Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates.

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells. FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers.

BACKGROUND: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. METHODS: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis. RESULTS: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected. CONCLUSIONS: The human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Transplacental Zika virus transmission in ex vivo perfused human placentas.

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.

Integration of Solid State and Submerged Fermentations for the Valorization of Organic Municipal Solid Waste.

Solid state fermentation (SsF) is recognized as a suitable process for the production of enzymes using organic residues as substrates. However, only a few studies have integrated an evaluation of the feasibility of applying enzymes produced by SsF into subsequent hydrolyses followed by the production of target compounds, e.g., lactic acid (LA), through submerged-liquid fermentations (SmF). In this study, wheat bran (WB) was used as the substrate for the production of enzymes via SsF by Aspergillus awamori DSM No. 63272. Following optimization, cellulase and glucoamylase activities were 73.63 ± 5.47 FPU/gds and 107.10 ± 2.63 U/gdb after 7 days and 5 days of fermentation, respectively. Enzymes were then used for the hydrolysis of the organic fraction of municipal solid waste (OFMSW). During hydrolysis, glucose increased considerably with a final value of 19.77 ± 1.56 g/L. Subsequently, hydrolysates were fermented in SmF by Bacillus coagulans A166 increasing the LA concentration by 15.59 g/L. The data reported in this study provides an example of how SsF and SmF technologies can be combined for the valorization of WB and OFMSW.

Biorefinery Concept Employing Bacillus coagulans: LX-Lignin and L-(+)-Lactic Acid from Lignocellulose.

A new biorefinery concept is proposed that integrates the novel LX-Pretreatment with the fermentative production of L-(+)-lactic acid. Lignocellulose was chosen as a substrate that does not compete with the provision of food or feed. Furthermore, it contains lignin, a promising new chemical building material which is the largest renewable source for aromatic compounds. Two substrates were investigated: rye straw (RS) as a residue from agriculture, as well as the fibrous digestate of an anaerobic biogas plant operated with energy corn (DCS). Besides the prior production of biogas from energy corn, chemically exploitable LX-Lignin was produced from both sources, creating a product with a low carbohydrate and ash content (90.3% and 88.2% of acid insoluble lignin). Regarding the cellulose fraction of the biomass, enzymatic hydrolysis and fermentation experiments were conducted, comparing a separate (SHF), simultaneous (SSF) and prehydrolyzed simultaneous saccharification and fermentation (PSSF) approach. For this purpose, thermophilic B. coagulans 14-300 was utilized, reaching 38.0 g L-1 LA in 32 h SSF from pretreated RS and 18.3 g L-1 LA in 30 h PSSF from pretreated DCS with optical purities of 99%.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

Induction of brain-infiltrating T-bet-expressing B cells in multiple sclerosis.

OBJECTIVE: Results from anti-CD20 therapies demonstrate that B- and T-cell interaction is a major driver of multiple sclerosis (MS). The local presence of B-cell follicle-like structures and oligoclonal bands in MS patients indicates that certain B cells infiltrate the central nervous system (CNS) to mediate pathology. Which peripheral triggers underlie the development of CNS-infiltrating B cells is not fully understood. METHODS: Ex vivo flow cytometry was used to assess chemokine receptor profiles of B cells in blood, cerebrospinal fluid, meningeal, and brain tissues of MS patients (n = 10). Similar analyses were performed for distinct memory subsets in the blood of untreated and natalizumab-treated MS patients (n = 38). To assess T-bet(CXCR3)+ B-cell differentiation, we cultured B cells from MS patients (n = 21) and healthy individuals (n = 34) under T helper 1- and TLR9-inducing conditions. Their CNS transmigration capacity was confirmed using brain endothelial monolayers. RESULTS: CXC chemokine receptor 3 (CXCR3)-expressing B cells were enriched in different CNS compartments of MS patients. Treatment with the clinically effective drug natalizumab prevented the recruitment of CXCR3high IgG1+ subsets, corresponding to their increased ability to cross CNS barriers in vitro. Blocking of interferon-γ (IFNγ) reduced the transmigration potential and antigen-presenting function of these cells. IFNγ-induced B cells from MS patients showed increased T-bet expression and plasmablast development. Additional TLR9 triggering further upregulated T-bet and CXCR3, and was essential for IgG1 switching. INTERPRETATION: This study demonstrates that T-bethigh IgG1+ B cells are triggered by IFNγ and TLR9 signals, likely contributing to enhanced CXCR3-mediated recruitment and local reactivity in the CNS of MS patients. ANN NEUROL 2019;86:264-278.

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

In vitro-Induced Human IL-10+ B Cells Do Not Show a Subset-Defining Marker Signature and Plastically Co-express IL-10 With Pro-Inflammatory Cytokines.

Regulatory B cells (Breg) have been described as a specific immunological subsets in several mouse models. Identification of a human counterpart has remained troublesome, because unique plasma membrane markers or a defining transcription factor have not been identified. Consequently, human Bregs are still primarily defined by production of IL-10. In this study, we sought to elucidate if in vitro-induced human IL-10 producing B cells are a dedicated immunological subset. Using deep immune profiling by multicolor flow cytometry and t-SNE analysis, we show that the majority of cells induced to produce IL-10 co-express pro-inflammatory cytokines IL-6 and/or TNFα. No combination of markers can be identified to define human IL-10+TNFα-IL-6- B cells and rather point to a general activated B cell phenotype. Strikingly, upon culture and restimulation, a large proportion of formerly IL-10 producing B cells lose IL-10 expression, showing that induced IL-10 production is not a stable trait. The combined features of an activated B cell phenotype, transient IL-10 expression and lack of subset-defining markers suggests that in vitro-induced IL-10 producing B cells are not a dedicated subset of regulatory B cells.

Genotypic variability and relationships between mite infestation levels, mite damage, grooming intensity, and removal of Varroa destructor mites in selected strains of worker honey bees (Apis mellifera L.).

The objective of this study was to demonstrate genotypic variability and analyze the relationships between the infestation levels of the parasitic mite Varroa destructor in honey bee (Apis mellifera) colonies, the rate of damage of fallen mites, and the intensity with which bees of different genotypes groom themselves to remove mites from their bodies. Sets of paired genotypes that are presumably susceptible and resistant to the varroa mite were compared at the colony level for number of mites falling on sticky papers and for proportion of damaged mites. They were also compared at the individual level for intensity of grooming and mite removal success. Bees from the "resistant" colonies had lower mite population rates (up to 15 fold) and higher percentages of damaged mites (up to 9 fold) than bees from the "susceptible" genotypes. At the individual level, bees from the "resistant" genotypes performed significantly more instances of intense grooming (up to 4 fold), and a significantly higher number of mites were dislodged from the bees' bodies by intense grooming than by light grooming (up to 7 fold) in all genotypes. The odds of mite removal were high and significant for all "resistant" genotypes when compared with the "susceptible" genotypes. The results of this study strongly suggest that grooming behavior and the intensity with which bees perform it, is an important component in the resistance of some honey bee genotypes to the growth of varroa mite populations. The implications of these results are discussed.

CD70-driven costimulation induces survival or Fas-mediated apoptosis of T cells depending on antigenic load.

Apoptosis plays an essential role in the removal of activated CD8 T cells that are no longer required during or postinfection. The Bim-dependent intrinsic pathway of apoptosis removes effector CD8 T cells upon clearance of viral infection, which is driven by withdrawal of growth factors. Binding of Fas ligand to Fas mediates activation-induced T cell death in vitro and cooperates with Bim to eliminate CD8 T cells during chronic infection in vivo, but it is less clear how this pathway of apoptosis is initiated. In this study, we show that the costimulatory TNFR CD27 provides a dual trigger that can enhance survival of CD8 T cells, but also removal of activated CD8 T cells through Fas-driven apoptosis. Using in vitro stimulation assays of murine T cells with cognate peptide, we show that CD27 increases T cell survival after stimulation with low doses of Ag, whereas CD27 induces Fas-driven T cell apoptosis after stimulation with high doses of Ag. In vivo, the impact of constitutive CD70-driven stimulation on the accumulation of memory and effector CD8 T cells is limited by Fas-driven apoptosis. Furthermore, introduction of CD70 signaling during acute infection with influenza virus induces Fas-dependent elimination of influenza-specific CD8 T cells. These findings suggest that CD27 suppresses its costimulatory effects on T cell survival through activation of Fas-driven T cell apoptosis to maintain T cell homeostasis during infection.

The costimulatory molecule CD27 maintains clonally diverse CD8(+) T cell responses of low antigen affinity to protect against viral variants.

CD70 and CD27 are costimulatory molecules that provide essential signals for the expansion and differentiation of CD8(+) T cells. Here, we show that CD27-driven costimulation lowered the threshold of T cell receptor activation on CD8(+) T cells and enabled responses against low-affinity antigens. Using influenza infection to study in vivo consequences, we found that CD27-driven costimulation promoted a CD8(+) T cell response of overall low affinity. These qualitative effects of CD27 on T cell responses were maintained into the memory phase. On a clonal level, CD27-driven costimulation established a higher degree of variety in memory CD8(+) T cells. The benefit became apparent when mice were reinfected, given that CD27 improved CD8(+) T cell responses against reinfection with viral variants, but not with identical virus. We propose that CD27-driven costimulation is a strategy to generate memory clones that have potential reactivity to a wide array of mutable pathogens.

Maternal effects on the hygienic behavior of Russian x Ontario hybrid honeybees (Apis mellifera L.).

Strains and hybrids of Russian and Ontario honeybees (Apis mellifera L.) were evaluated for hygienic behavior at both colony and individual levels. The objectives were to determine phenotypic and genotypic variability and to study the inheritance of this behavior. At the colony level, Russian bees uncapped and removed significantly more freeze-killed brood than Ontario bees. The most hygienic Russian colonies and the least hygienic Ontario colonies were selected to perform reciprocal crosses between the strains. Bees from the hybrid colonies as well as from the parental colonies were tagged and introduced into observation hives, where hygienic behavior was directly observed on a piece of frozen brood comb. Russian and hybrid bees of Russian mother had the highest percentages of workers uncapping cells and removing brood. Conversely, Ontario and hybrid bees of Ontario mother had the lowest percentages of individuals for these variables. Differences were also observed among the 4 genotypes for their degree of specialization on hygienic tasks. Russian and hybrid bees of Russian mother showed a significantly higher uncapping frequency per individual than Ontario and hybrid bees of Ontario mother. These results demonstrate phenotypic and genotypic variability for hygienic behavior and are suggestive of maternal effects in the inheritance of hygienic traits.

Occurrence of Aflatoxin in Hypoallergenic Milk Substitutes.

Aflatoxin B1 was detected, and its identity confirmed, in hypoallergenic milk substitutes composed, among other things, of the following ingredients susceptible to possible aflatoxin contamination: soya protein isolate, soy and coconut oils, cornstarch, and corn syrup. Except for one determination all findings were under 1 ng of aflatoxin/ml of formula at drinking concentration. Manufacturers' reserve samples of soya protein isolate, the ingredient thought to be the most likely source of the aflatoxin, were found to be uncontaminated. Reserve samples of other ingredients were not examined. Because hypoallergenic formula may be the major nutrient of an individual with a metabolic defect at infancy, a vulnerable stage of life, available control measures should be used to avoid aflatoxin-contaminated ingredients.