Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression.

In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

Applying valency-based immuno-selection to generate broadly cross-reactive antibodies against influenza hemagglutinins.

Conserved epitopes shared between virus subtypes are often subdominant, making it difficult to induce broadly reactive antibodies by immunization. Here, we generate a plasmid DNA mix vaccine that encodes protein heterodimers with sixteen different influenza A virus hemagglutinins (HA) representing all HA subtypes except H1 (group 1) and H7 (group 2). Each single heterodimer expresses two different HA subtypes and is targeted to MHC class II on antigen presenting cells (APC). Female mice immunized with the plasmid mix produce antibodies not only against the 16 HA subtypes, but also against non-included H1 and H7. We demonstrate that individual antibody molecules cross-react between different HAs. Furthermore, the mix vaccine induces T cell responses to conserved HA epitopes. Immunized mice are partially protected against H1 viruses. The results show that application of valency-based immuno-selection to diversified antigens can be used to direct antibody responses towards conserved (subdominant) epitopes on viral antigens.

Monoclonal antibodies lock down SARS-CoV-2 spike.

SARS-CoV-2 rapidly accumulated mutations in its immunodominant receptor-binding domain (RBD), rendering all clinically authorized monoclonal antibodies (mAbs) ineffective. Liu et al. unveil potent human mAbs that neutralize all tested SARS-CoV-2 variants by locking the Spike protein RBD in a downward conformation, thus inhibiting receptor engagement.

Pre-existing CD4 T cell help boosts antibody responses but has limited impact on germinal center, antigen-specific B cell frequencies after influenza infection.

The influenza virus is a persistent burden on global health, with seasonal vaccines providing incomplete protection. CD4+ T cells help shape B cell and antibody responses; however, the selectivity of help and the effect on various antigen-specific B cell populations have not been fully elucidated. Here, we studied the specificity, selectivity, and influence of nucleoprotein (NP) CD4+ T cells on the magnitude and quality of hemagglutinin (HA) and NP-specific B cells and antibody responses. We identified immunodominant peptides and showed that peptide immunization was sufficient to induce CD4+ cells with Th1 and Tfh phenotypes. Surprisingly, while preexisting CD4+ T cells enhanced the influx of total germinal center (GC) B cells in the mediastinal lymph node after infection, this was not reflected by an increase in the frequency of antigen-specific cells within the GC. Furthermore, we demonstrated that NP-specific help was able to accelerate the kinetics and magnitude of the Ab response for NP but not for HA. Overall, our results showed that pre-existing CD4+ T cells provide strong cognate help during immunization or infection to enhance Ab production but not antigen-specific GC or memory B cells.

Tr1 cell-mediated protection against autoimmune disease by intranasal administration of a fusion protein targeting cDC1 cells.

Curative therapies against autoimmune diseases are lacking. Indeed, most of the currently available treatments are only targeting symptoms. We have developed a novel strategy for a therapeutic vaccine against autoimmune diseases based on intranasal administration of a fusion protein tolerogen, which consists of a mutant, enzymatically inactive, cholera toxin A1 (CTA1)-subunit genetically fused to disease-relevant high-affinity peptides and a dimer of D-fragments from protein A (DD). The CTA1 R7K mutant - myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP) - DD (CTA1R7K-MOG/PLP-DD) fusion proteins effectively reduced clinical symptoms in the experimental autoimmune encephalitis model of multiple sclerosis. The treatment induced Tr1 cells, in the draining lymph node, which produced interleukin (IL)-10 and suppressed effector clusters of differentiation 4+ T-cell responses. This effect was dependent on IL-27 signaling because treatment was ineffective in bone marrow chimeras lacking IL-27Ra within their hematopoietic compartment. Single-cell RNA sequencing of dendritic cells in draining lymph nodes demonstrated distinct gene transcriptional changes of classic dendritic cells 1, including enhanced lipid metabolic pathways, induced by the tolerogenic fusion protein. Thus, our results with the tolerogenic fusion protein demonstrate the possibility to vaccinate and protect against disease progression by reinstating tolerance in multiple sclerosis and other autoimmune diseases.

Profiling of innate and adaptive immune cells during influenza virus infection reveals sex bias in invariant natural killer T (iNKT) cells.

BACKGROUND: Influenza A virus (IAV) infection leads to significant morbidity and mortality. Biological sex influences the immune responses to IAV infection, resulting in higher mortality in women of reproductive age. Previous studies revealed increased activation of T and B cells in female mice after IAV infection, but extensive analysis of sex differences in both innate and adaptive immune cells over time is lacking. Invariant natural killer T (iNKT) cells are fast-reacting forces and modulators of immune responses that are important to IAV immunity, but it is not known if the presence and function of iNKT cells differ between females and males. The aim of this study was to determine immunological mechanisms that contribute to the increased disease severity in female mice during IAV infection. METHODS: Female and male mice were infected with mouse-adapted IAV and monitored for weight loss and survival. Immune cell populations and cytokine expression in bronchoalveolar lavage fluid, lung, and mediastinal lymph node were determined at three time points after infection using flow cytometry and ELISA. RESULTS: The results reveal increased severity and mortality in adult female mice compared to age-matched males. Female mice show larger increases in innate and adaptive immune cell populations and cytokine production in lung compared to mock on Day 6 postinfection. On Day 9 postinfection, female mice express higher numbers of iNKT cells in lung and liver compared to males. CONCLUSIONS: This comprehensive analysis of immune cells and cytokines over time following IAV infection reveals increased leukocyte expansion and stronger proinflammatory cytokine responses in female mice during disease initiation. Furthermore, this is the first study to report a sex bias in iNKT cell populations after IAV infection. The data suggests that the process of recovery from IAV-induced airway inflammation is associated with increased expansion of several different iNKT cell subpopulations in female mice.

Plasmablasts in previously immunologically naïve COVID-19 patients express markers indicating mucosal homing and secrete antibodies cross-reacting with SARS-CoV-2 variants and other beta-coronaviruses.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin ß1, only some integrin ß7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

Memory B-cell diversity: From early generation to tissue residency and reactivation.

Memory B cells (MBCs) have a crucial function in providing an enhanced response to repeated infections. Upon antigen encounter, MBC can either rapidly differentiate to antibody secreting cells or enter germinal centers (GC) to further diversify and affinity mature. Understanding how and when MBC are formed, where they reside and how they select their fate upon reactivation has profound implications for designing strategies to improve targeted, next-generation vaccines. Recent studies have crystallized much of our knowledge on MBC but also reported several surprising discoveries and gaps in our current understanding. Here, we review the latest advancements in the field and highlight current unknowns. In particular, we focus on timing and cues leading to MBC generation before and during the GC reaction, discuss how MBC become resident in mucosal tissues, and finally, provide an overview of factors shaping MBC fate-decision upon reactivation in mucosal and lymphoid tissues.

Longitudinal single-cell analysis of SARS-CoV-2-reactive B cells uncovers persistence of early-formed, antigen-specific clones.

Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor-Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.

ADP-ribosylating adjuvant reveals plasticity in cDC1 cells that drive mucosal Th17 cell development and protection against influenza virus infection.

Migratory dendritic cells expressing CD103 are the targets for mucosal vaccines. These belong to either of two lineage-restricted subsets, cDC1 or cDC2 cells, which have been linked to priming of functionally distinct CD4 T cells. However, recent studies have identified plasticity in cDC2 cells with overlapping functions with cDC1 cells, while the converse has not been reported. We genetically engineered a vaccine adjuvant platform that targeted the cholera toxin A1 (CTA1) ADP-ribosylating enzyme to CD103+ cDC1 and cDC2 cells using a single-chain antibody (scFv) to CD103. Unexpectedly, intranasal immunization with the CTA1-svFcCD103 adjuvant modified cDC1 cells to effectively prime Th17 cells, a function previously limited to cDC2 cells. In fact, cDC2 cells were dispensible, while cDC1 cells, lacking in Batf3-/- mice, were critical. Following intranasal immunizations isolated cDC1 cells from mLN exclusively promoted Rorgt+ T cells and IL-17, IL-21, and IL-22 production. Strong CD8 T cell responses through antigen cross presentation by cDC1 cells were also observed. Single-cell RNAseq analysis revealed upregulation of Th17-promoting gene signatures in sorted cDC1 cells. Gene expression in isolated cDC2 cells was largely unaffected. Our finding represents a major shift of paradigm as we have documented functional plasticity in cDC1 cells.

Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

Temporal dynamics of persistent germinal centers and memory B cell differentiation following respiratory virus infection.

Following infection or immunization, memory B cells (MBCs) and long-lived plasma cells provide humoral immunity that can last for decades. Most principles of MBC biology have been determined with hapten-protein carrier models or fluorescent protein immunizations. Here, we examine the temporal dynamics of the germinal center (GC) B cell and MBC response following mouse influenza A virus infection. We find that antiviral B cell responses within the lung-draining mediastinal lymph node (mLN) and the spleen are distinct in regard to duration, enrichment for antigen-binding cells, and class switching dynamics. While splenic GCs dissolve after 6 weeks post-infection, mLN hemagglutinin-specific (HA+) GCs can persist for 22 weeks. Persistent GCs continuously differentiate MBCs, with "peak" and "late" GCs contributing equal numbers of HA+ MBCs to the long-lived compartment. Our findings highlight critical aspects of persistent GC responses and MBC differentiation following respiratory virus infection with direct implications for developing effective vaccination strategies.

Correction: Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders.

[This corrects the article DOI: 10.1371/journal.pone.0241104.].

Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.

Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.

Know your enemy or find your friend?-Induction of IgA at mucosal surfaces.

Most antibodies produced in the body are of the IgA class. The dominant cell population producing them are plasma cells within the lamina propria of the gastrointestinal tract, but many IgA-producing cells are also found in the airways, within mammary tissues, the urogenital tract and inside the bone marrow. Most IgA antibodies are transported into the lumen by epithelial cells as part of the mucosal secretions, but they are also present in serum and other body fluids. A large part of the commensal microbiota in the gut is covered with IgA antibodies, and it has been demonstrated that this plays a role in maintaining a healthy balance between the host and the bacteria. However, IgA antibodies also play important roles in neutralizing pathogens in the gastrointestinal tract and the upper airways. The distinction between the two roles of IgA - protective and balance-maintaining - not only has implications on function but also on how the production is regulated. Here, we discuss these issues with a special focus on gut and airways.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

CD4+ follicular regulatory T cells optimize the influenza virus-specific B cell response.

CD4+ follicular regulatory T (Tfr) cells control B cell responses through the modulation of follicular helper T (Tfh) cells and germinal center development while suppressing autoreactivity; however, their role in the regulation of productive germinal center B cell responses and humoral memory is incompletely defined. We show that Tfr cells promote antigen-specific germinal center B cell responses upon influenza virus infection. Following viral challenge, we found that Tfr cells are necessary for robust generation of virus-specific, long-lived plasma cells, antibody production against both hemagglutinin (HA) and neuraminidase (NA), the two major influenza virus glycoproteins, and appropriate regulation of the BCR repertoire. To further investigate the functional relevance of Tfr cells during viral challenge, we used a sequential immunization model with repeated exposure of antigenically partially conserved strains of influenza viruses, revealing that Tfr cells promote recall antibody responses against the conserved HA stalk region. Thus, Tfr cells promote antigen-specific B cell responses and are essential for the development of long-term humoral memory.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization.

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders.

BACKGROUND: To accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. This study aimed to describe serum-IgG responses to SARS-CoV-2 in a cohort of patients with both severe and mild COVID-19, including extended studies of patients who remained seronegative more than 90 days post symptom onset. METHODS: SARS-CoV-2-specific IgG antibody levels were quantified using two clinically validated and widely used commercial serological assays (Architect, Abbott Laboratories and iFlash 1800, YHLO), detecting antibodies against the spike and nucleocapsid proteins. RESULTS: Forty-seven patients (mean age 49 years, 38% female) were included. All (15/15) patients with severe symptoms and 29/32 (90.6%) patients with mild symptoms of COVID-19 developed SARS-CoV-2-specific IgG antibodies in serum. Time to seroconversion was significantly shorter (median 11 vs. 22 days, P = 0.04) in patients with severe compared to mild symptoms. Of the three patients without detectable IgG-responses after >90 days, all had detectable virus-neutralizing antibodies and in two, spike-protein receptor binding domain-specific IgG was detected with an in-house assay. Antibody titers were preserved during follow-up and all patients who seroconverted, irrespective of the severity of symptoms, still had detectable IgG levels >75 days post symptom onset. CONCLUSIONS: Patients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS-CoV-2-specific IgG than patients with mild symptoms. Of those patients who not develop detectable IgG antibodies, all have detectable virus-neutralizing antibodies, suggesting immunity. Our results showing that not all COVID-19 patients develop detectable IgG using two validated commercial clinical methods, even over time, are vital for the interpretation of COVID-19 seroprevalence surveys.