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Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are polymerase chain reaction (PCR)-cloned into expression vectors. Here, we exploited the rapid advances in single-cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice ( COR ona S pike E pitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single-cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation, and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV-2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

Background Chronic rhinosinusitis with nasal polyps (CRSwNP) is generally characterized by tissue‐infiltrating eosinophils. Various biologic treatments, targeting the inflammation, have demonstrated efficacy in reducing nasal polyp size and symptoms. However, their specific impact on granulocyte populations within polyps remains largely unclear. This study explores how different biological treatments modulate local nasal polyp inflammation by assessing changes in granulocyte presence and recruitment before and after treatment. Methods Type 2‐high CRSwNP patients received treatment with mepolizumab, benralizumab, omalizumab, or dupilumab. Immunohistochemistry and protein measurements were performed on their nasal polyp tissue. Bulk RNA‐sequencing was conducted on pre‐ and post‐treatment nasal samples, identifying differentially expressed genes. These results were integrated with single‐cell data from CRSwNP patients. Results Nasal polyp tissue from type 2‐high patients exhibited substantial eosinophil infiltration and limited neutrophils present. All tested biologics reduced eosinophil‐related proteins and genes in nasal tissue. However, our data suggest that benralizumab, mepolizumab and omalizumab could induce a concurrent upregulation of neutrophilic markers. In these patients, chemoattractant genes for neutrophils primarily originated from the epithelial cell cluster, whereas receptors for these biologics were expressed by plasma cells, dendritic cells, and mast cells. Conclusion Biological treatments effectively reduced eosinophilic inflammation in nasal polyps. However, most biologics could induce an eosinophil‐to‐neutrophil shift, indicating that solely targeting eosinophils may be insufficient as a treatment approach. Understanding these secondary effects on local immune pathways is critical for optimizing CRSwNP treatment strategies.

The LIN28B RNA binding protein exhibits an ontogenically restricted expression pattern and is a key molecular regulator of fetal and neonatal B lymphopoiesis. It enhances the positive selection of CD5+ immature B cells early in life through amplifying the CD19/PI3K/c-MYC pathway and is sufficient to reinitiate self-reactive B-1a cell output when ectopically expressed in the adult. In this study, interactome analysis in primary B cell precursors showed direct binding by LIN28B to numerous ribosomal protein transcripts, consistent with a regulatory role in cellular protein synthesis. Induction of LIN28B expression in the adult setting is sufficient to promote enhanced protein synthesis during the small Pre-B and immature B cell stages, but not during the Pro-B cell stage. This stage dependent effect was dictated by IL-7 mediated signaling, which masked the impact of LIN28B through an overpowering stimulation on the c-MYC/protein synthesis axis in Pro-B cells. Importantly, elevated protein synthesis was a distinguishing feature between neonatal and adult B cell development that was critically supported by endogenous Lin28b expression early in life. Finally, we used a ribosomal hypomorphic mouse model to demonstrate that subdued protein synthesis is specifically detrimental for neonatal B lymphopoiesis and the output of B-1a cells, without affecting B cell development in the adult. Taken together, we identify elevated protein synthesis as a defining requirement for early-life B cell development that critically depends on Lin28b . Our findings offer new mechanistic insights into the layered formation of the complex adult B cell repertoire.

Ebola virus disease remains a threat in different Sub-Saharan African countries more particularly in the Democratic Republic of Congo, where persistent outbreaks are driven by human populations living in close proximity to animal reservoirs. While vaccines like Ad26.ZEBOV and MVA-BN-Filo are safe and immunogenic, the dynamics of antibody responses after the two-dose regimen and booster vaccination are not fully understood. Within-host mathematical models offer valuable insights into disease dynamics and waning immunity, but data-driven mechanistic models of antibody kinetics remain scarce. The present study seeks to elucidate the processes involved in antibody kinetics after the two-dose vaccine regimen with Ad26.ZEBOV and MVA-BN-Filo vaccines, followed by a booster dose vaccination with Ad26.ZEBOV, addressing challenges in inference for and implementation of within-host approaches. By integrating established theoretical frameworks with recent empirical findings on antibody kinetics following Ebola vaccination, we illustrate how mechanistic modeling can enhance and refine our understanding of antibody dynamics. Specifically, we emphasize the distinction in the half-life of antibody responses at different vaccination time points and explore the role of vaccine antigens in eliciting an immunological response through the formation and activation of germinal center mediated response. Careful consideration was given to the development of a model that is both interpretable and practically feasible. The half-life of the antibody response was found to be longer after booster vaccination compared to after the second vaccine dose, indicating a steadier decay process. This may be due to the improved quality of antibodies generated, the formation of memory B cells sustaining antibody production, and antigen-antibody binding. This study highlights critical considerations for implementing within-host mechanistic models and the need for robust data to accurately estimate model parameters. Further research is essential to elucidate the decay dynamics of memory B cells and long-lived plasma cells, as these processes play a pivotal role in sustaining antibody-mediated immunity.

Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are polymerase chain reaction (PCR)-cloned into expression vectors. Here, we exploited the rapid advances in single-cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice ( COR ona S pike E pitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single-cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation, and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV-2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic T cell dynamics of smoking and a potential link to T cell infiltration in inflamed synovia. In this study, we, therefore, sought to study T cell features in lung and inflamed joints in smoking versus non-smoking patients.MethodsWe set up a framework to monitor T cells in paired bronchoalveolar lavage fluid, blood and inflamed synovium tissue samples from 17 new-onset treatment naïve anticitrullinated protein antibody+RA patients. T cell receptor (TCR) repertoire of index-sorted tissue residing in T cells was determined by single-cell TCR sequencing coupled with deep immunophenotyping.ResultsA significant enrichment of CD4+ and CD8+ T cells was seen in synovial samples from smoking versus non-smoking patients, along with an increase in expanded T cell clonotypes. This was particularly pronounced among SE+smokers, suggestive of a synergic gene-smoke effect. Strikingly, identical TCR clonalities were present in matched lung and joint samples of RA smokers, the majority being also detectable in circulation. This was mirrored by an increased clustering of lung and synovium TCRs across patients, suggesting a shared specificity by conserved motifs. The lung-joint shared T cell clonotypes showed a restricted TCR gene usage and exhibited a particular 4-1BB+CD57 hi effector profile within the inflamed synovium.ConclusionThe data indicate a profound interplay between a strong MHC predisposition, smoking and induction of autoimmunity by shaping the TCR repertoire.

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation.

Since the precursor frequency of naïve T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are PCR-cloned into expression vectors. Here, we exploited the rapid advances in single cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice (CORona Spike Epitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.

Given the ongoing evolution of SARS-CoV-2 and the historical emergence of other highly transmissible coronaviruses like SARS-CoV and HCoV-NL63, in vivo coronavirus research remains crucial even when the COVID-19 pandemic is receding. Due to restricted tropism of SARS-CoV-2 and other coronaviruses for mouse cells, model systems of infection rely on transgenic expression of the entry receptor human ACE2 (hACE2). Available hACE2 transgenic models using the Krt18 promotor express the receptor across multiple cell types and organ systems, leading to multiple disease features including pneumonia, vascular compromise and neuro-inflammation, that reflect the multi-organ nature of COVID-19. To disentangle the role of cell tropism in driving the clinical manifestations of the disease, we generated two new transgenic mouse models, Sftpa1-hACE2 and Cdh5-hACE2 transgenic mice, with hACE2 restricted to lung epithelium or endothelial cells respectively. In Sftpa1-hACE2 mice, with high expression of the hACE2 receptor in lung alveolar type 2 cells, SARS-CoV-2 infection led to rapidly progressing disease, characterised by a strong neutrophilic innate immune response in the lung, followed by viral neuro-invasion and early death. Krt18-hACE2 mice additionally recruited various dendritic cell subsets and gradually developed adaptive immunity. In Cdh5-hACE2 Tg mice with exclusive endothelial tropism of the virus, viral inoculation via the lung or systemic circulation did not lead to viral propagation or disease manifestations, despite endothelial expression of hACE2 in the lung. These results suggest that tropism for alveolar epithelial cells increases disease severity, while endothelial cell tropism per se does not drive the vascular consequences often seen in COVID-19 patients. Our new transgenic mouse models will be helpful to dissect how cell tropism contributes to the clinical manifestations of coronavirus infection.