text:
Protein chlorination
Preparation of bead-conjugated Ags
B16.F10 melanoma cells
Vaccination of mice with DCs pulsed with chlorinated B16.F10 melanoma cells
DCs and macrophage culture
Splenic DCs
Ag presentation assays
In vivo Ab responses
Preparation of fluorochrome-labeled Ags
Ag uptake in vitro
Ag uptake in vivo
Ag digestion
Separation of proteins by SDS-PAGE
HOCl modification facilitates processing and presentation to both CD4 and CD8 T cells
Chlorinated OVA enhances the Ab response in vivo
Enhancement of OVA via chlorination is not mediated via TLRs
Enhancement of OVA via chlorination is not mediated via aldehyde or chloramine functional groups, but requires the carbohydrate side chain
Chlorination enhances APC uptake of OVA
Chlorination facilitates proteolysis of OVA
Chlorination enhances APC binding of OVA via specific receptor(s)
Hypochlorous Acid: A Natural Adjuvant That Facilitates Antigen Processing, Cross-Priming, and the Induction of Adaptive Immunity
Zofia M. Prokopowicz;
Zofia M. Prokopowicz
*Division of Infection and Immunity, University College London, London, United Kingdom;
Frederick Arce;
Frederick Arce
Rafal Biedron;
Rafal Biedron
†Department of Immunology, Jagiellonian University Medical College; and
Cheryl L.-L. Chiang;
Cheryl L.-L. Chiang
Marta Ciszek;
Marta Ciszek
David R. Katz;
David R. Katz
Maria Nowakowska;
Maria Nowakowska
‡Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Kraków, Poland
Szczepan Zapotoczny;
Szczepan Zapotoczny
Janusz Marcinkiewicz;
Janusz Marcinkiewicz
Benjamin M. Chain
Address correspondence and reprint requests to Prof. Benjamin M. Chain, Division of Infection and Immunity, Windeyer Building, University College London, 46 Cleveland Street, London W1T4JP, U.K. E-mail address: B.chain@ucl.ac.uk.
Received: August 10 2009
Accepted: November 16 2009
Published: December 27 2009
Copyright © 2010 by The American Association of Immunologists, Inc.
© 2010 by The American Association of Immunologists, Inc.
J Immunol (2010) 184 (2): 824–835.
https://doi.org/10.4049/jimmunol.0902606
Zofia M. Prokopowicz, Frederick Arce, Rafal Biedron, Cheryl L.-L. Chiang, Marta Ciszek, David R. Katz, Maria Nowakowska, Szczepan Zapotoczny, Janusz Marcinkiewicz, Benjamin M. Chain; Hypochlorous Acid: A Natural Adjuvant That Facilitates Antigen Processing, Cross-Priming, and the Induction of Adaptive Immunity. J Immunol 15 January 2010; 184 (2): 824–835. https://doi.org/10.4049/jimmunol.0902606
The production of hypochlorous acid (HOCl) is a characteristic of granulocyte activation, a hallmark of the early phase of innate immune responses. In this study, we show that, in addition to its well-established role as a microbicide, HOCl can act as a natural adjuvant of adaptive immunity. HOCl enhances the T cell responses to the model Ag OVA, facilitating the processing and presentation of this protein via the class II MHC pathway. HOCl modification also enhances cross-presentation of the tumor Ag tyrosinase-related protein 2 via class I MHC. The adjuvant effects of HOCl are independent of TLR signaling. The enhanced presentation of HOCl-modified OVA is mediated via modification of the N-linked carbohydrate side chain rather than formation of protein aldehydes or chloramines. HOCl-modified OVA is taken up more efficiently by APCs and is degraded more efficiently by proteinases. Atomic force microscopy demonstrated that enhanced uptake is mediated via specific receptor binding, one candidate for which is the scavenger receptor lectin-like oxidized low-density lipoprotein receptor, which shows enhanced binding to chlorinated OVA. A function of HOCl is therefore to target glycoprotein Ags to scavenger receptors on the APC surface. This additional mechanism linking innate and adaptive immunity suggests novel strategies to enhance immunity to vaccines.
Innate immunity regulates the induction of adaptive immune responses via multiple interactions. The paradigm is based on the regulation of APC maturation and migration via ligation of pattern recognition receptors, but other examples include the ability of complement fragments to enhance B cell activation via CD21 (1) or direct effects of TLR ligation on lymphocyte activation (2). Typically, an innate immune response precedes and is necessary for strong and persistent adaptive immunity. In the absence of innate immunity, most Ags stimulate a weak and transient adaptive immune response, and this may result in tolerance rather than protection. Recent studies also suggest that many classical adjuvants work by promoting the innate/adaptive axis (3). A more complete understanding of the complex interaction between these two components is therefore a key to understanding and manipulating the vertebrate immune response.
Dendritic cells (DCs) play a pivotal role in facilitating the interaction between innate and adaptive immunity. DC uptake (4), processing (5), and loading of Ag onto MHC molecules, transport of Ag via migration from tissues to local lymph nodes or spleen (6), and finally presentation and costimulation of T cells all can be regulated by signals from innate immunity. Although earlier studies of innate immune regulation focused primarily on the regulation of migration and costimulation, more recent studies have focused on the earlier stages of uptake, processing, and MHC loading. DC maturation is accompanied by a complex sequence of changes in the Ag processing machinery (7), and recent studies have suggested that TLR signaling in DCs may be a prerequisite for efficient Ag loading (8). The response of the DCs to inflammatory cytokines can result in qualitative as well as quantitative changes in Ag processing, determining both the strength and the repertoire of the ensuing adaptive immune response (9).
There are several mechanisms by which innate immunity regulates DC Ag processing. At the site of infection, DCs are activated directly by ligation of their pattern recognition receptors by pathogen associated molecular patterns as well as by a variety of pro- and anti-inflammatory chemokines and cytokines. However, because activated neutrophils are recruited rapidly to most inflammatory sites, they are a likely component of the DC microenvironment early in immune activation. A major protein constituent of the neutrophil, found in the primary azurophil granules, is myeloperoxidase, an enzyme that catalyses the conversion of hydrogen peroxide to hypochlorous acid (HOCl) and to a lesser extent to hypobromous acid (10). These unstable strong oxidants were believed previously to act predominantly as bactericidal agents within the phagolysosome, but there is now extensive evidence that the enzyme and its products also act extracellularly, resulting in very specific oxidative modifications to lipids (11), enzymes (12), and extracellular matrix (13), all of which are components in the inflammatory microenvironment. Chlorinated lipids have been shown previously to induce DC maturation (14).
In this study, we identify a mechanism whereby protein chlorination targets glycoproteins to scavenger receptors on the APC, promoting Ag uptake and processing by APCs such as DCs. Therefore, we propose that in an inflammatory/immune milieu HOCl derived from neutrophils may represent a natural innate immune-derived adjuvant that can promote induction of adaptive immunity.
Six- to 8-wk-old C57BL/6 and BALB/c mice were obtained from Harlan (Bicester, U.K.) and maintained by Biological Services, University College London, London, U.K. Spleen cells from TCR transgenic mice expressing the OTII anti-OVA receptor were a kind gift from Prof. M. Collins (University College London, London, U.K.). Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)/MyD88 double-knockout mice were obtained from Cancer Research UK (London, U.K.). All of these experiments were carried out under U.K. Animal Project License authorization. In vivo Ab responses were determine using 8- to 10-wk-old BALB/c female mice housed at the Department of Immunology, Jagiellonian University Medical College, Kraków, Poland. The experimental procedures used were approved by the local Jagiellonian University Ethical Committee on Animal Experiments.
OVA (grade V; Sigma-Aldrich, Poole, U.K.) (2 mg/ml), endotoxin-free OVA (Endograd, Profos, Germany) (2 mg/ml), or hen egg lysozyme (HEL) (Boehringer Mannheim, Mannheim, Germany) (0.5–2 mg/ml) was dissolved in HBSS and mixed with NaOCl (Sigma-Aldrich) at various molar ratios. The reaction was incubated at 37°C for 1 h. The NaOCl concentration was determined before each reaction by absorbance measurements at 292 nm using a molar extinction coefficient of 350 M−1 cm−1. The modified protein was purified and transferred into cell culture medium by passage over a PD-10 Sephadex G-25 column (GE Healthcare, Chalfont, U.K.) according to the manufacturer's instructions. In some experiments, the chlorinated protein was reduced by addition of 50 mM NaBH4 (Sigma-Aldrich) and further incubation for 1 h at 37°C. Alternatively, protein chloramines were converted back to free amines by addition of 30 mM l-methionine (Sigma-Aldrich) and incubation for 30 min at room temperature. Samples were stored in −20°C. Carbonyl groups were quantified by reaction with 2,4-DNP‑hydrazine (Camlab, Cambridge, U.K.) (15). Amino groups were quantified by reaction with 2,4,6-trinitrobenzenesulfonic acid (Fluka, Sigma-Aldrich).
OVA and OVAClI were adsorbed onto fluorescent beads (1-μm sulfate microspheres, red fluorescent 580/605, Molecular Probes/Invitrogen, Eugene, OR) according to the manufacturer's instructions. Presence of the protein on the beads was confirmed by Western blot analysis.
The C57BL/6-derived mouse melanoma B16, subline F10, which expresses tyrosinase-related protein 2 (Trp2) was a kind gift from Dr. Jonathan Silk (Tumor Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, U.K.) and was cultured in complete RPMI 1640 medium at 37°C and 5% CO2.
B16.F10 melanoma cells (8 × 105 cells per milliliter) were incubated in the presence of HOCl (60 μM) in HBSS for 1 h at 37°C and excess HOCl removed by washing twice with HBSS. In some experiments, B16.F10 cells were incubated in HBSS at 56°C for 30 min and then washed twice with HBSS. Both procedures induced 100% cell death as measured by propidium iodide or trypan blue staining.
Bone marrow DCs were cocultured with B16.F10 cells (chlorinated or heat-killed) at a ratio of 1:1 or with medium alone for 24 h at 37°C and 5% CO2. The DCs were harvested, washed twice, and resuspended in HBSS. Each C57BL/6 mouse was injected in the tail vein with DCs (106 cells per mouse). Two weeks after injection, the mice were sacrificed, and the spleen cells were cultured with chlorinated B16.F10 (at a ratio of 1 tumor cell to 10 spleen cells) or medium without Ag for a further week in vitro. Viable splenocytes (>80% T cells, 106 cells per well) were evaluated for IFN-γ ELISpot (Millipore, Bedford, MA) responses against chlorinated B16.F10 cells (105 cells) or a peptide coding the major Trp2 CD8 epitope (1 μM) or medium alone.
The synthetic peptides coding for OVA 323–339 (ISQAVHAAHAEINEAGR) recognized by DO11.10 T cells and OTII cells, OVA 273–288 (MEERKIKVYLPRMKME) recognized by 3DO18.3 T cells, OVA 257–278 (SIINFEKLTEWTSSNVMEERKI) recognized by MF2.D9 T cells, and Trp2 (SVYDFFVWL) were synthesized and purified by the Cancer Research UK peptide synthesis facility.
Bone marrow cells were isolated from hind leg femurs and tibias of one mouse, and a single-cell suspension of bone marrow cells was plated in a six-well plate. The bone marrow cells were cultured in IMDM (Invitrogen) supplemented with 10% FCS (PAA Laboratories, Haidmannwerg, Austria), 5 × 10−5 M 2-ME (Life Technologies, Paisley, U.K.), 100 U/ml penicillin/streptomycin (Sigma-Aldrich), and 20 ng/ml rGM-CSF (PeproTech, Rocky Hill, NJ). On day 4, the rGM-CSF was renewed. On day 7 of culture, cells were harvested and purified by positive selection using magnetic CD11c+ beads according to the manufacturer's instructions (MicroBeads; Miltenyi Biotec, Auburn, CA).
Bone marrow-derived macrophages were cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and 10% supernatant from L929 cells, which was changed on day 3 of culture. On day 6, media was replaced with fresh DMEM supplemented with 10 ng/ml IFN-γ (PeproTech). After 2 d of culture, cells were harvested and used immediately.
Fresh spleens were obtained from mice injected previously with FITC-OVA, FITC-OVAClI, or HBSS and diced. Spleen fragments were incubated in collagenase type II (200 μg/ml; Sigma-Aldrich) and DNase I (500 μg/ml; Sigma-Aldrich) for 30 min at room temperature. Collagenase activity was stopped with EDTA (10 mM, 5 min; Sigma-Aldrich). The single-cell suspension was treated with RBC lysis buffer (Hybri-Max; Sigma-Aldrich) for 3 min at room temperature, washed twice with HBSS, and purified by positive selection using magnetic CD11c+ beads according to the manufacturer's instructions (MicroBeads; Miltenyi Biotec).
OVA-specific T cell hybridomas DO11.10, 3DO18.3, and MF2.2D9 (kind gift from Dr. K. Rock, University of Massachusetts, Worcester, MA) were cultured in DMEM supplemented with 10% FCS and 100 U/ml penicillin/streptomycin. The HEL T cell hybridoma Ad71–85 (kind gift from Dr. Eli Sercarz, University of California, Los Angeles, Los Angeles, CA) was cultured in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and 5 × 10−5 M 2-ME. CTLL-2 indicator cells were cultured in IMDM supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, 5 × 10−5 M 2-ME, and 10 ng/ml IL-2 (PeproTech). OTII cells were cultured from spleens of mice expressing the OTII TCR (16). Single-cell suspensions of spleen cells were cultured for 24 h in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, 5 × 10−5 M 2-ME, and 250 nM OVA 323–339 peptide and then expanded for 6 d in complete RPMI 1640 supplemented with 10 ng/ml IL-2. The cells were stored at −80°C until required. Before stimulation, cells were thawed rapidly and washed, then used immediately.
OVA-specific T cell hybridomas (2 × 104 cells per well) or HEL-specific hybridomas (5 × 104 cells per well) were cultured with purified DCs (5 × 103 or 104 cells per well) or with macrophages (104 cells per well) in the presence of Ag at the indicated concentrations. For some experiments, DCs (105 cells) were precultured with Ag in polypropylene tubes for different times. The excess Ag was removed by washing twice in HBSS, and the DCs were fixed with 0.05% glutaraldehyde (Sigma-Aldrich) for 30 s. Excess glutaraldehyde was removed by two more washes, and different numbers of fixed DCs were cocultured with T cell hybridomas (2 × 104 cells per well). After 24 h, the supernatants were harvested and frozen at −20°C. IL-2 in the supernatants was measured using the IL-2–sensitive CTLL-2 indicator cells as described (17). TCR transgenic T cells (2 × 104 OT-II cells per well), expanded as described above, were cultured with purified DCs (5 × 103 cells per well) in the presence of the Ag at the indicated concentrations. For some experiments, Ag was preincubated for 30 min at room temperature with 1 μg/ml polymyxin B (Invitrogen). Thymidine incorporation was measured after 18 h as described (18).
Mice were immunized with OVA or OVAClI (200 μg, LPS-free; Sigma-Aldrich) either alone or with LPS (1 μg per mouse) (Sigma-Aldrich) by i.p. injection (primary immunization). On day 14, all mice received 100 μg OVA and 1 μg LPS via i.p. injection (boost). After an additional 5–7 d, mice were sacrificed, and sera were tested by ELISA for specific IgG anti-native OVA Abs.
OVA and OVAClI were transferred to 0.1 M sodium carbonate buffer by passage over a PD-10 Sephadex G-25 column. FITC (2 mg/ml) (Sigma-Aldrich) and tetramethylrhodamine isothiocyanate (TRITC) (6 mg/ml) (Fluka) in anhydrous DMSO (Acros Organics, Geel, Belgium) were prepared fresh. Fluorochromes were added slowly to the protein (50 μl per milligram of protein) and left for 4 h at room temperature in the dark. The unconjugated fluorochromes were removed by passage over a PD-10 Sephadex G-25 column to HBSS buffer. Dye-to-protein molar ratios were determined by absorbance measurements at 493 nm using a molar extinction coefficient of 72,000 M−1 cm−1 for FITC and at 555 nm using a molar extinction coefficient of 65,000 M−1 cm−1 for TRITC. FITC-OVA, TRITC-OVA, FITC-OVAClI, and TRITC-OVAClI were stored at −20°C.
Purified bone marrow-derived DCs were incubated with different doses of FITC-OVA or FITC-OVAClI for the indicated time. Excess of Ag was removed by washing samples twice in ice-cold HBSS. Cells were fixed with 3.8% formaldehyde and analyzed on a FACScan (BD Biosciences, San Jose, CA) flow cytometer using CellQuest software or on a Leica confocal microscope (Leica Microsystems, Deerfield, IL) using the manufacturer's software.
C57BL/6 mice were injected i.v. with 1 mg of FITC-OVA, or FITC-OVAClI or an equal volume of HBSS. Spleens were collected at various time points, and CD11c+ DCs were purified as described above. DCs were stained for CD8a expression and then analyzed by flow cytometry.
Atomic force microscopy (AFM) tips were modified with a mixture of sulfonate-terminated thiol (sodium 2-mercapto-[1H]benzo[d]imidazole-5-sulfonate) and OVA or OVAClI. Force–distance measurements were performed using Veeco PicoForce AFM (Santa Barbara, CA) by cycling the sample on a piezo scanner over 500–1000 nm in the vertical direction, varying the speed of the movement (loading rate) and the time that tips stay in contact with the surface (contact time). The final measurements were performed for the optimized loading rate (1000 nm/s) and contact time (1 s). If not specified, then all of the measurements were performed in PBS. The curves were measured on different areas of the macrophages immobilized on glass and on the glass surface itself as negative controls. One hundred to 300 curves were collected on each spot; the data were processed and averaged for the number of spots. The measurements for each condition were performed at least twice, changing the tip and the set of cells. For the given set of the curves, the adhesion probability was determined, where
adhesion probability=number of curves with adhesion/number of all curves
Digestion was performed at 37°C for 18 h. For trypsin digestion, OVA or OVAClI (20 μg) was incubated with sequencing grade modified trypsin (1 μg) (Promega, Madison, WI) in 50 mM Tris-HCl and 10 mM CaCl2 (pH 7.8). For cathepsin E digestion, OVA or OVAClI (10 μg) was incubated with cathepsin E (R&D Systems, Minneapolis, MN) (10 ng) in 0.1 M NaOAc and 0.1 M NaCl (pH 3.5). For endoglycosidase F (PNGase F) digestion, OVA or OVAClI (10 μg) was incubated with PNGase F (BioLabs) (1000 U) in G7 reaction buffer (New England BioLabs, Hitchin, U.K.).
Proteins were separated using 4–12% NuPAGE Novex Bis-Tris minigels (Invitrogen). Samples were reduced by heating at 95°C for 5 min. Bands were visualized using silver (19) or Coomassie brilliant blue staining.
HOCl can cause a variety of covalent modifications to proteins (20, 21). One important reaction, which is straightforward to monitor, is the conversion of free α and ε amino side chains to the respective chloramines (Supplemental Fig. 1, Equation 1). These chloramines can be converted subsequently into free aldehydes (Supplemental Fig. 1, Equation 2) and ultimately oxidized irreversibly to carboxylic acids.
The model protein Ag OVA was reacted with various ratios of HOCl, and the extent of OVA modification was monitored by measuring the disappearance of free amines (Supplemental Fig. 1A) and the appearance of aldehydes (Supplemental Fig. 1B). Increasing amounts of HOCl induced a dose-dependent decrease in free amino groups. Concentrations of HOCl inducing a low (OVAClL), intermediate (OVAClI), or high (OVAClH) degree of free amino modification were tested in subsequent functional experiments. Increasing concentrations of HOCl also induced the appearance of free carbonyl groups, although these were produced at low frequency (maximum of 3 mol carbonyl/mol protein protein) and were only seen in OVAClI or OVAClH.
The effects of chlorination were tested initially in the well-characterized DO11.10 T cell hybridoma model (Fig. 1A). This hybridoma responds to intact OVA only after processing and presentation. DO11.10 responds poorly to native OVA, except at very high concentrations (>20 μM). In contrast, OVAClI induced a DO11.10 response even at concentrations of 0.2 μM. Both OVAClL and OVAClH were presented poorly. Efficient presentation of processed OVAClI could be detected after a lag phase of 2–3 h (Fig. 1B).
Modification with HOCl enhances the processing of OVA in vitro in an epitope-selective manner. A, DO11.10 hybridoma cells (which recognize the epitope sequence shown above the graph) were incubated with different concentrations of OVA or chlorinated OVA and bone marrow-derived DCs as described in 1Materials and Methods. The graph shows IL-2 release measured as thymidine incorporation of the CTLL-2 indicator cell line. B, OVAClI (250 μg/ml) was incubated with DCs for 1 or 3 h. Excess Ag was removed, and the DCs were fixed with glutaraldehyde, washed, and cocultured with DO11.10 hybridoma cells as in A. The graph shows IL-2 release measured as thymidine incorporation of the CTLL-2 indicator cell line. C, TCR transgenic OTII T cells were incubated with different concentrations of OVA or OVAClI and bone marrow-derived DCs as described in 1Materials and Methods. The graph shows OTII proliferation measured as thymidine incorporation. D, As for A, but using two other T cell hybridomas specific for different epitopes of OVA. E, DO11.10 hybridoma cells were incubated with different concentrations of OVA or OVAClI and bone marrow-derived macrophages as described in 1Materials and Methods. The graph shows IL-2 release measured as thymidine incorporation of the CTLL-2 indicator cell line. F, As for E, but using OVA or OVAClI adsorbed to 1-μm beads. Results are presented as the average ± SD [3H]thymidine incorporation of triplicate cultures. Experiments were repeated twice (B, E, F), three times (A, D), or more (C). G, Bone marrow-derived DCs were cocultured with chlorinated B16 melanoma cells, heat-killed B16 cells, or medium and then injected i.v. into C57BL mice (106 cells per mouse). Spleen cells were collected after 7 d, restimulated with medium or chlorinated B16 cells, and then assayed for IFN-γ responses to chlorinated B16 cells, Trp2 peptide (1 μg/ml), or medium by ELISpot as shown. The figure shows mean and SD of ELISpot values from triplicate wells from one representative experiment of three experiments.
To exclude the possibility that this result was peculiar to the DO11.10 hybridoma, the experiment was repeated with T cells from the TCR transgenic mouse, OTII, which recognize the same sequence of OVA as DO11.10 (323–339), presented by I-Ab instead of I-Ad, and with a 200-fold greater sensitivity (Fig. 1C). In this case, native OVA processing could be detected at concentrations of 2 μM or above. However, as for DO11.10, processing of OVAClI was at least 10-fold more efficient (Fig. 1C), whereas OVAClH was processed and presented poorly (not shown). To exclude the possibility that the enhancement was due to a direct effect of traces of HOCl remaining in the chlorinated OVA preparations, DCs were treated directly with 5 μM HOCl. At these concentrations, HOCl was not toxic to the cells, but failed to induce any enhancement in Ag processing of OVA (Supplemental Fig. 2). Higher concentrations induced cellular cytotoxicity.
Although both DO11.10 and OTII showed a large increase in response to chlorinated OVA, this effect was not universal for all of the OVA epitopes (Fig. 1D). Indeed, the responses of two other T cell hybridomas, MF2.D9 (specific for OVA 273–288) and 3DO18.3 (specific for OVA 257–278), were abrogated almost completely by chlorination even at intermediate levels. Therefore, chlorination of OVA modifies the T cell response in an epitope-specific manner.
There is considerable evidence that particulate Ags are taken up and processed via different pathways from soluble Ags. Therefore, to compliment these studies on soluble Ags, the influence of chlorination was explored using a particulate Ag model. OVA or OVAClI was coated onto 1 μm beads. To facilitate the removal of excess beads before the addition of T cells, these experiments were carried out with macrophages rather than DCs. These experiments confirmed that the enhancement was not DC specific, because chlorination enhanced the processing and presentation of both soluble and bead-associated OVA by macrophages (Fig. 1E, 1F).
All of the T cells tested above are CD4+ and recognize Ags after processing and loading onto class II MHC molecules. We wished to explore whether chlorination also facilitated cross-presentation via class I MHC molecules. However, preliminary experiments showed that the RF33 CD8 OVA-specific hybridoma behaved like the 3DO18.3 CD4 hybridoma (Fig. 1D) and did not recognize chlorinated OVA. The SIINFEKL epitope recognized by RF33 is within that recognized by the 3DO18.3 CD4 hybridoma and appears to be destroyed by chlorination.
Therefore, we used a different model for these cross-presentation experiments and studied the response to melanoma Ag Trp2, for which a CD8 class I epitope has been defined previously. DCs were incubated with B16.F10 melanoma cells expressing the Trp2 Ag (Supplemental Fig. 3), and injected i.v. into H2b mice. Two weeks later, splenic T cells were harvested and cultured in vitro, and the CD8 T cell response was measured by IFN-γ ELISpot (Fig. 1G). T cells from mice immunized with HOCl-modified B16.F10 cells, but not with B16.F10 cells killed by heat treatment or treatment with HCl (not shown) showed strong responses to the cross-presented Trp2 peptide coding for the major CD8 H2b epitope. Therefore, chlorination facilitates both direct processing of exogenous Ags to CD4 cells and cross-presentation of cell-associated Ag Trp2 to CD8 T cells.
Because chlorination enhanced presentation of some OVA epitopes to CD4 cells in vitro, we next explored the effect of this process on OVA Ab responses, which are CD4 T cell dependent. Because many previous studies have demonstrated that for an efficient Ab response an innate immune stimulant or adjuvant is required (22), mice were primed with OVA/OVAClI, with and without LPS. As expected, OVA without LPS stimulated a small IgG response (Fig. 2A). In contrast, OVAClI stimulated a significant Ab response, even in the absence of LPS. Immunization with OVA and LPS stimulated a much stronger response than OVA alone, and this was enhanced further by moderate chlorination (Fig. 2B). Immunization with OVAClH induced very little Ab response even in the presence of LPS. Interestingly, chlorination induced only a moderate (∼2-fold) increase of anti-OVA IgG1, which is the major default isotype in BALB/c mice (Fig. 2C), whereas the Ig2a OVA-specific response is enhanced >10-fold (Fig. 2D). The increased Ig2a/IgG1 ratio is characteristic of a Th2/Th1 shift.
Modification with HOCl enhances the anti-OVA Ab response in vivo. Groups of five mice (BALB/c) were immunized i.p. with OVA or OVAClI (200 μg) either alone (A) or with LPS (B–D) (1 μg per mouse) (primary immunization). On day 14, all of the mice were boosted with 100 μg of OVA and 1 μg of LPS. After an additional 5–7 d, mice were sacrificed, and sera were tested by ELISA for specific IgG anti-native OVA Abs. A and B show total IgG titers, whereas C and D show IgG1 and IgG2a isotype responses, respectively. All of the graphs are in arbitrary units in comparison with a standard anti-OVA Ig reference sample. p values for unmodified OVA versus OVAClI: A, p < 0.08; B, p < 0.002; C, p < 0.17; D, p < 0.04 (Student t test, n = 5).
There is considerable literature that emphasizes the need for cooperative stimulation of immunity between TLR ligands such as LPS and cognate Ags such as OVA. Trace amounts of TLR ligands, which are often found as contaminants of Ag preparations, may be sufficient to influence the outcome. Therefore, it is possible that chlorination might act via enhancing either the TLR response or the sensitivity of the OVA response to TLR ligation. Alternatively, chlorination might bypass the need for the TLR component. These alternative hypotheses were addressed in a number of ways.
Coculture of either OVA or OVAClI with DCs was used as a sensitive bioassay of LPS activity. Neither OVA nor OVAClI induced DC maturation (Fig. 3A), confirming the absence of any biologically active TLR ligand in the preparations.
HOCl adjuvant activity is independent of TLR signaling. A, Bone marrow DCs were incubated for 18 h with OVA or OVAClI (20 μg/ml, endotoxin-free) or LPS (100 ng/ml) and then analyzed for CD86 and MHC class II expression by flow cytometry. One representative experiment from three independent experiments is shown. B, TCR transgenic OT-II T cells were incubated with different concentrations of standard (grade V; Sigma-Aldrich) or endotoxin-free OVA or OVAClI and bone marrow-derived DCs as described in 1Materials and Methods. The graph shows OTII proliferation measured as thymidine incorporation. One representative experiment from three independent experiments is shown. C, As for B, but cultures were performed in the presence or absence of polymyxin B (1 μg/ml). D, As for B, but Ag presentation assays were carried out using DCs from TRIF/MyD88 double-knockout mice. Results are presented as the average ± SD [3H]thymidine incorporation of triplicate cultures. The experiment was repeated twice.
Next, the functional experiments were repeated with endotoxin-free OVA. However, the enhancement of the T cell response due to chlorination was equivalent irrespective of whether this endotoxin-free OVA or the conventional preparation was used (Fig. 3B). Furthermore, the enhancement was not affected by the addition of polymyxin B, an inhibitor of LPS (Fig. 3C). Finally, we tested OVA and OVAClI using DCs isolated from TRIF/MyD88 double-knockout mice (Fig. 3D), in which both major signaling TLR pathways are blocked. The absence of these two key TLR downstream signaling adaptors did not affect the enhanced processing and presentation of the chlorinated OVA.
Because HOCl-dependent enhancement of Ag processing was not mediated via TLRs, we examined other possible mechanisms that might underlie the phenomenon. One such mechanism is protein modification by introduction of aldehyde groups, which can enhance both uptake and processing (23, 24). As shown in Supplemental Fig. 1, HOCl treatment does result in the introduction of aldehyde groups via amine oxidation. However, reduction of aldehydes in OVAClI by treatment with NaBH4 (Fig. 4A) did not reverse the enhancement in T cell response (Fig. 4C). Thus, aldehyde groups were not required for HOCl-induced enhancement of processing.
HOCl adjuvant activity does not require aldehyde or chloramine groups. A, Reaction with NaBH4, but not methionine reduces the number of carbonyl groups on OVAClI. Mean ± SEM from three or more experiments. The carbonyl levels of OVAClI are significantly different from those of both OVA and OVAClI treated with NaBH4 (p < 0.01, Student t test). B, Reaction with methionine, but not NaBH4 restores free amine groups on OVAClI. Mean ± SEM from three or more experiments. The amine levels of OVAClI are significantly different from those of both OVA, and OVAClI treated with methionine (p < 0.01, Student t test). C, OVAClI was reacted with NaBH4 or methionine as in A and B and then tested for processing and presentation in the OTII model as described in Fig. 1C. Results are presented as the average ± SD [3H]thymidine incorporation (cpm) of the triplicate cultures. Experiment was repeated three times (C).
Chloramines, another major product of protein chlorination, were also not required, because reduction of chloramines back to amines by methionine did not reverse the enhanced T cell response (Fig. 4C). The number of free accessible amines in OVAClI after methionine reduction was greater than that for native OVA, suggesting that oxidation altered protein confirmation and allowed access to previously inaccessible amine side chains.
The OVA protein contains a single N-linked carbohydrate side chain at asparagine 293. The side chain is made up principally of mannose and N-acetyl glucosamine residues (25). More recent studies also suggest the presence of some terminal sialic acid residues (26). N-Acetyl glucosamine is a major target for HOCl modification, leading to the production of a whole variety of oxidized sugar products (27). Therefore, we explored the possibility that a component of the carbohydrate side chain was important for the adjuvant effects of HOCl. Both OVA and OVAClI were treated with PNGase F, which removes the entire carbohydrate side chain (Supplemental Fig. 4). Enzyme treatment had no effect on the presentation of native OVA or OVA peptide, but abolished the enhancement that resulted from chlorination (Fig. 5A, middle panel). In contrast, treatment with neuraminidase (which selectively removes sialic acid residues) had no effect on presentation of either OVA or OVAClI (not shown).
HOCl adjuvant activity requires the OVA carbohydrate side chain. A, OVA, OVAClI, or OVA 323–339 peptide was treated with PNGase F and then tested for processing and presentation in the OTII model as described in Fig. 2C. Results are presented as the average ± SD [3H]thymidine incorporation of triplicate cultures. The experiment was repeated twice. B, HEL was oxidized at low, intermediate, and high concentrations of HOCl and tested using the lysozyme-specific hybridoma Ad71–85 as described in 1Materials and Methods. The graph shows IL-2 release measured as thymidine incorporation of the CTLL-2 indicator cell line. The experiment was repeated three times.
To confirm the importance of the carbohydrate side chain, we repeated the chlorination experiments using a nonglycosylated protein Ag, lysozyme (Fig. 5B). Chlorination at low and medium ratios had no effect on presentation of lysozyme to a lysozyme-specific hybridoma. Similarly to OVA, chlorination using a high ratio of HOCl decreased the response.
The chemical modification of OVA, and specifically of its carbohydrate side chain, might alter the handling of Ag by the APC. We therefore examined the uptake of OVAClI by DCs. Bone marrow DCs were incubated with different concentrations of FITC-labeled OVA and OVAClI for 2 h (Fig. 6A). Chlorination enhanced uptake over a wide range of Ag concentrations. Confocal analysis of the DCs confirmed that both labeled OVA and OVAClI were taken up into endocytic vesicles inside the cell (Fig. 6B). Images of simultaneous uptake of OVA-FITC and OVAClI-TRITC showed extensive colocalization of the two fluorophores, suggesting that chlorination enhances the magnitude of Ag uptake, but does not substantially alter intracellular routing.
Modification with HOCl facilitates uptake and processing of OVA. A, Bone marrow-derived DCs were incubated with different concentrations of OVA or OVAClI conjugated with FITC for 2 h at 37°C. Free soluble Ag was removed, and the cells were fixed in 3.8% formaldehyde. Fluorescence was analyzed by flow cytometry. The graph shows median fluorescent intensity of the DC population in the FITC channel and is one representative experiment from three independent experiments. The two distributions are significantly different (p < 0.01, χ2 test). B, Bone marrow-derived DCs were incubated with OVA-FITC and OVAClI-TRITC (100 μg/ml) for 2 h at 37°C. Excess Ag was removed, and the cells were fixed in 3.8% formaldehyde. Fluorescence was analyzed by confocal microscopy. The figure shows a representative low-power (upper row) image and a high-power image of a cell that has taken up both Ags. The experiment was repeated three times. C, C57BL mice were injected i.v. with OVA or OVAClI conjugated with FITC (1 mg per mouse). Spleen cells were collected after 30 min or 18 h, and CD11c DCs were isolated by magnetic bead enrichment. The splenic DCs were stained for CD11c and CD8a. The plots show an overlay of OVA-FITC (green) and OVAClI-FITC (red) for CD8a+ and CD8− DCs. D, Confocal analysis of cells from the 30-min time point to demonstrate intracellular localization of OVA and OVAClI. Experiments were repeated three times; each time three mice per group were injected. One representative mouse from each group is shown (C, D).
The uptake of OVA and OVAClI also was investigated in vivo. Both OVA and OVAClI could be detected within splenic CD11c DCs following i.v. injection of Ag (Fig 6C, 6D). There was a moderate enhancement in the uptake of OVAClI at 30 min postinjection in both CD8+ and CD8− CD11c+ populations. Interestingly, at 18 h postinjection, the situation was reversed, and the OVAClI signal was close to that of the background, consistent with a more rapid processing and degradation of the chlorinated Ag.
On the basis of the findings shown in Fig. 6, the relative susceptibilities of OVA and chlorinated OVA to proteolysis were investigated. The 323–339 sequence recognized by the DO11.10 and OT-II hybridomas is buried within the hydrophobic core of the OVA molecule (Supplemental Fig. 5), and extensive proteolysis or fragmentation is required to release this sequence for MHC loading (28). Although HOCl itself at very high concentrations can cause protein fragmentation, no low molecular weight protein fragments could be detected with PAGE of OVAClI (Fig. 7A), consistent with earlier studies showing a lack of protein fragmentation at these levels of HOCl (29). Furthermore, OVAClI, like native OVA, could not be presented by fixed DCs (Fig. 7B), and processing of OVAClI was sensitive to the cathepsin D/E inhibitor MPC6, which has been shown to block processing and presentation of native OVA (Supplemental Fig. 6).
Modification with HOCl increases the proteolytic susceptibility of OVA. A, OVA and OVAClI (5 μg) were fractionated by 4–12% gradient PAGE, and the gel was stained with silver stain. No low molecular weight fragments were visible. The experiment was repeated three times. B, OVA (250 μg/ml), OVAClI (250 μg/ml), or OVA 323–339 peptide (1 μg/ml) were incubated with glutaraldehyde-fixed DCs and DO11.10 T hybridoma cells, and IL-2 release was measured as described in Fig. 1A. Results are presented as the average ± SD [3H]thymidine incorporation of triplicate cultures. The experiment was repeated twice. C and D, OVA and OVAClI were digested with cathepsin E (C) or trypsin (D) as described in 1Materials and Methods and analyzed by 4–12% gradient denaturing PAGE. Histograms show the degree of proteolysis quantified by comparing the intensity of the OVA bands before and after digestion using densitometry. Results are presented as the average ± SEM of three independent experiments. The difference between OVA and OVAClI are significant at p < 0.05 (Student t test, n = 3).
Nevertheless, although chlorination at low and intermediate ratios does not induce any extensive fragmentation by itself, it does alter the susceptibility of OVA to proteolysis. Native OVA itself is highly resistant to proteolytic degradation by either trypsin or cathepsin E, an aspartic proteinase required for OVA processing by DCs (18). In contrast, OVAClI is digested by both cathepsin E (Fig. 7C) and trypsin (Fig. 7D).
The flow cytometry and confocal studies demonstrate increased uptake, but cannot address the interaction between OVA and the APC surface. Lowering the temperature to 4°C completely blocked uptake. At this temperature, however, DCs gave only a very slight fluorescent signal when they were incubated with either labeled OVAClI or OVA (Supplemental Fig. 7). This technique therefore was not suitable for measuring binding parameters.
Instead, we used AFM (30) to measure directly the binding of OVA and OVAClI with fixed APCs. Macrophages rather than DCs had to be used for these studies, because the weak interaction of DCs with the substrate prevented us from obtaining reproducible results. OVA, OVAClI, or OVAClH were coated onto gold AFM probes, and the strength of the binding interaction to the surface of macrophages was quantified by measuring the adhesion probability as the probe was lowered and raised from the macrophage surface.
OVA binding to the macrophage surface was close to background values, whereas both OVAClI and OVAClH showed enhanced binding (Fig. 8A). Analysis of force‑distance graphs for individual binding events for OVAClI were consistent with a single OVA/receptor rupture event (Fig. 8B). In contrast, the graphs for OVAClH suggested that each binding event was made up of multiple rupture events (not shown), consistent with the model that high concentrations of HOCl induce protein fragmentation. The binding forces measured for OVAClI were on the order of several hundred piconewtons, consistent with a single or small number of Ag/receptor interactions per rupture event. Preincubation of macrophages in the presence of increasing concentrations of OVAClI decreased the adhesion probability down to ∼30% (Fig. 8C), the same value as that found for binding of OVAClI or OVAClH to fibroblasts. The enhanced binding of OVAClI to macrophages is therefore predominantly receptor-mediated. In contrast, the binding of OVAClH is likely mediated by numerous nonspecific interactions of fragmented or denatured protein.
Chlorinated OVA binds preferentially to receptors on the cell surface. A, The interaction of OVA, OVAClI, and OVAClH bound to the surface of a probe, with the surface of macrophages was measured by AFM as described in 1Materials and Methods. B, A representative force‑distance curve from one individual AFM measurement showing the characteristic profile of a single OVA/receptor rupture. C, The binding of OVAClI was measured by AFM as in A, but cells were preincubated in OVA (5 mg/ml) or different concentrations of OVAClI. D, OVA or OVAClI conjugated with FITC was incubated at different concentrations with Chinese hamster ovary cells expressing the Lox-1 scavenger receptor or control untransfected cells. After 30 min, the excess Ag was removed, and the cells were fixed in 3.8% formaldehyde and analyzed by flow cytometry. The results are expressed as median fluorescent intensity in the FITC channel and are one representative of three independent experiments. The difference between the oxidized and nonoxidized profiles is significant for the Lox-1–transfected cells (p < 0.01), but not the control cells (p = 0.15) (χ2).
One possible receptor that might be implicated in the binding of OVA and OVAClI is the lectin-like oxidized low-density lipoprotein receptor (Lox-1), which is expressed on both DCs and macrophages (31) and which has been implicated previously in binding to chlorinated macromolecules (32). To test whether Lox-1 mediates enhanced uptake of OVAClI, we incubated FITC-labeled OVA and OVAClI with Lox-1–expressing or control Chinese hamster ovary cells (Fig. 8D). The expression of Lox-1 enhanced uptake of both OVA and OVAClI, but OVAClI was taken up more efficiently (Supplemental Fig. 8). However, although we confirmed the presence of low levels of Lox-1 on bone marrow-derived DCs by flow cytometry, we were unable to block the presentation of either OVA or OVAClI using Abs against the Lox-1 receptor (not shown). Thus, Lox-1 may contribute to the enhanced uptake of OVAClI, but the uptake is likely to involve multiple redundant receptors on the DC surface.
Protein chlorination is a recognized hallmark of the inflammatory response (10) and contributes to neutrophil microbicidal activity as well as to inflammatory immunopathology. Chlorination also can lead to inflammatory enzyme inactivation (33) and thus regulate inflammation negatively. The influence of protein chlorination on adaptive immunity, however, has received relatively little attention. The hypothesis underlying this study, based on earlier studies (34, 35), was that chlorination might act as a natural adjuvant, facilitating recognition by APCs and hence enhancing the overall immune response.
The data presented in this article show that protein chlorination fulfills several of the criteria required for an adjuvant. Both DCs and macrophages processed and presented chlorinated OVA to DO11.10 and OTII T cells with increased efficiency. Indeed, in the case of the DO11.10 hybridoma, chlorination was essential to allow processing within physiological concentrations of Ag. The concentrations of HOCl present in vivo are difficult to determine, because they are confounded by the presence of scavengers, by rapid diffusion, and by spontaneous conversion into other species (36). However, the presence of protein modifications that result from myeloperoxidase-dependent HOCl oxidation have been extensively documented in vivo, both within the neutrophil phagolysosome (37, 38) and in the extracellular inflammatory milieu (38, 39). Thus, Ag modification by this route is a likely event during neutrophil activation.
Processing and presentation occurred independent of TLR signaling, which previously has been shown to be required to trigger Ag processing (8). Chlorination also enhanced Ag processing via the cross-priming class I pathway, although further studies will be required to determine how general this phenomenon is. Furthermore, the increased processing and presentation due to chlorination resulted in enhanced immune effector function, in terms of both Ab response and class I restricted IFN-γ–producing effector cells. Interestingly, the shift in isotype of the Ab response was consistent with a Th2/Th1 shift, which may reflect an increased epitope density on the surfaces of APCs resulting from enhanced uptake and processing (40).
The adjuvant effect of HOCl is tempered by its potential to damage proteins. It is therefore possible that epitopes that are recognized by the adaptive immune response could be destroyed by chlorination. In fact, the epitopes recognized by two CD4 (3DO18.3 and MF2.2D9) and one CD8 (RF33) TCRs tested are particularly sensitive to HOCl modification. This could be either because they contain methionines whose side chains are known to be the primary targets of HOCl (41–43) or because of their exposed position on the surface of the OVA molecule (Supplemental Fig. 5).
Higher HOCl-to-protein ratios reduced the response of all of the T cells tested as well as the Ab response. High concentrations of HOCl not only modify an increasing range of amino acid side chains, but also result in formation of active radical centers on nitrogens and carbons of the protein backbone (44). These radical centers induce further rearrangements that can result in extensive protein fragmentation and denaturation and associated loss of immunogenicity. The ability to enhance immune responses and in parallel to destroy epitope recognition is reminiscent of the effects of formaldehyde, whose adjuvant properties have long been incorporated into vaccine formulations (24). However, in contrast to formaldehyde, the adjuvant effect of HOCl is not mediated via protein aldehyde groups, and indeed aldehyde formation is limited at the ratios of HOCl giving optimum immune responses. Instead, HOCl adjuvant effects are dependent upon the presence of the asparagine-linked carbohydrate side chain. N-Acetyl glucosamine is a major component of this structure and is also a major target for HOCl (25), which converts this sugar into a whole range of oxidized products (45). Therefore, the carbohydrate side chain in OVAClI may contain a very high density of oxidation products and hence provide an efficient and accessible ligand for appropriate receptors.
At least two mechanisms have been identified that may contribute to the adjuvant effects of HOCl. Chlorinated OVA as well as chlorinated cells (46) are taken up more efficiently than native OVA. Enhanced uptake is mediated by stronger binding to the cell membrane, which is of too low affinity to be detected by flow cytometry, but could be detected by direct measurement using AFM. Furthermore, the binding is mediated via specific receptors, because it is inhibited by excess ligand. The scavenger receptor Lox-1 can contribute to binding, but several other members of the scavenger receptor family as well as receptor for advanced glycation endoproducts receptor families have been implicated in recognition of oxidized macromolecules, and the overall binding interaction is likely to show considerable redundancy.
In addition to enhanced uptake, HOCl also enhances the sensitivity of OVA to proteolysis. The known reactivity of HOCl with cystine disulfide bonds as well as the increased accessibility of ε amino groups in OVAClI suggest that this increased susceptibility results from a more relaxed open three-dimensional structure. It is tempting to speculate that this enhanced susceptibility to proteolysis also contributes to more efficient processing and presentation. However, one recent study has reported that enhanced proteolysis decreases immunogenicity by decreasing the t1/2 of Ag within the APC (47). Our data suggest that the overall efficiency of the processing pathway is determined by a balance between the efficiency with which epitopes are released from intact proteins and the rate at which the epitopes are destroyed by further degradation. Therefore, we suggest that the net effects of chlorination may vary between different Ags.
This study demonstrates that protein chlorination can act as a natural adjuvant of immune responses to proteins, leading to enhanced processing, presentation, and effector responses. It is becoming clear that man-made adjuvants, many of which have been used for over a century with little understanding of their mode of action, act predominantly by stimulating cross-talk between innate and adaptive immunity. In a similar way, HOCl, a product of innate immune activation, can be considered a natural adjuvant, facilitating the subsequent recognition and processing of Ags by APCs, and hence stimulating a more effective adaptive immune response. Further studies are in progress to harness the adjuvant activity of HOCl to improve the immune response to tumor-associated Ags and hence develop effective strategies for cancer immunotherapy (46, 48).
Disclosures The authors have no financial conflicts of interest.
This work was supported by the following: Atomic force microscopy experiments were supported by the Foundation for Polish Science Team Programme and cofinanced by the European Union European Regional Development Fund, PolyMed, TEAM/2008-2/6; Z.M.P. was supported by a Medical Research Council studentship; M.C. and J.M. were supported by Jagiellonian University Medical College Grant K/ZDS/001008; and B.M.C. was supported by a grant from the Biotechnology and Biological Sciences Research Council Selective Chemical Intervention in Biological Systems initiative.
The online version of this article contains supplemental material.
Abbreviations used in this paper:
dendritic cell
hen egg lysozyme
HOCl
Lox-1
lectin-like oxidized low-density lipoprotein receptor
TRIF
Toll/IL-1R domain-containing adaptor-inducing IFN-β
TRITC
tetramethylrhodamine isothiocyanate
Trp2
tyrosinase-related protein 2.
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Supplementary Figures S1-S8 (PDF, 485 Kb)
Description: Figure S1. The reaction of HOCl with OVA causes a dose dependent loss of free amines (equation 1) and the formation of aldehydes (equation 2). Figure S2. Exposure of DC to low concentrations of HOCl does not enhance antigen immunogenicity... Figure S3. The expression of the Trp-2 antigen in the B16-F10 melanoma. Figure S4. Removal of carbohydrate residues from OVA and OVAClI Figure S5. The three dimensional structure of OVA showing the three T cell epitopes explored in this study. Figure S6. Chlorinated OVA still requires processing by aspartic proteinase digestion. Figure S7. The uptake of OVA and OVAClI by DC is blocked at 40 C. Figure S8. Antigen uptake by CHO-LOX1 cells.
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tickets are $20.00 per ticket or 3 for $50.00. Drawing on March 8th at 7:00.Need not be present to win. Prizes are as follows:1st prize $1000.00, 2nd prize $500.00 and 3 3rd prizes $100.00.All proceeds benefit the operations of RLATo purchase tickets please contact Regina Luminis Academy.
We are geared towards protecting the innocence of children. Please help end child abuse.
Win $100,000! St. Andrew School Mega Raffle May 5, 2012. Over $180,000 in cash prizes will be given away in 75 drawings! Tickets are $100 each, and up to 10 people can purchase a ticket. Only 5000 tickets will be sold We accept checks, Mastercard and Visa. St. Andrew School is a Catholic PK-8 grade school in Murphysboro, Illinois. Call 866-862-7282 or go to saintandrew-school.org/raffle for more information and rules.
Sportsman Give-a-way - featuring 33 prizes. Top prize is $10,000 and second is $5,000. There will also be fifteen $400 prizes and fifteen assorted rifles, shotguns and black powder. Do not have to be present to win. Purchase tickets online at www.statonhousefire.com using Paypal. Games open at 1PM and drawings begin at 2PM continuing every 5 minutes! Free food and beverages for ticket holders. Drawing held at the Pitt County (NC) fairgrounds in Greenville, NC. Complete rules online.


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Q: Constrained Optimization Problem in Scipy: boolean index did not match indexed array along dimension 0 I'm having a constrained optimization problem, which i want to solve using the scipy.optimize package.
from scipy import optimize as opt
import numpy as np

def f(x):
    return (x[0]-5)**2 + (x[1]-6)**2

#Bounds and Linear Constraints
bounds = opt.Bounds([0,0],[np.inf, np.inf])
lin_const = opt.LinearConstraint([[1,2],[0,0]], [-np.inf, 0], [4,0])

#Nonlinear Constraints, Jacobian and Hessian
def cons_f(x):
    return [x[0]**2 - 4, np.exp(-x[0]) - 1]

def cons_J(x):
    return [[2*x[0], 0], [-np.exp(-x[0])]]

def cons_H(x, v):
    return v[0]*np.array([[2,0], [0,0]]) + v[1]*np.array([[np.exp(-x[0]), 0], [0,0]])

nonlin_const = opt.NonlinearConstraint(cons_f, -np.inf, 1, jac=cons_J, hess=cons_H)

#Solving the optimization problem
x0 = np.array([0.50, 0.75])
res = opt.minimize(f, x0, method='trust-constr',jac="2-point", hess=opt.SR1(), bounds=bounds, constraints=[lin_const, nonlin_const], options={'verbose': 1})
print(res.x)

I followed the Scipy doc closely but I get the typical numpy boolean index did not match indexed array along dimension 0; dimension is 1 but corresponding boolean dimension is 2 error message, what am I missing? Thanks for your help!
Here is the full Error Message:
IndexError                                Traceback (most recent call last)
Input In [32], in <cell line: 2>()
      1 x0 = np.array([0.50, 0.75])
----> 2 res = opt.minimize(f, x0, method='trust-constr',jac=cons_J, hess=opt.SR1(), bounds=bounds, constraints=[lin_const, nonlin_const], options={'verbose': 1})
      3 print(res.x)

File ~\anaconda3\envs\choquetclassifier\lib\site-packages\scipy\optimize\_minimize.py:634, in minimize(fun, x0, args, method, jac, hess, hessp, bounds, constraints, tol, callback, options)
    631     return _minimize_slsqp(fun, x0, args, jac, bounds,
    632                            constraints, callback=callback, **options)
    633 elif meth == 'trust-constr':
--> 634     return _minimize_trustregion_constr(fun, x0, args, jac, hess, hessp,
    635                                         bounds, constraints,
    636                                         callback=callback, **options)
    637 elif meth == 'dogleg':
    638     return _minimize_dogleg(fun, x0, args, jac, hess,
    639                             callback=callback, **options)

File ~\anaconda3\envs\choquetclassifier\lib\site-packages\scipy\optimize\_trustregion_constr\minimize_trustregion_constr.py:361, in _minimize_trustregion_constr(fun, x0, args, grad, hess, hessp, bounds, constraints, xtol, gtol, barrier_tol, sparse_jacobian, callback, maxiter, verbose, finite_diff_rel_step, initial_constr_penalty, initial_tr_radius, initial_barrier_parameter, initial_barrier_tolerance, factorization_method, disp)
    357     prepared_constraints.append(PreparedConstraint(bounds, x0,
    358                                                    sparse_jacobian))
    360 # Concatenate initial constraints to the canonical form.
--> 361 c_eq0, c_ineq0, J_eq0, J_ineq0 = initial_constraints_as_canonical(
    362     n_vars, prepared_constraints, sparse_jacobian)
    364 # Prepare all canonical constraints and concatenate it into one.
    365 canonical_all = [CanonicalConstraint.from_PreparedConstraint(c)
    366                  for c in prepared_constraints]

File ~\anaconda3\envs\choquetclassifier\lib\site-packages\scipy\optimize\_trustregion_constr\canonical_constraint.py:352, in initial_constraints_as_canonical(n, prepared_constraints, sparse_jacobian)
    350     finite_ub = ub < np.inf
    351     c_ineq.append(f[finite_ub] - ub[finite_ub])
--> 352     J_ineq.append(J[finite_ub])
    353 elif np.all(ub == np.inf):
    354     finite_lb = lb > -np.inf

IndexError: boolean index did not match indexed array along dimension 0; dimension is 1 but corresponding boolean dimension is 2

redpajama_set_name: RedPajamaStackExchange


text:
Real Time/Download Campaign Names Sigler Co-Chair
By Andrea Wells | September 9, 2011
Independent agent Joyce Sigler has been named co-chair of the Real Time/Download Campaign.
Sigler is vice president of administration at Jones & Wenner, an insurance agency in Fairlawn, Ohio. In her role with the campaign, she succeeds Cyndy Smith, vice president and director of technology at Haylor, Freyer & Coon, in Syracuse, N.Y., who co-led the campaign for several years. An active member of the Network of Vertafore Users (NetVU), Sigler has 17 years of insurance agency and carrier experience.
The Real Time/Download Campaign (www.getrealtime.org) advocates for workflow and technology efficiency in the independent agency distribution channel. Real-time is the ability to click on a button from a client file in the agency management system or comparative rater for immediate access to carrier information on that client. The transaction may be a quote, billing inquiry, claim inquiry/loss run, policy view, endorsement or a request for information.
'In today's marketplace, independent insurance agents and brokers need to find new ways to increase profitability and efficiency,' said Sigler. 'Real-time agent-carrier communication is one of the easiest and most impactful ways of doing that. It certainly has been in our office.
'We need to continue reaching those agents and brokers who are not using technology to its fullest,' Sigler noted, 'and we must keep advocating with carriers and vendors for continued improvements in their Real Time and Download interface functionality.'
While working for a carrier, Sigler helped agencies establish and upgrade their network infrastructures and provided management system and workflow training. She has held — and continues to hold — a number of volunteer roles with her users group and other industry organizations, and serves as a mentor to agencies seeking to improve their efficiency using advanced automation and workflows.
Sigler joins Campaign Co-Chair Karen Youngman, personal lines manager at Don Allen Agency, based in Rochester, N.Y.
About Andrea Wells
Andrea Wells is a veteran insurance editor and Editor-in-Chief of Insurance Journal Magazine. More from Andrea Wells
Categories: National NewsTopics: agency technology, Agents & Brokers
Applied Systems says:
Congratulations, Joyce! This is most definitely a key industry initiative that we support 100%. We recently published a report about the topic. "Real-Time Rating: The Most Pow... read more
LP in Nevada Adds Cantlon, Ennis, Garcia, Rossi to Ownership Group
Despite Pandemic, North Carolina Traffic Deaths Climb to 1,500 in 2020
2VP, Architecture ( Data & Analytics, Cloud, AI ) – Remote - Hartford, CT

redpajama_set_name: RedPajamaCommonCrawl


text:
Trey Songz woos Bay Area college students at free concert
Verification: What's 5 + 9?
Trey Songz takes off his shirt as he perfoms at a free concert hosted by Amazon Student and Pandora at the Fox Theatre in Oakland Sunday, Sept. 15, 2013. Photo by Benjamin Kamps / Xpress
R&B's "Mr. Steal Yo Girl," otherwise known as Trey Songz, didn't have to steal any young women at his back-to-school free performance. Still, Trey Songz managed to satisfy the mostly female audience with a stellar performance that rivaled a paid show.
Amazon Student and Pandora co-sponsored the Aug. 15 event at the Fox Theater in Oakland. About 1,000 Bay Area college students were invited to enjoy the free show for those who used Amazon to rent or buy textbooks during the month of August.
SF State biology major Brittany Wilson had already seen Trey Songz perform live during the tour of his last album "Chapter V" last year. Sunday's concert was a chance for her to come back for seconds.
Trey Songz perfoms at a free concert hosted by Amazon Student and Pandora at the Fox Theatre in Oakland Sunday, Sept. 15, 2013.
"He was electric," said Wilson. "The concert was amazing. He did it just like at the last show I saw him at."
Several SF State students in attendance said they found out about the event through flyers handed out on campus, and Twitter and Pandora ads that played between songs.
This was the first year Amazon launched its free Back to College concert series for students. Trey Songz performed another concert in the series Sept. 12 at the Electric Factory in Philadelphia before coming to the Bay Area.
"This was a way to kick off the back to school season, do something fun and let people know what we have to offer students," said Amazon Public Relations Manager Brittany Turner.
Although students from several other universities in the area came to see the show, the audience only filled about half of the entire Fox Theater, making the event feel like an invitation to a lavish MTV sweet 16 birthday party with a special guest performer. Goody bags weren't provided, but Amazon did give away Kindle Fire HD tablets to 20 lucky students at the top of the show.
"The goal is not to pack this place wall to wall; the goal is to get students to have a great experience and have it be exclusive," said Tommy Page, vice president of artists and brand partnerships.
According to Page, Pandora selected Trey Songz as the artist for the concert series after data from its music streaming app revealed that the artist had a large following in the Bay Area and Philadelphia.
During the hour-long set, an energetic Trey Songz beamed with a smile as he sang some of his sexy hits such as "Panty Droppa," "Say Ah" and "I Need A Girl."
Throughout the night, Trey Songz had the ladies in attendance shrieking with excitement as he sang one affectionate love song after another. At one point, Trey Songz made the entire theater erupt with screams when he teased them with his provocative hip-gyrations.
"I was waiting in line outside for about two hours, so by the time Trey Songz came out I was pretty tired, but it was a great free show and it was definitely worth it," said Meron Foster, a senior majoring in international relations.
Trey Songz closed the show with "Bottoms Up" as if to offer a toast to the women in the audience and let them know the night was all about them.
As the song ended, Trey Songz took off his shirt causing the entire theater to explode with cheers. He then wiped the sweat off his body and tossed his shirt into the crowd for one lucky audience member to enjoy who perhaps thought the night was actually all about her.
Stonestown to get Whole Foods, theater and more
Naked and unafraid
Pronouns matter, so learn them
"Mojada: A Medea in Los Angeles," a story of the common immigration experience
Lawsuit settlement between SF and the Academy of Art University rundown
Golden Gate Xpress
Queer Homecoming dances the night away
The Get Up Kids are finally adulting
Surviving finals with your mental health intact
Zombie comedy, coming of age and queerness rocks Roxie Theater.
Festival of Altars celebrates life and death
Annual Psoriasis Walk continues
'Addams Family' surprises
Feeling the 'Earfquake'
Tours of new cultural flavors of SF
California's Fire Season sparks people to question the use of N95 Respirators


Furniture — This furniture, is one of the 18 designs published under the gallery: Consulting Interior Home Designer. In the next page, we have the interior home designer of good home design interior photo of goodly designing fresh [ ... ]. While in	the previous page, we have the interior home designer of fine interior home designs of good home interior trend [ ... ].


We'll discuss the impact of North Carolina's airports on the state economy.
Plus, work has started on the rail line near Salisbury to improve travel times between Raleigh and Charlotte.
And, we'll unveil some safety tiles to keep motorists safe who drive through a major construction zone in Raleigh.
"NCDOT Now" is posted every week.


text:
Andy Ponsford
Head of Cash Product Management, EMEA, J.P. Morgan Treasury Services,
Published 1 April 2010 7 MIN READ
What Matters Today – #6 Centralisation & Rationalisation
#6: Centralisation & Rationalisation
by Andy Ponsford, Head of Cash Product Management, EMEA, J.P. MorganTreasury Services, and Seamus Desouza, Senior Product Manager for ForeignExchange, EMEA, J.P Morgan Treasury Services.
As we reach the end of J.P. Morgan's series on the issues that matter most to treasurers today, we take a look at some of the principal objectives that have been common to virtually every treasury both during and subsequent to the crisis. Firstly, companies are seeking to centralise their treasury activities, either physically or virtually, to achieve visibility and control over the company's cash, investment and borrowing activities. Secondly, they are rationalising bank relationships, accounts and systems, to realise greater efficiencies and leverage the advantages of centralisation more fully. This article looks at some of the current drivers and enablers of centralisation and rationalisation, and how these initiatives can benefit the company.
Treasurers are now recognising the implications and potential benefits of SEPA more clearly, particularly since the launch of SDD.
For many treasurers, centralisation and rationalisation have been long-standing objectives; however, the crisis has intensified the business case and reduced internal resistance to change. During the crisis, difficulties in securing access to finance and the need to optimise the use of cash meant that visibility and accessibility of cash was paramount. In addition, many optimisation projects had to be postponed or frozen while budgets were constrained, so initiatives such as SWIFT connectivity or Single Euro Payments Area (SEPA) migration were often held off. As we see a start to the recovery, and new industry opportunities such as SEPA Direct Debits (SDD) strengthen the business case for centralisation of collections and SEPA migration, we see greater momentum for treasury optimisation projects amongst our clients.
SEPA and centralisation
Treasurers are now recognising the implications and potential benefits of SEPA more clearly, particularly since the launch of SDD. Some issues still remain, particularly around reachability of SDD, and the use of existing direct debit mandates in countries such as Germany. However, these are nearing resolution as, for example, reachability will be mandatory from November 2010 across the Eurozone. This means that there is now an effective start date for SEPA, which is proving a catalyst for migration amongst large corporates. While many were waiting for a mandated end-date of existing domestic payment schemes on which to base their plans, treasurers recognise that with a meaningful start-date and a defined business case, there is now the incentive to migrate to SEPA. This is a catalyst for further centralisation of financial activities and rationalisation of both internal processes and external bank relationships.
Inside Save the Children International's Rapid Response Treasury
Visibility and efficiency improvements around several treasury processes has ratcheted up Save the Children's global crisis response several notches.
Closing in on Payments Nirvana
Instant Cross-border Treasury Transactions While domestic faster payments schemes have seen excellent traction over the past decade, obtaining the same visibility and control of cash flows in the cross-border world...
Relative Normality On Horizon — But Divergent Monetary Policy Pathways Likely
Exclusive insight for TMI subscribers! Northern Trust Asset Management share a monthly market commentary for treasurers.
Turning Cross-Border Payments from a Burden to a Benefit
Livia Benisty (Banking Circle) explores the current offering and limitations of cross-border payments, and what steps PSPs and banks can take to deliver them as an added value service.
Roche's Breakthrough for Cash Pooling in Saudi Arabia
Roche's group treasury worked with BNP Paribas and the local regulator to successfully implement a first-of-its-kind cash pooling solution in the country.
AR vs Inflation – Bringing Back Control
With cash flow concerns having risen rapidly up the agenda, a sharp but balanced approach to AR is essential.
UK Autumn Statement Restores Confidence in Sterling


text:
What breaks your heart?
What would you want people lining up to thank you for at the end of your life?
In this 3-part series, Andy Stanley challenges us to make a resolution that makes a difference.
DVD Contents:
3 Full-Length Messages
3 Sets of Discussion Questions in PDF Format
3 Audio Files of the Messages in MP3 Format
Message Titles
Something to Show For It
Pro-Motion
Communicator, author, and pastor, Andy Stanley founded Atlanta-based North Point Ministries in 1995. Today, NPM is comprised of six churches in the Atlanta area and a network 30 churches around the globe, collectively serving nearly 70,000 people weekly. As host of Your Move with Andy Stanley, with over 5 million messages consumed each month through television and podcast, and author of 20-plus books including The New Rules of Love, Sex, and Dating, Ask It, How to Be Rich, Deep & Wide, Visioneering, and Next Generation Leader, he is considered one of the most influential living pastors in America. Andy Stanley and his wife, Sandra, have three grown children and live near Atlanta.
You're reviewing: Re:Solution (DVD)


Incident responders are catching and remediating threats faster than ever, according to past SANS surveys. Much of their success can be attributed to improving technologies, such as threat intelligence—73 percent of respondents to last year's survey said they were using threat intelligence to enable more accurate investigations.
Andy Schmid, senior vice president of product, leads 1E's product strategy. He is responsible for 1E's go-to-market strategy including analyst relations, product marketing, product management, sales evangelism, and sales and technical enablement globally. Before joining 1E, Andy was responsible for Blue Coat's Asia Pacific/Japan product marketing team, after having led McAfee's Asia Pacific product and solution marketing team. Prior to that, Andy led Symantec's enterprise security product marketing team in the region for five years. He holds an MBA from the Australian Graduate School of Management and a bachelor's degree in computer science from the College of Higher Education in Regensburg, Germany.
Mike Stewart is the vice president of security consulting services for Fidelis Cybersecurity. He is responsible for professional services, consulting services, and the Fidelis managed detection and response (MDR) service. Mike, a retired Air Force chief, has more than three decades of experience in the information assurance and cybersecurity field. He possesses in-depth practical experience building and leading large classified security operations, facilities, personnel and resources. He has consulted with the FBI, NSA and DISA, delivering security solutions that were critical to national security. In the past 14 years, Mike has led some of the largest commercial cyber breach engagements around the globe, including organization of initial triage response and forensic support, remediation, expulsion, security engineering and security operations.
As CTO and co-founder of ThreatQuotient, Ryan Trost utilizes his 15-plus years of security experience focusing on intrusion detection and cyber intelligence to help drive thought leadership as well as innovative product discussion. As a recognized leader in the cyber industry, Ryan is a frequent speaker at industry conferences, an author and the developer of geospatial intrusion detection algorithms used to identify geolocation attack patterns. Prior to ThreatQuotient, Ryan managed several U.S. government and commercial security operations centers (SOCs) and was the senior director of security and privacy officer for a midsize healthcare company in Northern Virginia.


text:
Q: Check session for each controller and action in symfony2 How can we check session for each controller and action in Symfony2. If session is not found redirect it to home page. I can check session in each action but i think it is not the best way to do this.
Thanks.

A: Register your own listener which listens the kernel.request event and do your logic. Short example:
class KernelListener
{

    private $router;

    public function __construct(RouterInterface $router)
    {
        $this->router = $router;
    }

    public function onKernelRequest(GetResponseEvent $responseEvent)
    {
        $request = $responseEvent->getRequest();

        if (!$request->getSession()->has(YOUR_KEY)) {
            $responseEvent->setResponse(new RedirectResponse($this->router->generate('HOME_PAGE_ROUTE')));
        }
    }
}

A: I don't know there have a built-in special functionality to check this option in every controller and action. but i think its possible to do it by putting the session check code in each controllers constructor or in initialising method.
class ControllerName
{
   public function __construct()
   {
      //session checking code
   }
}