I wish to update the House on the progress made in securing the UK's continued participation in the WTO Agreement on Government Procurement (GPA) as we leave the EU. Yesterday (27 February 2019) in Geneva, the 19 parties to the GPA formally adopted the decision relating to the UK's accession to the Agreement as a party in its own right. The GPA Committee decision on the UK's accession refers to both the scenario where the UK and EU reach a deal on the terms of the UK's withdrawal, and one in which the UK is to leave the EU with no deal. If the UK leaves the EU without a deal, the UK will ratify the Agreement as soon as possible, once the process set out in Section 20 of the CRaG Act 2010 completes. This will ensure that our membership of the GPA - an agreement worth £1.3 trillion annually - continues, as now, in a no deal scenario. Should the Withdrawal Agreement be agreed between the EU and UK, a further decision of the GPA Committee would be required to allow for UK accession at the end of the Implementation Period, but the GPA would continue to apply to the UK as if it were a Member State of the EU during that Implementation Period. Leaving the EU with a deal remains the Government's top priority. This has not changed.
Yesterday's decision is a significant milestone for the UK, and it will ensure UK suppliers can continue to bid for government contracts overseas on substantially the same terms as currently provided to the UK as an EU Member State. It will also ensure that the UK continues to benefit from increased choice and value for money in areas where the UK's procurement opportunities are open to international competition, whilst importantly continuing to protect vital public services such as our NHS.
The Government has already begun the process set out in Section 20 of the Constitutional Reform and Governance (CRaG) Act 2010 for Parliamentary scrutiny of the Agreement. The Agreement was laid before both Houses of Parliament on 18 February. This includes text of the Agreement, the UK's market access schedules (consistent with our current offer as an EU member state) and the market access schedules of all Parties, alongside accompanying Explanatory Memoranda. In a 'no deal' Brexit scenario, the government is anticipating a short gap between the UK leaving the EU and its accession to the GPA becoming effective. This is to allow for the completion of the necessary processes that allow for a new GPA accession to become legally effective. The gap is expected to be very short and, as a result, the government is expecting the impact on UK businesses to be minimal.


text:
Home North America USA New York State Cheap flights to Buffalo, New York
Cheap Flights to Buffalo
Round-trip from AED 5,939 From Dubai to Buffalo
Which day is cheapest to fly to Buffalo?
Thursday is currently, on average, the cheapest day to fly to Buffalo. Flying on Friday will result in higher flight prices.
What time of day is cheapest to fly to Buffalo?
At the moment, flights in the evening are likely to offer the best value for money for your Buffalo trip. A flight in the morning will more often than not be of higher cost.
How long is the flight to Buffalo?
Flight times from UK to Buffalo, USA range from 10h to 52h. The shortest flight from the London Heathrow Airport to Buffalo Airport is 10h 25m. The shortest flight from the Edinburgh Turnhouse Airport to Buffalo Airport is 10h 23m. The shortest flight from the Manchester Airport to Buffalo Airport is 10h 48m. Some flights can take as long as 52 hours, based on the layover periods and number of stops.
Where can you get direct flights to Buffalo from UK?
Unfortunately, there aren't any direct flights from the UK to Buffalo, New York. However, the majority of flights available are one-stop flights, with a layover at New York John F Kennedy Intl., Newark Airport, or Philadelphia Intl. Both American Airlines and United Airlines offer a one-stop flight between London Heathrow and Buffalo. The stopover for American Airlines is Philadelphia, PA and the one for United Airlines is Newark, NJ. There are also several 2-stop flights between these cities. The majority of flights between Manchester and Buffalo involve utilising more than one airline, with the exception being a one-stop flight offered by American Airlines with a stopover in Philadelphia. To get to Buffalo from Edinburgh, you have a choice of American Airlines with a stopover in Philadelphia or United Airlines with a stopover at Washington Dulles Intl., as well as several 2-stop options using multiple airlines.
How do you get from Buffalo airport to the city centre?
There are several rental car companies located on the ground level of the short-term car park across from arrivals, including National, Hertz, Enterprise, Avis, Budget, and Alamo. There is also a bus stop located outside arrivals with Metro buses running every 30 minutes to take you to various places throughout Buffalo and beyond, such as to Niagara Falls or Toronto. There are also taxis available that you can get to take you to the city centre which is about 16 km away via the Kensington Expy., roughly a 16-minute drive.
What are some things to do in Buffalo?
Buffalo is the home of the Buffalo wing, so no visit is complete without rolling up your sleeves and digging into a bucket of them. An ideal way to experience them is with a bike ride wing tour that starts where these wings were first made and travels through the Broadway Market and Buffalo Central Terminal. Another way to see the city is on a double-decker bus tour with stops at the Buffalo & Erie County Naval and Military Park, The Barrel Factory, and Old Erie County Hall. Other places you'll want to visit include the Buffalo & Erie County Botanical Gardens created in the late 1800s on 155 acres, Forest Lawn Cemetery which is the final resting place of President Fillmore and other famous people, Buffalo Zoo, Coca-Cola Field, and Theodore Roosevelt Inaugural National Historic Site.
Do I need a passport or visa to fly to Buffalo?
To travel from the UK to Buffalo, NY, you will need your British Citizen passport, as well as a Visa Waiver Programme (VWP). Most passport holders can get an Electronic System for Travel Authorisation (ESTA) which allows travel for up to 90 days. If you do not qualify for entry under the VWP, you must apply for a visa at a US embassy or consulate before traveling.
Cheap tickets to Buffalo found by our users in United Arab Emirates
Dubai to Buffalo flights from AED 5,939 pp
Buffalo, New York airports
Flights to Buffalo Niagara International
Cheapest flights to USA
Dubai to New York flights from AED 2,516 pp
Dubai to Miami flights from AED 2,755 pp
Abu Dhabi to New York flights from AED 2,781 pp
Sharjah to New York flights from AED 2,828 pp
Dubai to Washington flights from AED 2,832 pp
Dubai to Boston flights from AED 2,876 pp
Dubai to Chicago flights from AED 2,880 pp
Abu Dhabi to Washington flights from AED 2,968 pp
Sharjah to Washington flights from AED 2,979 pp
Dubai to Los Angeles flights from AED 3,137 pp
Dubai to William P. Hobby flights from AED 3,152 pp
Dubai to Houston flights from AED 3,152 pp
Abu Dhabi to Chicago flights from AED 3,170 pp
Abu Dhabi to Tampa flights from AED 3,427 pp
Abu Dhabi to Los Angeles flights from AED 3,478 pp
Sharjah to Atlanta flights from AED 3,578 pp
Flights from Dubai International to Newcastle International
Flights to Masterton
Flights to Karachi
Flights to Mombasa
Flights to Burbank
International departures to Buffalo
Buffalo flights from Hong Kong
Buffalo flights
Buffalo flights from India
Buffalo flights from South Africa
Buffalo flights from Malaysia
Buffalo flights from New Zealand
Buffalo flights from Qatar
Buffalo flights from Australia
Buffalo flights from Singapore
Buffalo flights from Vietnam
Buffalo flights from Philippines
Buffalo flights from Indonesia
Buffalo flights from UK & Ireland
Buffalo flights from Canada
Buffalo flights from USA

redpajama_set_name: RedPajamaCommonCrawl


Time Commitment / Availability: Flexible, non-traditional schedule.
The Development // Events Intern will assist with and learn how to plan and manage large-scale museum events. Internal Museum events include The Museum Ball, Art on the Rocks, exhibition openings, curatorial support group art acquisition events and salons, upper level donor dinners, and other fundraising events.
Assist with invitation mailings and RSVP event registration.
Assist with all event logistics, including but not limited to: site walk throughs, budget management, vendor and artist relations, scheduling meetings, valet, florals, rentals, catering, layouts, and other aspects as needed.


This document describes how Scanfil Sweden AB and its affiliates in Swedish and its staff process the personal data of job applicants applying to Scanfil. Scanfil acts as a controller to such personal data.
As a controller, we are responsible for the personal data of job applicants, and for the pro-cessing of such personal data. Protecting your privacy and your personal data is of utmost importance to Scanfil. We are committed to complying with the requirements of data protec-tion regulation applicable to us in the processing of your personal data. The means and pur-poses of processing the personal data of job applicants are described in more detail in this Privacy Notice.
The EU General Data Protection Regulation (EU) 2016/679 ("GDPR") applies to all processing of job applicants' personal data as a general law. Furthermore, certain provisions of the Swe-dish personal data in accordance with what is stipulated in the present document and other-wise in line with the terms of the Swedish Personal Data Act (GDPR) and any other applicable legislation.
Processing of personal data must always be based on a legal basis set forth in the GDPR.
(ii) Ensuring and improving data security or the security of premises and data net-work; preventing and investigating suspected fraud or misuse; and protection of Scanfil's property.
Data relating to job applicants shall primarily be collected from the job applicant him/herself. In addition, data is collected also from other permitted sources, such as from data systems to which personal data of the applicant is recorded in connection with the job applicants use such data system.
In addition, Scanfil may collect data from other sources based on the job applicant's con-sent, unless it collects personal credit data in order to establish the employee's reliability, in which case consent is not required.
Your personal data will be used as the basis for automated decision-making. For each indi-vidual post, we use a requirement profile/requirement specification as a framework for the decisions made. Based on your answers to the select questions on the application form, your application may not be processed further if you do not meet the SHALL requirements in the requirement specification. SHALL requirements for each specific position are shown in the relevant job advert. In our recruitment process, automated decision-making is necessary for entering into, or performance of, a contract between you and Scanfil. Scanfil has implement suitable measures to safeguard your rights and freedoms and legitimate interests in the au-tomated decision-making. For example, the tool used by us in performing the initial qualifying of the applicants uses transparent decision-making based on the SHALL requirements deter-mined for each position. In addition, you have right to obtain human intervention on our part to express your point of view and to contest the initial qualification.
Our recruitment process makes use of one or more occupational psychological tests. Those candidates that are, on the basis of the first interview, suitable for the position, may be re-quired to participate to an occupational psychological test / personal and aptitude assess-ment. Such assessments are subject to your consent and are performed in accordance with the applicable compelling legislation. Your responses in the tests are compared with a prede-termined occupational psychological profile for the post. The occupational psychological profile is based on competences and other parameters derived from the requirement profile. The results of the occupational psychological tests form the basis for deciding whether you will progress further in the process or not. A person who is to be evaluated is always provid-ed with a copy of the statement given.
Scanfil may transfer job applicant's personal data to third parties in the following manner in order to fulfil the processing purposes described in this Privacy Notice. When personal data are transferred to an entity which processes personal data on behalf of Scanfil (i.e. data pro-cessor) Scanfil has through contractual arrangements ensured that personal data is pro-cessed only in accordance with Scanfil's written instructions and only for the purposes de-scribed in this document and that access to personal data is restricted to persons who need access to data based on their tasks.
Some of the data processors used by Scanfil are located outside the EU or EEA area. Scan-fil has contractually ensured that these entities undertake to apply an appropriate level of da-ta protection in their processing practices, and thus the data transfers are subject to appro-priate safeguards. More information on cross border transfers of personal data and on the appropriate safeguards applied thereto from time to time is available from the contact person mentioned under Section 2 of this Privacy Notice.
Scanfil retains the personal data of the job applicants in accordance with the applicable legis-lation only for as long as the retention of data is necessary for processing purposes speci-fied in this Privacy Notice. When Scanfil no longer needs personal data for the specified pur-poses, the data will be deleted from the data systems of Scanfil as well as from other files.
The general maximum retention period of personal data of job applicants is two year from the relevant recruitment decision, based on the limitation period for raising discrimination charge under the Gender Equality Act (LAS 1998).


Lowboy has dense broad-mounded habit with the ultimate size being from 3-4' h x 3-4'w in 10 years. Bronze to metallic bluish green to reddish purple winter color foliage, and a minimal fruit set. Considered an improvement over the numerous strains of Compacta. Introduced by Rushing Nursery in 1983. The first nandina to be patented in the US.


Music in playlist doesn't export to iTunes.
After I create a playlist, I export it to iTunes. When I click on the playlist in iTunes, no music, nothing. I'm using Express 7 which I downloaded a couple weeks ago onto my new Mac. I used Express 6 on my PC before and never had a problem.
This is just one of a few problems I'm having, but the most inconvenient. I am able to burn the playlist to a cd, then import that into iTunes, but I'm limited to the number of songs I can burn.


text:
Les Deux Colonels () est un film italien réalisé par Steno, sorti en 1962.

Synopsis

Fiche technique
 Titre original :
 Titre français : Les Deux Colonels
 Réalisation : Steno
 Scénario : Bruno Corbucci et Giovanni Grimaldi
 Photographie : Tino Santoni
 Musique : Gianni Ferrio
 Production :
 Pays d'origine : Italie
 Format : Noir et blanc - Mono
 Genre : comédie
 Date de sortie : 1962

Distribution
 Totò : colonel Di Maggio
 Walter Pidgeon : colonel Timothy Henderson
 Nino Taranto : sergent Quaglia
 Scilla Gabel : Iride
 Toni Ucci : Mazzetta
 Adriana Facchetti : Penelope
 Nino Terzo : soldat La Padula
 John Francis Lane : sergent McIntyre

Autour du film
Les scènes de combat du film sont tournées dans le petit village de Civita di Bagnoregio, près la ville de Viterbe.

Liens externes

Film italien sorti en 1962
Comédie italienne
Film sur la résistance italienne
Film réalisé par Steno
Film scénarisé par Bruno Corbucci
Film scénarisé par Giovanni Grimaldi
Film avec une musique composée par Gianni Ferrio
Film se déroulant en Grèce
Film tourné dans le Latium
Film italien en noir et blanc

redpajama_set_name: RedPajamaWikipedia


With events like New York Champagne Week, which is only in its second year and has already experienced an incredible amount of notoriety, it brings to mind an important question about the future of champagne as not just a celebratory beverage on its own, but a key player in the cocktail world.
The French 75 and the Kir Royale – both cocktails that feature champagne – are staples in the classic cocktail category, but as we've seen from events such as this one on Nobember 4th, versatility is the name of the game.
Champagne and cocktail lovers alike came together for the first consumer event of New York Champagne Week 2014 to see bartenders from around New York City compete for the title of best champagne cocktail. Sponsored by Nicolas Feuillatte, these competitors took over Evelyn Drinkery and created some truly outstanding tipples for people to sip on and enjoy as they talked about all things bubbly.
The competition ended with a bit of a twist; two competitors came away with the first place trophy, that trophy being a magnum bottle of Nicolas Feuillatte and the possibility of having their cocktail featured at Tales of the Cocktail 2015. Marlo Gamora of Jeepney and James Menite of The Plaza Hotel were crowned the two winners of the evening, with Micaela Piccolo of Distilled NYC coming away with the People's Choice award.
A tie in a cocktail competition is fairly unheard of, but it signaled something exciting that industry insiders have known for a while; that Champagne cocktails are on the rise. It also means that competitions like these are finally being seen as ways to showcase the creative use of a base spirit like Champagne, and not just for bartender bragging rights or awareness of a certain spirit brand. In a display of excellent sportsmanship, Gamera and Menite accepted their equal first place win with joy, hugging it out and congratulating each other on a job extremely well done.
Whereas most champagnes ring in with a fairly hefty price tag, making working them into a drink menu not the economical choice for using in cocktails, Bigelow believes it's brands like Nicolas Feuillette that end up coming out on top based on the low price point and the younger and hipper personality.
Cocktail innovation tends to be the focus for many of these competitive events, but there is also an element of education that comes in as well. Mixologists of this caliber know what makes a good cocktail great, and by discovering that champagne serves as the perfect balancing agent, it's only a matter of time before the bubbly becomes a regularly featured ingredient in many a cocktail.
The cocktail industry sees its fair share of changing trends, with a particular drink being all the rage one day and yesterday's news the next day, but when it comes to champagne cocktails, the attitude is fairly clear; they aren't going anywhere.
www.hannaleecommunications.com) and Alia Akkam of Beverage Media (www.bevnetwork.com) and I shared with the USBG New York Chapter members recently at a monthly meeting held at Macao Trading Company (www.macaonyc.com) as Lee introduced her PR 101 program. In short…RETURN MEDIA QUERIES!
Seriously, people. Wonder why so and so is getting all the press and you've worked longer or harder than they have and never get featured? Hmmm, maybe that's because when the media calls, you don't respond. Or maybe it's because they don't know how to find you. Well, that stinks. But it's easy enough to change.
If you walk away with only two things from this story, remember this: 1) Return media queries (as noted above) and 2) Content is king.
Oh sure, sometimes it's glamorous; famous people coming in for your drinks, keeping the party going and being recognized as the ringleader of it all, phone numbers and propositions flying across the bar. But the truth is, you work hard for your money…long shifts with wet hands, satisfying customers who don't know what they want but know they don't like gin until you make them a gin drink without telling them it's gin and they tell you it's delicious, hours spent perfecting a cocktail recipe, planning that drink menu, batching five gallons of cocktails for a charity event that you graciously donate your time to, keeping a straight face when the liquor salesman comes in to sell you something that has absolutely no application at your bar and it's obvious he hasn't a clue about his product, the list goes on and on.
And it's all chronicled in Bar Diaries with Naren Young and Simon Ford; taking you behind the glitz and glamour of tending bar.
Mix It Up NYC with Don Q from Francine Cohen on Vimeo.
Leave it to DonQ rums (www.donq.com) to hold their cocktail competition in a space (almost) nobody had seen before, revealing the country's best rum cocktails that (almost) nobody had tasted before in New York.
Though New York City's fire department officially frowns on Albert Trummer's pyrotechnic show at his Chinatown cocktail den, Apotheke, thankfully they were nowhere to be found on May 17th during the Manhattan Cocktail Classic (www.manhattancocktailclassic.com) as the judges for the DonQ cocktail competition's US finals dodged workmen busily putting the finishing touches to Trummer's latest venture, Theater Bar.
After an hour or so of ducking out of the way as wallpaper was smoothed and lighting fixtures affixed to the wall, a sneak peak taste of the soon to be released Blackbeard Spiced Rum was shared before the taste tests began.
15 cocktails were sipped and assessed; each featuring DonQ Rum. The competitors were evaluated on their use of the spirit, drink flavor, and presentation by judges Tony Abou Ganim, Francine Cohen, James Menite and Albert Trummer. The prize? Five winners were to head to Puerto Rico in June to represent the United States in a cocktail throw down against the Puerto Rican team.
The fabulous five selected to represent the United States: Tony Devencenzi of San Francisco; Noah Heaney of Denver, CO; Charles Joly of Chicago; Brian Matthys of New York City; Darren West of Las Vegas.
Ultimately, the overall competition winner was from Puerto Rico, Hiram Avila.
In a cocktail shaker, add rums, ginger syrup, fresh cucumber juice and ice.
Shake vigorously and strain over martini glass.
Garnish with cucumber slice and a sprinkle of sparkly sugar.
To make cucumber juice, use 2 cucumbers and a juicer. If a juicer is not available, add 2 unpeeled cucumbers and ¼ oz. of water to a blender. Blend on high speed and strain mixture.
Cheers to an amazing Manhattan Cocktail Classic (www.manhattancocktailclassic.com)! Five days and evenings of parties, seminars, tastings, luncheons, dinners…and of course, lots of cocktails. I couldn't resist the opportunity to ask many of the notable bartenders, chefs, panelists and event hosts in attendance about their favorite things to do in New York City during the summertime.
Mario Batali (Celebrity Chef, Television Personality and Former James Beard Chef of the Year): "Pier 40 (www.pier40.org) . I really love going there to watch my kids play ball." The Chef makes an excellent choice. My husband plays rec. soccer here, too, and it's also fun (and free) to kayak through the Downtown Boathouse (www.downtownboathouse.org).
Jon Bignelli (Chef de Cuisine, wd-50 www.wd-50.com and Food Network "Chopped" Winner): "I love anywhere that lets you sit outside and enjoy an ice cold beer. Gowanus Yacht Club, for instance, or any beer garden." When it comes to cocktails, he and others from wd-50 love PDT and Death and Co.
Richard Boccato (Proprietor, Painkiller, Co-owner, Dutch Kills): Richie offered another vote for Prospect Park, and spending a lazy afternoon relaxing there.
Michael McIlroy (Panelist, Manhattan Cocktail Classic and Bartender, Milk and Honey, Little Branch): Winter, Spring, Summer or Fall, Michael always enjoys paying homage to his heritage. And what's the best way to do that? "Drink at McSorley's," he says (the renowned Irish pub dating back to 1854).
Sasha Petraske (Proprietor, Milk and Honey, Little Branch, White Star, Dutch Kills, The Varnish [Los Angeles] and cocktail consultant): Sasha honed in on a classic New York City spot: "Grand Central Station or honestly, a cab to JFK" (admitting to another trend among New Yorkers who escape to avoid the city heat). He also enjoys strolling the semi-deserted streets of the Financial District post-closing bell. As a former Battery Park resident, I tend to agree — it feels like the city is all yours after Wall Streeters and tourists head out of town.
Lesley Townsend (Founder, Manhattan Cocktail Classic): "I love bringing my dog during off-leash hours to Prospect Park." Lesley also noted the stellar picnic potential. "To avoid using plastic cups, I bring Reidel stemless glassware and a bottle of champagne" (so she doesn't have to worry about a wine corkscrew).
Phil Ward (Proprietor, Mayahuel): Phil didn't name one spot or activity in particular, but discussed how he loved the freedom of spending more time outside and meandering around the city's streets.
My pick? For me, it's all about the private rooftop. I admittedly tend to cringe when friends suggest visiting public roof bars, only because they're usually packed and come with poor drink quality plus high prices. Of course, having your own outdoor space is ideal, but you can become an honorary deck guest with just about anyone by bringing quality drinks and / or tasty foods if you're ever invited over (please note, I enjoy bubbly and chocolate covered-strawberries).
What are your favorite NYC Summer picks?
You've toiled in the front of the house for how many years now, listening to management and biting your tongue as you know that that paycheck means eventual freedom and you just need to mark time until you can open your own place and do it right?
But can you? Do you have what it takes? Do you even know what it takes to run a profitable bar and avoid the pitfalls and traps that cause a high percentage of new bar businesses to fail within the first 18 months?
Hedge your bets and join us for Booze 2.0: Beyond Slinging Drinks (5/17, 11:30 a.m.) – The Manhattan Cocktail Classic's insightful panel discussion with some of this country's most successful bar owners and managers. For ticket information and to purchase yours for the event please click here!
In a discussion moderated by INSIDE F&B Editor in Chief Francine Cohen (aka yours truly) Jason Littrell (Death & Company www.deathandcompany.com, Dram www.drambar.com), James Moreland (Bombay Sapphire www.bombaysapphire.com) , Gianfranco Verga (Louis 649 www.louis649.com), and Dushan Zaric (Employees Only www.employeesonlynyc.com, Macao Trading Company www.macaonyc.com) will tell you how to get liquor on the shelves, put butts in seats, and keep engaged, and enthusiastic bartenders behind the bar.
Hip Hostess NYC sits down with Inside F&B editor-in-chief, Francine Cohen.
Click the link below to read the full article!
INSIDE F&B Editor-In-Chief, Francine Cohen, along with a panel of some of NYC top cocktail and culinary minds gathered together on May 19th for the Manhattan Cocktail Classic to discuss some of NYC best summer spots.
Read the release below to see what Francine and other minds had to say about their favorite NYC Summer Spots!


text:
Article| June 29 2009
Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant
M. Paula Longhi,
M. Paula Longhi
Laboratory of Cellular Physiology and Immunology and Chris Browne Center, The Rockefeller University, New York, NY, 10065
Christine Trumpfheller,
Christine Trumpfheller
Juliana Idoyaga,
Juliana Idoyaga
Marina Caskey,
Marina Caskey
Ines Matos,
Ines Matos
Courtney Kluger,
Courtney Kluger
Andres M. Salazar,
Andres M. Salazar
Oncovir, Inc, Washington, DC 20008
Marco Colonna,
Marco Colonna
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Ralph M. Steinman
CORRESPONDENCE Ralph M. Steinman: steinma@mail.rockefeller.edu
Abbreviations used: cDC, conventional DC; poly IC, polyinosinic:polycytidylic acid; PRR, pattern recognition receptor; TLRs, Toll-like receptor.
Received: January 30 2009
© 2009 Longhi et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
M. Paula Longhi, Christine Trumpfheller, Juliana Idoyaga, Marina Caskey, Ines Matos, Courtney Kluger, Andres M. Salazar, Marco Colonna, Ralph M. Steinman; Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant. J Exp Med 6 July 2009; 206 (7): 1589–1602. doi: https://doi.org/10.1084/jem.20090247
Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4+ T cell responses to a dendritic cell (DC)–targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205+ DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4+ immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow–derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.
Vaccines induce protective immunity against numerous infectious diseases. However, current vaccines have limited efficacy against challenging infections like tuberculosis, malaria, and HIV, where Th1-type T cell immunity likely has a potentially important role (Seder and Mascola, 2003). Proteins represent a potential route to safe new generation vaccines (Ulmer et al., 2006), but, typically, these are poorly immunogenic for T cells when administered alone. Therefore, special attention is being given to additional adjuvants or enhancers of immunity. Adjuvants activate innate immunity and, in particular, the maturation of antigen-presenting immunostimulatory DCs (Pulendran and Ahmed, 2006; Kool et al., 2008). To understand adjuvant action, and to identify optimal ones, it is necessary to move beyond the current focus on in vitro assays and selected monoclonal or TCR transgenic T cells to the intact animal and patient. This avenue of research should also help to identify mechanisms that link innate responses to adaptive immunity in vivo.
Adjuvants can stimulate innate immunity by interacting with specialized pattern recognition receptors (PRRs), including Toll-like receptors (TLRs; Akira et al., 2006; Kawai and Akira, 2009) and nucleotide-binding oligomerization domain receptors (Li et al., 2008). DCs express a repertoire of PRR, allowing the recognition of a range of pathogen constituents. The engagement of PRR on DCs leads to de novo transcription and secretion of cytokines and chemokines, enhanced antigen presentation capacity, and migration to lymphoid tissues where the DCs interact with T cells and B cells to initiate and shape the adaptive immune response. The type of stimulus conditions DCs to adopt a Th1- or Th2-polarizing function (Mazzoni and Segal, 2004; Kwissa et al., 2007). However, many cell types, including nonhematopoietic cells, express PRR and produce cytokines during innate immunity (Nolte et al., 2007). To understand adjuvant action, it will likely be necessary to understand the contribution of cytokines and different cellular sources of cytokines to the initiation of immunity from the naive polyclonal repertoire.
Among innate inflammatory cytokines, type I IFNs are considered to be major players for linking innate to adaptive immunity. First uncovered for their innate antiviral activity (Nagano and Kojima, 1954; Lindenmann et al., 1957), type I IFNs also enhance adaptive responses. They activate DCs, critical antigen-presenting cells for initiating immunity (Blanco et al., 2001), by promoting the expression of costimulatory molecules (Luft et al., 1998; Gallucci et al., 1999; Ito et al., 2001). This includes human blood monocytes, where type I IFN can stimulate differentiation into DCs (Paquette et al., 1998; Santini et al., 2000). Type I IFN–treated DCs prime T cells in vitro more effectively. The DCs also up-regulate expression of lymph node–homing CCR7 and exhibit stronger migratory capacity compared with DCs differentiated with other cytokines (Paquette et al., 1998; Parlato et al., 2001). Type I IFN further promotes cross-priming of CD8+ T cells by direct stimulation of DCs (Le Bon et al., 2003). Le Bon et al. (2001) showed that type I IFNs act on ex vivo–derived DCs to become better stimulators of antibody immunity. Nevertheless, the effects of type I IFN on DCs in situ need definition to understand the link between the innate production of cytokines to adaptive immunity.
Type I IFNs also have direct effects on T cells, particularly CD8+ T cells (Tough et al., 1996), by extending their survival during antigen-driven clonal expansion (Kolumam et al., 2005; Le Bon et al., 2006). In contrast, the importance of type I IFNs in the generation of antigen-specific CD4+ T cell responses is uncertain. Type I IFN inhibits secretion of Th2 cytokines (IL-4 and IL-5) and stimulates type II IFN or IFN-γ production (Brinkmann et al., 1993; Wenner et al., 1996). Type I IFNs inhibit the death of activated CD4+ T cells (Marrack et al., 1999), a finding which was later extended to the adoptive transfer of antigen-specific CD4+ T cells lacking the expression of type I IFN receptors (Havenar-Daughton et al., 2006). However, although it was originally believed to induce STAT4 activation, type I IFN failed to promote Th1 commitment in human and mouse CD4+ T cells in vitro. Despite induction of detectable STAT4 tyrosine phosphorylation, the activation was transient and insufficient to drive Th1 development in the absence of IL-12 (Berenson et al., 2004; Ramos et al., 2007). Therefore, the effect of type I IFNs on the generation of antigen-specific CD4+ T cells in vivo is unclear from these observations.
Several TLR agonists are currently being evaluated as adjuvants for vaccine development. Among them, synthetic double-stranded RNA, polyinosinic:polycytidylic acid (poly IC), induces inflammation and long-lasting T cell immunity (Salem et al., 2006; Trumpfheller et al., 2008; Stahl-Hennig et al., 2009). Poly IC is recognized by TLR3 located mostly in endosomal membranes. Also, poly IC binds the cytoplasmic RNA helicase MDA5, resulting in activation of IRF-3 and representing a major pathway for the production of type I IFNs in a TLR-independent fashion (Kato et al., 2006). Poly IC is a strong inducer of type I IFN. Although DCs and macrophages produce type I IFN upon poly IC stimulation in vitro, many nonhematopoietic cell types potentially could provide a source of this cytokine in vivo (Alexopoulou et al., 2001; Gitlin et al., 2006). In addition, the critical question of whether type I IFN is required for the adjuvant effect of poly IC and its mechanism of action in situ has not been addressed.
In this paper, we study the mechanism whereby poly IC adjuvants CD4+ Th1 immunity for an HIV gag protein vaccine that is targeted in vivo to DCs within a monoclonal antibody to the uptake receptor DEC-205/CD205 (Trumpfheller et al., 2008). We found that poly IC is a superior adjuvant and then we made several findings on its effects in vivo. First, type I IFN is the dominant driver of two innate cellular responses: the maturation of DCs and the activation of NK cells to produce type II IFN. Second, type I IFN must act directly on DCs to induce their maturation into immunostimulatory cells and also is necessary for the generation of a Th1 CD4+ adaptive T cell response, whereas IL-12 and type II IFN-R are not. Third, both bone marrow–derived and stromal cells play essential roles in providing type I IFN. These data illustrate that poly IC adjuvants Th1 CD4+ T cell immunity by replicating the early systemic effects of viral infection through its superior and widespread induction of type I IFN.
Poly IC is a superior adjuvant for CD4+ T cell responses to a protein vaccine
We had previously shown that poly IC was a superior adjuvant to monophosphoryl lipid A for inducing strong protective CD4+ T cell responses to HIV gag p24 delivered within an anti-DEC antibody (Trumpfheller et al., 2008). To find the most appropriate adjuvant for this new approach to protein-based vaccines, we extended our analysis to other TLR ligands, including MALP-2 (TLR2/TLR6), Pam3Cys (TLR1/TLR2), poly IC (TLR3), poly ICLC (TLR3-complexed poly IC with carboxymethylcellulose and poly-l-lysine to improve resistance to ribonucleases), LPS (TLR4), R-848 (TLR7/TLR8) delivered as an aqueous compound or within a topical cream, and CpG (TLR9). Mice were immunized with α-DEC-gag p24 mAb and specific adjuvants twice i.p. over 4 wk. 1 wk after the final injection, antigen-specific responses were evaluated by IFN-γ secretion in response to gag p24 peptides, primarily by multicolor flow cytometry.
Poly IC, together with its analogue poly ICLC, induced stronger gag-specific CD4+ T cell responses compared with the other adjuvants (Fig. 1, A and B) with frequencies of >1% of IFN-γ producing CD3+CD4+ T cells after i.p. injection. The improved CD4+ T cell responses with poly IC and poly ICLC were also evident at the single cell level when IL-2 and TNF-α were measured (Fig. S1). ELISA assays showed that IL-4 and IL-17 were not induced during the adjuvant role of poly IC or poly ICLC, verifying a polarized Th1 response (Fig. 1 C). The second most active adjuvant was R-848 applied in a cream to the skin, but the other adjuvants were weak (Fig. 1, A and B). To verify the activity of the adjuvants, we also measured antibody responses to HIV gag and found that all the adjuvants were active, with LPS being at least as active as poly IC (Fig. 1 D). These results encouraged us to compare the innate response to poly IC relative to other agonists to help explain its superior adjuvant role in inducing Th1 type CD4+ T cell immunity.
The innate response to poly IC uniquely leads to high levels of serum type I IFN
To assess the innate response to poly IC and poly ICLC, we studied the systemic release of inflammatory cytokines, a prototype response to agonists for PRR. We did ELISA assays on serum at 1, 3, 6, or 20 h after administration of different TLR ligands i.p., measuring IL-6, TNF-α, IL-12p40, IFN-γ, IFN-α, and IFN-β. All TLR agonists, to different extents, induced production of IL-6, TNF-α, and IL-12 p40 (Fig. 2), whereas serum IL-12p70, IL-1β, and IL-10 were not detected (not depicted). Interestingly, production of type I and type II IFNs was more restricted, being detected primarily with poly IC, poly ICLC, and R-848. Detection of IFN-α and IFN-β was transient, peaking at 3–6 h, and was highest with both poly IC and poly ICLC (Fig. 2). Thus, strong type I IFN production is a special feature of poly IC, suggesting a role for IFN in its adjuvant effect.
Poly IC induces activation of DCs
Poly IC matures both mouse and human DCs (Verdijk et al., 1999; Blanco et al., 2001; Lopez et al., 2004; Tsujimoto, 2006), and this maturation was shown to depend on type I IFN (Honda et al., 2003). However, these experiments were performed in vitro. To assess DC maturation in vivo, mice were injected with 50 µg poly IC, and the effect of type I IFN was blocked by simultaneous injection of IFNAR1 (anti–IFN-αβ receptor) antibody or isotype control. After 12 h, expression of MHC-II and costimulatory molecules (CD40 and CD86) on DEC+ and DEC− DCs was analyzed by flow cytometry (Fig. 3 A). Blocking type I IFN receptor effectively reduced DC maturation, as monitored by the up-regulation of these immunostimulatory molecules in response to poly IC.
To directly evaluate the stimulatory capacity of DCs after poly IC stimulation with or without blockade of type I IFN receptors, we isolated the CD11c+ DC fraction from the spleen, fixed the cells with formaldehyde to block further differentiation in culture, and added the DCs in graded doses to allogeneic T cells in an MLR. DCs from poly IC–treated mice became active stimulators of the allogeneic MLR, whereas blockade of type I IFNs abrogated this effect (Fig. 3 B), showing that this cytokine was essential for DC maturation in response to the poly IC adjuvant.
Direct IFNAR signaling is critical for DC activation in vivo
To determine if type I IFN had to act directly on DCs to bring about maturation, we made mixed bone marrow chimeras in which the injected bone marrow suspension was a 50:50 mixture of CD45.1 WT and CD45.2 IFN-AR−/− cells (Fig. 4 A). When poly IC was given to these chimeric mice, only the CD45.1 WT cells were able to up-regulate costimulatory molecules and MHC class II (Fig. 4 B) and exhibit enhanced MLR stimulatory activity (Fig. 4 C and Fig. S3). Therefore, Type I IFN directly promotes maturation of DCs to link innate to adaptive immunity.
Poly IC induces IFN-γ production by NK cells
An additional component to the innate response to the in vivo administration of poly IC is the rapid activation of NK cells to produce IFN-γ (Salem et al., 2006). Because we had observed an innate IFN-γ response to poly IC, we assessed NK cells as a possible source. We found that CD11cintDX5+ NK cells were major producers of IFN-γ, whereas other cells, including CD11chighDX5− DCs were inactive (Fig. 5, A and B).
Human NK cells respond directly to poly IC and CpG to produce IFN-γ (Lauzon et al., 2006) but, in contrast, purified mouse NK cells fail to respond directly to TLR agonists and require the presence of DCs or IL-12 (Sawaki et al., 2007). To assess the latter possibility, highly purified splenic NK cells were incubated overnight in the presence of poly IC, and IFN-γ production was measured by ELISA. As a positive control, the NK cells were stimulated with rIL-12 and rIL-15 (Walzer et al., 2005). In concordance with previous studies, poly IC failed to directly activate mouse NK cells (Fig. 5 C). We then considered that NK activation was indirect and a result of stimulation by type I IFNs. To test this, mice were injected with poly IC 1 h after injection of anti-IFNAR1 or isotype control. Antibody treatment dramatically reduced IFN-γ production by NK cells (Fig. 5 D). Collectively, these results highlight the importance of type I IFN during the innate responses of both DCs and NK cells in the intact animal.
Poly IC directly activates multiple cell types to produce type I IFN
Type I IFNs are rapidly produced by many cell types in response to immune and/or inflammatory stimuli. Poly IC was previously shown to stimulate mouse conventional DCs (cDCs), bone morrow–derived DCs, and macrophages to produce type I IFN (Gitlin et al., 2006; Kumagai et al., 2007). Splenic cells were sorted into several different cell types, stimulated in vitro with poly IC, and tested for the production of type I IFN. First, spleen cells were preenriched by magnetic depletion of B, T, and erythroid cells with antibodies to CD19, CD3, and TER119. The depleted cells were then sorted based on the expression of a set of specific markers. NK cells were CD3−DX5+, plasmacytoid DCs (pDCs) were CD11cintDX5−B220+, and cDCs were CD11chiDX5−CD8− or CD8+DEC-205+ (Fig. 6 A; Asselin-Paturel et al., 2005; Blasius et al., 2007; Dudziak et al., 2007). To sort granulocytes and monocytes, we gated for CD11bhi cells and then isolated LyC6hi granulocytes and CD115+ monocytes. Macrophages were defined as CD11blowF4/80+ cells (Fig. 6 A; Taylor et al., 2005; Randolph et al., 2008).
When each of the sorted cell subsets (3 × 105 cells/well) were stimulated overnight with poly IC, both CD8+DEC+ DCs and monocytes produced type I IFN, whereas CD8− DCs, pDCs, NK, granulocytes, and macrophages did not (Fig. 6 B). To test the capacity of nonhematopoietic cells to produce IFN-β, we used splenic stromal cells (105 cells/well) that had been expanded from CD45-negative spleen cells. Similar to monocytes and DEC+ DCs, stromal cells were able to produce type I IFN (Fig. 6 B).
Although production of type I IFN by pDCs requires activation of the TLR system, cDCs and fibroblasts were shown to secrete type I IFNs in response to viral infection in a cytosolic helicase-dependent fashion (Kato et al., 2005). Because poly IC can be recognized by the RNA helicase mda5, we next tested if poly IC–induced IFN-β required this PRR. Monocytes, DEC+ DC, and stromal cells from WT, mda5−/−, or TLR3−/− mice were stimulated in vitro with poly IC as described in the previous paragraph. Interestingly, the production of IFN-β by all cell types was comparable between WT and TLR3−/− cells, whereas it was severely impaired in mda5−/− cells (Fig. 6 C). Similar results were obtained when we measured the release of type I IFN into the serum of mice genetically deficient in these PRRs after poly IC injection (Fig. S2), confirming prior observations (Gitlin et al., 2006). DEC+ DCs were also able to produce small amounts of IFN-β in an mda5-independent fashion (Fig. 6 C), but this was ablated in mice doubly deficient in TLR3 and mda5 (not depicted).
To investigate the extent to which type I IFN in vivo required hematopoietic cells, e.g., DEC+ DCs and monocytes, and nonhematopoietic cells, e.g., endothelia, stromal bone marrow chimeras were generated by reconstituting mda5−/− mice with WT bone marrow or, alternatively, WT mice were reconstituted with mda5−/− marrow. The different chimeras were injected i.p with poly IC, and serum was collected after 6 h. Interestingly, WT recipients of mda5−/− marrow produced high levels of IFN-α/β, whereas mda5−/− recipients of WT marrow were severely compromised (Fig. 6 D). These data demonstrated that stimulation of radioresistant cells, but not bone marrow–derived cells, was required for the bulk of type I IFN production after poly IC treatment.
Production of type I IFNs by poly IC, but not IL-12 p40, is needed for Th1 immunity
To determine if type I IFN could influence the development of adaptive Th1 immunity, we first analyzed gag-specific CD4+ T cells responses after IFNAR1 blocking of mice immunized with either α-DEC-p24 or nontargeted gag p41 protein. In both cases, the adjuvant effect of poly IC was markedly reduced by IFNAR1 blocking compared with isotype control (Fig. 7 A and Fig. S4 A). TNF-α and IL-2 production likewise was also severely reduced (unpublished data). Similar results were obtained with IFNAR−/− mice, which were unable to develop adaptive Th1 immunity in response to α-DEC-p24 plus poly IC (Fig. 7 B). To extend the evidence for a critical role of type I IFNs, we studied mice with a genetic deletion of STAT1, the major signal transducer for type I IFNs. As anticipated, immunization with either α-DEC-p24 or p41 protein was totally ablated in STAT1−/− mice (Fig. 7 C and Fig. S4 B). Thus, the stimulation of type I IFNs by poly IC is required for its adjuvant role.
Type I IFNs may regulate Th1 responses by indirect mechanisms. IFN-αβ signaling can up-regulate IFN-γ production by DCs, promoting the ability of naive T cells to respond to IL-12 (Wenner et al., 1996; Montoya et al., 2002), whereas type I IFN can up-regulate expression of IL-12Rβ2 on human CD4+ T cells, increasing responsiveness to IL-12, a pivotal cytokine for Th1 responses (Rogge et al., 1997). To determine if type I IFN might additionally require either increased IFN-γ or IL-12 signaling, CD4+ T cell responses to DEC-p24 and poly IC were analyzed in IFN-γR (IFN-γ receptor) and IL-12 p40–deficient mice. Both IFN-γR−/− and IL-12p40−/− mice mounted similar responses to WT mice (Fig. 7, D and E and Fig. S4, C and D), implying that type I IFN, but not type II IFN or IL-12, was directly required for poly IC to induce polarized Th1 immunity.
IFN from both bone marrow–derived and nonhematopoietic cells is needed for the adjuvant function of poly IC
Because mda5 and TLR3 are the major PRRs for type I IFN production in response to poly IC, and both receptors were needed to induce gag-specific CD4+ T cell responses (Trumpfheller et al., 2008), we analyzed chimeric mice in which either the bone marrow donor or recipient were lacking both mda5 and TLR3. The B and T cells in these mice were >98% donor in origin after typing with anti-CD45.1 and anti-CD45.2 (unpublished data). In each case, i.e., when either the hematopoietic cells or nonhematopoietic cells could not make type I IFN via poly IC, its adjuvant effect was reduced by >80% (Fig. 8). These results indicate that a systemic innate response, and not only a local response by antigen-capturing DCs, accounts for the adjuvant role of poly IC.
Poly IC as a clinically feasible adjuvant for protein-based vaccines
Vaccines, particularly those that elicit protective T cell immunity, are comprised of antigens and adjuvants, with the latter influencing the quantity and quality of the specific immune response. Vaccine science can be extended by understanding the mechanism of adjuvant action, particularly in the setting of the intact animal and a polyclonal T cell repertoire. In this paper, we have analyzed the adjuvant mechanism of poly IC in an experimental system in which HIV gag is the antigen and the elicited immune response is durable, broad (many peptides recognized), and capable of providing protective immunity (Trumpfheller et al., 2008). In contrast to the findings in the current paper, many prior studies of adjuvants, like CpG and LPS, have analyzed isolated cells and the clonal expansion of TCR transgenic T cells (Lore et al., 2003; Napolitani et al., 2005; Thompson et al., 2005; He et al., 2007; Mata-Haro et al., 2007).
Our interest in poly IC was initially based on two sets of findings. Poly IC has been used in humans to induce type I IFN in cancer patients and had a good safety record (Robinson et al., 1976). These studies were directed purely to the innate IFN response, rather than adjuvanting adaptive Th1 immunity. Also, Stahl-Hennig et al. (2009) have shown that poly IC is a more potent adjuvant than other agonists for responses to protein antigens in rhesus macaques. We find that poly IC is superior to several other candidate adjuvants and have identified important mechanistic aspects. For example, although both monocytes and DEC+ DCs are able to make type I IFN in response to poly IC, we found in bone marrow chimera experiments that the main source of type I IFN in vivo after administration of poly IC emanates from nonhematopoietic radioresistant cells. We proceeded to analyze the contribution of the type I IFN response to adjuvant action.
Type I IFN but not type II or IL-12 p40 mediates the adjuvant action of poly IC
A recognized feature of adjuvants is the induction of inflammatory cytokines through PRR (Kool et al., 2008; Li et al., 2008). We find that one type of cytokine, type I IFN, dominated the adjuvant role of poly IC during CD4+ Th1 immunity in vivo. Although type I IFN has been proposed to have a role during clonal expansion and survival of CD8+ T cells, its role in CD4+ Th1 development was unclear (see Introduction). However, prior experiments were done in vitro with recombinant cytokines and using CD3 antibody stimulation or transgenic models, making it difficult to extrapolate in vivo. We did consider the possibility that type I IFN could act indirectly by promoting secretion of other Th1-enhancing cytokines, IL-12 and IFN-γ. IL-12 plays a crucial role in Th1 priming (Trinchieri, 1994), whereas IFN-γ increases responsiveness to IL-12 and, in addition, activates the Th1-specific transcription factor T-bet (Lighvani et al., 2001; Afkarian et al., 2002). Nevertheless, our findings in vivo show that deletion of IL-12 p40 or IFN-γ receptor genes did not abrogate the CD4+ T cell response to a protein plus poly IC vaccine, which predominantly depends on type I IFN to be an adjuvant.
The relevance of IL-12 in vivo seems to depend on the nature of the infecting or immunizing agent. IL-12 is a major cofactor for Th1 differentiation during infection with several bacteria and parasites (Gazzinelli et al., 1994; Cooper et al., 1997) as well as soluble antigens together with adjuvant, e.g., MPLA, Quil A, and CpG (Magram et al., 1996; Smith et al., 1999; Puggioni et al., 2005; Iborra et al., 2008). Yet for some virus infections, e.g., lymphocytic choriomeningitis virus and mouse hepatitis virus, the lack of IL-12 has little or no effect on the induction of Th1 immunity (Schijns et al., 1998; Oxenius et al., 1999). One criterion for the selection of a suitable Th1 adjuvant has been the production of IL-12 by DCs in vitro, in particular, when searching for synergistic effects of multiple TLR ligands (Napolitani et al., 2005). In contrast, we could not detect systemic production of IL-12 p70 after administration of poly IC, whereas poly IC was superior at inducing systemic type I IFNs. Our findings indicate that poly IC mimics infections with certain viruses, rather than bacteria and parasites, and does not require the presence of IL-12 for the induction of Th1 immunity.
Type I IFN activates NK cells to produce type II IFN in vivo, but the latter is not required for the adjuvant action of poly IC
Another major pathway to Th1 immunity involves NK cells. NK cells are rapidly recruited to lymph nodes and splenic white pulp upon stimulation with mature DCs or some adjuvants like R-848 and poly IC, providing an early source of IFN-γ required for Th1 polarization (Martin-Fontecha et al., 2004; Grégoire et al., 2008). In addition, the production of proinflammatory cytokines by NK cells has been associated with the adjuvant effect of poly IC (Salem et al., 2006). Specifically, engagement of NKp30 by DC induces secretion of proinflammatory cytokines by NK cells, e.g., TNF-α, which has been identified as an active inducer of DC maturation (Vitale et al., 2005). NK cells may have additional roles in inducing DC maturation, e.g., by direct cell contact and by selective killing of mature DCs that do not express optimal levels of MHC class I molecules (Ferlazzo et al., 2001). Several inflammatory cytokines stimulate NK cell proliferation and IFN-γ production. For example, IL-12 synergizes with IL-18 or IL-15 to induce IFN-γ, whereas IFN-αβ promotes NK proliferation and cytotoxicity (Okamura et al., 1995; Yoshimoto et al., 1998; Fehniger et al., 1999; Kim et al., 2000; Chakir et al., 2001). In this paper, we have found that poly IC induces strong IFN-γ production by NK cells and that this is fully dependent on the production of type I IFN in vivo by direct NK stimulation or though synergism with other cytokines, i.e., IL-18 (Matikainen et al., 2001). Yet IFN-γ was not needed for CD4+ Th1 responses or DC maturation, which was normal in mice genetically lacking IFN-γ receptor. It remains possible that NK cells serve other roles in the adjuvant effects of poly IC, i.e., independent of type II IFN.
Innate type I IFNs in vivo induce DCs to stimulate adaptive immunity
The major challenge to understanding adjuvant function in vivo is, by definition, to understand the induction of adaptive immunity and how an early innate response to an adjuvant leads to adaptive immunity. Immunity induced by vaccination requires two broad functions at the level of DCs but needs to be studied in vivo. One is antigen presentation, which we enhanced by targeting the HIV gag protein to the DEC-205 antigen uptake receptor on a subset of DCs. Nevertheless, our mechanistic findings were similar when we administered higher doses of the antigen as an isolated gag p41 protein. The second function is that antigen-capturing DCs need to mature in vivo to induce immunity, as shown initially with another adjuvant, α-galactosyl ceramide (Fujii et al., 2003). Additionally, maturation is required to avoid T cell tolerance upon targeting of antigens to DCs (Hawiger et al., 2001; Bonifaz et al., 2002; Kretschmer et al., 2006; Yamazaki et al., 2008). Our experiments show that type I IFN is important for DC maturation after poly IC. Poly IC and type I IFNs were each known to induce DC maturation, but their interdependence had not previously been tested in vivo. Likewise, Le Bon et al. (2001) found that type I IFNs could act on ex vivo–derived DCs, which then stimulates antibody immunity. We noted that poly IC not only up-regulates DC costimulatory molecules but allows DCs to become better inducers of MLR. The latter assay remains valuable to document that DCs have matured in the critical functional sense and not only by altering their cell surface phenotype. Both changes in DCs, i.e., up-regulation of CD86 and increased MLR stimulation, were abrogated by blockade of type I IFN receptors. Although ligation of PRRs has previously been associated with some features of DC maturation, we showed that this is not sufficient by itself and, additionally, the development of immunostimulatory function requires direct IFNAR signaling of the DCs.
Both bone marrow–derived and nonhematopoietic cells need to respond to poly IC
Elegant studies have shown that antigen-presenting cells must themselves respond to TLR agonists to induce strong clonal expansion of antigen-specific TCR transgenic T cells (Spörri and Reis e Sousa, 2005; Nolte et al., 2007), but the response of naive mice and the contribution of radioresistant cells was not fully evaluated. Unexpectedly, we found that the adjuvant role of poly IC for a primary CD4+ T cell response was severely reduced when either bone marrow–derived or radioresistant cells lacked the mda-5 and TLR3 PRRs. Radioresistant cells could include endothelial, epithelial, and stromal cells. The Langerhans type of DC is also radioresistant (Merad et al., 2002), but we found that depletion of these cells did not alter the levels of type I IFN detected in the serum. In contrast, we showed that stromal cells in vitro produced type I IFN as effectively as DCs upon poly IC stimulation, and in ongoing experiments, we find that poly IC markedly increases type I IFN mRNA in liver and lung cells suspensions that are depleted of CD11c+ cells. These findings indicate that adjuvants become more effective when they induce a systemic inflammatory response during the time that the antigen is captured and presented by DCs. In experiments that were not published, we found that the Th1 response was reduced if the administration of poly IC either preceded or followed the administration of protein antigen by 6 h. The systemic production of type I IFNs enhances two key innate responses in vivo: the maturation of DCs and the activation of NK cells, as well as key adaptive lymphocytes mediating delayed type hypersensitivity (Gallucci et al., 1999), humoral immunity (Le Bon et al., 2001), and CD8 T cell memory (Ferrantini et al., 1994). The further development of adjuvants may well need to consider the contributions of both hematopoietic and nonhematopoietic cells, which make innate cytokines that work directly on DCs so that they can act as initiators of antigen-specific adaptive immunity.
C57BL/6, BALB/c, and CxB6 F1 were obtained from Harlan, 129/SvJ, IFNγR−/−, and IL-12 p40−/− were obtained from The Jackson Laboratory, STAT1−/− were provided by D. Levy (New York University, NY, NY), IFNAR−/− were provided by K. Murali-Krishna (University of Washington, Seattle, WA), and TLR3−/− were provided by S. Akira (Osaka University, Osaka, Japan). Mice were maintained under specific pathogen-free conditions and used at 7–8 wk of age according to Institutional Animal Care and Use guidelines. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC).
In vivo cytokine secretion.
Mice were injected i.p. with 50 µg poly IC (InvivoGen), 50 µg Pam3cys (InvivoGen), 0.5 µg Malp2 (Enzo Biochem, Inc.), 10 µg LPS (E. Coli; Sigma-Aldrich), 50 µg poly ICLC (Oncovir, Inc.), 50 µg CpG ODN1826 (InvivoGen), or R-848 cream (2% topically applied to ∼5 cm2 of back skin; Celldex Therapeutics, Inc.), and serum was collected at the indicated times. Production of TNF-α, IL-12p40, IL-12p70, IL-6, IFN-γ (eBioscience), and IFN-α and IFN-β (R&D Systems) was determined by ELISA according to the manufacturer's instructions.
Cell sorting and stromal cell isolation.
Spleens were cut in small pieces and incubated at 37°C for 30 min in Hank's medium supplemented with 400 U/ml collagenase d (Roche). EDTA (5 mM in final concentration) was added for the last 5 min. Cells were stained with biotinylated anti-CD3, anti-CD19, and anti-Ter119 mAbs (BD) for 20 min, washed, incubated with Streptavidin-coated magnetic beads, and passed through LD columns (Miltenyi Biotec). The negative fraction was collected and stained with Live/Dead Fixable Aqua viability dye (Invitrogen), FITC–anti-DX5, PE–anti-CD11c, PercPCy5.5-B220, Pacific Blue–anti-CD3, Alexa Fluor 750–anti-CD8 (eBioscience), and Alexa Fluor 647–DEC-205 (produced in house). Alternatively, cells were stained with Fixable Aqua viability dye, FITC–anti-Ly6G, PE–anti-CD115, PerCP–anti-F4/80, PE-Cy7–anti-CD11c, and Alexa Fluor 750–CD11b (eBioscience). Different populations were isolated by sorting on a FACSAria with DiVa configuration (BD). Splenic stromal cells were obtained as previously described (Cheng et al., 2007). In brief, spleens were cut into small fragments, placed in a 48MW plate, and cultured in RPMI 1640 medium supplemented with 20% FCS and 1% MEM nonessential amino acids. After 1 mo, cells were further purified by CD45 negative selection.
ELISA for antibody response.
To detect gag-specific antibody, high-binding ELISA plates (BD) were coated with 1 µg/ml of gag protein overnight at 4°C, washed with PBS/0.1% Tween 20, and blocked with PBS/5% BSA for 1 h at 37°C. Serial dilutions of serum were added to the plates and incubated for another 1 h at 37°C. Plates were washed and incubated 1 h at 37°C with secondary HRP goat anti–mouse IgG antibody (Jackson Immunoresearch Laboratories). TMB (3,3′,5,5-tetramethylbenzidine) substrate solution (Thermo Fisher Scientific) was used as substrate. OD was measured in an ELISA reader at 450 nm.
DC maturation and function.
Mice were injected i.p. with 100 µg of anti-IFNAR blocking antibody or isotype control (Leinco Technologies) and 1 h later with PBS or 50 µg poly IC. Spleens were collected 12 h later and collagenase digested. Maturation was monitored by increased expression of MHC-II (I-Ek), CD86, and CD40 after gating on CD11chiDEC− or CD11chiDEC+ DCs. In mixed bone marrow chimera mice, expression of maturation markers was analyzed after gating on CD11chiCD45.2+ (IFNAR−/−) or CD11chiCD45.2− DCs. To test DC stimulatory capacity, spleen CD11c+ DCs were isolated 12 h after administration of poly IC with anti-IFNAR blocking antibody or isotype control. Alternatively, for mixed chimera mice, WT and IFNAR-deficient DCs were FACS sorted based on the expression of CD45.2. BALB/c DCs were fixed with 1% paraformaldehyde for 10 min on ice and graded numbers were added to 2 × 105 CFSE-labeled (Invitrogen) C57BL/6 T cells. After 5 d of culture, samples were stained with Live/Dead Fixable Aqua viability dye and Pacific blue–anti-CD3 and acquired on an LSR II flow cytometer (BD).
NK activation.
2 h after 50 µg poly IC i.p., spleen cell suspensions were prepared and incubated for 3 h with 10 µg/ml brefeldin A (Sigma-Aldrich). Cells were stained with Live/Dead Fixable Aqua viability dye, FITC–anti-DX5, PE–anti-CD11c, and Pacific Blue–anti-CD3 mAbs. Cells were then fixed, permeabilized, and stained with APC–anti–IFN-γ antibody. Sorted NK cells (CD3−DX5+) were cultured (3 × 105) with 50 µg/ml poly IC or 2 ng/ml rIL-12 and 1 ng/ml rIL-15 (eBioscience).
Bone marrow chimeras.
Recipient mice were γ irradiated twice with 500 rad, 3 h apart. 3 h later, they were reconstituted with 3 × 106 marrow cells that had been harvested from the femurs and tibias of age-matched mice. After 6 wk, mice were tested for chimerism by RT-PCR for mda5 on CD11c+ cells (InvivoGen) or by flow cytometry, based on the expression of CD45.1 and CD45.2 (>98%).
Assays for HIV-specific CD4+ T cells.
Mice were immunized twice i.p. at 4-wk intervals with 5 µg HIV gag-p24 and 10 µg HIV gag-p41 (Trumpfheller et al., 2008) together with adjuvant, which was 50 µg poly IC, 50 µg Pam3cys, 0.5 µg Malp-2, 10 µg LPS, 50 µg poly ICLC, and R-848 cream (topic, 5 cm2) or 50 µg CpG ODN1826. For IFNAR1 blocking, mice were simultaneously injected days 0 and 2, with 100 µg of anti-IFNAR blocking antibody or isotype control. 1 wk later, splenocytes were restimulated with 2 µM p24 or negative control peptide mix, along with 2 µg/ml of costimulatory α-CD28 (clone 37.51) for 6 h. 10 µg/ml brefeldin A was added for the last 5 h. Cells were washed, incubated 10 min at 4°C with 2.4G2 mAb to block Fcγ receptors, washed, and stained with Live/Dead Fixable Aqua viability dye, Pacific blue–conjugated anti-CD3, PerCP-conjugated anti-CD4, and Alexa Fluor 750–conjugated anti-CD8 mAbs for 20 min at 4°C. Cells were permeabilized (Cytofix/Cytoperm Plus; BD) and stained with APC–anti–IFN-γ, PE–anti–IL-2, and PE-Cy7 anti–TNF-α mAbs for 15 min at room temperature (BD). Samples were acquired on an LSR II flow cytometer and analyzed with FlowJo Software (Tree Star, Inc.).
Statistical significance was evaluated using a two-tailed Student's t test with a 95% confidence interval. Results are expressed as means ± SD. In the figures, p-values of 0.001 are labeled with a triple asterisk. Analysis was performed with a Prism 3 program (GraphPad Software, Inc.).
Fig. S1 shows the frequency of IL-2 and TNF-α–producing CD4+ T cells after DEC-p24 immunization with different adjuvants. Fig. S2 demonstrates the mda-5 dependence of the production of type I IFN in serum. Fig. S3 shows the gating strategy for the MLR assays. Fig. S4 shows representative dot plots from Fig. 7.
This research was supported by the National Institute of Allergy and Infectious Diseases (grants AI13013 and AI40874 to R.M. Steinman), by a Center for AIDS Vaccine Discovery grant from the Bill and Melinda Gates Foundation, and by a grant from the Human Frontiers of Science Program.
R.M. Steinman is a scientific advisor to Celldex Therapeutics, which is developing DEC-targeted vaccines for use in humans. A.M. Salazar heads Oncovir, which produces poly ICLC. The authors have no additional financial interest.
Afkarian
Sedy
Cereb
T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells
Pathogen recognition and innate immunity
Alexopoulou
Medzhitov
Flavell
Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3
Asselin-Paturel
Boonstra
O'Garra
Type I interferon dependence of plasmacytoid dendritic cell activation and migration
Berenson
Frontline: absence of functional STAT4 activation despite detectable tyrosine phosphorylation induced by murine IFN-α
Palucka
Pascual
Banchereau
Induction of dendritic cell differentiation by IFN-α in systemic lupus erythematosus
Barchet
Development and function of murine B220+CD11c+NK1.1+ cells identify them as a subset of NK cells
Bonifaz
Bonnyay
Mahnke
Nussenzweig
Steinman
Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance
Alkan
Heusser
Interferon α increases the frequency of interferon γ–producing human CD4+ T cells
Chakir
Cytokine expression by murine DX5+ cells in response to IL-12, IL-18, or the combination of IL-12 and IL-18
Cell. Immunol.
Nefedova
Gabrilovich
Regulation of dendritic-cell differentiation by bone marrow stroma via different Notch ligands
Magram
Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with Mycobacterium tuberculosis
Dudziak
Kamphorst
Heidkamp
Trumpfheller
Cheong
Differential antigen processing by dendritic cell subsets in vivo
Fehniger
Vandeusen
Wechser
Goodsaid
Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response
Ferlazzo
Semino
Melioli
HLA class I molecule expression is up-regulated during maturation of dendritic cells, protecting them from natural killer cell-mediated lysis
Immunol. Lett.
Ferrantini
Giovarelli
Modesti
Venditti
Lollini
P.L.
IFN-α1 gene expression into a metastatic murine adenocarcinoma (TS/A) results in CD8+ T cell-mediated tumor rejection and development of antitumor immunity. Comparative studies with IFN-γ-producing TS/A cells
Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a co-administered protein
Lolkema
Natural adjuvants: endogenous activators of dendritic cells
Gazzinelli
R.T.
Wysocka
Denkers
Hieny
Caspar
Parasite-induced IL-12 stimulates early IFN-γ synthesis and resistance during acute infection with Toxoplasma gondii
Gitlin
Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus
Chasson
Coupet
C.-A.
Dalod
Reboldi
Intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation
Havenar-Daughton
Kolumam
G.A.
Murali-Krishna
Cutting edge: the direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection
Hawiger
Ravetch
Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo
L.Z.
Mendoza-Ramirez
X.T.
Petromilli
Antigenic targeting of the human mannose receptor induces tumor immunity
Sakaguchi
Yanai
Ohteki
Kaisho
Takaoka
Selective contribution of IFN-α/β signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection
Iborra
Abanades
Bonay
Prates
F.O.
Barral-Netto
Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice
Amakawa
Ikehara
Differential regulation of human blood dendritic cell subsets by IFNs
Yoneyama
Tsujimura
Cell type-specific involvement of RIG-I in antiviral response
Reis e Sousa
Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses
The roles of TLRs, RLRs and NLRs in pathogen recognition
Weissman
I.L.
In vivo natural killer cell activities revealed by natural killer cell-deficient mice
Sprent
Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection
Soullie
Van Nimwegen
Willart
Muskens
Hoogsteden
H.C.
Hammad
B.N.
Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells
Heng
Von Boehmer
De novo production of antigen-specific suppressor cells in vivo
Nat. Protoc.
Kumagai
Morii
Aozasa
Alveolar macrophages are the primary interferon-α producer in pulmonary infection with RNA viruses
Kwissa
Kasturi
Pulendran
The science of adjuvants
Expert Rev. Vaccines.
N.M.
Ashkar
The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity
Le Bon
Bulfone-Paus
Kalinke
D.F.
Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming
Schiavoni
D'Agostinio
Gresser
Belardelli
Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo
Etchart
Gewert
Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon
Willingham
Inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3
Lighvani
Jankovic
Yamane
Aliberti
Hissong
Nguyen
Gadina
O'Shea
T-bet is rapidly induced by interferon-γ in lymphoid and myeloid cells
Isaacs
Studies on the production, mode of action and properties of interferon
Br. J. Exp. Pathol.
Moltedo
TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses
Brenchley
Kuruppu
Khojasteh
Koup
Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses
Hertzog
D.N.J.
Cebon
Type I IFNs enhance the terminal differentiation of dendritic cells
Connaughton
Warrier
Carvajal
C.-Y.
Gately
IL-12-deficient mice are defective in IFN-γ production and type 1 cytokine responses
Marrack
Kappler
Type I interferons keep activated T cells alive
Martin-Fontecha
Thomsen
Induced recruitment of NK cells to lymph nodes provides IFN-γ for TH1 priming
Mata-Haro
Cekic
The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4
Matikainen
Paananen
Kurimoto
Timonen
Julkunen
Sareneva
IFN-α; and IL-18 synergistically enhance IFN-γ; production in human NK cells: differential regulation of Stat4 activation and IFN-γ gene expression by IFN-α; and IL-12
Controlling the Toll road to dendritic cell polarization
Merad
Karsunky
Wagers
Cyster
Engleman
Langerhans cells renew in the skin throughout life under steady-state conditions
Montoya
Type I interferons produced by dendritic cells promote their phenotypic and functional activation
Pouvoir immunisant du virus vaccinal inactivé par des rayons ultraviolets
C R Seances Soc. Biol. Fil.
Napolitani
Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells
Leibundgut-Landmann
Joffre
Dendritic cell quiescence during systemic inflammation driven by LPS stimulation of radioresistant cells in vivo
Tsutsui
Yutsudo
Hakura
Tanimoto
Torigoe
Nukada
Cloning of a new cytokine that induces IFN-γ production by T cells
Oxenius
Karrer
IL-12 is not required for induction of type 1 cytokine responses in viral infections
Paquette
Kiertscher
Glaspy
Interferon-α and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells
Parlato
Lapenta
Di Pucchio
Logozzi
Giammarioli
Malorni
Fais
Expression of CCR-7, MIP-3β, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities
Puggioni
Monophosphoryl lipid A (MPL) promotes allergen-induced immune deviation in favour of Th1 responses
Translating innate immunity into immunological memory: implications for vaccine development
IFN-α is not sufficient to drive Th1 development due to lack of stable T-bet expression
Jakubzick
Antigen presentation by monocytes and monocyte-derived cells
Levine
A phase I-II trial of multiple-dose polyriboinosic-polyribocytidylic acid in patients with leukemia or solid tumors
J. Natl. Cancer Inst.
Barberia-Maino
Passini
Presky
Gubler
Sinigaglia
Selective expression of an interleukin-12 receptor component by human T helper 1 cells
El-Naggar
Kadima
Gillanders
The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu
Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in hu-PBL-SCID mice
Sawaki
Tanizawa
Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways
Schijns
V.E.
Haagmans
Wierda
Kruithof
Horzinek
Mice lacking IL-12 develop polarized Th1 cells during viral infection
Mascola
Basic immunology of vaccine development
The Vaccine Book
B.R.
P.-H.
Donachie
Grdic
Lycke
Mowat
Immune-stimulating complexes induce an IL-12-dependent cascade of innate immune responses
Spörri
Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function
Stahl-Hennig
Jasny
Rzehak
Tenner-Racz
Uberla
Kleinschmidt
Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques
PLoS Pathog.
Martinez-Pomares
Macrophage receptors and immune recognition
The low-toxicity versions of LPS, MPL(R) adjuvant and RC529, are efficient adjuvants for CD4+ T cells
Induction of bystander T cell proliferation by viruses and type I interferon in vivo
Interleukin-12: A cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes
Caskey
Nchinda
Longhi
Mizenina
The microbial mimic polyIC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine
Tsujimoto
R.F.
Abouhamze
Mochizuki
Moldawer
Maturation of murine bone marrow-derived dendritic cells with poly(I:C) produces altered TLR-9 expression and response to CpG DNA
Rappuoli
Vaccine manufacturing: challenges and solutions
Nat. Biotechnol.
Verdijk
Mutis
Esendam
Melief
Goulmy
Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells
Della Chiesa
Pende
NK-dependent DC maturation is mediated by TNFα and IFNγ released upon engagement of the NKp30 triggering receptor
Zitvogel
Natural-killer cells and dendritic cells: "l'union fait la force"
Wenner
Guler
Macatonia
Roles of IFN-γ and IFN-α in IL-12-induced T helper cell-1 development
Fiorese
CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells
Ohkusu
Kashiwamura
IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-γ production
Supplemental Material- pdf file
Poly IC is a superior adjuvant to elicit CD4+ T cell immunity. (A) CxB6 F1 mice were primed and boosted 4 wk apart with 5 µg α-DEC-p24 and 50 µg poly IC, 50 µg Pam3cys, 0.5 µg Malp2, 50 µg poly ICLC, 10 µg LPS, 50 µg CpG, or R-848 cream. R-848 cream was applied topically over 5 cm2 on the back of shaved mice. IFN-γ secretion in gated CD3+CD4+ splenic T cells in response to HIV gag p24 peptides was measured 1 wk after boost. (B) As in A, but means ± SD from five (poly IC), three (poly ICLC, R-848, and CpG), and two (Malp-2, Pam3cys, and LPS) experiments, with three mice per group. (C) CxB6 F1 mice were primed and boosted 4 wk apart with 5 µg α-DEC-p24 and 50 µg poly IC. 1 wk after boost, bulk splenocytes were stimulated with gag p24 peptide mix or control peptide mix for 3 d. The concentrations of the indicated cytokines and chemokines were measured in cell culture supernatants by luminex. Samples were acquired in duplicate and analyzed by ELISA. Bars represent the mean ± SD from two experiments (six mice total). (D) Mice were immunized as in A. 1 wk after boost, gag-specific antibodies in serum samples were measured by ELISA. Data are given as OD mean ± SD from two independent experiments (five mice total).
Induction of inflammatory cytokines in the serum by TLR ligands. Mice were treated i.p. with 50 µg poly IC, 50 µg Pam3cys, 0.5 µg Malp2, 10 µg LPS, 50 µg poly ICLC, 50 µg CpG, and 5 cm2 of 0.2% R-848 cream. IL-6, TNF-α, IL-12p40, IFN-α, IFN-β, and IFN-γ were analyzed in serum at different time points (means of four mice). Error bars indicate the mean ± SD. Data are representative of two similar independent experiments.
Type I IFN signaling controls DC maturation. (A) 1 h after 100 µg of anti-IFAR1 blocking antibody or isotype control, mice were stimulated with 50 µg poly IC for 12 h and analyzed for cell surface MHC-II, CD86, and CD40 on DEC+ and DEC− DCs. (B) As in A, but graded numbers of fixed splenic DCs from BALB/C mice were added for 5 d to 2 × 105 allogeneic C57BL/6 T cells. T cell proliferation was detected by CFSE dilution of CD3+ cells. Data are representative of three experiments.
Direct type I IFN signaling of DCs is required for their maturation. Mixed bone marrow chimeras were prepared with a 50:50 mixture of bone marrow from WT (CD45.2−) and IFNAR−/− (CD45.2+) mice. (A) Gating strategy for WT (CD45.2−) and IFNAR−/− (CD45.2+) DCs 4–6 wk after chimerism. (B) The chimeras were stimulated with 50 µg poly IC or PBS for 12 h. Expression of CD86, MHC-II, and CD40 on WT and IFNAR−/− DCs, was analyzed by FACS. Arrows show increased up-regulation of costimulatory molecules in IFNAR−/− DCs. (C) The chimeras were stimulated as in B. After CD11c enrichment, WT and IFNAR−/− CD11chi were FACS sorted as in A. Graded numbers of fixed B6.WT or B6.IFNAR−/− splenic DCs were added for 5 d to 2 × 105 allogeneic Balb/C T cells. T cell proliferation was detected by CFSE dilution of CD3+ cells. Cells numbers are indicated as 103. Data are representative of two similar independent experiments.
NK cells are major producers of IFN-γ after poly IC. Mice were injected i.p. with 50 µg poly-IC or PBS. After 2 h, spleens were collected and a single cell suspension of splenocytes was incubated with 10 µg/ml BFA for 3 h before intracellular staining for IFN-γ. (A) Five color flow cytometry to identify IFN-γ–positive splenocytes. (B) As in A, but production of IFN-γ by NK cells gated as CD3− DX5+ cells. Means ± SD of four experiments. (C) Highly purified CD3− DX5+ NK cells. 3 × 105 cells/well were stimulated with 50 µg/ml poly IC or, as a positive control, a combination of rIL-12 and rIL-15. After 12 h, IFN-γ in culture supernatants was detected by ELISA. Means ± SD of three independent experiments are shown. (D) Mice were injected with poly IC and anti-IFAR1 blocking antibody or isotype control. Production of IFN-γ was detected by intracellular staining. Mean ± SD from three experiments is shown.
Production of IFN-β by multiple cell types. (A) Splenocytes from WT mice were depleted with Ter119−, CD3−, and CD19− biotinylated antibodies using streptavidin magnetic beads before cell sorting. For monocytes, macrophages, and granulocytes, splenocytes were additionally depleted of DX5+ cells. cDCs (CD3−DX5−CD11chiCD8− and CD8+DEC205+), pDCs (CD3−DX5−CD11clowB220+), NK cells (CD3−DX5+), macrophages (CD11blowF4/80+), monocytes (CD11bhiCD115+), and granulocytes (CD11bhiLy6G+) were sorted based on their expression of specific markers (see text). Numbers 1–6 indicate the different cell types sorted. (B) Highly purified cells were plated at 3 × 105 cells/well in a round-bottomed 96-well plate and incubated with 50 µg/ml poly IC or medium alone. Splenic stromal cells, obtained as described in the Materials and methods, were plated at 5 × 104 cells/well in a 48-well plate. Supernatants were collected after 12 h and production of Type I IFN was detected by ELISA. (C) As in B, DEC+ DCs, monocytes, stromal cells from WT, TLR3−, and mda5-deficient mice were stimulated in vitro with 50 µg/ml poly IC or medium alone. (D) WT and mda5−/− mice were lethally irradiated and injected with bone marrow cells from WT or mda5−/− mice. After 6 wk, chimeras were injected with 50 µg poly IC. Serum was collected 6 h later and analyzed by Type I IFN ELISA kit. Error bars show the means ± SD from at least two experiments. ND, not detectable.
The adjuvant role of poly IC is dependent on type I IFNs and independent of IL-12 p40. (A) CxB6 F1 mice were injected i.p. with 5 µg α-DEC-p24 or 10 µg gag-p41 and 50 µg poly IC together with anti-IFNAR1 or isotype control. Mice were boosted with the same conditions at 4 wk. 1 wk later, HIV gag-specific CD3+ CD4+ splenic T cells were analyzed for IFN-γ. (B–E) As in A, the percentage of CD3+ CD4+ T cells producing IFN-γ was measured in WT, IFNAR−/−, STAT-1−/−, IFN-γR−/−, and IL-12 p40−/− immunized mice, respectively. Means ± SD are shown of two independent experiments with a total of six mice. ***, P = 0.001.
Both hematopoietic and nonhematopoietic cells contribute to the adjuvant effect of poly IC. (A) Gag p24-specific CD4+ T cell responses were evaluated in bone marrow chimeras. WT and double TLR3.mda5−/− mice were lethally irradiated and injected with bone marrow cells from WT or deficient mice. After 6 wk, chimeras were primed and boosted 4 wk apart with 5 µg α-DEC-p24 and 50 µg poly IC. After 1 wk, HIV gag-specific CD3+CD4+ splenic T cells were analyzed for IFN-γ secretion. Means ± SD from two experiments are shown; n = 6. (B) Chimerism was assessed by expression of mda5 on CD11c+ splenic cells. ***, P = 0.001.


Search Engine Optimisation - This is the art of boosting your website to the top of get MUCH higher Google rankings, build massive credibility in your chosen niche, generate leads like never before, and put your business in front of thousands upon thousands of eyeballs across the world. Their custom made services range from Website development Search services but not so good at marketing your business. Branding - This is a standard marketing term and applies to creating an image or picture logo particular topic as more and more people start blogs as experts in their fields of business. Incentive based traffic -Some websites offer incentives for visitors and unlimited hosting you can have many micro sites directing anywhere you choose. Make the vital move by securing a package for your business and wait with banking websites to ensure the safe transfer of private and sensitive information. However, care should be taken to keep the advertisements as for beginners to use and allows for integration into a website.
Internet Marketing Help - H Hubpages - This online free by searching the internet, unless they are selling a piece of internet marketing software. In winter more time might be spent on internet marketing, otherwise their interest will surely die out, and you may end up giving away too much information! each They Need To Set Key Dates For Ensuring That The Strategy Is Running According To The Plan. business is different and only the business with one page doorway sites with links to other websites. If you want to make your job even easier and save lots or business heard about, now FaceBook and Twitter are becoming prime areas where people spread the news about different companies. There are many other internet marketing methods, all of log in and check account information and other features. There is so much email marketing going on that is clogging up the email End products whic are usually the repeat sales or main sale products.


Systems and methods for providing related commercial content to a user. Systems and methods can include extraction or development of data from metadata and searching for related commercial content based upon the extracted or developed data.
MX2008011223A (en) 2008-11-04 Mining web search user behavior to enhance web search relevance.


text:
Home News Michelle Hinchey gets to work as new state senator
Michelle Hinchey gets to work as new state senator
Photo provided.
ALBANY – Michelle Hinchey, the new senator from New York's 46th District, is ready to carry the environmental mantle of her late father, former Assemblyman and Congressman Maurice Hinchey.
The Democratic Hinchey has been appointed chairwoman of the Senate Agriculture Committee. She sees agriculture as "a strong pillar for New York's economic recovery and essential to the preservation of our natural resources."
Governor Cuomo has now renewed his call for the legalization of recreational marijuana, something Hinchey supports as an economic and revenue generator.
As for herself, Hinchey said she doesn't use it at present.
"I don't now, but I am from Woodstock and I know a lot of people who do," she said. When asked if she would use marijuana if and when it becomes legalized, she said, "we will see. I don't know."
Hinchey's senate district includes Greene and Montgomery counties and parts of Ulster, Albany, and Schenectady counties.
A Brief History of Saratoga Race Course
Kingston takes wraps off city 2030 climate action plan
Orange County Sheriff's Police Reform and Reinvention session scheduled
Dutchess County to open COVID-19 vaccination sites
Close to 2,000 new positives, 22 more deaths in latest one-day...
Competition Subaru of Smithtown
CBC Computer Services
Truck and Trailer Depot
Martucci declares victory in 42nd State Senate race


text:
Ambrosini AR – włoski, bezzałogowy samolot sterowany radiem wybudowany w okresie II wojny światowej.

Historia
13 sierpnia 1942 roku, włoski bombowiec Savoia-Marchetti SM.79 wypełniony materiałami wybuchowymi zaatakował brytyjskie okręty stojące w porcie w Algierze. Nie byłoby w tym nic dziwnego gdyby nie fakt, że na pokładzie bombowca nie było sterującej nim załogi. Lot samolotu kontrolowany był drogą radiową i była to pierwsza w czasie II wojny światowej próba bojowego użycia bezzałogowego samolotu bombowego. Co prawda atak nie został uwieńczony sukcesem ale samą próbę uznano za obiecującą. Do idei tego typu maszyn powrócono w wytwórni SAI Ambrosini, w której inżynier Sergio Stefanutti zaprojektowała i zbudował samolot wykorzystujący tą samą technikę nazwany Ambrosini AR (AR od Assalto Radioquidato). Samolot startował i dolatywał w rejon celu będąc kierowanym przez pilota znajdującego się na pokładzie. W rejonie celu pilot opuszczał maszynę na spadochronie a kontrolę nad samolotem przejmował operator znajdujący się na pokładzie samolot towarzyszącego. Operator drogą radiową sterował bezzałogową maszyną w drodze do celu. Na pokładzie Assalto Radioquidato przewidziano miejsce na dwie 1000 kg bomby. Dziewiczy lot odbył się 13 czerwca 1943 roku, za sterami samolotu siedział pilot doświadczalny Nello Valziana. Lot przebiegł pomyślnie i samolot skierowano do dalszych badań w ośrodku technicznym lotnictwa w Guidonia Montecelio niedaleko Rzymu. Wyprodukowano cztery egzemplarze (niektóre źródła podają pięć), które jednak przed kapitulacją Włoch nie weszły do bojowego użycia.

Konstrukcja
Samolot był wolnonośnym średniopłatem o drewnianej, prostej konstrukcji. Zastrzałowe, stałe podwozie główne i kółko ogonowe. Usterzenie klasyczne, wolnonośne. Otwarta kabina pilota znajdująca się nad centropłatem z niewielką owiewką. Samolot napędzany był silnikiem Fiat A-80 o mocy 1000 KM z trójłopatowym śmigłem.

Bibliografia
 Samolot bezzałogowy Ambrosini AR, "Skrzydlata Polska", nr 37 (1984), s. 11, .

Bezzałogowe statki powietrzne
Włoskie samoloty wojskowe II wojny światowej
AR


"Make phone calls safely and conveniently through the bluetooth enabled audio system. This Brand New Peugeot 2008 SUV has air conditioning, making those summer drives a breeze. It makes the journey stylish and economical. This car has 6 airbags to protect you and your family with an ANCAP safety rating of 5. This car has front & rear power windows, driver airbag, brake assist and speed dependant volume stereo. Collision avoidance system. Park assist will give you that peace of mind. Answer calls listen to music using the multi-function steering wheel. This car has leather steering wheel, USB audio input, 16\" alloy wheels, hill holder, central locking, passenger airbag and side airbags. Fog lights. Strong ABS brakes. This car has LED tail lamps, cruise control, remote central locking, rear vision camera and engine start stop system.
Enquire on this car to speak to a dealer. This car has 6 airbags fitted for your safety with an ANCAP safety rating of 5.
Offer available whilst stocks last at participating Peugeot dealers on new MY18 2008 Active models sold between 1 March 2019 and 30 April 2019 and delivered by 30 April 2019. Price is the manufacturer's recommended retail driveaway price (RDP) on a new vehicle. Final driveaway costs may vary according to individual circumstances and from dealer to dealer. Savings calculated based on the pre-offer manufacturers list price plus registration, stamp duty and dealer delivery charges, and is the minimum saving depending on the state or territory of purchase. Offer excludes demonstrator models, fleet, government and rental buyers. Metallic/Premium paint not included. Not available in conjunction with any other offer. Peugeot Australia reserves the right to change, supersede or extend these offers at its discretion.


On April 14th 2015 Canadian premiers released an outcome statement after the Quebec Summit on Climate Change. Here is an unedited version of their declaration.
Disseminate this statement to the various bodies involved in the fight against climate change, both in Canada and internationally, in order to demonstrate the leadership of the provinces and territories of Canada in this regard.


text:
NYPD Fatally Shoots 14-Year-Old Shaaliver Douse in the Bronx
Sarah Fruchtnicht
An armed teenager in the Bronx was shot by police Sunday after he allegedly refused to drop his gun.
According to the New York Police Department, two rookie police officers responded to gunfire near East 151st Street and Cortlandt Avenue at about 3 a.m. The officers reportedly saw Shaaliver Douse carrying a 9mm semi-automatic handgun and chasing another male.
Authorities say Douse was ordered to drop his weapon, but he did not.
"I heard the police ask him to disarm himself," said Danilo Batista, who lives across the street from the crime scene, to Newsday. "He was chasing another kid."
An officer shot Douse one time in the lower left jaw. He was pronounced dead at the scene.
NYPD Commissioner Ray Kelly said he does not think there is anything the officers could have done differently. The two officers joined the force in January this year.
Douse was not unknown to police, CBS 2 reported. He was arrested May 16 in connection with a gang shooting at a gas station. He was charged as an adult in the crime, according to Newsday. Witnesses did not cooperate and in the end those charges were later dropped.
Sources: CBS, Newsday
nycBronxNYPDteen shot
NYPD Fatally Shoots 86-Year-Old Gunman
14-Year-Old Fatally Shoots Older Sister Over The Laundry
Suicidal Woman Suing the NYPD For Shooting Her After She Lunged at Them With a Knife
NYPD Seizes Fewer Guns Since Controversial Stop-And-Frisk Ruling
Bronx Borough President Outraged by "Ghetto" Bus Tour
Drunk NYPD Officer Allegedly Shoots Random Man Six Times
Bronx Man Fatally Shot In Head By Hecklers For Defending His Mother's Singing
Family Claims Botched NYPD Raid Caused Carlos Alcis' Fatal Heart Attack


Adding a new countertop to your kitchen is one of the best ways to elevate the look and feel of your kitchen without undergoing an entire kitchen renovation. At Custom Craft Cabinets & Millwork in Burnaby, BC, we have the high quality materials and exceptional craftsmanship you need to get the countertops of your dreams, built to last for years to come. Our team will help you with every step of the process, from selecting the right material for your kitchen to crafting the countertop pieces to the final installation.
As you work to decide which kitchen countertop material is right for your home, there are many factors to consider. How much are you willing to spend on a countertop? What level of maintenance do you want? What overall aesthetic are you going for? And how hard are you on your countertops?
Quartz: Also known as engineered stone, quartz is an extremely popular kitchen countertop choice that has a similar appearance to granite with better stain resistance and less maintenance. Quartz countertops are made up of about 90% to 95% ground quartz, with the remainder comprised of resin that holds the particles together. This makes the countertops very resistant to stains and scratches. It also comes in a wide range of colors, so you can select the right look for your space.
Stone: Natural stone countertops made from soapstone, limestone, or marble, are extremely popular options due to their natural beauty and heat resistance. Soapstone is typically dark grey or dark green in color, with a smooth, soft feel. Limestone is lighter in color and requires sealing to prevent stains. Marble provides a luxurious, veined look, but comes with a higher price tag.
Laminate: Laminate countertops have long been a staple as affordable, quality countertops. Made of plastic-coated synthetics, they come in a wide range of colors and are easy to clean and maintain, and resist stains better than some natural stones.
Granite: For a high-end option, many homeowners turn to granite. It's an extremely beautiful contribution to any kitchen and holds up well to use, including resistance to heat and scratching.
When you work with our team at Custom Craft Cabinets & Millwork in Burnaby, BC, you'll get high quality customer service and craftsmanship throughout the entire process. Whether you already have a specific vision for your new countertops or you're just starting to consider different countertop types, our designers will work with you to hone your idea and turn it into reality. We provide reliable, beautiful installation, leaving you with a kitchen that looks and feels brand new.
Call us at (604) 435 2422 today to find your new countertops!