The Autoimmune Disorder Susceptibility Gene CLEC16A Restrains NK Cell Function in YTS NK Cell Line and Clec16a Knockout Mice.

CLEC16A locus polymorphisms have been associated with several autoimmune diseases. We overexpressed CLEC16A in YTS natural killer (NK) cells and observed reduced NK cell cytotoxicity and IFN-γ release, delayed dendritic cell (DC) maturation, decreased conjugate formation, cell-surface receptor downregulation and increased autophagy. In contrast, siRNA mediated knockdown resulted in increased NK cell cytotoxicity, reversal of receptor expression and disrupted mitophagy. Subcellular localization studies demonstrated that CLEC16A is a cytosolic protein that associates with Vps16A, a subunit of class C Vps-HOPS complex, and modulates receptor expression via autophagy. Clec16a knockout (KO) in mice resulted in altered immune cell populations, increased splenic NK cell cytotoxicity, imbalance of dendritic cell subsets, altered receptor expression, upregulated cytokine and chemokine secretion. Taken together, our findings indicate that CLEC16A restrains secretory functions including cytokine release and cytotoxicity and that a delicate balance of CLEC16A is needed for NK cell function and homeostasis.

Tumor-priming converts NK cells to memory-like NK cells.

Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity. Here, we provide substantial evidence that priming of naive human NK cells with pediatric acute B cell leukemia or acute myeloid leukemia specimens induces a functional conversion to tumor-induced memory-like (TIML)-NK cells displaying a heightened tumor-specific cytotoxicity and enhanced perforin synthesis. Cell cycles analyses reveal that tumor-priming sustainably alters the balance between NK cell activation and apoptosis in favor of survival. In addition, gene expression patterns differ between TIML- and cytokine-induced memory-like (CIML)-NK cells with the magnitude of regulated genes being distinctly higher in TIML-NK cells. As such, the tumor-induced conversion of NK cells triggers the emergence of a so far unacknowledged NK cell differentiation stage that might promote GvL effects in the context of adoptive cell transfer.

Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

Antibody-mediated inhibition of ricin toxin retrograde transport.

Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake. In this report, however, we describe a neutralizing monoclonal antibody (MAb) against ricin's binding subunit (RTB) that not only associates with ricin after the toxin has bound to the cell's surface but actually enhances toxin uptake into host cells. Following endocytosis, the antibody-toxin complexes are then routed for degradation. The results of this study are important because they reveal a previously unappreciated role for B-subunit-specific antibodies in intracellular neutralization of ricin toxin.

Neutralizing activity and protective immunity to ricin toxin conferred by B subunit (RTB)-specific Fab fragments.

SylH3 and 24B11 are murine monoclonal antibodies directed against different epitopes on ricin toxin's binding (RTB) subunit that have been shown to passively protect mice against ricin challenge. Here we report that Fab fragments of SylH3 and 24B11 neutralize ricin in a cell based assay, and in a mouse challenge model as effectively as their respective full length parental IgGs. These data demonstrate that immunity to ricin can occur independent of Fc-mediated clearance.

FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

Sub-domains of ricin's B subunit as targets of toxin neutralizing and non-neutralizing monoclonal antibodies.

The B subunit (RTB) of ricin toxin is a galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, ß, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicity assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2ß, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB's high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB's sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

Immunity to ricin: fundamental insights into toxin-antibody interactions.

Ricin toxin is an extraordinarily potent inducer of cell death and inflammation. Ricin is also a potent provocateur of the humoral immune system, eliciting a mixture of neutralizing, non-neutralizing and even toxin-enhancing antibodies. The characterization of dozens of monoclonal antibodies (mAbs) against the toxin's enzymatic (RTA) and binding (RTB) subunits has begun to reveal fundamental insights into the underlying mechanisms by which antibodies neutralize (or fail to neutralize) ricin in systemic and mucosal compartments. This information has had immediate applications in the design, development and evaluation of ricin subunit vaccines and immunotherapeutics.

Protective immunity to ricin toxin conferred by antibodies against the toxin's binding subunit (RTB).

The B subunit (RTB) of ricin toxin is a galactose-/N-acetyl galactosamine-specific lectin that promotes attachment and entry of ricin into host cells. RTB is also the archetype of the so-called R-type lectin family, whose members include haemagglutinins of botulinum neurotoxin (BoNT) progenitor toxins, as well as the binding subunits of cytolethal distending toxins. Although RTB is an appealing subunit vaccine candidate, as well as a potential target for immunotherapeutics, the degree to which RTB immunization elicits protective antibodies against ricin toxin remains unresolved. To address this issue, groups of mice were immunized with RTB and then challenged with 5×LD(50)s of ricin administered intraperitoneally. Despite high RTB-specific serum antibody titers, groups of RTB immunized mice were only partially immune to ricin challenge. Analysis of a collection of RTB-specific B cell hybridomas suggested that only a small fraction of antibodies against RTB have demonstrable neutralizing activity. Two RTB-specific neutralizing monoclonal IgG(1) antibodies, 24B11 and SylH3, when passively administered to mice, were sufficient to protect the animals against a 5×LD(50) dose of ricin. Both 24B11 and SylH3 blocked ricin attachment to terminal galactose residues and prevented toxin binding to the surfaces of bone marrow-derived macrophages (BMM), suggesting that they function by steric hindrance and recognize epitopes located on RTB's carbohydrate recognition sub-domains (1α or 2γ). These data raise the possibility of using specific RTB sub-domains, rather than RTB itself, as antigens to more efficiently elicit neutralizing antibodies and protective immunity against ricin.

Identification of ciprofloxacin resistance by SimpleProbe, High Resolution Melt and Pyrosequencing nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis.

The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro(r)) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe and High Resolution Melt (HRM) dye chemistries and Pyrosequencing genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe and Pyrosequencing successfully identified all known mutants, the HRM assay identified all but those resulting from G<-->C or A<-->T substitutions.