Visualizing germinal center B cell dynamics at single cell transcriptome resolution
ABSTRACT: During a germinal center (GC) response, B cells diversify their immunoglobulin (Ig) genes by somatic hyper-mutation (SHM) and undergo clonal expansion and positive selection thereby enabling the generation of higher affinity antibodies. We have analyzed the genomic states underlying GC B cell dynamics by single cell RNA-Seq. Profiling of antigen specific GC B cells during the peak of the response, revealed four distinctive genomic states characterized by antigen presentation, apoptotic, mitochondrial and mitotic gene expression modules. Intersection of genomic states and Ig heavy-chain (Igh) class-switch trajectory suggested that mitochondrial machinery is utilized to support class-switch recombination (CSR). Furthermore, by analyzing the transcriptomes of B cells with varying affinity BCR sequences that assembled from single-cell RNA-seq data through a novel algorithm, we show that high affinity GC B cells manifest enhanced mitotic and BCR signaling transduction, but compromised antigen processing and presentation gene expression modules. Thus, we are developing a comprehensive framework of the genomic states and molecular pathways underlying GC B cell dynamics. Overall design: C56BL/6J (Jax 664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) mixed with 50% (v/v) Alum (Thermo Scientific) and 1 μg LPS (Sigma). Splenocyte suspensions were prepared in MACS buffer (pH 7.4 PBS plus 1% FBS and 5 mM EDTA) on day 13 post immunization and blocked with 25 μg/ml 2.4G2 (BD) for 15 min on ice. Then cells were labeled with 2 μg/ml biotin anti-CD3, 1 μg/ml biotin anti-CD4, 1 μg/ml biotin anti-CD8, 2.5 μg/ml biotin anti-CD11C and 2.5 μg/ml biotin anti-IgD for 25 min at 4°C. After washing four times, cells were further labeled with anti-biotin beads (Miltenyi Biotec) for 20 min at 4°C. The magnetic columns were used to deplete non-GC B cells according to standard protocols (Miltenyi Biotec). The effluent cells were further labeled with NP-PE, B220, Fas and GL7 antibodies (Supplementary Table 1) for 30 min at 4 °C. GC B cells were sorted as 7AAD–B220+Fas+GL7hi with FACSAria II (BD) with 70 μm nozzle. Single GC B cells were prepared using the C1TM Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions. 182 scRNA Seq libraries were sequenced per HiSeq 2500 gel with 75bp paired-end sequencing.
Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories. Naïve B cells were isolated from wild type (WT) mice spleen and activated in vitro with 10μg/ml LPS (Sigma). ChIP was performed by using anti-IRF4, -IRF8 antibodies (Santa Cruz Biotech). For massively parallel sequencing, 10-20 μg of chromatin fragments from indicated samples were immunoprecipitated by using anti-IRF-4 and anti-IRF8 antibodies, and DNA libraries were prepared with Illumina Kit. DNA was sequenced by using the Illumina HiSeq2500. Reads were aligned to the mouse genome (mm9) by using Taphat2 and peak calling were performed by homer 2. GC B cells were sorted from WT mice on dpi 13 post NP-KLH immunization. Cells were flash frozen immediately and process by Active Motif for IRF8 ChIP-Seq. Reads were aligned to mm9 by using BAM and peak calling were performed by using MACS. More details are provided in the manuscript.
Project description:Streptococcus pyogenes (S. pyogenes) ZUH1 was isolated and characterized using morphological, cultural and biochemical methods. The results showed that the marker genes (namely spyCEP, ssa, sic, sdaB and speG) indicating group A streptococci (GAS) were detected in the S. pyogenes genome. The results showed that the S. pyogenes strain was inhibited by Crocus sativus methanol extract (CSME), bee honey (BH) and catfish glycoprotein (CFG). The inhibitory activity of these natural agents were compared with standard antibiotics such as Ceftazidime (30 μg/mL), Cefoperazone (75 μg/mL), Cefoxitin (30 μg/mL) and Imipenem (10 μg/mL). There was a synergistic effect between certain antibiotics and CSME. GC-MS and IR analysis of CSME showed different cyclic ketones, aldehydes, esters, alcohols and acids. The main compounds were tetradecanoic acid, safranal and isophorone. Transmission electron microscopy (TEM) images of S. pyogenes cells treated with CSME showed signs of an irregular wrinkled outer surface, fragmentation, adhesion and aggregation of damaged bacterial cells or cellular debris. The marker genes (spyCEP, ssa, sic, sdaB and speG) could be used as a rapid diagnostic tool for GAS. CSME, BH and CFG showed distinctive anti-streptococcal activity either alone or in combinations with antibiotics; their action on S. pyogenes cells was studied by TEM. There was a synergistic effect between antibiotics and Crocus sativus, bee honey, and glycoprotein against S. pyogenes ZUH1. The action of natural agents on the pathogenic cells was shown using TEM.
Project description:Malaria remains a major challenge to global health causing extensive morbidity and mortality. Yet, there is no efficient vaccine and the immune response remains incompletely understood. Apical Membrane Antigen 1 (AMA1), a leading vaccine candidate, plays a key role during merozoite invasion into erythrocytes by interacting with Rhoptry Neck Protein 2 (RON2). We generated a human anti-AMA1-antibody (humAbAMA1) by EBV-transformation of sorted B-lymphocytes from a Ghanaian donor and subsequent rescue of antibody variable regions. The antibody was expressed in Nicotiana benthamiana and in HEK239-6E, characterized for binding specificity and epitope, and analyzed for its inhibitory effect on Plasmodium falciparum. The generated humAbAMA1 shows an affinity of 106-135 pM. It inhibits the parasite strain 3D7A growth in vitro with an expression system-independent IC50-value of 35 μg/ml (95% confidence interval: 33 μg/ml-37 μg/ml), which is three to eight times lower than the IC50-values of inhibitory antibodies 4G2 and 1F9. The epitope was mapped to the close proximity of the RON2-peptide binding groove. Competition for binding between the RON2-peptide and humAbAMA1 was confirmed by surface plasmon resonance spectroscopy measurements. The particularly advantageous inhibitory activity of this fully human antibody might provide a basis for future therapeutic applications.
Project description:Objective:Considering the emergence of biofilm-associated pathogens with multidrug resistance, the objective(s) of the present study was to evaluate the anti-virulence property of Syzygium aromaticum extracts/fractions against 2 multidrug-resistant catheter isolates. Materials and Methods:Pulverized clove buds were subjected to bioactivity-guided isolation to identify the bioactive extract/fraction(s) with significant anti-virulence property. The clove bud powder was subjected to Soxhlet extraction and all these extracts were investigated in terms of anti-virulent efficacy using initial readout assays. Bioassay-guided partial purification was performed through column and size exclusion chromatography. Detailed testing of the anti-virulence and anti-biofilm activity of sub-minimum inhibitory concentration (sub-MIC) levels of the active fraction, was performed besides the cytotoxicity profiling in RAW 264.7 cells. Results:Gas chromatography-mass spectrometry (GC-MS) analysis of the clove bioactive fraction-2 (CBF-2), done after the bioassay-guided fractionation, revealed eugenol as the major bioactive ingredient present in CBF-2. Reduced mRNA levels of virulence factor genes after CBF-2 (700 μg/ml) treatment correlated well with the respective phenotypic assays (p<0.001). Similarly, CBF-2 (700 μg/ml) treatment exhibited significantly low mRNA levels of quorum sensing (QS) receptor genes compared to their cognate synthase genes (p<0.001). Crystal violet staining and scanning electron micrographs of CBF-2-treated biofilms showed lesser macrocolonies with remarkably simplified architecture. Cytotoxic evaluation of CBF-2 suggested a minute reduction in viability even at the highest tested concentration (1600 μg/ml, p<0.05). Conclusion:The present study indicated that eugenol-rich CBF-2 has potent anti-virulence and anti-biofilm activity against urinary catheter isolates and can be regarded as an alternative for treatment of catheter-associated urinary tract infections.
Project description:Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.
Project description:An improved synthesis of biotinol-5'-AMP, an acyl-AMP mimic of the natural reaction intermediate of biotin protein ligase (BPL), is reported. This compound was shown to be a pan inhibitor of BPLs from a series of clinically important bacteria, particularly Staphylococcus aureus and Mycobacterium tuberculosis, and kinetic analysis revealed it to be competitive against the substrate biotin. Biotinol-5'-AMP also exhibits antibacterial activity against a panel of clinical isolates of S. aureus and M. tuberculosis with MIC values of 1-8 and 0.5-2.5 μg/mL, respectively, while being devoid of cytotoxicity to human HepG2 cells.
Project description:Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation. We investigated the role of the human GC receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells. Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t(1/2) = 8 ± 1 min versus 62 ± 17 min in DMSO-treated cells) and CCL7 mRNA (t(1/2) = 15 ± 4 min versus 114 ± 37 min), but not that of CCL5 mRNA (t(1/2)=231 ± 8 min versus 266 ± 5 min) in the BEAS-2B cell line. This effect was inhibited by preincubation with an anti-GR Ab, indicating that GR itself plays a role in the turnover of these transcripts. Coimmunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells. Association of the GR was localized to the 5' untranslated region of CCL2 mRNA and further mapped to nt 44-60. The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR. Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro. This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein.
Project description:Chimeric antigen receptor T cells (CAR-Ts) are promising cancer therapeutics. However, since cancer cells can lose the CAR-targeted antigen and avoid destruction, targeting multiple antigens with multiple CARs has been proposed. We illustrate here a less cumbersome alternative, anti-tag CARs (AT-CARs) that bind to tags on tumor-targeting antibodies. We have created novel AT-CARs, using the affinity-enhanced monomeric streptavidin 2 (mSA2) biotin-binding domain that when expressed on T cells can target cancer cells coated with biotinylated antibodies. Human T cells expressing mSA2 CARs with CD28-CD3? and 4-1BB-CD3? signaling domains were activated by plate-immobilized biotin and by tumor cells coated with biotinylated antibodies against the tumor-associated antigens CD19 and CD20. Furthermore, mSA2 CAR T cells were capable of mediating cancer cell lysis and IFN? production in an antibody dose-dependent manner. The mSA2 CAR is a universal AT-CAR that can be combined with biotinylated tumor-specific antibodies to potentially target many different tumor types.
Project description:Background: Cordyceps militaris is a medicinal mushroom and has been extensively used as a folk medicine in East Asia. In this study, the separation of constituents involved in xanthine oxidase (XO) inhibitory, antioxidant and antibacterial properties of C. militaris was conducted. Methods: The aqueous residue of this fungus was extracted by methanol and then subsequently fractionated by hexane, chloroform, ethyl acetate and water. The ethyl acetate extract possessed the highest XO inhibitory and antioxidant activities was separated to different fractions by column chromatography. Each fraction was then subjected to anti-hyperuricemia, antioxidant and antibacterial assays. Results: The results showed that the CM8 fraction exhibited the strongest XO inhibitory activity (the lowest IC50: 62.82 μg/mL), followed by the CM10 (IC50: 68.04 μg/mL) and the CM7 (IC50: 86.78 μg/mL). The level of XO inhibition was proportional to antioxidant activity. In antibacterial assay, the CM9 and CM11 fractions showed effective antibacterial activity (MIC values: 15⁻25 mg/mL and 10⁻25 mg/mL, respectively). Results from gas chromatography-mass spectrometry (GC-MS) analyses indicated that cordycepin was the major constituent in the CM8 and CM10 fractions. Conclusions: This study revealed that C. militaris was beneficial for treatment hyperuricemia although in vivo trials on compounds purified from this medicinal fungus are needed.
Project description:The importance of follicular T helper (TFH) cells and the germinal center (GC) reaction in the humoral immune response has become clear in recent years, however the role of TFH cells and the GC in an HIV vaccine strategy remains unclear. In this study, we primed mice with gp120-encoding DNA and boosted with gp120 protein, a regimen previously shown to induce high titers of high affinity and cross-reactive anti-gp120 Abs. Priming with gp120 DNA caused increased TFH cell differentiation, GC B cells, and antigen-specific antibody titers, compared with priming with gp120 protein. Priming with DNA also caused more activated CD4(+) T cells to become TFH cells and more GC B cells to become memory cells. Deletion of BCL6 midway through the vaccine regimen resulted in loss of TFH cells and GCs, and, unexpectedly, increased anti-gp120 IgG titers and avidity. Our data suggests vaccination with gp120-encoding DNA elicits a stronger and more rapid TFH and GC response than gp120 protein. Furthermore, we demonstrate that the GC reaction may actually limit antigen-specific IgG secretion in the context of repeated immunizations.