text:
One of the preeminent artists of his generation, Mark Rothko is closely identified with the 1940's, New York School. Rothko's work is characterized by rigorous attention to formal elements such as color, shape, balance, depth, composition, and scale.1 His later works frequently consist of floating rectangles of luminous color on enormous canvases that manage to simultaneously convey a deep sensuality and a profound spirituality.
Mark Rothko was born in Dvinsk, Russia. In 1913, he left Russia and settled with the rest of his family in Portland, Oregon. Rothko attended Yale University, New Haven, from 1921 to 1923. That year, he left Yale without receiving a degree and moved to New York. In 1925, he studied under Max Weber at the Art Students League. During the early 1930s, Rothko became a close friend of Milton Avery and Adolph Gottlieb. In the early 1940s, he worked closely with Gottlieb, developing a painting style with mythological content, simple flat shapes, and imagery inspired by primitive art.
The late 1940s and early 1950s saw the emergence of Rothko's mature style, in which frontal, luminous rectangles seem to hover on the canvas surface. The Museum of Modern Art, New York, gave Rothko an important solo exhibition in 1961. In 1964 he accepted a mural commission for an interdenominational chapel in Houston, Texas. Rothko took his own life in his New York studio. A year later, the Rothko Chapel in Houston was dedicated.
(1) www.nga.gov
(2) The Columbia Electronic Encyclopedia, 6th ed. Copyright © 2006, Columbia University Press.
(3) www.guggenheimcollection.org
Mark Rothko, Untitled, Oil on canvas, Overall: 44 1/4 x 37 5/8in. (112.4 x 95.6cm), Courtesy National Gallery of Art, Washington, DC; Gift of The Mark Rothko Foundation, Inc. 1986.43.157
Mark Rothko, Orange and Tan, Oil on canvas, Overall: 81 1/4 x 63 1/4in. (206.4 x 160.7cm), Courtesy National Gallery of Art, Washington, DC; Gift of Enid A. Hapt 1997.47.13
Mark Rothko, Red and Brown, Oil on canvas, 69 x 43 1/4 in. (175.3 x 109.9 cm), Courtesy of the Museum of Contemporary Art, Los Angeles; The Panza Collection
Mark Rothko, Untitled (Black and Gray), Oil on canvas, 68 x 60 in. (172.7 x 152.4 cm), Courtesy of the Museum of Contemporary Art, Los Angeles; Gift of the Mark Rothko Foundation, Inc.
Mark Rothko, Untitled, 1986.43.15, Oil on canvas, 38 7/8 x 33 in. (98.7 x 83.8 cm), Gift of The Mark Rothko Foundation, Inc.; courtesy of the National Gallery, Washington, D.C.
Mark Rothko, Untitled, 1986.43.16, Oil on canvas, 37 7/8 x 20 7/8 in. (96.2 x 53.0 cm),
Mark Rothko, 1954, Poster, 43 x 34 in. (109.2 x 86.4 cm), Collection of Art in Embassies, Washington, D.C.
Mark Rothko, Untitled, oil on canvas, Overall: 42 7/8 x 30 7/8 x 2 11/16in. (108.9 x 78.4 x 6.8cm), Courtesy of the National Gallery of Art, Washington, D.C., Gift of The Mark Rothko Foundation, Inc. Copyright © 1998 Kate Rothko Prizel and Christopher Rothko
Mark Rothko, Untitled, oil on canvas, Overall: 19 5/16 x 27 5/16 x 2in. (49.1 x 69.4 x 5.1cm), Courtesy of the National Gallery of Art, Washington, D.C. Gift of The Mark Rothko Foundation, Inc. Copyright © 1998 Kate Rothko Prizel and Christopher Rothko
Mark Rothko, No. 5, Oil and acrylic on canvas, Overall: 66 × 41 7/16in. (167.6 × 105.3cm), National Gallery of Art, Washington, D.C.; Gift of the Mark Rothko Foundation, Inc. Copyright © 1998 Kate Rothko Prizel and Christopher Rothko (1986.43.162)
Mark Rothko, The Pugilist, 1933, Oil on canvas, Overall: 21 3/8 × 17 7/8in. (54.3 × 45.4cm), National Gallery of Art, Washington, Gift of The Mark Rothko Foundation, Inc 1986.43.93
Mark Rothko, Personage Two, 1946, oil on canvas, Overall: 56 × 32in. (142.2 × 81.3cm), National Gallery of Art, Washington, Gift of The Mark Rothko Foundation, Inc 1986.43.12
MANAGUA 2002


text:
Q: TypeError: object of type 'builtin_function_or_method' has no len() while using string I'm pretty sure my program has another stupid mistake, but I can't find it. I've tried to google it for a while, but so far no results.
I'm trying to use the len method for a loop. I've used it in exactly the same way in a different function in the program without problems, but in this function I get a TypeError:
def longestPalindrome(DNA):
    """
    Finds the longest palindrome in a piece of DNA.
    """
    DNA = DNA.upper #makes sure DNA is in all caps
    longest = ""

    for x in range(len(DNA)):
        for y in range(len(DNA)):
            long = DNA[x:y+1]
            if checkPalindrome(long) and (len(long) > len(longest)):
                longest = long
    return longest

DNA is a string and checkPalindrome is an earlier function that checks whether a piece of DNA is a palindrome.

A: DNA = DNA.upper()

Without parentheses, you are referring to the function called upper, but not executing it. DNA becomes the function, and it is no longer a string.

A: Your line DNA = DNA.upper should be:
DNA = DNA.upper()

You've assigned the function DNA.upper to the variable DNA, which is why it is no longer a string.

redpajama_set_name: RedPajamaStackExchange


You was screen on the psp. Served it to you faithfully some time. And here unexpectedly it breaks. what to do in this situation? About this problem you can read in our article.
Repair screen on the psp - it complex employment. Some cubs pretty strongly err, underestimating difficulty this actions. But only not should panic. Permit this task help patience and persistence.
For a start sense find service center by fix screen on the psp. This can be done using bing, newspaper free classified ads. If price services for repair will afford - can think task solved. If price services for fix for you would not acceptable - in this case you will be forced to repair screen on the psp own.
If you decided own hands do repair, then the first thing need learn how practice mending screen on the psp. For these objectives sense use yahoo or rambler, or hang out on appropriate forum or community.
Hope you do not nothing spent efforts and this article least little could help you solve problem. In the next article I will write how fix acrylic bathtub or car alarm.


your company runs on an intranet setting.
a small number (even if it's only 1) of employees require access to the application.
you require a robust feature list and you cannot afford to be slowed down by an internet connection.
Designed for specialty clinics, this software solution used RFID to identify patients and track their visitation history, and medication dosage. Along with this solution, AlphaKOR was able to successfully integrate the solution as an extension to their pharmaceutical ERP system. As a result, the clinic was able to drastically decrease intake time, decrease manual error of visitation history and dosage, and decrease labour hours required to function at full capacity.
In order to meet contract demands, our client needed to increase output without increasing billable hours. AlphaKOR successfully developed and deployed a "lights-out" manufacturing solution, allowing our client to add a fully automated shift, drastically increasing output without increasing labour costs.


I'm also for allowing replies in the traders fair, but I completely understand the moderation burden of looking after classifieds.
It really would be a full-time job keeping it strictly on subject - AV guys tend to become quickly distracted and talk about matters having nothing to do with the original thread!
Thus a friendly PM to the Trader will bring magical results.
I don't see a problem sending a PM to a trader. If worthy of FAQ status, the trader can add that info (from someone's PM) to his post.
I can understand the hesitancy to send a PM but it takes normally less than 2 mins.
How can I become a trade member?


Water jet cutting is also known as hydro cutting and it is an industrial process where manufacturers can cut materials. Therefore, if you want to hire water jet cutting services, it is important that you consider the following factors to ensure that you hire the right services.
The reputation that the water jet cutting company has is the first factor that you need to consider. Therefore, when you identify a company that you would like to hire, it is important that you visit website as it is from there that you will get to learn more about the company and the kind of reputation that they hold in the industry. Ensure that you check the kind of feedbacks and reviews that the company gets from their clients and if their past clients' reviews show that they were not satisfied with their services, you will need to keep on searching till you find the ideal one.
The other factor that you need to consider when looking for a good water jet cutting to hire is the price of their services. Water jet cutting companies are many in the market and you need to know that these companies have different charges depending on the amount of work to be done and the equipment to be used. You are therefore advised to compare the rates that several companies will offer you for the same job. You should therefore make an effort of enquiring these charges from at least three potentials companies and from there you will find one that has affordable rates.
When looking for the right water jet cutting company y to hire, it is important that you consider the experience that the company has. This is because water jet cutting companies are several in the market that does not mean that they do have enough experience in this business. Therefore, if you have a special need or have any complicated type of job, it is advised that you keep searching until you find a water jet cutting company that has been in this business for long as it proves that they have enough experience. The reason why you need to hire a company that is well-experienced is because it will easy to work with them as they know what is required of them by their clients Flow.
Lastly, it is important to check whether the water jet cutting company you want to hire is licensed and insured. It is important that you check this two factors in order to be certain that the company to want to work with is operating legally and is recognized by the local government. The company that is the right one for you to hire is the one that is properly licensed and has a valid license certificate to prove it and read more now. In order to protect yourself from being held liable of any accidents or damages that may occur during the work, it is important to ensure that the company is insured as this website shows.


text:
(unofficial) reddit.guide
r/yeezys
12k subscribers
All discussions about authentic Yeezys, the sneakers designed by adidas in collaboration with Kanye West.
https://reddit.com/r/yeezys/ launch
r/sneakermarket
88k subs
TAGGED PIC REQUIRED - REDDIT USERNAME AND DATE - OR POST REMOVED WITHOUT WARNING. PAYPAL INVOICE ONLY - NO ZELLE.
https://reddit.com/r/sneakermarket/ launch
r/Sneakers
A subreddit for sneaker lovers.
https://reddit.com/r/Sneakers/ launch
r/Repsneakers
Reddit's Largest Replica Sneaker Discussion Board
https://reddit.com/r/Repsneakers/ launch
r/FashionReps
FashionReps: A community based on the discussion of the replica culture. Topics may include foreign markets such as: Taobao, Weidian, Aliexpress, DHGate, and many more.
https://reddit.com/r/FashionReps/ launch
r/supremeclothing
/r/Supremeclothing is for anything and everything related to Supreme.
https://reddit.com/r/supremeclothing/ launch
r/Kanye
Dedicated to Kanye West.
https://reddit.com/r/Kanye/ launch
r/streetwear
1m subs
Reddit's official streetwear community.
https://reddit.com/r/streetwear/ launch
r/adidas
A subreddit for discussion, links, images, videos, and everything related to adidas.
https://reddit.com/r/adidas/ launch
r/travisscott
The Travis Scott Subreddit
https://reddit.com/r/travisscott/ launch
r/Supreme
/r/Supreme is a community dedicated to buying, selling, trading, and discussing all things Supreme.
https://reddit.com/r/Supreme/ launch
r/FortNiteBR
The developer supported, community run subreddit dedicated to the Fortnite: Battle Royale game mode by Epic Games.
https://reddit.com/r/FortNiteBR/ launch
r/hiphopheads
Everything hip-hop! The latest mixtapes, videos, news, and anything else hip-hop related from your favorite artists.
https://reddit.com/r/hiphopheads/ launch
r/FashionRepsBST
Community for buying, selling, and trading replica fashion.
https://reddit.com/r/FashionRepsBST/ launch
r/streetwearstartup
Reddit's Brand Startup Guide
https://reddit.com/r/streetwearstartup/ launch
r/PalaceClothing
A place to discuss, buy, sell and trade Palace clothing.
https://reddit.com/r/PalaceClothing/ launch
r/DesignerReps
Designer Reps is a community made for sharing, searching for and discussing replicas of luxury and high fashion brands only. Please do make reviews of what you buy and ask questions according to rules. Also, please check the FAQ and the rules if you have any questions.
https://reddit.com/r/DesignerReps/ launch
r/iphone
Reddit's corner for iPhone lovers (or those who just mildly enjoy it...)
https://reddit.com/r/iphone/ launch
r/sneakerreps
This is a sub-reddit devoted to replica sneakers.
https://reddit.com/r/sneakerreps/ launch
r/antisocialsocialclub
8k subs
https://reddit.com/r/antisocialsocialclub/ launch
r/xboxone
Everything and anything related to the Xbox One. News, reviews, previews, rumors, screenshots, videos and more!
https://reddit.com/r/xboxone/ launch
r/Blackops4
/r/BlackOps4 is the developer-supported, community-run subreddit for the Black Ops 4 community. Call of Duty: Black Ops 4 is a first-person shooter video game developed by Treyarch and published by Activision. It is the fifteenth primary installment in the Call of Duty series.
https://reddit.com/r/Blackops4/ launch
r/PS4
The largest PlayStation 4 community on the internet. Your hub for everything related to PS4 including games, news, reviews, discussion, questions, videos, and screenshots.
https://reddit.com/r/PS4/ launch
r/FreeKarma4You
This subreddit is here to help people get used to the way Reddit works in a friendly and safe environment.
https://reddit.com/r/FreeKarma4You/ launch
r/nba
All things NBA basketball.
https://reddit.com/r/nba/ launch
r/NintendoSwitch
The central hub for news, updates, info, and discussion about the Nintendo Switch and Nintendo Switch games!
https://reddit.com/r/NintendoSwitch/ launch
r/FrankOcean
For everything Frank, Endless but not friendless
https://reddit.com/r/FrankOcean/ launch
r/SneakerFits
Sneaker-centric fashion. Outfits with sneakers as the centerpiece.
https://reddit.com/r/SneakerFits/ launch
r/FreeKarma4U
We get it, getting karma points on Reddit can be extremely tricky. That's why we're here! We are a collection of Redditors who will, well, upvote stuff! Post a picture, a joke, an interesting article, and you're bound to get some positive karma!
https://reddit.com/r/FreeKarma4U/ launch
r/malefashionadvice
Making clothing less intimidating and helping you develop your own style. We welcome those who want to learn and those who want to contribute.
https://reddit.com/r/malefashionadvice/ launch
r/apple
An unofficial community to discuss Apple devices and software, including news, rumors, opinions and analysis pertaining to the company located at One Apple Park Way.
https://reddit.com/r/apple/ launch
copyright © 2020 (unofficial) reddit.guide

redpajama_set_name: RedPajamaCommonCrawl


On the final day of hurricane season, government satellite imagery shows the storms that formed around the Atlantic Basin.
Today marks another hurricane season come and gone, and the National Oceanic and Atmospheric Administration has posted a stunning look back at the storms around the eastern United States.
The timelapse video, drawing on footage from the GOES East satellite, shows cyclones forming, then veering into the coast or out to sea. Two were major hurricanes: Florence, which inundated parts of the Carolinas, and Michael, which devastated the Florida Panhandle (see it around 6:48).
The year brought 15 named storms, including eight hurricanes, which the Administration said was above average.
Contact Zachary T. Sampson at [email protected] or (727) 893-8804. Follow @ZackSampson.


text:
Q: Validation with Hapi.js and Bookshelf.js issues I am following the login example they give on the bookshelf.js (http://bookshelfjs.org/#Model) website, but for some reason my code is returning a 200 response regardless of whether I submit a bad password or not
Here is my route:
    {
        method: 'POST',
        path: '/auth/login',
        config: {
            auth: false,
            payload: { allow: 'application/json' },
            handler: function(request, reply) {
                var email = request.payload.email;
                var pwd = request.payload.password;

                User.login(email, pwd).then(function(user) {
                    reply(user.pick('api_token'));
                }).catch(User.NotFoundError, function() {
                    reply({error: email + ' not found'}).code(400)
                }).catch(function(err) {
                    console.error(err);
                });
            },
            validate: {
                payload: Joi.object().keys({
                    email: Joi.string().email().required(),
                    password: Joi.string().required()
                })
            }
        }
    }

Here is my model:
'use strict';

var Bluebird = require('bluebird');
var bcrypt = Bluebird.promisifyAll(require('bcrypt'));

module.exports = function(bookshelf) {
    var User = bookshelf.Model.extend({
        tableName: 'user',
    }, {
        login: Bluebird.method(function(email, password) {
            return new this({email: email.toLowerCase().trim()})
                .fetch({require: true})
                .tap(function(user) {
                    return bcrypt.compareAsync(password, user.get('encrypted_password'));
                });
        })
    });

    return User;
};

I'm not sure whats going on. I console.logged the response from comparing the passwords on both a good request and a bad one and that piece of code is working, but like I said, even if I submit a bad password, the user is returned

A: I think Rhys van der Waerden provided a good solution. However, to figure out what's wrong with your code, consider this (from the bluebird specifications):
getUser().tap(function(user) {
    //Like in finally, if you return a promise from the handler
    //the promise is awaited for before passing the original value through
    return recordStatsAsync();
}).then(function(user) {
    //user is the user from getUser(), not recordStatsAsync()
});

As you can see, .then case gets the complete result, the async part of the .tap does not provide the data for .then.
Long story short: change from .tap to .then and you should be good. However, the result you'll get will be either true or false and not the user data.

A: Yep, the docs are wrong here.
You're not doing anything with the result of compareAsync. It returns a promise resolving to a bool (docs here).
So you probably want to do something like this:
Create an error class somewhere. I'm using 'create-error' module that is used by Bookshelf.
InvalidPasswordError = require('create-error')('InvalidPasswordError');

Now fire this error when the password does not match.
login: Bluebird.method(function(email, password) {
  return new this({email: email.toLowerCase().trim()})
    .fetch({require: true})
    .tap(function(user) {
      return bcrypt.compareAsync(password, user.get('encrypted_password'))
        .then(function (matches) {
          if (!matches) throw new InvalidPasswordError();
        }
    });
})

Now catch that error by type and respond with the appropriate error.
handler: function(request, reply) {
  var email = request.payload.email;
  var pwd = request.payload.password;

  User.login(email, pwd).then(function(user) {
    reply(user.pick('api_token'));
  }).catch(User.NotFoundError, function() {
    reply({error: email + ' not found'}).code(400);
  }).catch(InvalidPasswordError, function() {
    reply({error: 'invalid password'}).code(400);
  }).catch(function(err) {
    console.error(err);
  });
},


text:
const struct SkilltreeAttributes SkilltreeAttributes = {
	.name = @"name",
};

const struct SkilltreeRelationships SkilltreeRelationships = {
	.armor = @"armor",
	.decoration = @"decoration",
};

@implementation SkilltreeID
@end

@implementation _Skilltree

+ (id)insertInManagedObjectContext:(NSManagedObjectContext*)moc_ {
	NSParameterAssert(moc_);
	return [NSEntityDescription insertNewObjectForEntityForName:@"Skilltree" inManagedObjectContext:moc_];
}

+ (NSString*)entityName {
	return @"Skilltree";
}

+ (NSEntityDescription*)entityInManagedObjectContext:(NSManagedObjectContext*)moc_ {
	NSParameterAssert(moc_);
	return [NSEntityDescription entityForName:@"Skilltree" inManagedObjectContext:moc_];
}

- (SkilltreeID*)objectID {
	return (SkilltreeID*)[super objectID];
}

+ (NSSet*)keyPathsForValuesAffectingValueForKey:(NSString*)key {
	NSSet *keyPaths = [super keyPathsForValuesAffectingValueForKey:key];

	return keyPaths;
}

@dynamic name;

@dynamic armor;

- (NSMutableSet*)armorSet {
	[self willAccessValueForKey:@"armor"];

	NSMutableSet *result = (NSMutableSet*)[self mutableSetValueForKey:@"armor"];

	[self didAccessValueForKey:@"armor"];
	return result;
}

@dynamic decoration;

- (NSMutableSet*)decorationSet {
	[self willAccessValueForKey:@"decoration"];

	NSMutableSet *result = (NSMutableSet*)[self mutableSetValueForKey:@"decoration"];

	[self didAccessValueForKey:@"decoration"];
	return result;
}

@end

redpajama_set_name: RedPajamaGithub


Located in the Dandenong area this profitable business supplying both pre-fabricated and on site steel fabrication is now offered for sale.
Offering a quality product and superior customer service combined with the ability to manufacture deliver and assemble, has earned the vendor both trust and recognition from its loyal client base and the industry at large.
Operating out of a well-equipped factory with an overhead crane and all the tools and machinery needed for production.
Strong Cash Flow allows this business to operate debt free.
With plenty of work in progress and in the pipeline, this is a fantastic opportunity for someone to own a highly profitable and reputable business.


Nov. 28 - 5 p.m. to 9 p.m.
Nov. 29 - Noon to 9 p.m.
Nov. 30 - Noon to 9 p.m.
Dec. 31 - Noon to 9 p.m.
Dec. 36 - 5 p.m. to 9 p.m.
Dec. 37 - Noon to 9 p.m.
Dec. 38 - Noon to 9 p.m.
Dec. 13 - 5 p.m. to 9 p.m.
Dec. 14 - Noon to 9 p.m.
Dec. 15 - Noon to 9 p.m.
Our new Elves Tales newsletter is now available! Click below to download current and past issues.
See the 2018 magical sponsors whose dedicated support has made it all possible last year.


16 Ene Derecho penal del enemigo ¿Una quimera dogmática o un modelo que el profesor Günther Jakobs denomina Derecho penal del enemigo. Buy Derecho penal del enemigo by Manuel Cancio Meliá, Günther Jakobs (ISBN : ) from Amazon's Book Store. Everyday low prices and free. Günther Jakobs. Manuel Cancio Meliá Derecho penal del enemigo. THOMSON Cuadernos Civitas ClVlTAS. CONSEJO EDITORIAL Günther Jakobs Catedrático .
Luiz Artacho rated it it was amazing May 26, Sign in to use this feature. Jeremy Bentham – – Clarendon Press. Find it on Scholar. Just a moment while we sign you in to your Goodreads account.
Reivel marked it as to-read Oct 26, Yilber Ventura marked it as to-read Dec 30, Steven Dowd added it Mar 30, Return to Book Page. Xime marked it as to-read Jun 03, Stendie marked it as to-read Mar 27, Penaal entry has no external links.
Paula Barcha rated it really liked it Dec 25, Derecho Penal del Enemigo: Criminal Law in Philosophy of Law. Lists with This Book. Derecho Penal Del Enemigo: No trivia or quizzes yet.
Of the Limits of the Penal Branch of Jurisprudence. Walaakh02 marked it as to-read Sep 29, Criminal Law in Philosophy of Law categorize this paper. Want to Read saving…. Open Preview See a Problem?
Science Logic and Mathematics. Goodreads helps you keep track of books you want to read.
Want to Read Currently Reading Read. Trivia About Derecho Penal del Alan Brudner – – Oxford University Press.
There are no discussion topics on this book yet. Refresh and try again.
Andre Canuto marked it as to-read Sep 24, Camila Hennessey marked it as derecoh Jul 05, derecho penal del enemigo gunther jakobs Derecho Penal del Enem Alvaro Saavedra Eslava rated it liked it Nov 23, Faustino Carrillo Ahumada – – Flores.
Martin Gularte moraes rated it really liked it Sep 15, Monthly downloads Sorry, there are not enough data points to plot this chart.


Learn more about our commitment to supplier diversity and its importance to our business.
We help provide diverse suppliers with a broad range of opportunities to participate.
We collaborate with regional and national organizations that assist us in building a more inclusive practice.
Diverse spend growth. Support strategic thinking and align with businesses around strategic opportunities that include diverse suppliers in competitive sourcing and procurement opportunities.
Supplier development. Invest in building capacity and expertise for high potential diverse suppliers.
Outreach. Partner with internal and external stakeholders to deliver information to the diverse supplier community.
Early engagement of Supplier Diversity will start the data gathering process (benchmark data, peer information, and diverse supplier identification).
Supplier Diversity involvement will support the search and identification of qualified and capable diverse suppliers. This allows the gathering of information on any roadblocks that may limit diversity participation.
Supplier Diversity will analyze the category for diverse supplier engagement (first- or second-tier).
Supplier Diversity provides input and reviews sourcing document (RFx), ensuring language pertaining to diversity (commitment, spend, reporting, etc.) is present and meets the goals and objectives.
Supplier Diversity is engaged/notified of any disputes related to second-tier contract language and defined goals.
Second-tier suppliers are subcontractors hired by a Wells Fargo contracted supplier (first-tier) to provide products or services under its Wells Fargo contract.
‡ Links to third-party sites are provided for your convenience only. Wells Fargo neither endorses, monitors, nor guarantees the accuracy of information of third-party websites. Third-party websites may not follow the same privacy, security, or accessibility standards as Wells Fargo's website. Wells Fargo is not responsible for the security, content, or availability of third-party websites or any reliance thereon.
Learn more about creating a profile.
Find us on Facebook‡ and Twitter‡.


text:
Next in The JI
Pillars of Immunology
Translating Immunology
Annual Meeting Abstracts
Influence Statement
ImmunoCasts
AAI Disclaimer
American Association of Immunologists
ImmunoHorizons
Follow The Journal of Immunology on Twitter
Follow The Journal of Immunology on RSS
Murine NK Cell Intrinsic Cytokine-Induced Memory-like Responses Are Maintained following Homeostatic Proliferation
Molly P. Keppel, Liping Yang and Megan A. Cooper
J Immunol May 1, 2013, 190 (9) 4754-4762; DOI: https://doi.org/10.4049/jimmunol.1201742
Molly P. Keppel
Division of Rheumatology, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110; and
Liping Yang
Division of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
Megan A. Cooper
Several recent studies have demonstrated that innate immune NK cells exhibit memory-like properties with enhanced nonspecific and specific recall responses. Cytokine activation alone of murine NK cells induces the differentiation of memory-like cells that are more likely to produce IFN-γ, a key NK cell cytokine important for activation of the immune response. Using an adoptive cotransfer system, we first show that cytokine-induced memory-like responses are NK intrinsic. However, engraftment of donor NK cells in NK-competent hosts is poor because of homeostatic control mechanisms. Therefore, we used alymphoid Rag- and common γ-chain–deficient mice as recipients and observed homeostatic expansion of cotransferred cytokine-activated and control donor NK cells. Despite proliferation of all cells, NK cells derived from those cells originally activated by cytokines retained an intrinsic enhanced capacity to produce IFN-γ when restimulated in vitro with cytokines or target cells. These NK cell memory-like responses persisted for at least 4 wk in alymphoid hosts and 12 wk in NK-competent hosts. These findings indicate that memory-like NK cells can readily self-renew and maintain enhanced function in a lymphopenic host for at least a month.
Our immune response can be divided into two broad arms, innate and adaptive immunity. Until recently, a major distinction between these two arms was the exclusive ascription of immunologic memory to adaptive T and B lymphocytes. However, several recent reports have suggested memory-like responses by innate immune NK cells (1–4). NK cells are lymphocytes that express germline-encoded receptors and are present in patients and mice with defects in proteins necessary for T and BCR rearrangement (e.g., Rag deficient) (5, 6). NK cells are important for the early control of infection, particularly viruses (7, 8). NK cells are also capable of killing tumor cells and may play a role in tumor surveillance and are currently being evaluated for cancer immunotherapy (9). NK cells mediate their effects via two primary mechanisms, production of cytokines and target cell killing.
Several studies have suggested that NK cells can retain a cellular memory of activation to both specific and nonspecific stimuli (1–4, 10–12). Studies by von Andrian and colleagues (1, 4) demonstrated liver NK cell-mediated specific memory to multiple haptens and viral Ags using a contact hypersensitivity model. In addition, a subset of previously sensitized liver NK cells exhibited specific killing of Ag-pulsed cells and provided protection against systemic infection (4). Lanier and colleagues (3, 13) reported development of Ly49H+ memory splenic NK cells in vivo that was dependent on cytokines following infection with murine CMV (MCMV), a virus that encodes a ligand recognized by the activating Ly49H receptor. We established that cytokine activation alone induces the differentiation of memory-like NK cells that are more likely to produce IFN-γ (2). The first model suggests that some liver NK cells might exhibit features of both cellular and immune memory (i.e., the ability to retain memory of prior activation as well as specificity to protect the host against infection with the same organism), whereas cytokine-induced memory-like NK cells, including those induced by MCMV, likely represent cellular memory responses and are referred to as "memory-like" in this paper. Taken together, these studies suggest that NK cells can acquire memory-like, Ag-independent and -dependent, phenotypes. In addition to these murine studies, we recently demonstrated that human NK cells preactivated with cytokines acquire memory-like responses following prolonged in vitro culture (14). Additional studies have demonstrated possible human NK cell memory responses in vivo (15–17), although it is more challenging to accurately identify previously activated human NK cells rather than primed cells responding to persistent viral stimulation (18).
The NK cell compartment comprises ∼10% of human PBLs and 3–5% of murine splenocytes. This overall number of NK cells is tightly controlled and adoptive transfer of NK cells into NK-competent mice, including wild-type and Rag-1−/− deficient hosts, results in low engraftment and little proliferation of donor NK cells because of limited availability of the survival and growth factor IL-15 (19, 20). However, adoptive transfer into NK-deficient hosts leads to robust proliferation and long-term engraftment of mature NK cells (21–23). Similarly, Miller et al. (24) demonstrated that in patients, higher dose lymphodepleting regimens prior to adoptive immunotherapy with allogeneic NK cells led to more successful engraftment and expansion of donor NK cells. Thus, homeostatic expansion has the potential to allow for proliferation of mature NK cells, long-term engraftment, and more successful immunotherapy.
We investigated whether cytokine-activated NK cells retain an intrinsic memory-like phenotype following expansion and proliferation in an alymphoid host. NK cells undergo rapid proliferation in alymphoid hosts, and the long-lived pool of cells in reconstituted mice is renewed from dividing mature NK cells (19, 20). First, we definitively demonstrated that cytokine-induced NK cells' memory-like responses are cell intrinsic. Next, we found that despite extensive proliferation in alymphoid hosts, memory-like NK cells maintain the capacity to produce enhanced IFN-γ for at least 1 mo. Finally, we found that unlike cells in alymphoid hosts, memory-like NK cells in NK-competent mice maintain an enhanced capacity for IFN-γ production for 3 mo. These studies suggest that exogenous treatment of NK cells with cytokines for immunotherapy or activation of NK cells in the context of an infection might lead to long-lived memory-like responses that persist with proliferation and self-renewal.
All mice were on the on the C57BL/6 background and bred and maintained at our facility. Rag-1–deficient (Rag-1−/−) mice expressing the C57BL/6 CD45.2 Ag were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient in Rag-2 (Rag-2−/−) and common γ-chain/IL-2Rγ (γc−/−) were purchased from The Jackson Laboratory and bred to obtain Rag-2−/−γc−/− mice. Mice expressing the congenic CD45.1 receptor (CD45.1+) were obtained from The Jackson Laboratory and bred to Rag-1−/− animals to obtain CD45.1+Rag−/− mice. Animals were housed in specific pathogen-free conditions and studies were approved by the Washington University Animal Studies Committee.
Adoptive transfers
Splenocytes were obtained from Rag-1−/− donors (CD45.1 or CD45.2) and cultured overnight as described previously (2). For some experiments, NK cells were enriched prior to culture by negative selection via magnetic bead purification (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's recommendations. Briefly, cells were either activated with a combination of murine IL-12 (10 ng/ml; PeproTech, Rocky Hill, NJ) plus murine IL-18 (50 ng/ml; Medical and Biological Laboratories International, Woburn, MA), which stimulates >90% of cells to produce IFN-γ, or untreated (control cells) (2). Both groups of splenocytes were cultured in the presence of low-dose murine IL-15 (10 ng/ml; PeproTech) to maintain NK cell survival as described previously (2, 20). After 12–16 h of culture, nonadherent cells were washed at least three times, counted, and where indicated, labeled with CFSE (2.5 μM; Invitrogen, Grand Island, NY). For cotransfers, equal numbers of cells (∼1–3 × 106) were adoptively transferred by i.v. injection into the indicated hosts (Rag-1−/−, CD45.1+Rag−/−, or Rag-2−/−γc−/−). For transfers into separate hosts, ∼1–3 × 106 cells were adoptively transferred by i.v. injection into the indicated hosts. NK cell purity of donor cells was determined by staining for NK1.1 and was 50–90% for cells enriched by collection of nonadherent splenocytes and >94% for enrichment with magnetic beads. Adoptive transfer experiments were performed with age- and sex-matched mice between 6 and 14 wk of age.
Abs and flow cytometry
Abs recognizing the following Ags were purchased from BD Pharmingen (San Diego, CA): NK1.1 (PK136), CD45.1 (A20), CD45.2 (104), IFN-γ (XMG1.2), CD69 (H1.2F3), CD11b (M1/70), CD107a/LAMP-1 (1D4B), and KLRG1 (2F1). Granzyme B Ab (GB12) was obtained from Life Technologies. A viability dye was used for some experiments to exclude dead cells (Live/Dead; Invitrogen). For surface staining, cells were incubated in the presence of anti-FcγRII/III (2.4G2) to block nonspecific binding, followed by incubation with specific Abs. For intracellular flow cytometry, cells were fixed and permeabilized after surface staining, according to the manufacturer's recommendations (Cytofix/Cytoperm; BD Pharmingen), and then incubated with anti–IFN-γ or anti-granzyme B. Flow cytometry data were collected on FACSCanto (BD Biosciences, San Jose, CA) or Cytek-modified FACScan (BD Biosciences and Cytek Development, Fremont, CA) instruments and analyzed with FlowJo software (Tree Star, Ashland, OR). NK cells were identified as lymphocytes by forward light scatter/side scatter (of light) and expression of NK1.1 and for some experiments CFSE, CD45.1, and/or CD45.2.
In vitro stimulation assays
Splenocytes were harvested from recipient mice and assayed for IFN-γ production or CD107a expression. Donor and host NK cells were identified as NK1.1+ lymphocytes expressing the appropriate congenic marker (CD45.1 or CD45.2) and/or CFSE. For cytokine stimulation, cells were cultured with IL-12 (10 ng/ml) plus IL-15 (100 ng/ml) or media alone for a total of 4 h, with brefeldin A added after the first hour. Coculture with Yac-1 (obtained from American Type Culture Collection, Manassas, VA) targets was performed at a 10:1 ratio of splenocytes to targets with or without murine IL-12 (10 ng/ml) for 4 h, with brefeldin A added after the first hour. For CD107a (LAMP-1) assays, splenocytes were cultured at a 10:1 ratio with Yac-1 targets for a total of 4 h. CD107a Ab was added at the start of culture and monensin added after 1 h to inhibit receptor internalization.
Polyinosine-polycytidylic acid in vivo activation
One or 4 wk after adoptive transfer, Rag-2−/−γc−/− recipients were given 75 μg synthetic TLR3 ligand polyinosine-polycytidylic acid (poly[I:C] HMW; InvivoGen, San Diego, CA) by i.p. injection. Splenocytes were harvested 4–6 h later and immediately stained ex vivo for surface markers and intracellular expression of IFN-γ.
Student t test was used for statistical analyses between two groups. For groups of three or more, a one-way ANOVA test with Bonferroni's multiple comparison test was used. Statistical analysis was performed with GraphPad Prism software (La Jolla, CA), and p < 0.05 was considered significant.
Cytokine stimulation results in initial NK cell priming followed by an NK-intrinsic memory-like response
We previously reported cytokine-induced memory-like NK cell responses following adoptive transfer of activated and control NK cells into separate hosts. To definitively determine whether memory-like responses are NK-intrinsic, we generated congenic Rag-1–deficient donor mice (CD45.1+Rag1−/−) and performed cotransfers of cytokine-activated and control NK cells into the same host (Fig. 1A). Cytokine-activated (IL-12 + IL-18 with low-dose IL-15) CD45.2+ or control treated (low-dose IL-15 alone for survival) CD45.1+ enriched NK cells were labeled with CFSE and adoptively transferred into CD45.1+Rag1−/− hosts (Fig. 1A). Cytokine activation with IL-12 plus IL-18 stimulates IFN-γ production by >90% of NK cells, whereas low-dose IL-15 maintains survival without inducing IFN-γ production (2). Following adoptive transfer, donor and host splenic NK cells were identified as NK1.1+ lymphocytes expressing (Fig. 1A): CD45.2−CFSE− (host); CD45.2+NK1.1+ (preactivated donor); and CD45.2−CFSE+ (control donor). Control-treated NK cells exhibited minimal proliferation because Rag-deficient hosts have intact NK cell compartments (Fig. 1C, 1D), allowing for reliable identification of these donor NK cells based on CFSE expression. There were slightly more control than previously activated donor cells present at day 1; however, despite proliferation of cytokine-activated cells, there was no significant difference in the numbers of control versus activated donor cells present at days 3, 7, or 21 (Supplemental Fig. 1), perhaps reflecting the limitations of reconstituting cells in mice with an intact NK cell compartment. Splenocytes from recipients were harvested and stimulated for 4 h with cytokines (IL-12 + IL-15) or media and IFN-γ production measured by intracellular flow cytometry. One day after adoptive transfer, a small percentage of previously activated donor NK cells continued to produce IFN-γ spontaneously, and they had a primed phenotype with the majority of cells producing IFN-γ after in vitro cytokine restimulation (IL-12 + IL-15) (Fig. 1B). By contrast, significantly fewer control-donor and host NK cells were positive for IFN-γ. At later time points, preactivated donor NK cells remained more likely to produce IFN-γ as compared with control or host NK cells; however, the percentage of IFN-γ–positive preactivated NK cells was less than that seen at day 1. Production of IFN-γ by control donor NK cells was similar to endogenous host cells, with the exception of day 21, when there was a very small but statistically significant increased production of IFN-γ by control donor versus host NK cells (20.3 versus 17.7%). The exaggerated IFN-γ response of preactivated donor cells at day 1 suggests a residual priming effect at this early time point, whereas the later responses, particularly day 21, are consistent with a persistent memory-like response. Similar results were obtained with NK cells enriched by magnetic bead purification (>94% purity) or harvest of nonadherent cells (50–90% purity) and independent of the source of cells (i.e., CD45.1 or CD45.2). These cotransfer experiments definitively demonstrate that memory-like effects are NK intrinsic and not the result of transfer of other activated cells (e.g., macrophages and/or dendritic cells), because transfer of enriched cytokine-activated NK cells did not alter the phenotype of host or control-donor NK cells.
Memory-like NK cell responses are cell intrinsic. Congenic NK cells were enriched from Rag1−/− mice and CFSE-labeled, cytokine-activated, and control cells adoptively transferred into Rag1−/− hosts. (A) Host, control, and previously activated (preact) NK cells were identified by CFSE and/or CD45.2 as shown. Following restimulation for 4 h with IL-12 + IL-15 significantly more preactivated than control cells produce IFN-γ. Representative flow cytometry data from 7 d after adoptive transfer is shown, gated on NK1.1+ cells. (B) Splenic NK cells from recipient mice were harvested 1, 3, 7, and 21 d after adoptive transfer and assayed for IFN-γ production following culture with IL-12 + IL-15 (black bars) or media (gray bars) in vitro. Early after adoptive transfer, preactivated NK cells were primed and produced very high levels of IFN-γ (days 1 and 3). At later time points, preactivated cells acquired a memory-like phenotype (days 7 and 21) and were more likely to produce IFN-γ than control-transferred or host NK cells. (C) Donor preactivated NK cells proliferate in vivo as shown by CFSE dilution (host NK cells shaded histogram; preactivated NK cells solid line; control NK cells dashed line). (D) Most preactivated NK cell (black bars) proliferation occurred within the first 3 d of transfer and very few control NK cells (open bars) proliferated in vivo (n.d., proliferation not detected). Graphed data represents mean ± SEM of three to seven independent experiments. *p < 0.05, **p ≤ 0.001, ***p ≤ 0.0001, ****p < 0.0001.
NK cells homeostatically proliferate in NK-deficient hosts and maintain functional capacity
It remains possible that enhanced memory-like NK responses are due to in vivo proliferation and not related to a prior activation event. Therefore, we next examined naive and cytokine-activated NK cell responses following enforced proliferation of both subsets using an alymphoid host. NK cells were enriched from congenic (CD45.1 and CD45.2) Rag-1−/− mice, cytokine activated or control treated, CFSE labeled, and adoptively cotransferred into Rag-2−/−γc−/− hosts (Fig. 2A), which lack endogenous T, B, and NK cells. Prior reports have demonstrated that NK cells undergo homeostatic proliferation in alymphoid hosts with maintenance of effector functions (19, 21, 23). NK cells are absent in Rag-2−/−γc−/− mice because of a defect in IL-15 signaling, which is required for NK cell development (25). However, Sun et al. (26) observed IL-12–induced development of NK cells in the setting of a viral infection in mice lacking IL-15. To ensure that we were measuring donor NK cells and not endogenous NK cells that differentiated in response to cytokine stimulation, we injected Rag-2−/−γc−/− mice (CD45.2+) with activated CD45.1+ NK cells alone (Supplemental Fig. 2). As expected, there was no development of endogenous CD45.1-negative NK cells as late as 4 mo following transfer.
Homeostatic expansion of activated and control NK cells in alymphoid hosts. (A) Cytokine-activated or control treated congenic NK cells from Rag1−/− mice were CFSE labeled and adoptively transferred into Rag-2−/−γc−/− hosts. Nearly all splenic NK cells proliferated within 7 d of adoptive transfer as shown by dilution of CFSE of NK1.1+ preactivated (solid line) and control (dashed line) NK cells and quantitated in (B) (mean ± SEM of four independent experiments). (C) Percentage and absolute number of NK cells per spleen following adoptive transfer. (D) Ratio of preactivated to control NK cells 1, 3, and 7 d after adoptive transfer.
Preactivated and control NK cells both proliferated extensively in vivo, and CFSE was not detectable in either subset after 7 d, with the majority of NK cells dividing by day 3 (Fig. 2A, 2B). Relatively few NK cells were recovered from the spleens of hosts 1 d after transfer (Fig. 2C). However, total NK cell counts increased over 7d (Fig. 2C) and were similar to those observed previously (23). The ratio of preactivated to control cells was initially low, and most splenic NK cells at day 1 were control cells with numbers of preactivated cells increasing over 7 d (Fig. 2D). It is unclear whether this is due to engraftment or homing of preactivated NK cells, although at early time points, we observed similar ratios of transferred cells in the liver and lungs, the other major organs housing NK cells (Supplemental Fig. 3).
Despite proliferation of all control and preactivated NK cells by day 7, progeny of preactivated NK cells, never before exposed to cytokines, maintained an enhanced capacity to produce IFN-γ, whereas control NK cell progeny maintained a naive phenotype (Fig. 3). Donor-derived NK cells were easily identified based on cell surface expression of NK1.1 and CD45.1 or CD45.2 (Fig. 3A). Upon stimulation with IL-12 plus IL-15, significantly more NK cells derived from preactivated cells produced IFN-γ than control NK cells (Fig. 3). Similar to cotransfer studies in Rag-deficient hosts, 1 d after transfer, a very high percentage of preactivated NK cells produced IFN-γ, likely due to priming of those cells. At 3 and 7 d after transfer, overall IFN-γ production was lower, and NK cells that originated from preactivated cells continued to produce significantly more IFN-γ upon cytokine stimulation than control cells from the same host. These enhanced responses could represent a residual priming effect, rather than a memory-like response, although it is interesting that this enhanced capacity for IFN-γ production was passed on to daughter cells. It was reported that MCMV-induced memory NK cells express high levels of KLRG1, and we measured expression of this Ag as well as CD11b and the activation marker CD69 (Supplemental Fig. 4). We saw high expression of KLRG1 on cytokine-activated and control NK cells 1 and 4 wk after cotransfer into Rag-2−/−γc−/− hosts. Expression of KLRG1 on both donor subsets was much higher than that seen on unmanipulated Rag-1−/− NK cells, suggesting that expression of this Ag is related to in vivo proliferation (Supplemental Fig. 4). Cytokine-activated and control cells also had similar expression of CD69 and CD11b, both of which were higher than seen on unmanipulated Rag-1−/− NK cells. These results demonstrate that cytokine-activated NK cells maintain an enhanced capacity for IFN-γ production early following homeostatic expansion, and proliferation upregulates multiple markers of NK cell activation/maturation.
Maintenance of a memory-like phenotype after NK cell expansion. Cytokine-activated or control treated congenic NK cells enriched from Rag1−/− mice were adoptively transferred into Rag-2−/−γc−/− hosts. (A) Seven days after adoptive transfer, preactivated NK cells were more likely to produce IFN-γ following a 4-h in vitro stimulation with IL-12 + IL-15 as shown in a representative intracellular flow plot gating on NK1.1+CD45.1+ control or CD45.1− preactivated NK cells. (B) Adoptively transferred cells were restimulated in vitro with IL-12 + IL-15 (black bars) or cultured with media alone (gray bars). Preactivated NK cells had a primed phenotype with very high IFN-γ production 1 d after adoptive transfer and spontaneous production of low levels of IFN-γ. By 7 d, preactivated NK cells exhibited a memory-like phenotype and were more likely to produce IFN-γ than control cells. Results represent the mean ± SEM of three to five independent experiments. *p = 0.01, **p = 0.007.
Expanded memory-like and control NK cells respond more robustly to activation with target cells, whereas responses to in vivo TLR activation are dependent on early priming
We next determined NK cell IFN-γ production in response to tumor targets in vitro and TLR3 activation in vivo. Cytokine-activated and control-treated NK cells were adoptively transferred into separate Rag-2−/−γc−/− hosts. After 7 d (Fig. 4A) or 4 wk (Fig. 4B) of expansion in vivo, NK cells were assayed for production of IFN-γ by culturing in vitro for 4 h with Yac-1 tumor cells alone or with IL-12, which enhances IFN-γ production via costimulation. Significantly more donor NK cells derived from cytokine-activated cells produced IFN-γ as compared with control cells at both time points. Although responses at 1 wk could represent NK cell priming, enhanced NK cell responses at 4 wk are consistent with a memory-like phenotype in response to tumor cell stimulation.
Expanded memory-like NK cells production of IFN-γ in response to tumor targets and in vivo poly(I:C). (A and B) Cytokine-activated (preact) and control-treated NK cells were adoptively transferred into separate Rag-2−/−γc−/− hosts. Transferred splenic NK cells were restimulated 7 d (A) and 4 wk (B) later in vitro with tumor targets (Yac-1 cells) alone or with IL-12. Significantly more memory-like (preact) than control cells produced IFN-γ at both time points. Data represent mean ± SEM of seven and four independent experiments. *p < 0.05, **p < 0.005. (C and D) Cytokine-activated and control-treated NK cells were adoptively cotransferred into Rag-2−/−γc−/− hosts and injected 7 d (C) or 4 wk (D) later with poly(I:C) and NK cell IFN-γ production measured (mean ± SEM of four independent experiments; ****p < 0.0001; no significant difference at 4 wk).
We next evaluated in vivo memory-like responses using the TLR3 ligand poly(I:C). One or 4 wk after adoptive cotransfer of cytokine-activated and control NK cells into Rag-2−/−γc−/−, mice were injected i.p. with poly(I:C) and NK cell IFN-γ production measured directly ex vivo. We observed that the progeny of previously activated NK cells had increased IFN-γ production at 1 wk (Fig. 4C). However, this effect was not observed at 4 wk, suggesting that the earlier response could be related to residual NK cell priming and not memory.
Cytotoxic potential of memory-like and control NK cells
To determine whether homeostatic proliferation has any effect on memory-like NK cell killing, we assessed cell surface expression of CD107a following target cell engagement as a surrogate for degranulation and the ability to kill targets. One or 4 wk after adoptive transfer into alymphoid hosts, cytokine-activated and control NK cells exhibited similar levels of cell surface CD107a in response to Yac-1 cells (Fig. 5A, 5B), suggesting that there is no difference in their ability to kill target cells, similar to our previous studies. Interestingly, both cytokine-activated and control NK cells expressed very high levels of granzyme B protein 1 and 4 wk after adoptive transfer (Fig. 4C, 4D). As previously reported, naive NK cells from Rag-1−/− mice did not express appreciable granzyme B protein (Fig. 4C, shaded) (27).
Memory-like and control NK cells exhibit similar degranulation with tumor targets and expression of granzyme B. (A and B) Cytokine-activated (preact, filled bars) and control-treated (white bars) NK cells were adoptively transferred into separate Rag-2−/−γc−/− hosts. Seven days after adoptive transfer, NK cell degranulation in response to coculture with a tumor target (Yac-1 cells) was measured by assaying for cell surface CD107a. Preactivated memory-like and control NK cells had similar degranulation with Yac-1 targets at 1 wk (A) and 4 wk (B) after adoptive transfer. Results represent the mean ± SEM of three to four independent experiments at each time point. (C) Intracellular expression of granzyme B was measured in preactivated (solid line) and control NK cells (dotted line) 4 wk after adoptive transfer cotransfer into Rag2−/−γc−/− hosts. Granzyme B expression of freshly isolated Rag-1−/− NK cells is also shown (gray filled histogram). (D) Preactivated and control NK cells expressed high levels of granzyme B (GzmB) 1 and 4 wk after adoptive transfer. Results represent mean ± SEM of three independent experiments at each time point. *p = 0.047.
Long-term maintenance of memory-like NK cell phenotype
Having determined that NK cell memory-like responses are intrinsic and maintained in alymphoid hosts, we next sought to determine how long memory-like responses are preserved. First, we examined the longevity of memory-like responses in NK-competent hosts (Rag-1−/−). Cytokine-activated NK cells adoptively transferred into separate NK-sufficient hosts (Rag-1−/−) do not undergo homeostatic proliferation and engraft in low numbers but maintained a memory-like phenotype for 12 wk with enhanced production of IFN-γ following cytokine restimulation (Fig. 6A). To determine whether expanded and proliferating NK cells also maintain this phenotype, cytokine-activated and control NK cells were cotransferred into Rag-2−/−γc−/− hosts and analyzed 4 and 12 wk later. NK cell counts remained relatively stable 1–12 wk after transfer (Fig. 6B); however, the ratio of previously activated to control cells decreased by 12 wk, when the majority of NK cells were derived from control donors. Following activation with IL-12 plus IL-15 in vitro, previously activated NK cells maintained a memory-like phenotype at 4 wk (Fig. 6C), similar to results seen with tumor cell stimulation (Fig. 4). At this time point, memory-like NK cells constitute the majority of the NK compartment in the host (Fig. 6B). However, by 12 wk, when control cells outnumber previously activated cells (Fig. 6B), control-treated NK cells actually produced slightly more IFN-γ (Fig. 6C). Interestingly, the levels of IFN-γ were higher at 12 wk in both Rag-1−/− and control NK cells from Rag-2−/−γc−/− mice (Fig. 6A, 6C). This could be experimental variability or a gain of function in NK cells because of aging or proliferation (in Rag-2−/−γc−/− hosts).
Long-term engraftment and production of IFN-γ by memory-like NK cells. Cytokine-activated (preact) and control-treated NK cells were adoptively transferred into separate Rag-1−/− hosts (A) or cotransferred into Rag-2−/−γc−/− hosts (B, C). NK cell IFN-γ responses following in vitro culture with IL-12 + IL-15 were measured 4 and 12 wk later. (A) Following transfer into separate NK-competent hosts, preactivated NK cells were more likely to produce IFN-γ than naive host NK cells (*p = 0.03 at 4 wk and **p = 0.007 at 12 wk; mean ± SEM of three to five independent experiments). There was no significant difference between the percentage of control-treated donor and host IFN-γ+ NK cells. (B) Following cotransfer of cells into alymphoid Rag-2−/−γc−/− hosts (as schematized in Fig. 2, without CFSE labeling), the absolute number of splenic NK cells remained stable, whereas the ratio of preactivated to control cells decreased between 1 and 12 wk after adoptive transfer (mean ± SEM of three to eight mice per time point; data shown for 1 wk is also shown in Fig. 2C, 2D). (C) Four weeks after adoptive transfer, significantly more preactivated than control NK cells produced IFN-γ. Twelve weeks after cotransfer into alymphoid hosts, slightly more control donor NK cells produce IFN-γ (mean ± SEM of four to five independent experiments; black bars, IL-12 + IL-15 stimulation; gray bars, media; **p < 0.004).
Although immunologic memory has traditionally been the hallmark of adaptive immunity, several recent studies have demonstrated that innate immune NK cells have the capacity for memory-like responses (1–4). In this study, we found that cytokine-induced memory-like NK cells expand in alymphoid hosts via homeostatic proliferation and maintain their phenotype for at least a month despite extensive proliferation of all NK cells. As expected, activated NK cells made very high amounts of IFN-γ shortly after adoptive transfer in to alymphoid hosts, likely because of a primed phenotype. However, both cytokine-activated and control NK cells quickly proliferated, and by day 7, nearly all cells present in the hosts had never before been primed in vitro. Memory-like NK cells had enhanced IFN-γ responses to restimulation with cytokines and tumor cells in vitro up to 4 wk after adoptive transfer. In vivo TLR activation showed enhanced responses by preactivated NK cells at early (7 d) but not later (4 wk) time points. No difference in expression of granzyme B or the ability to degranulate in response to tumor cells was observed. Interestingly, all NK cells, regardless of prior activation, expressed high levels of granzyme B after transfer into alymphoid hosts, perhaps because of proliferation or other host factors.
Our data demonstrate that only a portion of daughter cells from previously activated NK cells retain a capacity for enhanced IFN-γ production, because the majority of cells did not produce IFN-γ, and identification of a cell surface marker to identify memory-like NK cells will be important for future studies. As shown in this study and based on expression profiling (M.A. Cooper, unpublished data), we have not found a cell surface marker that distinguishes memory-like from naive splenic NK cells. KLRG1, an inhibitory cell surface receptor (28), has been proposed as a marker of splenic NK cell memory and was elevated on the cell surface of MCMV-induced memory NK cells (3). In this study, we found that KLRG1 was highly expressed by both cytokine-induced memory-like and naive expanded NK cells (Supplemental Fig. 4), suggesting that KLRG1 may be a marker of mature NK cell proliferation, as was previously suggested (28, 29), rather than memory.
NK cell numbers are normally tightly regulated in healthy hosts. Homeostatic expansion occurs in the setting of lymphopenia, and mature NK cells can proliferate and expand to "fill" the empty NK compartment (21–23). In patients, this can occur as the result of infection or chemotherapy-induced lymphopenia. Following adoptive transfer into alymphoid hosts, both cytokine-activated and control NK cells proliferated extensively within a week of adoptive transfer. Cytokine-activated NK cells maintained a memory-like phenotype for 12 wk in NK-competent Rag-1−/− hosts, whereas memory-like cells lost this phenotype by 3 mo in alymphoid hosts. The observed differences may be related to NK cell homeostasis in the two models. In alymphoid, Rag-2−/−γc−/− hosts, mature NK cells undergo homeostatic proliferation driven by a lack of endogenous lymphocytes. Whereas in NK-competent, Rag-1−/− hosts, only a subset of memory-like cells proliferate 3–7 d after adoptive transfer. Thus, one explanation for the loss of memory in Rag-2−/−γc−/− hosts at 12 wk is that multiple rounds of proliferation ultimately alters the memory-like phenotype of NK cells, whereas NK cells with low turnover in NK-competent hosts maintain a memory-like phenotype. An alternative hypothesis is that there is a gain of function in all (or only control) NK cells related to homeostatic proliferation, analogous to the memory-phenotype observed in T cells following homeostatic proliferation in a lymphopenic host (30). It will be interesting to determine whether NK cells exhibit a similar type of proliferation-driven memory–phenotype differentiation at late time points.
Regardless, the maintenance of clear memory-like effects after 4 wk of proliferation demonstrates that cytokine-induced memory is stable and hereditary. From a therapeutic standpoint, clinically relevant lymphopenia in patients (i.e., infection or chemotherapy induced) and inhibition of NK cell development is likely temporary, lasting from a few days to months (31). Thus, transient lymphopenia in patients might allow for the expansion of adoptively transferred or infection-induced memory-like NK cells that, with resumption of normal NK cell differentiation and resolution of lymphopenia, are subsequently maintained in a quiescent state. On the basis of the model in this study, such non-dividing memory-like NK cells would be predicted to maintain their phenotype long-term.
There are currently three experimental models of murine NK cell memory: 1) cytokine-induced splenic NK cell memory-like function (2), as discussed in this study; 2) splenic NK memory conferred via stimulation with cytokines and a germline-encoded NK cell receptor following recognition of a pathogen-encoded ligand (3, 13); and 3) Ag-specific liver NK cell memory not mediated by a known germline-encoded receptor, which was the first type to be described previously (1, 4). Taken together, these models are complementary, rather than mutually exclusive, and definitively demonstrate that NK cells have the capacity for recall responses. Our model suggests that, after an initial cytokine activation event and production of IFN-γ, NK cells retain the capacity for enhanced IFN-γ responses to multiple stimuli. Thus, cytokine-induced NK cell memory responses are predicted to provide nonspecific protection. This is physiologically relevant, because NK cells express only a limited number of activation receptors and often rely upon cytokine signals to mediate effector function (6).
Studies from the Lanier group (3) using an adoptive transfer system demonstrated that MCMV-induced expansion of long-lived Ly49H+ NK cells with enhanced cytokine production and the ability to provide superior protection against MCMV infection in neonatal mice. Similar to our system, this model was also dependent on cytokines, in particular IL-12 (13). Additional studies by Schlub et al. (32) in an intact host found that the kinetics of Ly49H+ NK cell expansion and contraction are different from T cells and there may not be a physiologically significant pool of memory-like NK cells that can expand in a secondary response, perhaps more consistent with the idea of enhanced cellular function rather than long-lived protective immunologic memory. It is not known whether Ly49H+ MCMV-induced memory NK cells are virus specific; however, because they respond more robustly to nonviral stimuli (i.e., anti-NK1.1) (3), it is possible that they might also provide enhanced protection against other infections. Indeed, it may be the inherent nonspecific nature of cellular memory-like responses in NK cells that could allow them to better protect the host, rather than specific immunologic memory to infections.
Studies from von Andrian's group (1) were the first to demonstrate the capacity for liver NK cell immunologic memory with the finding that NK cells can mediate hapten-specific contact hypersensitivity (CHS) responses in the absence of T cells. Subsequent studies demonstrated a small subset of liver lymphocytes from Rag-deficient mice expressing NK1.1, CXCR6, Thy1, and other markers had the capacity for Ag-specific intrinsic memory but not splenic cells expressing the same markers (4). This type of memory appears more distinct from the two previous models based on its Ag specificity and limitation to lymphocytes from the liver. Memory NK1.1+ liver cells were present in genetically diverse strains of mice (BALB/c and C57BL/6); capable of recognizing haptens and viral-like particles from HIV, influenza, and vesicular stomatitis virus; and conferred specific protection against viral infection in vivo (4). The recognition of such a diverse pool of Ags, and the fact that no Ag failed to induce NK cell memory responses (4), is reminiscent of T and B lymphocyte diversity and suggests that these NK1.1+ cells have the capacity for receptor editing, either via posttranscriptional modification or non–Rag-mediated recombination events. A study from another group demonstrated that the character and quality of CHS responses mediated by NK cells may be distinct from T cell CHS, suggesting a unique mechanism for liver NK1.1+ cell memory (33). It will be interesting to determine the mechanism of specific Ag recognition in these NK cells.
Collectively, these studies demonstrate that NK cells "learn" from prior experiences. In all of the experimental systems, the memory phenotype appears to be heritable and persists longer than the estimated 7- to 17-d half-life of NK cells (34, 35). In the case of cytokine and MCMV-induced memory, NK cells were more responsive not only to the original stimuli but also other activation signals (e.g., cytokines, receptor ligation, and tumor target cells), suggesting a generalized enhanced state of activation that could be exploited for immunotherapy (2, 3). Indeed, a recent study by the Cerwenka laboratory (36) demonstrated that IL-12, -15, and -18 preactivation of NK cells prior to adoptive transfer into sublethally irradiated tumor-bearing hosts led to significant reduction in established tumors. In this study, we discovered that cytokine-induced memory-like NK cells proliferate and expand in alymphoid hosts. There are currently multiple studies investigating NK cell adoptive therapy for cancer (9), and homeostatic expansion may be critical for treatment success as evidenced by the finding that patients treated with more intense conditioning regimens have enhanced donor NK cell engraftment (24). A recent study from Gill et al. (37) found that murine NK cells adoptively transferred into Rag-2−/−γc−/− hosts completely lost the ability to produce IFN-γ within 5 d. They concluded that these NK cells become exhausted because of homeostatic proliferation and that caution should be used with NK cell adoptive therapy (37). However, we and others (19, 21, 23) clearly demonstrate that adoptively transferred NK cells retain the capacity to produce IFN-γ following proliferation in alymphoid hosts. The cause for discrepancy between these studies is unclear; however, the murine NK cells used by Gill et al. (37) were expanded with IL-2 prior to adoptive transfer, which may result in NK exhaustion, whereas we demonstrate that control (low-dose IL-15) or IL-12 plus IL-18–activated NK cells retain the capacity to produce IFN-γ following in vitro and in vivo stimulation. This suggests that thorough investigation of growth factors that support optimal NK cell function, such as IL-12 and IL-18, is critical for successful NK cell immunotherapy.
Many questions regarding the nature of NK cell memory responses remain. Can memory-like NK cells provide enhanced protection to a variety of infections and tumors? What are the mechanisms of heritable memory-like responses and are they present in humans? NK cell memory has only recently been recognized, and as we gain a better understanding of these and other fundamental questions, it will be interesting to see how innate immune memory can be exploited for NK cell vaccination and adoptive therapy strategies.
The authors have no financial conflicts of interest.
We thank W. Yokoyama and T. Fehniger for thoughtful discussion and review of the manuscript.
This work was supported by the National Institutes of Health–National Institute of Allergy and Infectious Diseases Grant K08AI085030, Child Health Research Center of Excellence at Washington University Grant K12-HD01487, and the Children's Discovery Institute of Washington University and St. Louis Children's Hospital.
The online version of this article contains supplemental material.
Abbreviations used in this article:
contact hypersensitivity
γc
common γ-chain
MCMV
murine CMV
poly(I:C)
polyinosine-polycytidylic acid
Rag-1−/−
Rag-1–deficient.
Accepted February 25, 2013.
Copyright © 2013 by The American Association of Immunologists, Inc.
O'Leary J. G.,
M. Goodarzi,
D. L. Drayton,
U. H. von Andrian
. 2006. T cell- and B cell-independent adaptive immunity mediated by natural killer cells. Nat. Immunol. 7: 507–516.
Cooper M. A.,
J. M. Elliott,
P. A. Keyel,
L. Yang,
J. A. Carrero,
W. M. Yokoyama
. 2009. Cytokine-induced memory-like natural killer cells. Proc. Natl. Acad. Sci. USA 106: 1915–1919.
Sun J. C.,
J. N. Beilke,
L. L. Lanier
. 2009. Adaptive immune features of natural killer cells. Nature 457: 557–561.
Paust S.,
H. S. Gill,
B. Z. Wang,
M. P. Flynn,
E. A. Moseman,
B. Senman,
M. Szczepanik,
A. Telenti,
P. W. Askenase,
R. W. Compans,
. 2010. Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat. Immunol. 11: 1127–1135.
Yokoyama W. M.
2008. Mistaken notions about natural killer cells. Nat. Immunol. 9: 481–485.
Vivier E.,
D. H. Raulet,
A. Moretta,
M. A. Caligiuri,
L. Zitvogel,
L. L. Lanier,
W. M. Yokoyama,
S. Ugolini
. 2011. Innate or adaptive immunity? The example of natural killer cells. Science 331: 44–49.
Biron C. A.,
L. Brossay
. 2001. NK cells and NKT cells in innate defense against viral infections. Curr. Opin. Immunol. 13: 458–464.
French A. R.,
. 2003. Natural killer cells and viral infections. Curr. Opin. Immunol. 15: 45–51.
Cooley S.,
D. S. Weisdorf
. 2010. Natural killer cells and tumor control. Curr. Opin. Hematol. 17: 514–521.
. 2010. Memory-like responses of natural killer cells. Immunol. Rev. 235: 297–305.
. 2011. Natural killer cell memory. Nat. Immunol. 12: 500–508.
S. Lopez-Verges,
C. C. Kim,
J. L. DeRisi,
. 2011. NK cells and immune "memory". J. Immunol. 186: 1891–1897.
S. Madera,
N. A. Bezman,
M. H. Kaplan,
. 2012. Proinflammatory cytokine signaling required for the generation of natural killer cell memory. J. Exp. Med. 209: 947–954.
Romee R.,
S. E. Schneider,
J. W. Leong,
J. M. Chase,
C. R. Keppel,
R. P. Sullivan,
M. A. Cooper,
T. A. Fehniger
. 2012. Cytokine activation induces human memory-like NK cells. Blood 120: 4751–4760.
Lopez-Vergès S.,
J. M. Milush,
B. S. Schwartz,
M. J. Pando,
J. Jarjoura,
V. A. York,
J. P. Houchins,
S. Miller,
S. M. Kang,
P. J. Norris,
. 2011. Expansion of a unique CD57+NKG2Chi natural killer cell subset during acute human cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 108: 14725–14732.
Björkström N. K.,
T. Lindgren,
M. Stoltz,
C. Fauriat,
M. Braun,
M. Evander,
J. Michaëlsson,
K. J. Malmberg,
J. Klingström,
C. Ahlm,
H. G. Ljunggren
. 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208: 13–21.
Foley B.,
S. Cooley,
M. R. Verneris,
J. Curtsinger,
X. Luo,
E. K. Waller,
C. Anasetti,
D. Weisdorf,
J. S. Miller
. 2012. Human cytomegalovirus (CMV)-induced memory-like NKG2C+ NK cells are transplantable and expand in vivo in response to recipient CMV antigen. J. Immunol. 189: 5082–5088.
White D. W.,
T. A. Reese,
J. Coder,
J. E. Payton,
T. J. Ley,
H. W. Virgin,
. 2010. Latent herpesvirus infection arms NK cells. Blood 115: 4377–4383.
Ranson T.,
C. A. Vosshenrich,
E. Corcuff,
O. Richard,
W. Müller,
J. P. Di Santo
. 2003. IL-15 is an essential mediator of peripheral NK-cell homeostasis. Blood 101: 4887–4893.
J. E. Bush,
T. A. Fehniger,
J. B. VanDeusen,
R. E. Waite,
Y. Liu,
H. L. Aguila,
M. A. Caligiuri
. 2002. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood 100: 3633–3638.
Prlic M.,
B. R. Blazar,
M. A. Farrar,
S. C. Jameson
. 2003. In vivo survival and homeostatic proliferation of natural killer cells. J. Exp. Med. 197: 967–976.
V. Laloux,
A. Lehuen,
. 2003. IL-15 availability conditions homeostasis of peripheral natural killer T cells. Proc. Natl. Acad. Sci. USA 100: 2663–2668.
. 2011. Homeostatic proliferation generates long-lived natural killer cells that respond against viral infection. J. Exp. Med. 208: 357–368.
Miller J. S.,
Y. Soignier,
A. Panoskaltsis-Mortari,
S. A. McNearney,
G. H. Yun,
S. K. Fautsch,
D. McKenna,
C. Le,
T. E. Defor,
L. J. Burns,
. 2005. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105: 3051–3057.
Fehniger T. A.,
. 2001. Interleukin 15: biology and relevance to human disease. Blood 97: 14–32.
A. Ma,
. 2009. Cutting edge: IL-15-independent NK cell response to mouse cytomegalovirus infection. J. Immunol. 183: 2911–2914.
S. F. Cai,
X. Cao,
A. J. Bredemeyer,
R. M. Presti,
A. R. French,
T. J. Ley
. 2007. Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs. Immunity 26: 798–811.
Robbins S. H.,
K. B. Nguyen,
N. Takahashi,
T. Mikayama,
C. A. Biron,
. 2002. Cutting edge: inhibitory functions of the killer cell lectin-like receptor G1 molecule during the activation of mouse NK cells. J. Immunol. 168: 2585–2589.
Huntington N. D.,
H. Tabarias,
K. Fairfax,
J. Brady,
Y. Hayakawa,
M. A. Degli-Esposti,
M. J. Smyth,
D. M. Tarlinton,
S. L. Nutt
. 2007. NK cell maturation and peripheral homeostasis is associated with KLRG1 up-regulation. J. Immunol. 178: 4764–4770.
Jameson S. C.
2002. Maintaining the norm: T-cell homeostasis. Nat. Rev. Immunol. 2: 547–556.
Castelino D. J.,
P. McNair,
T. W. Kay
. 1997. Lymphocytopenia in a hospital population—what does it signify? Aust. N. Z. J. Med. 27: 170–174.
Schlub T. E.,
J. C. Sun,
S. M. Walton,
S. H. Robbins,
A. K. Pinto,
M. W. Munks,
A. B. Hill,
L. Brossay,
A. Oxenius,
M. P. Davenport
. 2011. Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection. J. Immunol. 187: 1385–1392.
Rouzaire P.,
C. Luci,
E. Blasco,
J. Bienvenu,
T. Walzer,
J. F. Nicolas,
A. Hennino
. 2012. Natural killer cells and T cells induce different types of skin reactions during recall responses to haptens. Eur. J. Immunol. 42: 80–88.
Jamieson A. M.,
P. Isnard,
J. R. Dorfman,
M. C. Coles,
D. H. Raulet
. 2004. Turnover and proliferation of NK cells in steady state and lymphopenic conditions. J. Immunol. 172: 864–870.
Koka R.,
P. R. Burkett,
M. Chien,
S. Chai,
F. Chan,
J. P. Lodolce,
D. L. Boone,
A. Ma
. 2003. Interleukin (IL)-15Rα‑deficient natural killer cells survive in normal but not IL-15Rα‑deficient mice. J. Exp. Med. 197: 977–984.
Ni J.,
M. Miller,
A. Stojanovic,
N. Garbi,
A. Cerwenka
. 2012. Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors. J. Exp. Med. 209: 2351–2365.
Gill S.,
A. E. Vasey,
A. De Souza,
J. Baker,
A. T. Smith,
H. E. Kohrt,
M. Florek,
K. D. Gibbs Jr..,
K. Tate,
D. S. Ritchie,
R. S. Negrin
. 2012. Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells. Blood 119: 5758–5768.
The Journal of Immunology
Thank you for your interest in spreading the word about The Journal of Immunology.
You are going to email the following Murine NK Cell Intrinsic Cytokine-Induced Memory-like Responses Are Maintained following Homeostatic Proliferation
Message Subject (Your Name) has forwarded a page to you from The Journal of Immunology
Message Body (Your Name) thought you would like to see this page from the The Journal of Immunology web site.
Molly P. Keppel, Liping Yang, Megan A. Cooper
The Journal of Immunology May 1, 2013, 190 (9) 4754-4762; DOI: 10.4049/jimmunol.1201742
Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α
Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization
The Orphan Immune Receptor LILRB3 Modulates Fc Receptor–Mediated Functions of Neutrophils
Show more INNATE IMMUNITY AND INFLAMMATION
Print ISSN 0022-1767 Online ISSN 1550-6606


Hey folks, Cabby here! Just wanted to tell you a little about my equipment and features installed by the Team at TRAC-USA. Well, as you are likely aware, I joined TRAC-USA to help demonstrate the latest equipment to improve operator safety and comfort in all types of work environments.
For the dusty environments, I am equipped with the latest Sy-Klone RESPA-CF cab pressurization system and pressure monitor. The RESPA-CF with Vortex Hyper Flow pressurizes the cab, which reduces HVAC system maintenance and reduces the operator's exposure to the outside elements -- all while meeting the current regulations for exposure limits and making operators more productive!
I am also equipped with a self-contained Red-Dot R9727 rooftop A/C unit with 22,000 BTU cooling. This unit is renowned for its quality and reliability, as well as its serviceability. Red-Dot offers a complete line of heavy duty Off-Road A/C units and heaters for all types of rugged applications. Give the folks at TRAC-USA a call with your specific requirements, and they will help design the system best suited for your needs.
For the extreme cold environments, I am equipped with a diesel-fired auxiliary heater to keep my operator comfortable on those wintry days. TRAC–USA offers both air and coolant heaters from the top manufacturers in the world, including Espar and Webasto. TRAC-USA also handles the appliances by Arctic-Fox to help heat the fuel and hydraulics on your specific machine.
This is just a summary of the equipment I have, but TRAC-USA can help you with all types of mobile climate control, as they represent the finest manufacturers in the industry. Custom designed systems are our specialty. Contact us with your requirements, and we will do our best to find you a solution!!
The Occupational Safety and Health Administration (OSHA) is amending its existing standards for occupational exposure to respirable crystalline silica. OSHA has determined that employees exposed to respirable crystalline silica at the previous permissible exposure limits face a significant risk of material impairment to their health. The evidence in the record for this rulemaking indicates that workers exposed to respirable crystalline silica are at increased risk of developing silicosis and other non-malignant respiratory diseases, lung cancer, and kidney disease. This final rule establishes a new permissible exposure limit of 50 micrograms of respirable crystalline silica per cubic meter of air (50 μg/m3) as an 8-hour time-weighted average in all industries covered by the rule. It also includes other provisions to protect employees, such as requirements for exposure assessment, methods for controlling exposure, respiratory protection, medical surveillance, hazard communication, and recordkeeping.
When TRAC customers asked us for help in remaining compliant with tightening controls for work-place air quality, we sought out and found today's cutting edge technology to clean cabin air. We are now certified Sy-Klone Solutions Providers - offering vortex hyperflow technology that purges 95% of particulates in the air – down to 5 microns in size – before it hits the filter." The unique patented Self Cleaning Filter strips particulate out of the air down to .3 microns at 95+% efficiency. Filter life is extended, downtime is reduced, the HVAC is protected and maintenance costs are significantly reduced.
We will analyze your particular equipment, design, install and monitor a right-sized, custom solution that will keep you compliant with the most restrictive legislation, reduce your exposure to litigation, and, most important of all, protect the health of your valued operators.
Sy-Klone engine air pre-cleaners use the same state-of-the-art technology to pre-clean engine air, increasing maintenance intervals, extending engine life and lowering your overall cost of ownership.


text:
Q: Npm not found when running docker container from node image # Dockerfile
FROM node:7-alpine
RUN mkdir -p /src/app
WORKDIR /src/app
COPY package.json /src/app/package.json
RUN npm install
COPY . /src/app
EXPOSE 3000
CMD ['npm', 'start']

I'm trying to complete a katacoda.com exercise for Dockerizing nodejs applications with the Dockerfile above. The build completes but running the image quits immediately and in the docker logs I see:
/bin/sh: [npm,: not found

I tried running the container in interactive mode with docker -it nodeapp /bin/bash which raised the error docker: Error response from daemon: oci runtime error: container_linux.go:262: starting container process caused "exec: \"/bin/bash\": stat /bin/bash: no such file or directory". So I'm not sure what is going on here.

A: I had the same symptom but the problem was slightly different. Writing here in case google leads others in my situation to this link For me the issue was forgetting commas in the CMD. So the solution was going from CMD ["npm" "start"] to CMD ["npm", "start"].

A: The reason it doesn't work is single quotes
CMD ['npm', 'start']

should be
CMD ["npm", "start"]

When you don't use double quotes, docker will remove the single quotes and process the command as [npm, start]
That is why you see error [npm, : not found


text:
Jester King Brewery Adds Notable Georgetown Chef for New Restaurant
The Hollow is now closed
by Nadia Chaudhury Mar 19, 2018, 2:34pm CDT
Share All sharing options for: Jester King Brewery Adds Notable Georgetown Chef for New Restaurant
Flatbread with vegetables and chorizo from The Hollow
The Hollow/Facebook
The Hollow, the Georgetown bistro, is now closed. Chef Jacob Hilbert announced this via a lengthy, rambling letter sent to Eater Austin, as well as other news outlets. Buried within the note was the announcement that he is joining Jester King Brewery's forthcoming restaurant.
Hilbert has worked with Jester King Brewery co-founders Jeffrey Stuffings and Michael Steffing before, on various beer dinners. Together, they want to open "one of the best restaurants in the world," as Hilbert wrote.
Hilbert described it as "a more casual accessible restaurant built on the cuisine of nomads and ancient peoples." The restaurant will use grains and produce grown on the farm land, as well as animals raised on the property. It'll serve up charcuterie, bread, roasted vegetables, dairy, and more.
Austin 360 confirmed the collaboration. Stuffings revealed to the publication that there will be two restaurants, the first will be a casual one, whereas the second will be more narrow in menu scope.
The unnamed Jester King restaurant is pegged to open this year. The brewery is already offering tours of its farmland with goats. Stuffings and Steffing purchased the property next door to the Dripping Springs brewery in early 2016, with the goal of turning it into farmland, vineyard space, and a restaurant.
Elsewhere in Hilbert's letter, the chef opines on his childhood, baseball, his restaurant background starting in New Mexico, how his late mother informed his cooking style, his drug addiction and rehabilitation, how he met his wife Lynda, and the history of The Hollow, which he opened in 2013. The full letter follows below, in its original, unedited form.
Jacob Hilbert's Letter
Many years ago I walked away from baseball. I was good, I was strong, I loved the game. So distant in years that the memories are captured frozen daguerreotype with captions written Bernard Malamud across the bottom of my youth. Like many things we are rarely privy to know the last time. I did not know when I crossed that baseline the threshold I was crossing, I did not know I would not stand again on the pitcher's mound a bottacelli gladiator facing down demons and insecurities with my strongest gift, a 95 mile an hour fastball moving Baryshnikov across grass and clay. Then, like the lightning bolt giving way to wonder boy there was a tear in my shoulder, a pain across my chest and a broken heart…it was over. The memories vestigial.
For years after I could not watch baseball, the history books of the game collected dust, my teams went unwatched and unloved. It was too much to watch, like I was witnessing the theft of dreams and I was mute to scream, to let the authorities know. Occasionally I would pass by a little league game, a youth parade, and I would pull over and watch. For a few moments I could walk with them, become a listener for the banter of the game, a voyeur not for the love of watching but for the want of being.
My love for baseball was slowly supplanted by a love for cooking and a disdain for my college studies. I was to be a therapist, like my mother, father, stepmother and stepfather. I was to attain a doctorate and begin a path riddled with Jungian academia, a Jewish archetype, professorial and argumentative. The last weeks of graduate school, thesis in mind, sleepless nights informing daydreams, I quit. I had been making salsas at a little Mexican restaurant in Las Cruces New Mexico, I had become drunk in vats of menudo and posole, my thoughts were irrational, my studies lost in the steam of simmering pots, and I quit. Telling my parents, monoliths of academic discipline, purveyors of footpaths to success as determined by most, was akin I believe to coming out of the closet. In fact, to have been gay would have been an easier conversation. My mother would have been thrilled and I have often wondered if her love of reading me Allen Ginsberg as a child was some sort of conversion therapy to discourage me from becoming part of oppressive patriarchal social constructs, to open me up to the infinite erotic and ontological joys of homosexuality. To take a bite out of The Night Apple. But it was not to be, sadly I was straight, and I wanted to be a cook.
My mother died before I ever took control of a kitchen. I cooked for her a few times. She enjoyed the cuisine of my mentor, it was a timeless cuisine familiar in images of relative stasis. The gastronomique remained, more or less, unchanged for decades trading nothing but still life for kinetic visages of artichokes in boiling water, meant all the same for the overcooked plates that represented haute cuisine for generations. My cooking originated in this style, one that moved in a direct line from Escoffier to Troisgros. Mom recognized these dishes borrowed from the pages of the day's Bon Apetit being the same dishes from yesterday's Bon Apetit. French cuisine was, at the time, just post nouvelle and was different in totality from the days of Careme but remained so for decades as though the museum had been built and curated for the capturing of an epoch in which evolution was a non starter or even a blasphemy. It is not unusual for resistance to revolution as the conservative machines rely on inertia to attain autocracy and even divine right supremacy. So it is with food as it is with all things human, art, politics, literature. My mother liked rack of lamb, overcooked with tomate provencal.
I have been enchanted by tradition, I am reverential in many ways. Love for tradition does not mean distaste for change, it is the exercising of curiosity, it is the building of time machines where one can sit at ancient tables and dine with ghosts. Who amongst lovers of cooking could resist dining with Elizabeth David, M.F.K. Fisher or James Beard? Who amongst lovers of cooking could resist watching them experience the new, imagine dinner with Adria and Richard Olney?
I tasted fame, it was a demi tasse spoon, but it changed me. It was written once that I was to be the next great Southwestern chef, I was sexy and brash, I was stupid and immoral, I was arrogant and intoxicated…all the time. I believed it was a rite of passage, I believed in cocaine, I believed in fucking into oblivion, I believed that I was great, I believed that everything was as it was supposed to be, that I was Henry Miller with a wooden spoon and hydrocolloids, that the whisk was mightier than the sword, that the experiences given to the guests were the currency exchanged for a harem of women and sycophantic men, that I was wielding Freudian weaponry. Until I died, which I did. When I died there was no light, I did not see my mother or my grandfather with hand oustretched to pull me into the warmth, I was not overcome with salvation, I was simply dead, my heart had stopped. I had walked to a denny's in Santa fe with nothing on my body but a chef coat and shoes, in my pocket was a thermometer, a sharpie
and a bag of cocaine. I walked the street without consideration for my nudity, without a care. I stopped to blow a rail on a newspaper stand. I wanted a moons over my hammy. When I entered the denny's I was not seated as there seemed to be a problem with my attire or lack thereof. I went into the bathroom, blew another rail off of the back of the toilet and shortly thereafter died.
28 days later I emerged from rehab, homeless, jobless, directionless, hopeless. I took a job at a shitty restaurant back in Las Cruces. I made bad desserts and terrible pasta. I had not learned much really. death had not taught me anything yet. I was still an inculcation of competing viewpoints. I had been a very ugly child, curious, thoughtful, and deeply unpopular. I don't know if I was ugly but I was told so. Children can be beautiful and cruel and the indelible fingerprint of anti-semitism and social caste can impregnate a world, a world where a child will grow into adolescence and adulthood informed by little other than what people say about him/her. Baseball was my Christ, it was the field on which I was forgiven the sins of existence. Then it was soup. When the world decided I was valuable, wanted, needed I believed it in the Newtonian sense of law. These physical laws of being are immutable to the sufferer of gravity. The calculus of circumspect yields only to the caprice of critics with equation-like differentiation. Love becomes a given, unearned and without causality indulging an infinite mass of asshole moving loudly to a point of singularity where abyss and a black hole await, where hands do not reach out from the evernight, where you are forgotten by your most intimate ghosts. At this moment I was gone, nothing, just an undeniable piece of shit, a child who lost his toy, a toy that was the people in his world who had packed up their hearts and gone home. Then I met Lynda.
Lynda believed in me when she should not have, she loved me when I did not deserve it. She did not give up. She did not give up through lies, infidelity, grotesque dedication to my lost craft, absent fatherhood, narcissistic rants. I took her to hell, and she stood among the perils of tectonic consequence. I did not ask her to be stoic, but she was, and I am now just a slight reward for her suffering, the miracle being that I may just be enough for her, as I am, without dazzling plates or articles extrapolating my character, without my be anything to anyone other than her. She is a hand that reaches out in the breathing light of day. For this there was an endeavor for anonymity. Difficulties, however, seek us out no matter the light we live in. There are all kinds of lies, lies we tell and lies we live being just a couple.
When The Hollow opened it was to be a French bistro, it was to be provincial French cuisine run through processor of Chez Pannise and Pastis, decidedly unelectric delicious cuisine playing host to years of venerable dishes made slightly more modern. It was a lie that I could not tell. I did not know it at first. The goal was to cook en obscura. To disappear into the kitchen to make sauces and braises without a name and with only a point of view that could be subtly introduced in such a manner as the alterations could only be detected by the transcendent aspects of minimal adulteration. In that pomp I could hide in the cuisine quieted by derision self imposed. Know one would know me, know one would care, know one could judge me as there would be little to judge. Lynda and I had children, we bought a house, we had two cars and a project truck, we built tree houses and planted a garden, and we watched seeds grow and vines fruit. I cooked hamburgers and schnitzel and made the occasional foam, met the media on uncontroversial terms with an occasional foam thrown in, an aspic, a terrine and a sphere. I had created cuisine born of neuroses and pathos weighted down by the feeling of dying. I was deeply unoriginal and uninspired by my own pans and sick of my own rhetoric. I woke up one morning to a mirror, my beard was grey, my hair was thinning, my smile was dull and my children were fed, all was well, all was expected, all was inevitable and nothing was sacred.
The Hollow is closed, and it is with a whimper. A most inconsequential restaurant, a humble restaurant and a beautiful restaurant. The people that I worked with inspired me, taught me, the struggles to speak a new language were present in every menu, my inability to change and bend was painful to watch I am sure. I hope that the young people that I met will come with me, will want to embrace this new journey. The closing of the doors at The Hollow felt more like a closing down of a school, like I walked out of class and bid my students goodbye. I felt like I walked out of class and bid my teachers goodbye. So I did what anyone does, I cried a bit, and felt sorry for myself, and then I decided to let Lynda cut my hair.
When she cut my hair, I was not tied to the porch chair, there were no thrones, from my lips I drew no hallelujah. I was not changed and there were no intended biblical metaphors, I just wanted a change, a release, a vanquishing of body parts playing a role in mediocrity. Since cutting off my head would have been inconvenient, and my hands are necessary, the hair was all that I could think off. In the days after closing the restaurant came spring and the fields and forests were imbued with mis en place, flora ignited the frontier and the trees in renaissance showed timely vulnerability, the brontosaur's baby lettuce salad, the forager's cue. I was saddened as the dishes ran through my head that would not be this year, the things I would not do and only think of, the mistakes I would not make, the sicknesses I would endeavor and fail to avoid. The Texas fields are bitter, aromatic and serrated, sometimes sweet, always changing and forever in need of catalogue. These are the first steps I think, to begin a canon, to figure a starting point, nearly a prayer, for finding something new and different, an exposition of a new cuisine that will either have value or not. It is my thought that good cooking is objective, this thought will be challenged in a world without precedent. So the realization of this particular goal is in chiaroscuro, hidden, beautiful, not very well lit, much like twilight. When I first met with Jeffrey & Michael from Jester King there had been much talking prior, however the question or the statement had not been made. I said "I know this is crazy, but I want to make one of the best restaurants in the world." Without so much as a hesitation and in harmony the response was "we want that too."
So this letter begins a story, a story that asks a question. How do you build one of the greatest restaurants in the world? I suppose we'll find out together.
This year the farmhouse will open, a more casual accessible restaurant built on the cuisine of nomads and ancient peoples, there will be fire and spit roasting and vegetables cooked in mud. We will muddle sauces in ancient ways and walk the land looking for tomorrow, we will bake bread and we will preserve things. The goal is to have the restaurant completely self-sustained, growing all of the produce, milking the cows and goats, making the cheese, hanging the charcuterie. Over the next two to three years we will be building accum. The restaurant that will change everything about the cooks working there, about the service and will make every effort to be great, even if it fails.
Jester King Brewery Is Now Offering Farm Tours With Goats [EATX]
Jester King Brewery Plans on Creating a Farm Wonderland [EATX]
All Coverage of Jester King Brewery [EATX]
Jester King Craft Brewery
13005 Fitzhugh Road, Austin, TX 78736 512 537 5100 Visit Website
The Hollow [Closed]
708 South Austin Avenue, Georgetown, Texas 78626 (512) 868-3300 Visit Website
Austin's Restaurant Closings of 2018
North Austin Pub Is Still In Danger of Closing After 31 Years
Guadalupe Japanese Restaurant Is Turning Into a New Korean Spot
12 Most Romantic Restaurants in Austin For cozy corners and memorable dinners, look no further