

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

A scalable method for parameter-free simulation and validation of mechanistic cellular signal transduction network models.

The metabolic modelling community has established the gold standard for bottom-up systems biology with reconstruction, validation and simulation of mechanistic genome-scale models. Similar methods have not been established for signal transduction networks, where the representation of complexes and internal states leads to scalability issues in both model formulation and execution. While rule- and agent-based methods allow efficient model definition and execution, respectively, model parametrisation introduces an additional layer of uncertainty due to the sparsity of reliably measured parameters. Here, we present a scalable method for parameter-free simulation of mechanistic signal transduction networks. It is based on rxncon and uses a bipartite Boolean logic with separate update rules for reactions and states. Using two generic update rules, we enable translation of any rxncon model into a unique Boolean model, which can be used for network validation and simulation-allowing the prediction of system-level function directly from molecular mechanistic data. Through scalable model definition and simulation, and the independence of quantitative parameters, it opens up for simulation and validation of mechanistic genome-scale models of signal transduction networks.

Using rxncon to Develop Rule-Based Models.

We present a protocol for building, validating, and simulating models of signal transduction networks. These networks are challenging modeling targets due to the combinatorial complexity and sparse data, which have made it a major challenge even to formalize the current knowledge. To address this, the community has developed methods to model biomolecular reaction networks based on site dynamics. The strength of this approach is that reactions and states can be defined at variable resolution, which makes it possible to adapt the model resolution to the empirical data. This improves both scalability and accuracy, making it possible to formalize large models of signal transduction networks. Here, we present a method to build and validate large models of signal transduction networks. The workflow is based on rxncon, the reaction-contingency language. In a five-step process, we create a mechanistic network model, convert it into an executable Boolean model, use the Boolean model to evaluate and improve the network, and finally export the rxncon model into a rule-based format. We provide an introduction to the rxncon language and an annotated, step-by-step protocol for the workflow. Finally, we create a small model of the insulin signaling pathway to illustrate the protocol, together with some of the challenges-and some of their solutions-in modeling signal transduction.

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

UTX - moonlighting in the cytoplasm?

The X-linked histone demethylase UTX has a pivotal role in cellular and developmental processes including embryogenesis, hematopoiesis and cancer. UTX removes di- and trimethyl groups on histone H3 lysine 27, thereby regulating gene expression. But there is growing evidence that UTX displays biological functions independent of its histone demethylase activity. To elucidate these novel functions, it is of great interest to define subcellular localizations of UTX. Here we show for the first time that native UTX is primarily localized in the cytoplasm whereas ectopic GFP and Flag-tagged UTX display nuclear and cytoplasmic localization. While its epigenetic function is exerted in the nucleus, its cytoplasmic localization points to a novel function.

Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

RNAi profiling of primary human AML cells identifies ROCK1 as a therapeutic target and nominates fasudil as an antileukemic drug.

Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling.

Cellular reporter systems for high-throughput screening of interactions between bioactive matrices and human mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) and factors secreted by them are essential components of the hematopoietic stem cell (HSC) niche within the bone marrow microenvironment. It has been shown that the extracellular matrix (ECM) can influence HSC-supportive potential of MSC and is a prerequisite for the proper signaling of morphogens. Therefore, we aimed at the identification of ECM components and candidate morphogens capable of enhancing the expression of HSC-supportive proteins in human MSC, namely, angiopoietin-1 (Ang-1) and stromal cell-derived factor 1 (SDF-1). For this purpose, highly sensitive secreted dual reporter constructs for Ang-1 and SDF-1 were established. These newly designed dual reporter systems enable continuous monitoring of the Ang-1 and SDF-1 promoter activity in an immortalized human MSC line cultured on ECM/morphogen microarrays. Reporter arrays showed that Ang-1 and SDF-1 expression can be induced by different ECM/morphogen combinations. In addition, continuous monitoring of promoter activity allows delineating time-dependent effects of the ECM and morphogens. Thus, we identified that collagen I and vitronectin in combination with Wnt3a favored SDF-1 expression over time, while only transiently inducing the expression of Ang-1. Taken together, the newly developed reporter systems allow for the monitoring of Ang-1 and SDF-1 promoter activity induced by morphogens and the ECM in a combinatorial and high-throughput manner. This technology might therefore be helpful to optimize culture conditions, which favor the activity of MSC as feeder cells for various types of stem and progenitor cells.

Overexpression of Jagged-1 and its intracellular domain in human mesenchymal stromal cells differentially affect the interaction with hematopoietic stem and progenitor cells.

Mesenchymal stromal cells (MSC) are an important component of the bone marrow microenvironment. Notch ligands expressed by MSC are known to play a regulatory role for hematopoietic stem and progenitor cells (HSPC) and in support of bone marrow homeostasis. While the role of Notch signaling in HSPC, their progeny, and MSC has been relatively well studied, little is known about the Notch-independent regulatory impact of Notch ligands on MSC themselves. In the present study, we used genetically engineered bone marrow-derived human MSC to study the function role of Jagged-1 and the Jagged-1 intracellular domain (JICD) with regard to the interaction with HSPC. We demonstrate that Jagged-1 in human MSC undergoes cleavage to produce an intracellular domain that translocates into the nucleus. JICD but not Jagged-1 overexpression was associated with an increased expression of stromal cell-derived factor-1. Short-term co-culture (7 days) of HSPC with JICD-overexpressing MSC, but not with Jagged-1-overexpressing MSC, led to increased proliferation of CD34(+) progenitors. In contrast, long-term co-culture of HSPC with Jagged-1-overexpressing MSC (up to 6 weeks) led to a significantly better support of cobblestone area-forming cells and long-term culture-initiating cells (LTC-ICs) compared with JICD-overexpressing MSC. Taken together, results of this study indicate that full-length Jagged-1 and JICD have differential effects on MSC and on their interaction with HSPC ex-vivo. JICD-overexpressing MSC induce proliferation of HSPCs in short-term culture at the expense of immature precursors (LTC-ICs), whereas Jagged-1 overexpressing MSC favor LTC-IC formation.

Information content and scalability in signal transduction network reconstruction formats.

One of the first steps towards holistic understanding of cellular networks is the integration of the available information in a human and machine readable format. This network reconstruction process is well established for metabolic networks, and numerous genome wide metabolic reconstructions are already available. Extending these strategies to signalling networks has proven difficult, primarily due to the combinatorial nature of regulatory modifications. The combinatorial nature of possible protein-protein interactions and post translational modifications affects both network size and the correspondence between the reconstructed network and the underlying empirical data. Here, we discuss different approaches to reconstruction of signal transduction networks. We divide the current approaches into topological, specific state based and reaction-contingency based, and discuss their different information content and scalability. The discussion focusses on graphical formats but the points are in general applicable also to mathematical models and databases. While the formats have complementary strengths especially for small networks, reaction-contingency based formats have a number of advantages in the light of global network reconstruction. In particular, they minimise the need for assumptions, maximise the congruence with empirical data, and scale efficiently with network size.

Generation of inducible immortalized dendritic cells with proper immune function in vitro and in vivo.

Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1ß, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4(+) T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells.

The histone demethylase UTX regulates stem cell migration and hematopoiesis.

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

Genome Fusion Detection: a novel method to detect fusion genes from SNP-array data.

MOTIVATION: Fusion genes result from genomic rearrangements, such as deletions, amplifications and translocations. Such rearrangements can also frequently be observed in cancer and have been postulated as driving event in cancer development. to detect them, one needs to analyze the transition region of two segments with different copy number, the location where fusions are known to occur. Finding fusion genes is essential to understanding cancer development and may lead to new therapeutic approaches. RESULTS: Here we present a novel method, the Genomic Fusion Detection algorithm, to predict fusion genes on a genomic level based on SNP-array data. This algorithm detects genes at the transition region of segments with copy number variation. With the application of defined constraints, certain properties of the detected genes are evaluated to predict whether they may be fused. We evaluated our prediction by calculating the observed frequency of known fusions in both primary cancers and cell lines. We tested a set of cell lines positive for the BCR-ABL1 fusion and prostate cancers positive for the TMPRSS2-ERG fusion. We could detect the fusions in all positive cell lines, but not in the negative controls.

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

P67-phox (NCF2) lacking exons 11 and 12 is functionally active and leads to an extremely late diagnosis of chronic granulomatous disease (CGD).

Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000 + 2T → G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage.

Massively parallel RNA sequencing identifies a complex immune gene repertoire in the lophotrochozoan Mytilus edulis.

The marine mussel Mytilus edulis and its closely related sister species are distributed world-wide and play an important role in coastal ecology and economy. The diversification in different species and their hybrids, broad ecological distribution, as well as the filter feeding mode of life has made this genus an attractive model to investigate physiological and molecular adaptations and responses to various biotic and abiotic environmental factors. In the present study we investigated the immune system of Mytilus, which may contribute to the ecological plasticity of this species. We generated a large Mytilus transcriptome database from different tissues of immune challenged and stress treated individuals from the Baltic Sea using 454 pyrosequencing. Phylogenetic comparison of orthologous groups of 23 species demonstrated the basal position of lophotrochozoans within protostomes. The investigation of immune related transcripts revealed a complex repertoire of innate recognition receptors and downstream pathway members including transcripts for 27 toll-like receptors and 524 C1q domain containing transcripts. NOD-like receptors on the other hand were absent. We also found evidence for sophisticated TNF, autophagy and apoptosis systems as well as for cytokines. Gill tissue and hemocytes showed highest expression of putative immune related contigs and are promising tissues for further functional studies. Our results partly contrast with findings of a less complex immune repertoire in ecdysozoan and other lophotrochozoan protostomes. We show that bivalves are interesting candidates to investigate the evolution of the immune system from basal metazoans to deuterostomes and protostomes and provide a basis for future molecular work directed to immune system functioning in Mytilus.