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SSC Stenographer Result 2016 – Steno Grade C & D Cut off Marks, Merit List Available at ssc.nic.in :
SSC Stenographer Result 2016 for Grade C & D
Staff Selection Commission has successfully conducted recruitment examination for Stenographer Grade C & D vacancies. Written test was conducted 31.01.2016 at various examination centers across India. Lots of applicants had participated in Steno Grade C & D Examination in order to grab jobs in Ministries / Department of Central Department. Now all aspirants who were appeared for Written Examination are looking for their SSC Stenographer Result 2016 (Grade C & D).
Latest Update (30th November, 2016) As per latest report Staff Selection Commission will publish Result of SSC Stenographer Written Exam 2016 on 01st December, 2016 in this month Stay tuned with us to monitor entire recruitment process. Candidate who has qualified as per result of SSC Stenographer 2016 written exam is called for the skill test.
SSC Stenographer 2015 Final Result will be declared today evening 30 Nov 2016
Staff Selection Commission has released SSC Stenographer Grade C & D Exam Answer key at its official website..!
Earlier Staff Selection Commission has published an advertisement in order to fill up 1064 posts of Stenographer. The massive number of candidates had applied for the post. Now application process is over and the candidates are waiting for the SSC Result 2016 for Stenographer Grade C & D Exam.
SSC Stenographer Result 2016 Expected Date
Result will be declared on the official website very soon. Applicant should download the SSC Stenographer Grade C & D Result 2016 from the official website ssc.nic.in. Candidates are requested to keep touch with website to get the all latest updates about result. Staff Selection Commission will disclose list of shortlisted candidates or SSC Steno Result 2016 on 01.12.2016. SSC will shortlisted candidates who meet the cut off marks in written exam. Shortlisted applicant will be called for next stage of selection process which is skill test.
ssc.nic.in Stenographer Result 2016
- Organization : Staff Selection Commission
- Examination : SSC Stenographer Grade C & D Examination
- Name of Post : Stenographer Grade C & D
- Total Vacancies : 1064
- Exam Date : 31st January, 2016
- Announcement of Result : 30.11.2016 (expected date)
- Selection Process : Written Exam, Skill Test
- Website : ssc.nic.in
SSC Stenographer Skill Test Details
Candidates who score at least cut off marks in written examination will qualify for skill test. Selected candidates have to appear for Skill test. In the skill test the candidates will be given one dictation for 10 minutes in English/Hindi at the Speed of 100 w.p.m. for the post of Stenographer Grade 'C' and 80 w.p.m. for the post of Stenographer Grade 'D'.
|Steno. Grade C
|Steno. Grade D
|Dictation speed
|100 wpm
|80 wpm
|Transcription time for English
|40 minutes
|50 minutes
|Transcription time for Hindi
|55 minutes
|65 minutes
SSC Stenographer Cut Off Marks 2016 Grade C & D
Here we have given last year cut off marks for Stenographer Grade C & D exam. This year also you have to obtain at least cut-off marks to in order to participate in further selection process.
Applicant should download the SSC Stenographer Result 2016 for Grade C & D Posts from the official website ssc.nic.in and then you can check out your result by following Roll Number or Registration No.
|Steno. Grade 'C'
|Steno. Grade 'D'
SSC Stenographer Merit List 2016
Staff Selection Commission is an Indian organization to recruit staff for various posts in the various Ministries and Departments of the Government of India and in Subordinate Offices. It was set up on 1 July 1976 to make recruitment on zonal basis for non-technical Group C posts under the Central Government, except the post for which recruitment was made by Railway Service Commission and the industrial establishments.
The functions of the Staff Selection Commission have been enlarged from time to time and now it carries out the recruitment for Group B posts also under the Central Government. The Commission has been provided with a Regional set up in order to ensure smooth conduct of its recruitment examinations through a large network of examination centers situated in different parts of the country for the convenience of the candidate. The examinations are held as far as possible at different centers and successful candidates posted, to the extent possible, to their state/region. The interviews conducted by the Commission are structured in such a manner that they are effectively uniform and objective, keeping in view the diverse backgrounds of the candidates.
SSC Stenographer Final Result 2015


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For now, I'm putting my camera on the shelf. In other words, I'm going on a photography hiatus of sorts鈥攁聽sabbatical.聽
My relationship with photography began not too long after meeting my husband Brad. He actually introduced me to the camera and the idea of freezing time. Something supernatural would happen when I would pick up my camera and bring it to mine open eye. I would see things I wouldn't ordinarily see, things I felt only I could聽see. And when I would look at the film for the first time, what I felt was happening, did in fact happen. There was proof in the emulsion of silver on the film. And it was only I who originally saw it.
It was that duplication of what only mine eyes saw that lead me to pursue photography. I felt I had a perspective all my own and to share it with others was my life's purpose. Or so I thought. Until my my life was forever changed. My perspective changed. My eyes opened. No longer did I need my camera to see these things. My eyes alone could see what only my eyes and camera could see before. But now the camera was blocking my view.
My photography hiatus of sorts will involve using my eyes to experience life as it should be seen鈥攕eeing all of the potential and聽possibilities聽in others.聽No longer will I be a spectator but a participator. It was the good Lord who gifted me these eyes and聽the good Lord will sustain me.聽He has shown me that my perspective will continue to shift for the better, in only seeing the good, seeing all of the potential and聽possibilities聽in others, similar to the eyes He looks at us with. That shift in my perspective, in seeking only the light in others, will only聽strengthen my sight and bring me to take up the camera once again, refreshed.

subset: commoncrawl


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Click to Watch in HD > The Devil Wears Prada - Assistant To The Regional Manager [OFFICIAL VIDEO]
Watch The Devil Wears Pradas music video for Assistant To The Regional Manager from the album, With Roots Above And Branches Below - available now on Ferret Music. Download it at http://smarturl.it/withroots Site: http://tdwpband.com Store: http://tdwpstore.com Facebook: http://facebook.com/tdwp Twitter: http://twitter.com/tdwpband Instagram: http://instagram.com/tdwpband Tumblr: http://tdwpband.tumblr.com LYRICS These writings are to those who have wept. These writings are to those who havent looked deepley enough into the correct resolution. Wrong again but stronger now, we can face this. Fight life with life. All glory to the one in existence. Bring upon your name, your grace, your everything. Hold hands and stare into the circle. what are we looking at? Rumors and rumors and rumors. Assembly: Disintegration. Perish. Tombstones serve as mirrors and the graves are infinite. Take a look through the lens and through your eyes. End the partitioning. wrong again but stronger now, we can face this. fight life with life. Enemies, well go for your throat.
Youtube Channel / Ferret Music
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All the information you need to know about Waste Services in Wollondilly is listed here.
Customer Request
Wollondilly Shire Council takes Customer Requests from the community relating to requests, services and issues about Council facilities and infrastructure. A Customer Request can be made in person, by telephone or in writing. If you would like to lodge a Customer Request with Wollondilly Shire Council you can complete the...
Animals and Pets
Find out all relevant information relating to animals and pets in Wollondilly.
Emergency Information
Emergency Management is the term that covers the Planning & Preparation for, Response to, and Recovery from, incidents that have the potential to harm the lives and property of our community.
Council maintains cemeteries in the townships of Bargo, Picton and Thirlmere. All cemeteries cater for the internment of ashes and grave burials.
Information relating to your rate account can be found here.
Children's Services
Wollondilly Shire Council offers a number of children service's to people living in the Shire.
Council has created an interactive online mapping system for the public to access information about our local area.
New Residents
Wollondilly Shire Council extends a warm welcome to all new and future residents in the Shire.
Council Sportsgrounds
Council has a number of sportsgrounds in the Shire for the many sports and recreation activities available to the community.
Community Halls
Council has a number of halls across the Shire that are available for hire by the community.
Storm Recovery Information
A significant amount of business and residents, throughout Wollondilly, were severely effected by a storm event occurring on the 5th of June. Council is committed to providing the community with important information and support to those effected.


text: Montana Outing By mffc20145 Other Areas, Outings West Fork Madison West Fork Madison 7 Mile Yellowstone River Falls Group Photo at Lamar Elk in the River Yellowstone Madison River Madison River Madison River Yellowstone Yellowstone Three weeks of fishing in and around the Yellowstone Park area! The dates for the Montana Fly Fishing Trip are shown below. Week #1: Saturday, 29 July to Saturday 5 August Week #2: Saturday 5August to Saturday 12 August Week#3: Saturday 12 August to Saturday 19 August Price includes room and breakfast and lunch for six days of the visit Travel: Your arrangements Contact: Al Haxton – firstname.lastname@example.com


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Tell us how The Honest Kitchen has helped your pet and receive 100 rewards points!
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Welcome to True Stories!
True Stories testimonials are for informational purposes only. They are written by actual customers, and represent their own observations. Results are not guaranteed, are not medically substantiated, and may not be typical for other pets. The information is not a substitute for expert veterinary care. Please consult your veterinarian if your pet has health concerns.
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Please remember that True Stories content has been generated by customers in their own words, and may not represent the thoughts or opinions of The Honest Kitchen or its staff. The Honest Kitchen's products are not intended to diagnose, treat or cure any disease. Please consult your veterinarian if your pet has health concerns.


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login with
feeding baby
I found out its best to feed Andrew baby food then the bottle. Or he spits up some of the food he eats when he burps. he eats about a half a jar and then 4 oz of his bottle. He might be a little piggy lol
Comment deleted
Mango and peaches
That's what Lucas eats!!! Twice a day we do a 4oz bottle with half a jar of food...sometimes a whole jar lol. We both have little piggies!!! :D
lol thats for sure
Aw.. grow Andrew grow!!!
it is great that you figured out what works for your guy! Does he have any favorite foods...or snack foods yet?
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Louisiana's business community has won legislative battle after legislative battle during Gov. Bobby Jindal's seven-plus years in office.
But business lobbies have been on the defensive this year as lawmakers propose to plug Louisiana's massive $1.6 billion budget deficit by taking away hundreds of millions of dollars in tax breaks and subsidies given to companies over the years.
Republicans who traditionally have been the business lobby's biggest champions in the Capitol are sounding like Democrats this year.
"Corporate welfare" is Jindal's phrase to describe $526 million in tax rebates that businesses get from state government for paying 12 different taxes.
"We're giving away the ranch in tax breaks," said state Sen. Robert Adley, R-Benton. "We can't keep doing that."
The effort to trim businesses' tax breaks has pitted the business community against the state's public colleges and universities and health care providers, who lawmakers say face crippling budget cuts unless business starts paying more in taxes. Their argument is especially potent now because the Jindal administration and the Legislature have sharply cut state spending on higher education and hospitals, nursing homes and the developmentally disabled in recent years.
In trying to protect their tax breaks, the business lobbies, led by the Louisiana Association of Business and Industry, are not swayed by arguments that using the money to fund hospitals, universities and other programs would create thousands of jobs and benefit Louisiana's economy.
"The discussion now is to lower (tax) exemptions to fund large government," said Stephen Waguespack, who is LABI's president. "The focus has been on raising taxes on business."
LABI is also opposing a 10-cents-per-gallon increase in the gasoline tax that supporters say would help the economy by fixing roads and bridges whose deficiencies leave drivers stuck in traffic.
LABI instead is supporting two measures that would reduce business taxes. One would repeal the inventory tax that businesses pay to local governments, but it has been stalled by opposition from sheriffs, police juries, mayors and school boards.
A proposal to phase out the corporate franchise tax, House Bill 828, is alive and will be heard before the House Ways and Means Committee on Tuesday. The tax cost businesses $140 million last year, so eliminating it would offset some of the higher taxes that businesses seem likely to have to absorb.
A series of numbers explains why lawmakers are targeting business tax breaks this year.
One is the conclusion by legislative leaders that they need to raise $1 billion more in tax revenue, along with cutting about $600 million in spending through such measures as not letting state employees' paychecks keep pace with inflation.
Another is that the state gave away nearly $1.1 billion in taxpayer money in 2014 to subsidize six different business activities, as The Advocate reported last year. The incentives subsidize investment in blighted areas, the creation of jobs for low-income workers, producing movies, installing solar energy systems and drilling oil wells. Those same tax breaks cost much less, $200 million, in 2004, the newspaper found.
Two more key statistics: Tax collections for the corporate income and franchise taxes totaled $400 million last year, down from a peak of $1 billion in 2008.
And of the 87 corporations with the highest revenue in Louisiana in 2012, only 22 of them actually paid corporate income taxes in Louisiana, a study by the state Department of Revenue found.
Corporate tax breaks awarded by lawmakers over the years have exempted dozens of activities from taxation. To cite just three: Businesses don't have to pay 1 cent of the 4-cent sales tax when they pay utilities bills, credit unions are exempt from most taxes and certain milk producers get a tax credit.
"The state could have collected $1.97 billion in corporate income taxes in 2013," state Rep. Lance Harris, R-Alexandria, said just before the 2015 session began. "We collected $288 million, only 15 percent. That's why I'm going to look at some of these credits. The Republican delegation is very concerned about the health care and higher education cuts."
The Republicans in charge of the House showed their concern May 7 when they voted on 11 different measures that would raise the cigarette tax, scale back tax subsidies for filming movies and installing solar energy systems in Louisiana, and trim a variety of business tax breaks by 20 percent.
Appealing to his colleagues before the votes began, House Speaker Chuck Kleckley, R-Lake Charles, made a rare floor speech in which he said the members would be closing "corporate loopholes," an effort he called "long overdue."
"We are making tough votes for higher ed," he added, because 2015 is an election year.
Kleckley was followed by Rep. Joel Robideaux, R-Lafayette, chairman of the tax-writing Ways and Means Committee. Robideaux said he had sponsored some of the business tax breaks in past years, but now he appealed to his colleagues to put a higher tax burden on business.
"There's no greater incentive to businesses that are considering moving here than having an excellent higher ed system," Robideaux said, "having an excellent health care system and providing the infrastructure and roads we need to make this state grow the way it needs to grow."
With the support of the Republican leadership and most Democrats, the House passed bills that would raise $615 million in new revenue.
LABI's lobbyists sent in notes to lawmakers on the floor expressing their opposition to the five bills that would make business pay the most, to no avail.
Founded in 1975, LABI's mission, according to its website, is "to foster a climate for economic growth by championing the principles of the free enterprise system."
Following that principle could have led LABI to lobby to eliminate the tax subsidies for the film producers and for solar energy that cost taxpayers a total of $300 million last year. Both of those subsidies distort the free enterprise system, critics say. But the group took no position on those bills.
The business community's point man in playing defense has been Waguespack, who spends most of his days at the Capitol, plotting strategy with other business lobbyists, holding quick conversations with lawmakers and meeting with senior Jindal aides.
An affable 41-year-old lawyer from Gonzales, Waguespack was part of Jindal's inner circle, first as executive counsel and then as the governor's chief of staff. He left that job in 2012, spent a year working as a lobbyist for a law firm and then took LABI's top job 18 months ago.
Waguespack has a team of lobbyists, and LABI works in conjunction with a phalanx of other lobbyists from the Louisiana Chemical Association, the Louisiana Mid-Continent Oil and Gas Association (which represents major oil and companies), the Louisiana Oil and Gas Association (which represents smaller oil and gas companies) and numerous individual companies.
In two interviews defending the groups' anti-tax stance, Waguespack said legislators should be more focused on cutting state spending on pensions and so-called statutory dedications, by which nonprofit groups and local government entities automatically get state money without having to go through the appropriations process.
He also said colleges and universities would have more money if the Legislature allowed them to set their own tuition levels. Even if freed to raise tuition by, say, 10 percent, however, they would collect $70 million more, when the Legislature is threatening a $573 million cut in state money for them.
"There has been a lack of discussion about how to reform the budget," Waguespack said, "about pension reform and about the statutory dedications. Making these changes can make a big impact on the budget."
Waguespack rejected the notion that businesses are not paying their fair share, saying business tax collections have dropped because more and more companies are filing individual tax returns as limited liability companies or as S-Chapter corporations.
If so, why aren't individual income tax collections rising?
Because of tax cuts given by Jindal and the Legislature when they repealed tax increases that were part of the Stelly tax plan, Waguespack explained.
Jan Moller, who is director of the Louisiana Budget Project, a left-leaning nonprofit based in Baton Rouge, said it's time for businesses to pony up more.
"Legislators are asking only for a small haircut for a sector of the state that has not been asked to sacrifice one iota while colleges and health care providers and others have suffered deep cuts," Moller said.
F. King Alexander, LSU's chancellor and president, also seems to think businesses need to do more.
"In order to be a partner with the business community, you have to have a higher education system," he said in an interview Thursday. "Colleges and universities need to be funded adequately so we can meet the business and industry demands of us. I'm getting ready to graduate 9,100 students. Most of them are going into business. That relationship needs to be firm and consistent. You can't have one without the other."
Follow Tyler Bridges on Twitter, @TegBridges. For more coverage of the State Capitol, follow Louisiana Politics at http://blogs.theadvocate.com/politicsblog/.


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I've been working on new site that's still under serious development. I post it here in case any translator might be intereted in checking it out:
New site
A discussion of the peculiarities of languages and the differences between them.
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The elements may be familiar ones, but the finished product is something relatively distinctive. That's the feeling I always have when listening to an Ahmet Sisman production. Over the last eighteen months or so Sisman has become one of the more vital producers of deep, hedonistic, funk-fuelled house music. Think Hot Natured after a heavy session of magic mushrooms crossed with the tribal techy sound that emerged out of California at the turn of the century on labels such as Siesta.
'Playing With Acid' is a deep, squelchy stomp of a track that motors along to the accompaniment of ghostly disembodied voices that rise and fall in synthesised wails, it's got funk in spades and it defies you to keep still. It's obvious tendencies towards eighties black synth soul are very much in vogue at the moment and they align it with the aforementioned Hot Natured crew, as well as other kindred spirits like Soul Clap and Wolf and Lamb.
'Feel Today Think Tomorrow' makes similar use of deep-in-the-mix vocals, but even though there's a similar tempo to the track, the musical elements are slightly different and serve to emphasise a guitar lick that is expertly used to create a euphoric window that, on the one hand had me scratching my head, but on the other grinning like an idiot.
'No Return No Regret' is a bittersweet, bordering on melancholic, track that again uses a subdued cut and spliced vocal to devastating effect, along with a harpsichord-like break that evokes, amongst other things, the theme tune to "The Persuaders." After that it plunges into the darkness once more.
What I like about this record is that even though the three tracks present are all made up of the same principal parts: driving beat, disembodied vocals, an individual flourish or two on top; they each evoke so much and work on so many different levels. Ahmet Sisman has hit upon a winning formula, the time of which has most certainly come.
|Artist: Ahmet Sisman
Title: Playing With Acid EP
Label: Slash
Our Rating: 8/10


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A LONE female was left shaken and upset after a man made an obscene gesture.
Police are searching for the suspect following the incident at about 11.30am on Friday on Lightship Way, The Hythe, Colchester.
A spokeman for Essex Police said: "The victim, who is in her 20s and lives in the town, described the man as white, aged approximately 30-years-old, of stocky build, around 5ft 10ins tall, with a stubbly beard, brown eyes and straight brown hair.
"He was wearing a navy blue fleeced top and a pair of dark trousers.
"The victim was left shaken after the incident."
Anybody with any information is asked to contact PC Andy Gudgin at Colchester police station on 101 or Crimestoppers anonymously on 0800 555 111.


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If you signed up at a hotel or by phone, activate your SPG account.
We've got the perfect place to get together with friends or co-workers and enjoy a great meal.
Fueled by passion, our Sheraton Park Grill is a great place to connect with your family, team, or clients. We use the freshest ingredients, which are innovatively prepared and offer great variety.


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The wonderful advice in this forum led me to FindNFix, CWShredder, and wonderful people and extraordinary talent and information. The people who perpetrate this selfish, thoughless, sick activity have no power compared to the wonderful, bright, caring people who HELP instead of hinder and damage for small, selfish motives.
I KNOW I am misusing this forum to express these thanks but I am overcome with gratitude and awe about this phenomenal experiment in cooperation. G-d bless all who cooperate and help. To those who stoop to low levels for a few ill-gotten bucks - you don't know what you're missing. Kindness and true accomplishment are better that small, devious gains.
I know I shouldn't but ... I want to post THANKS!
Started by Cardinal23, Jun 28 2004 11:12 PM
1 reply to this topic
Posted 28 June 2004 - 11:12 PM
Posted 28 June 2004 - 11:13 PM
Yep these people are awesome
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learn more
SaferKid alerts you if your child downloads any of 200,000 apps of concern
Get alerts when SaferKid detects:
Ways to secretly meet strangers
Sexting Risks
Age-Inappropriate Violence
Bullying Risks
Get What You Need to Parent
Our app rating system was developed after extensive research into what the real app risks are, and what you need to know.
learn more
Our reviews include possible developmental risks, links to parent's guides and tips on how to talk to your children about the apps.Sign Up
What We Do
We scour apps looking for dangerous risks that parents would want to know about. We already have 200,000 apps in our database, and like an anti-virus company, we are always learning as systems out there change and as new apps are released.
As we keep an eye out for new threats, we also monitor what's on your child's device and let you know if we find something. If we do, we send you the details of the app and why it's a concern, including parent's guides with developmental information you may need for the situation.
What We Don't Do
We don't compromise the trust between you and your child. As a result, we don't spy on what they do inside of apps.
We also can't provide 100% protection for every child. Nothing can. But we do cover over 200,000 apps, and we add more every week. So, we're pretty far ahead of the game :)
What You Do
At the right moment in time, your job is to parent. You check the alert, read the quick summary and info, and then sit down and have a talk with your child.
You also need to set up SaferKid on your child's device. It's very easy, and we have step-by-step instructions to help you through it.
Protection Is a 3 Step Process
Enter each child's name and birthdate for age-specific coverage.
Follow the set-up guide to connect any iOS or Android device. This enables remote monitoring.
If we find a risky app, we'll send an alert with the info you need to understand the concern and how to step in and parent.


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Kenwood TK-880
From InterceptRadio.com Wiki
Kenwood TK-880 / TK880 information
TK880 450-490mhz model
Out of the box, the stock radio bottoms out TX/RX at 443 mhz.
The radio is capable of going down to about 436 mhz.
Here is how to do it:
Program radio for 441, 442, and 443 mhz.
At 442mhz: Radio shouldn't receive. TX should unlock PLL (radio beeps after mic keyed for about 2 seconds).
To fix: Open radio Locate two holes (pll adjustment) The bottom hole is TC109 (towards front of radio). Go 1/4 turn ccw (brings tx down to 440mhz) The lowest the TX VCO will go is 434.5. TX range will be 434.5 to 470
The Middle hole is TC106. Go 1/4 turn cw (brings rx down to 440mhz) Lowest RX VCO will go is 436.0. RX range will be 436.0 to 470
Use KPG49D programming software and set "Sensitivity" to 60 (low), 135 (medium), 200 (high) These settings control the varactor diodes in the front end preselector.
Turn the L203 and L207 slugs fully clockwise. Further adjust by feeding 400-500mhz into the antenna with a tracking generator and put a spectrum analyzer on TP2.
Other info: The top hole is VR1 (tx power) The right hole (under speaker) is L5 or L6 (discriminator?)
The 4 inductors on left (top to bottom) are: L203(top), L203(bottom), L207(top), L207(bottom)
The receiver circuit goes from antenna thru L203, thru an RF Amp transistor,
then thru L207.


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Trend Setters: Preppy Pets Rock . . . Tennis Chic
It's been a while since I've gave you a preppy update, but here I am with some tennis-inspired dog gear to celebrate the US Open.
If your dog's ready to play a game of fetch (and not a fan of dressing up), why not sub a simple prop like a collar or accessory? I especially love these visors – I thought I found every possible hat back on National Hat Day, but guess not . . . it's totally tennis prep for pups!


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Please upload a photo that has a resolution of at least 300 dpi at its print size and is in one of the following file formats: ai. The file must not exceed 10MB. Beau-coup will not crop or retouch your photo and is not responsible for the quality of uploaded files. By uploading an image, you agree to our terms and conditions.
1. Customer is responsible for owning the copyright or having permission from the copyright holder for all images uploaded to this website. Customer agrees to hold Beau-coup harmless from any claim arising out of the use of copyrighted material.
2. Beau-coup may choose not to publish any images found to be offensive.
Add a festive touch to your holiday soiree with these winter snowglobe place card holders. These adorable little snowglobes do double-duty as party supplies as well as favors. Each measures 2.75"L x 2"H and is available with add-on personalized clear labels (2 lines of text; up to 14 characters on the first line and 16 characters on the second).
Add more holiday party supplies with our mini ornament wine charms and laser carved snowflake tea light holders.
Processing time is the time from when the order is placed to when it leaves our warehouse. This varies by product.
Shipping time is the time from when the package leaves our warehouse to when it arrives at your door. This varies by shipping method.
In a rush? See items that ship same day or next business day. Click here.
Shipping internationally? Read our International Orders FAQ for information on custom duties, export restrictions and more.
* Shipping options and estimated delivery dates apply to orders shipping within the 48 contiguous United States only.
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Wrist Coil with Trigger Snap, Blue, PK5
LUCKY LINE PRODUCTS| Zoro #: G0374561| Mfr #: 4133005
Zoro #: G0374561Mfr. Part #: 4133005
$6.81 /pk
Orders over $50.00 ship free. DetailsStandard ground shipping
Technical Specifications
|Zoro #: G0374561 Mfr #: 4133005
|Includes: Split Key Ring
|Item: Wrist Coil
|Type: Wrist Coil w/Trigger Snap
|Material: Polyurethane
|Color: Blue
|Overall Length (In.): 4
|Ring Dia. (In.): 7/8
|Country of Origin (subject to change): Taiwan
Product Description
Wrist Coil, Mini Designer Coil, Blue, Ring Dia. 7/8 In., Overall Length 4 In, Polyurethane, Includes Split Key Ring, Package Quantity 5


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
BillingAsked by dfdoughe on February 23, 2017 at 11:54 AM
We recently upgraded to the $19.00 a month charge but have come to realize that the credit card on the account is a former employee's personal one. I have upgraded our account to have our business credit card listed. It is showing though that the $19.00 was already charged to the old credit card. Is there any way we can get that amount refunded to the old card and charged to the new business card that is listed on the account now. If you can email me back at firstname.lastname@example.com or I can be reached at 610-478-6920 ext. 3924 in regards to this matter. Thanks!
JotForm Support Manager
I have refunded the latest payment of $19.00, please logout and login back again. Then re-upgrade the account with a new card.


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|Re: GroovyMarkup syntax works!
|Thu, 15 Apr 2004 16:20:49 GMT
On 15 Apr 2004, at 17:16, Bertrand Delacretaz wrote: > Le 15 avr. 04, à 18:01, email@example.com a écrit : > >> ...BTW you could just create the Sql object with a DataSource which >> is-a JDBC connection pool, then the Sql object takes care of all the >> pooling for you. Is there support for DataSource in Cocoon or some >> kinda adapter between DataSource and ConnectionProvider?... > > Dunno about javax.sql.DataSource support, I'll have to check. > > ConnectionProvider does not exist yet, I just made it up with the > following idea: Ah OK :) > public class ConnectionProvider { > // Groovy scripts which need a Connection will use this to get > // it from the Cocoon pool > public Connection getConnection(String connectionName); > > // then, once the Groovy script has been executed, ScriptGenerator > // calls this to return all connections provided by getConnection to > the pool > public void releaseConnections() > } > > In this way, the Groovy script can access any Connections that are > configured, and does not have to care about releasing them. It's easy > to implement in case we don't have DataSource support. You might as well just use DataSource then as this is pretty close to all DataSource is. (Or at the least get ConnectionProvider to implement DataSource) There's an implementation in Jakarta Commons dbcp and various JDBC drivers come with an implementation of DataSource for you already (e.g. Oracle & Axion). If nothing else just provide a DataSource facade to whatever connection pool you use and the groovy.sql.Sql will work fine with your pool. James ------- http://radio.weblogs.com/0112098/
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This kid wins KC a Superbowl
Join Date: May 2006
Location: Springfield
Casino cash: $64486
May be going to jail for awhile
Actually just got out yesterday morning after spending a night but depending on the judge's mood I could go back for a long time.
I put my ex-wife's boyfriend in the hospital Thursday evening for slapping my 11 year old daughter across the face. She called me crying about 7PM saying he slapped her for telling her mom to shut up. She also informed me he spanked my 6 year old son about a month before that but didnt tell me because her mom made her promise she wouldnt. I went over there and confronted him about it. He denied it, then my daughter came outside and I could still see the hand print on her face. The whole thing got bad quick. I used no restraint and by the time my ex's neighbor pulled me off of him he was coughing up blood. My lawyer informed me that he had a collapsed lung, broken nose, broken orbital bone near his left eye, broken collar bone, and a ruptured spleen. I only hope the judge has a daughter and can be reasonable and understand my side. I go back to court March 8th.
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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
Gardener / Landscape gardeners
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have a corner of the garden, about 12 x 12 feet, to lay patio and then erect a timber gazebo...
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Someone to dig up old posts and replace posts and 5 lap panels
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Services required: replace / install a fence Approximate length of fence: 10m-20m Type of...
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Services required: decking, laying turf, paving Approximate length of garden: 10m to 20m /...
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Very honest, our fence had been blown over, David suggested repairing the panels rather than replacing with new. Would highly recommend. Chelmsford May 2015.
Excellent throughout. Clear quote well presented, open always to questions. Work carried out on time, and as required. Would not hesitate in recommeding, one of the best tradesman I have dealt with
Jason and his team were absolutely brilliant at transforming our field into a beautiful garden. They were extremely professional at all times and their work was done to an extremely high standard. Nothing was too much trouble. Would certainly recommend their services.
Good communication. Supplied list of materials required. arrived on time and delivered perfect work within the quote.
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additional fees
Can someone tell me the other fees besides the ffl fee that are in florida or atleast help me with finding somewhere that tells me thank u in advance.
OK- your communciation skills need work. FEES FOR WHAT?:confused:
Now, making an assumption that you need a FFL to recieve a firearm for you- go to gunbroker.com. On their home page left sides, under the green bar that says "areas of interest" is a "find a FFL" function. Click on that, enter your zip code, and it will pull up ffls in your area that have rgistered as being willing to act as a transfer dealer. Most have what they charge listed.
Ok I already know there will be an ffl fee that not what I asked I'm just asking in general if there are other fees I will need to pay (not including tax)
Usually when you buy a weapon from Gun Broker or a dealer out of state. You * pay them for the weapon and *shipping costs. You will need to find an FFL source in your state and area to receive it for you. The Companies or Dealers must exchange FFLs first. Then when the weapon arrives at your designated dealer's location and you go to pick it up. The dealer who received it for you will do the transfer usually *charging you $25-$50 for the transfer. You fill out the Federal Form and answer the questions and they will do your records check and you are on your way. Unless, there is a waiting period in your area. If so you would need to return after that period and pick the weapon up. But there are no additional charges or cost that I know of! :)
Sometimes the FFL will charge a NICS fee (that's your background check) but if varies from FFL to FFL. Usually your pawnshops, home based dealers will be lower. The FFL holders of brick and motar gun shops want you to buy there and some can charge upwards of $200(just seen a dealer on the fflonly forum do that) so it varies to say the least.
Thanks and yea in florida we have a cool down period of only 3 days
Okay, here goes....
You want me to order a firearm that my supplier has listed for $300.
In my county, add 7.5% sales tax on the firearm ($300 + 22.50 = 322.50)
My supplier charges me for shipping (2nd day) and for the fun of it, say it costs $45.00 ($300 + $45 = $345.00)
My supplier charges me an additional 3% for credit card ($345 + $10.35 = $355.35)
My transfer fee is $20.00 for NICS check and other expenses I have.
So, let's add up
$300.00 + $22.50 + $45.00 + $10.35 + $20.00 = $397.85
Now because I'm doing you a favor and charging only 5% ($19.89) on the deal, add that to the 397.85, your total cost would be $417.74
I have to make 10 such sales a day to make it as a firearms sales shop only.
Thanks now I know a ball park of what I'm going to pay
If you noticed, by going rock bottom on my prices, I am not making a good living.
If I had to rely on sales only, I would be closing shop. This would make me no longer available to you, and you would have to deal with other dealers who charge more.
By supporting the local LGS, the money spent stays in the local economy, you have a source who is willing to help you, and he/she can support his/her family.
Always put yourself into the local LGS shoes. Consider how much you would have to get from the deal to make it worth your while. (like a waiter in a local restaurant, the local LGS knows who the better customers are.)
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Aliso Viejo is the first city in California to have an outlet of a Denver-based Mediterranean restaurant that hopes to expand nationwide.
Garbanzo Mediterranean Grill is a choose-your-own-ingredients establishment similar in a way to Chipotle Mexican Grill. The difference is that Garbanzo's three main choices are stuffed pita, a wrap called a laffa, and a plate of ingredients with pita on the side. It also offers kabobs and salads.
"It really is create your own, right on the spot," said Bill Gunstream, president and chief executive of Banzo Bean Inc., a franchisee of Garbanzo Mediterranean Grill. "Mediterranean food is something people can really enjoy by selecting the food they like."
Gunstream, a 30-year veteran of family dining, worked with breakfast giants such as IHOP and Village Inn before beginning the Garbanzo location in Aliso Viejo as a franchise. Gunstream hopes to open 10 locations in Orange County in the next four to five years with business partner David Clouse, Banzo Bean's vice president.
"I get to try my experience as a pancake guy, trading in pancakes, butter and syrup for hummus, vegetable salad and shawarma," Gunstream said.
Gunstream and Clouse have lived in Denver for years, but both grew up in California. They expressed excitement about the market.
"We just felt like Southern California was ideal," Gunstream said. "We love this concept. We think this concept will be so exciting in California because people know Mediterranean food of sorts."
Clouse, who has a background in online business, approached Gunstream with the concept and together they combined their skills to get things rolling.
"Bill is definitely the restaurateur," Clouse said. "I am not in the restaurant business, but I am an entrepreneur.
"We're really glad to be in Aliso Viejo and are looking forward to meeting a lot of people who like Mediterranean food or introducing people to Mediterranean food who haven't had it before," Clouse said. "I think they'll really like it."
For Gunstream, the style of restaurant might be different from what he's used to, but his goals are the same.
"Taking care of the guest is all the same," he said. "It's all about creating a pleasant environment, providing customer service and high-quality products and doing it in a timely manner. … That's what I stress most of all."
Contact the writer: 949-366-9473 or firstname.lastname@example.com


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Pierre Gimonnet has long been considered a benchmark for quality in Chardonnay-based wines in Champagne. An elegant Champagne, with layers of flavour including apple sauce, honey, smoke, toast and quince. Shows well-integrated acidity that provides a subtle juiciness throughout and brings you back for another sip.
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Austin and Phillip become the real cum sluts in this massive cock sucking party! The boys are hanging out at the pool when dicks start to throb, and before you know it they're sharing an 8-guy cock pleasuring session with so much meat to stroke and suck! Austin and Phillip get splashed with hot juices from all their horny friends before sucking the loads out of each other to finish things off! We would all love to be there to join in
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Dynamic Running Clinics - Intermediate Runners' Clinics
Cairns, Queensland or online
Tue 01/28/2014
These clinics are incorporated into our regular Tuesday night session where we alternate between speed work, fartlek or hill running. We do a 10-15minute warm up and then the regular runners will start session. The clinic participants are provided with instructions on why and how to do speed work, and how to run up (and down hills). We also talk about energy systems and how it is linked with running. Participants then join in with the other runners to complete the session. Week 1: Speed work Week 2: Hill running Week 3: Fartlek Dates for 2014 Wet Season sessions Tuesdays, 5.30-6.00pm 7th, 14th and 21st January 28th January, 4th and 11th February 18th, 25th February and 4th March 18th, 25th March and 1st April 29th April, 6th and 13th May For your payment you receive ? one of the 15 spots available ? three ?-hour group sessions on how to run better and faster ? a booklet with notes on interval training, running tips and advice to keep you motivated ? on-going support at the Tuesday night sessions *Three consecutive Tuesday nights from 5.30-6.00pm. Conditions of entry You must be signed up as a Dynamic Running participant. You need to already comfortably run 5km or more on a weekly basis. It is recommended that you also complete the Technique sessions held on Monday nights, as there is no time for technique training on Tuesday nights. It is also recommended that you complete the Beginners2Intermediate Clinics on Thursday nights to build up your speed and distance before signing up for these Intermediate Runners? Clinics. A minimum of 4 participants are required for the Clinics to go ahead. No refunds once the clinics have started.
- Email:
- Phone:0417 798 444 or 07 4
- Address:Greenslopes Street Edge Hill
Cairns, queensland 4870


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Appropriate development of regulatory T cells (Tregs) is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg development. It is unclear what these missing factors are. However, signals emanating from the TCR, CD28 and γc-dependent cytokine receptors are required for Treg development. Herein we demonstrate that expression of a constitutively-active STAT5b transgene (STAT5b-CA) allows for Treg development in neonatal mice and restores Treg numbers in CD28−/− mice. Sequence analysis of TCR genes in STAT5b-CA Tregs indicates that ectopic STAT5 activation results in a TCR repertoire that more closely resembles that of naïve T cells. Using MHCII tetramers to identify antigen-specific T cells, we demonstrate that STAT5 signals divert thymocytes normally destined to become naïve T cells into the Treg lineage. Our data support a two-step model of Treg differentiation in which TCR/CD28 signals induce cytokine responsiveness; STAT5-inducing cytokines then complete the program of Treg differentiation.
Autoimmune disease is a consequence of the generation of self-reactive T cells. While most self-reactive T cells are deleted in the thymus via negative selection, some self-reactive T cells escape this process. Therefore, a second mechanism involving the generation of CD4+ regulatory T cells (Tregs) in the thymus has evolved to prevent autoimmunity (Shevach, 2000). Although several distinct types of T cells capable of exerting suppressive activity have now been described, the most well characterized are CD4+ T cells that express the transcription factor Foxp3 (referred to hereafter as Foxp3+ Tregs). Signals required for Foxp3+ Treg differentiation have been shown to include high affinity interactions between the T cell receptor (TCR) and major histocompatability class II (MHC II):peptide complexes (Bensinger et al., 2001; Aschenbrenner et al., 2007). For example, several mouse models have been developed in which both a specific TCR and its respective antigen are co-expressed as transgenes. A large percentage of T cells that develop in those mice were found to be Foxp3+ Tregs that exhibit suppressor activity (Jordan et al., 2001; Apostolou et al., 2002; Aschenbrenner et al., 2007). Likewise, work from three different groups has demonstrated that the TCR repertoire of Foxp3+ Tregs overlaps extensively with that of self-reactive T cells; in contrast, only limited overlap was seen with naïve peripheral T cells (Hsieh et al., 2006; Pacholczyk et al., 2006; Wong et al., 2007). Other signals required for Foxp3+ Treg development include CD28:B7 costimulatory signals (Tang et al., 2003; Tai et al., 2005). This has been attributed, in part, to a role for CD28 in generating IL2 required for Foxp3+ Treg homeostasis. However, CD28 appears to play an additional role in Foxp3+ Treg development that has not yet been defined (Tai et al., 2005). Taken together, these studies have led to the conclusion that effective Foxp3+ Treg differentiation requires both relatively high affinity TCR interactions with MHCII:peptide complexes as well as CD28:B7 costimulatory signals.
In contrast to the clearly accepted importance of TCR- and CD28-dependent signals for Foxp3+ Treg differentiation, the role of IL2 in this process has been quite controversial. Early studies by Malek and colleagues using IL2Rβ transgenic mice supported a role for IL2Rβ-dependent signals in Foxp3+ Treg development in the thymus (Malek et al., 2000; Malek et al., 2002). In contrast, studies by Lafaille and colleagues demonstrated that transfer of CD4+ T cells from IL2−/− mice could prevent autoimmune disease in a mouse model that normally develops spontaneous experimental autoimmune encephalomyelitis (EAE)(Furtado et al., 2002). Transfer of IL2Rα−/− CD4+ T cells did not prevent EAE in this model, suggesting a requirement for IL2 to support Foxp3+ Treg suppressive capacity but not for Foxp3+ Treg development. Additional studies from the Rudensky and Klein labs independently demonstrated that young IL2−/− mice have relatively normal numbers of thymic and peripheral Foxp3+ T cells. These latter findings seemed to convincingly rule out a requirement for IL2 in Foxp3+ Treg differentiation (D'Cruz and Klein, 2005; Fontenot et al., 2005b). However, unlike Foxp3+ Treg development in IL2−/− or IL2Rα −/−mice, we have recently found that Foxp3+ Treg development in IL2Rβ −/− and IL2−/− x IL15−/− mice is dramatically impaired (Burchill et al., 2007). Similar findings were reported by Ziegler and colleagues (Soper et al., 2007). IL2 appears to play the primary role in this process based on competitive mixed bone marrow chimera studies using IL2Rα+/+ and IL2Rα −/− donors; in those studies 5-fold more Foxp3+ Tregs in the resulting chimeric mice were derived from IL2Rα+/+ as opposed to IL2Rα −/− donor cells (Fontenot et al., 2005b). Thus, IL2Rβ-dependent signals play a key role in effective Foxp3+ Treg development.
Several pieces of evidence indicate that IL2 (and IL15) entrain Foxp3+ Treg differentiation via activation of STAT5. For example, transgenic mice expressing a constitutively active form of STAT5 (called STAT5b-CA) have a significant increase in the percentage and total number of Foxp3+ Tregs in both the thymus and spleen (Burchill et al., 2003). Furthermore, crossing the STAT5b-CA transgene onto the IL2Rβ −/− background restores Foxp3+ Treg development in the thymus. Likewise, IL2Rβ mutants that have been engineered to signal exclusively through STAT5 also restore Foxp3+ Tregs (Burchill et al., 2007). Conversely, mice lacking STAT5 in developing thymocytes fail to generate Foxp3+ Tregs (Burchill et al., 2007; Yao et al., 2007). Thus while multiple cytokines can induce Foxp3+ Treg differentiation, they all do so via STAT5 activation.
Although we now have some appreciation of the signals required to induce Foxp3+ Treg differentiation, the process this still remains somewhat enigmatic. Work from the Rudensky and Chatila labs has demonstrated that mice lacking functional Foxp3 exhibit precursors that can induce transcription of a GFP cDNA knocked into the foxp3 gene locus. These cells exhibit many other hallmarks of Foxp3+ Tregs including increased CD25 expression, but do not exhibit suppressor activity or prevent autoimmune disease in vivo (Gavin et al., 2007; Lin et al., 2007). Likewise, Fontenot et al reported that neonatal mice, which lack Foxp3+ Tregs, do express a population of CD4+CD25+Foxp3− thymocytes (Fontenot et al., 2005a). It was unclear at that time what these cells were, although it was speculated that they might be Foxp3+ Treg progenitors that failed to complete differentiation due to the absence of as yet uncharacterized factors. Thus, a key question that remains is whether high-affinity TCR/CD28 signals induce an autonomous program that leads to Foxp3+ Treg differentiation or whether this process occurs via multiple steps and requires the influence of other factors such as cytokines. Herein, we present data supporting a model of Foxp3+ Treg lineage commitment in which high affinity TCR/CD28 signaling leads to the generation of thymocytes with enhanced cytokine responsiveness, and that a second step, involving cytokine/STAT5-dependent signals completes the process of Foxp3+ Treg differentiation. Moreover, this linkage of TCR/CD28- and cytokine/STAT5-dependent signals plays an important role in shaping the TCR repertoire of Foxp3+ regulatory T cells.
To test potential cooperation between TCR and IL2R signaling pathways in promoting Foxp3+ Treg development, we utilized a transgenic mouse model in which a mutant form of the transcription factor STAT5 (referred to herein as STAT5b-CA) is expressed throughout lymphocyte development (Burchill et al., 2003). Although the STAT5b-CA construct we used is typically referred to as constitutively active, it still requires phosphorylation on Tyr-699 for activation (Onishi et al., 1998). In the absence of exogenous cytokines, the STAT5b-CA transgene results in low levels of constitutive STAT5 activity ex vivo. Phosphorylation of STAT5b-CA is further increased following exposure to STAT5 activating cytokines; thus, the transgene is best described as a weakly constitutively active, but hyperactivateable form of STAT5b ((Will et al., 2006) and Fig. 5C). Using these mice, we examined whether STAT5-dependent signals could induce Foxp3+ Treg development in one day old neonatal mice. Previous studies have demonstrated that such mice are essentially devoid of CD4+Foxp3+ Tregs (Fontenot et al., 2005a). We reasoned therefore that if STAT5 signals are sufficient to drive Foxp3+ Treg development, then we should see Foxp3+ Tregs in neonatal mice which normally lack such cells. Consistent with previous findings, we observed that newly generated CD4+HSAhiFoxp3+ Tregs are virtually absent in the thymus of LMC mice; in contrast, we observed a significant population of CD4+HSAhi Foxp3+ thymocytes in one day old STAT5b-CA mice (Fig. 1). Thus, STAT5b-CA expression promotes the development of Foxp3+ Tregs under conditions which are normally not permissive for the differentiation of such cells.
Although STAT5b-CA expression clearly induces Foxp3+ Treg differentiation in neonatal mice, it is unclear what upstream factors it is compensating for. Previous studies have demonstrated that neonatal dendritic cells express much lower levels of the costimulatory ligands B7-1 and B7-2; furthermore, these cells are quite poor at inducing immune responses (Muthukkumar et al., 2000). Thus, one possibility is that neonatal mice lack sufficient expression of B7-1/B7-2 on thymic antigen presenting cells to induce Foxp3+ Treg differentiation. An alternative possibility is that neonatal mice may lack γc-dependent cytokines (most likely IL2) that are required for Foxp3+ Treg differentiation. To test these two possibilities, we introduced the STAT5b-CA transgene onto CD28−/− and γc−/− backgrounds to examine whether constitutive STAT5 signals can drive Foxp3+ Treg development in the absence of costimulatory- or γc-cytokine-dependent signals. As illustrated in figure 2A, the STAT5b-CA transgene restored Foxp3+ Treg numbers in the absence of γc indicating that STAT5 activation can restore Foxp3+ Treg development in the absence of all γc-dependent cytokines. Importantly, the Foxp3+ Tregs in STAT5b-CA x γc−/− mice are functional suppressor cells. Specifically, in γc−/− mice, which lack Foxp3+ Tregs, the small number of T cells that remain show an activated phenotype as determined by upregulated CD69 and downregulated CD62L expression. This phenomena is reversed in STAT5b-CA x γc−/− mice (Sup. Fig. 1). Moreover, T cell numbers are restored in STAT5b-CA x γc−/− mice, and the mice remain healthy up to at least 9 months of age, suggesting that they have a functional Foxp3+ Treg population. More surprisingly, we found that thymic Foxp3+ Treg numbers were also restored in STAT5b-CA x CD28−/− mice (Fig. 2B,C). Foxp3+ thymocytes from STAT5b-CA x CD28−/− mice retained high expression of additional markers indicative of a Foxp3+ Treg phenotype such as CD25 and GITR suggesting that we had restored a normal population of Foxp3+ regulatory T cells (Fig. 2D). A potential caveat to the above rescue experiments is that it is difficult to determine whether these findings result from induced Foxp3+ Treg differentiation or just enhanced proliferation/survival of the few Tregs that emerge in CD28−/− mice. Nevertheless, these data provide evidence that CD28 signals may be upstream of STAT5 activation and suggest the possibility that CD28 signals induce cytokine responsiveness during Foxp3+ Treg differentiation.
We next wanted to determine whether STAT5 activation could restore Foxp3+ Treg development in the absence of high-affinity TCR signals. MHCII−/− mice are defective in positive selection of all CD4+ thymic T cells. Thus, crossing STAT5b-CA mice with MHCII−/− mice is likely to be uninformative. In addition, we wished to be able to unambiguously differentiate between STAT5 effects on post-selection Foxp3+ Treg expansion versus pre-selection induction of Foxp3+ Treg development. To circumvent these issues, we examined whether the repertoire of TCRs expressed in the Foxp3+ Treg pool was perturbed in STAT5b-CA mice. Specifically, recent studies have demonstrated that both thymic and peripheral Foxp3+ T cell populations have diverse TCR repertoires that exhibit very limited overlap with the TCR repertoire of CD4+Foxp3− naïve T cells (Hsieh et al., 2004; Hsieh et al., 2006; Pacholczyk et al., 2006). To test if constitutive activation of STAT5 alters the TCR repertoire of Foxp3+ Tregs we utilized a novel strategy (Moon et al., 2007) employing tetramers of the MHC class II molecule, I-Ab, covalently bound to the 2W1S variant of peptide 52–68 of the MHC class II I-Eα molecule (Crawford et al., 1998; Rees et al., 1999) (referred to hereafter as the 2W1S-tetramer). We used this 2W1S-tetramer in conjunction with magnetic bead enrichment to isolate all 2W1S-specific T cells in the spleen and lymph nodes of a single mouse (Sup. Fig. 2). Enriched cells were subsequently stained with a panel of additional markers to characterize the 2W1S-tetramer specific population (Sup. Fig. 3). Staining was specific as the 2W1S-tetramer-positive population is not observed in mice restricted to a single TCR specificity such as TEa TCR Tg x Rag−/− (Grubin et al., 1997) or SM1 TCR Tg x Rag−/− mice (McSorley et al., 2002)(Sup. Fig. 3). As expected, virtually all 2W1S-tetramer specific CD4+ T cells are naïve (CD44lo) in mice that have not been previously exposed to the 2W1S peptide (Sup. Fig. 3). Importantly, we found that CD4+2W1S+ T cells from B6 mice are almost entirely CD25− and Foxp3− (Fig 3A), demonstrating that this TCR specificity is strikingly underrepresented in the Treg pool. In contrast, approximately 46 ± 9.2 % of 2W1S-tetramer-specific T cells from STAT5b-CA mice are Foxp3+ (Fig 3A,B). Furthermore, the percentage of 2W1S-specific Foxp3+ T cells is not significantly different (p=0.24) than the average distribution of all other TCR specificities in the Foxp3+ Treg lineage (64.2% ± 5.9%) (Fig 3B). These results demonstrate that constitutive activation of STAT5 diverts 2W1S-tetramer-specific cells that would normally develop into naïve CD4+ T cells into the Foxp3+ Treg lineage. Thus, the presence of the STAT5b-CA transgene alters the TCR selection bias that typically characterizes Foxp3+ Treg differentiation.
Although the above findings show that expression of the STAT5b-CA transgene results in selection of a large fraction of 2W1S-tetramer specific T cells into the Foxp3+ Treg lineage, the conclusions drawn from these experiments are limited due to the fact that only one TCR specificity was assayed. Therefore, we examined the ability of the STAT5b-CA transgene to divert T cell progenitors into the Foxp3+ Treg lineage by analyzing the TCR sequence diversity in Foxp3+ and Foxp3− populations from STAT5b-CA and LMC mice. To address this issue, we made use of mice expressing both a TCRβ transgene and a Foxp3-GFP reporter (Hsieh et al., 2006); the TCRβ transgene fixes TCRβ chain diversity while the Foxp3-GFP reporter allows us to specifically isolate Foxp3+ Tregs. Previous studies have documented that Foxp3+ Tregs in the thymus and spleen show considerable overlap in TCR gene usage; in contrast, there is little overlap in TCR gene usage between naïve T cells and Foxp3+ Tregs in either the spleen or thymus (Hsieh et al., 2004). To examine the effect of ectopic STAT5 activation on the Foxp3+ Treg TCR repertoire, we sequenced over 1000 productively rearranged TCR Vα2 genes from STAT5b-CA and LMC CD4+Foxp3+ and CD4+Foxp3− thymocytes. Consistent with previous reports, we found that TCR Vα2 gene usage in CD4+Foxp3+ thymocytes from LMC mice is different from that observed in CD4+Foxp3− naïve T cells. Specifically, we found that only 5% of unique TCR Vα2 sequences in the CD4+Foxp3− thymocytes from LMC mice or STAT5b-CA mice were also present in the CD4+Foxp3+ Tregs from LMC mice (Fig. 4A, black bars). In contrast, a much greater percentage of the unique TCR Vα2 rearrangements in LMC and STAT5b-CA non-Tregs were found in CD4+Foxp3+ thymocytes from STAT5b-CA mice (15% and 20%, respectively)(Fig. 4A, gray bars). We also examined skewing of the Foxp3+ Treg TCR repertoire by comparing the relative frequency at which specific TCR Vα2 sequences could be found in a larger database of TCR sequences obtained from splenic Foxp3+ Tregs, Foxp3−CD44lo naïve T cells, and Foxp3−CD44hi memory or effector T cells (Hsieh et al., 2006). To analyze these results we plotted individual TCR sequences obtained from CD4+Foxp3+ thymocytes on the x-axis and the relative frequency at which this particular sequence could be found in wild type splenic CD4+Foxp3+ T cells (green), CD4+Foxp3−CD44lo naïve T cells (blue), and CD4+Foxp3−CD44hi memory or effector T cells (pink) on the y-axis (Fig. 4B). This analysis demonstrated an increase in the number of TCR Vα2 sequences found in STAT5b-CA versus LMC CD4+Foxp3+ thymocytes that were also observed in splenic CD4+Foxp3− naïve T cells (Fig. 4B). To quantitate the magnitude of this difference more precisely, we took the area represented by the green, blue, and pink frequency distributions in Fig. 4B and plotted them as a bar graph (Fig. 4C). From this graph it is clear that the presence of the STAT5b-CA transgene greatly expands the number of TCR sequences in the Foxp3+ Treg repertoire that are more typically found in the naïve T cell repertoire (Fig. 4C – compare blue bars for Foxp3+ Tregs in LMC versus STAT5b-CA mice). These findings demonstrate that expression of a constitutively active form of STAT5 alters the TCR selection bias that is normally associated with Foxp3+ Treg development and subsequently expands the repertoire of sequences found in Tregs to include many more non-Treg specific TCR sequences.
To further test the ability of STAT5 activation to drive Foxp3+ Treg differentiation, we made use of a recent observation by Lio and Hsieh who identified CD4+CD25+Foxp3− thymocytes as penultimate progenitors of Foxp3+ Tregs that can be rapidly differentiated into Foxp3+ Tregs by IL2 plus IL7 addition (manuscript submitted). In contrast, they found that the CD4+CD25−Foxp3− thymocyte subset could not be converted into Tregs indicating that they are not Foxp3+ Treg progenitors. We predicted that if STAT5 activation downstream of TCR-dependent selection events is a key second step in Foxp3+ Treg differentiation, then we should be able to convert CD4+CD25−Foxp3− non-Treg progenitors into Foxp3+ Tregs in STAT5b-CA but not LMC mice. To examine this issue, we used Foxp3-GFP reporter mice (Fontenot et al., 2005c) to identify Foxp3+ and Foxp3− thymocyte subsets. We then sorted CD4+Foxp3-GFP−CD25− thymocytes from STAT5b-CA and LMC mice and placed them in culture overnight in either media alone, IL7, or IL7 and IL2. Consistent with reports from Lio and Hsieh, we found that CD4+Foxp3-GFP−CD25− thymocytes neither spontaneously converted into Foxp3+ Tregs, nor could be converted into Foxp3+ Tregs by cytokine treatment. In contrast, CD4+Foxp3-GFP−CD25− thymocytes from STAT5b-CA mice could be converted into Tregs following addition of either IL7, or IL7 and IL2 (Fig. 5A,B); stimulation of these cells with IL2 alone also worked (Sup. Fig. 4) but was substantially less effective as CD4+Foxp3-GFP−CD25− thymocytes not only fail to express the IL2Rα chain but express very low levels of the IL2Rβ chain as well (KBV and MAF, unpublished results) making theses cells very poorly responsive to IL2. In contrast, the synergistic affect of IL7 and IL2 appears to be due to the ability of IL7 to modestly upregulate CD25 in CD4+Foxp3-GFP− thymocytes from STAT5b-CA mice thereby rendering them more responsive to IL2.
The observation that the STAT5b-CA transgene only converts CD4+Foxp3-GFP−CD25− thymocytes into Tregs in vitro following addition of cytokines suggests that a relatively high threshold of STAT5 signaling is required for this process. To test this hypothesis we measured levels of phospho-STAT5 expression in both WT and STAT5b-CA CD4+Foxp3-GFP−CD25− thymocytes in the Treg conversion assay. As shown in Fig. 5C, unstimulated LMC CD4+Foxp3− thymocytes exhibited low levels of phospho-STAT5 expression that was increased following exposure to cytokines. Unstimulated CD4+Foxp3− thymocytes from STAT5b-CA mice exhibited a higher basal level of phospho-STAT5 staining; this is consistent with the constitutively activated nature of the STAT5b-CA transgene. Following stimulation of CD4+Foxp3− T cells from STAT5b-CA mice with IL2 and IL7 we observed two distinct patterns of phospho-STAT5 expression (Fig. 5C). Cells that remained negative for Foxp3-GFP did not exhibit an increase in phospho-STAT5 relative to unstimulated cells. In contrast, cells that induced Foxp3-GFP expression demonstrated a significant increase in phospho-STAT5 staining. These findings indicate that a threshold of STAT5 activation is required for CD4+Foxp3−thymocytes to convert into CD4+Foxp3+ Tregs. Taken together, our findings strongly support a model in which high-affinity TCR/CD28 signals induce the ability to respond to γc-dependent cytokines; subsequent encounter with such cytokines results in the final conversion of Foxp3− Treg progenitors in the thymus into Foxp3+ Tregs.
The conversion of CD4+CD25+Foxp3-GFP− thymocytes into CD4+Foxp3-GFP+ Tregs resembles, at least superficially, the conversion of mature splenic T cells into Tregs following anti-CD3, TGFβ, and IL2 stimulation that has been well described by others (Chen et al., 2003; Davidson et al., 2007; Zheng et al., 2002). Therefore, we examined whether TGFβ produced by converted Tregs, or potentially present in bovine calf serum, might be required for this process. As shown in Fig. 6A, neutralizing TGFβ had only a modest effect on the conversion of CD4+CD25−Foxp3-GFP− progenitor cells derived from STAT5b-CA mice. Conversely, addition of exogenous TGF-β resulted in minimal enhancement of the conversion process (ranging from no effect up to a 10% increase; data not shown). Moreover, using CD4+CD25+Foxp3-GFP− Treg progenitor cells obtained from wild type mice Lio and Hsieh found that neither adding exogenous TGFβ or neutralizing TGFβ antibodies, affected the conversion process (manuscript submitted). Thus, the cytokine-dependent conversion of the thymic Treg progenitors that we have identified differs significantly from the conversion of splenic CD4+ T cells because it does not require TGFβ. This is consistent with other published data on the requirements for thymic development of natural Tregs which have been shown to be TGFβ independent (Marie et al., 2005).
STAT5 associates with both histone acetyltransferases (HATs) and histone deacetylases (HDACs) and these interactions have been suggested to play an important role in regulating STAT5-dependent responses (Nakajima et al., 2001; Pfitzner et al., 1998). Therefore, we examined whether blocking histone deacetylase activity would augment or inhibit the cytokine-dependent Treg conversion process. For these studies, we inhibited HDACs with either the pan-HDAC inhibitor trichostatin A (TSA), or a more selective inhibitor, apicidin, which preferentially targets HDAC2 and HDAC3 (Khan et al., 2007). TSA functioned as expected by increasing global histone H3 and H4 acetylation in CD4+ thymocytes (Sup. Fig. 5). However, when added into the conversion assay both TSA and apicidin completely blocked the ability of CD4+CD25−Foxp3-GFP− progenitors from STAT5b-CA mice to convert into CD4+CD25+Foxp3-GFP+ Tregs (Fig. 6B). These results suggest that STAT5-dependent recruitment of HDACs is required for transcription of the foxp3 gene. Moreover, given the specificity of apicidin for HDAC2 and HDAC3 it is likely that one of these two HDACs is the relevant target.
Two of the fundamental questions remaining in the field of Foxp3+ Treg development are what governs this developmental process and how is it that the Foxp3+ Treg TCR repertoire is matched to that of self-reactive T cells. Herein we provide data demonstrating that linked TCR/CD28 and STAT5-dependent signals play a key role in both of these processes. Specifically, we demonstrate that constitutive activation of STAT5b induces Foxp3+ Treg development in newborn mice; such animals typically lack Foxp3+ Tregs due to the absence of as yet unidentified factors. Both CD28 and γc-dependent cytokines are potential factors required for Foxp3+ Treg differentiation that may be absent in neonatal mice. Supporting this hypothesis we found that STAT5-dependent signals were sufficient to restore Foxp3+ Treg development in both γc−/− and CD28−/− mice. Finally, we demonstrate that STAT5 signals are sufficient to convert thymocytes that would typically be selected into the naïve T cell pool into the Foxp3+ regulatory T cell lineage. For example, we found that a subset of CD4+Foxp3−CD25−non-Treg progenitors from STAT5b-CA, but not LMC mice, can be converted into Foxp3+ Tregs in vitro. Furthermore, using a novel strategy to identify antigen-specific T cells, we demonstrate that ectopic STAT5 signals can convert cells typically destined to become naïve T cells into the Foxp3+ Treg lineage in vivo. These findings were confirmed by sequencing of the TCR repertoire in Foxp3+ Tregs from STAT5b-CA and LMC mice; the TCR repertoire of Foxp3+ Tregs from STAT5b-CA mice exhibited much greater overlap with the naïve TCR repertoire than that observed for Foxp3+ Tregs in LMC mice. These findings definitively address the ongoing controversy regarding the role of the IL2R and STAT5 signaling in Foxp3+ Treg development. Previous studies using IL2Rβ −/−, γc−/−, and STAT5a/b−/− mice have suggested a role for these molecules in Foxp3+ Treg differentiation (Burchill et al., 2007; Fontenot et al., 2005b; Malek et al., 2000; Yao et al., 2007). However, in those studies it was difficult to distinguish between a role for these factors in post-selection expansion of Foxp3+ Treg numbers versus preselection induction of Foxp3+ Treg differentiation. The findings presented herein that STAT5 signals can divert non-Treg progenitors into the Foxp3+ Treg lineage clearly demonstrate that STAT5 signals play an important role in the pre-selection differentiation Foxp3+ Tregs. Taken together our data support a two step model of Foxp3+ Treg differentiation in which TCR/CD28 signals first enhance the ability of cells undergoing negative selection to respond to IL2R/STAT5 signals; subsequent encounter with STAT5-inducing ligands (most likely IL2) completes the differentiation process that drives Foxp3+ Treg development.
The above model is supported by significant amounts of other data. First, we and others have found that STAT5 binds to the foxp3 promoter in Foxp3+ Tregs, suggesting that STAT5 may induce foxp3 expression (Burchill et al., 2007; Yao et al., 2007). Second, previous reports have documented that thymocytes undergoing negative selection upregulate expression of the IL2Rα and IL2Rβ chains (Bassiri and Carding, 2001). Importantly, IL2Rα upregulation occurs only in CD4 but not CD8 destined thymocytes as the IL2Rα chain is upregulated during negative selection in transgenic mice expressing a MHC class II (HEL) but not a MHC class I (OT-I) restricted TCR (Baldwin and Hogquist, 2007). This may explain why CD4+ thymocytes can differentiate into Tregs but CD8+ thymocytes typically do not. Third, IL2 has also been found selectively in the medulla of the thymus (Yang-Snyder and Rothenberg, 1998), a region which correlates with Treg development ( Fontenot et al., 2005a ; Aschenbrenner et al., 2007). Finally, in work by Lio and Hsieh (manuscript submitted), a CD4+CD25+Foxp3− Treg precursor has been identified that can be converted to CD4+CD25+Foxp3+ Tregs by administration of IL2 or IL15 in vitro. Taken together with our in vivo data, these studies support a role for IL2/STAT5-dependent signals in driving Treg development.
A remaining question is why all CD4+ thymocytes in STAT5b-CA mice are not converted into Foxp3+ Tregs. One potential explanation for this observation is provided by recent studies examining the methylation status of the foxp3 gene. For example, changes in DNA CpG methylation and histone H4K3-trimethylation have been found in developing CD4+Foxp3+ thymocytes (Floess et al., 2007). Likewise, TCR/CREB-dependent signals induce changes in the methylation status of histones associated with the foxp3 gene locus (Kim and Leonard, 2007). A second potential explanation is that relatively strong TCR/CD28 signals may be required to induce a state of heightened cytokine responsiveness. Only cells in which both the IL2Rα and IL2Rβ chain have been upregulated, as well as other positive regulators of IL2R signaling such as Socs2 (Tannahill et al., 2005), will likely be capable of augmenting STAT5 signals to the threshold level required to complete Treg differentiation in WT mice. We suggest that in STAT5b-CA mice, the other factors contributing to enhanced cytokine signaling play a lesser but still important role. Based on these findings we propose a model in which high-affinity TCR signals both open the foxp3 gene locus and induce the potential for cytokine responsiveness; only cells that subsequently encounter a STAT5 inducing cytokine will induce foxp3 expression thereby completing the program of Foxp3+ Treg differentiation.
An important question is how STAT5 signals result in Treg differentiation. STAT5 binds to several regions within the foxp3 gene (Burchill et al., 2007; Yao et al., 2007) and is known to interact with both histone acetyltransferases and histone deacetylases. A common perception is that histone deacetylases (HDACs) inhibit gene transcription by deacetylating histones and thereby reducing gene accessibility (Kuo and Allis, 1998). However, mounting evidence suggests that HDACs can also deacetylate other factors (Nusinzon and Horvath, 2005). Along these lines, recent work from at least two groups has demonstrated that inhibition of HDACs prevents the induction of STAT5-dependent gene transcription (Rascle et al., 2003; Xu et al., 2003). Perhaps the best example of this is work by Sun and colleagues in which they characterized transcription of the id1 gene in pro-B cells. Id1 expression is regulated by a pro-B cell specific enhancer that contains critical binding sites for C/EBPβ and STAT5. In this system STAT5 was shown to recruit a histone deacetylase that deacteylated C/EBPβ. Deacetylation of C/EBPβ resulted in enhanced DNA binding and subsequent induction of id1 gene transcription. Importantly, inhibiting HDAC activity blocked the ability of STAT5 to cooperate with C/EBPβ to induce id1 transcription; conversely, introduction of a mutant form of C/EBPβ that could no longer be acetylated removed the requirement for STAT5 in the induction of id1 transcription (Xu et al., 2003). Although we cannot rule out all other interpretations, at this point the simplest explanation for our findings is that STAT5 recruits a HDAC that acts on a second factor to induce foxp3 transcription.
We believe our data also provide an explanation for several other outstanding questions in the field. For example, work from several labs has demonstrated that transgenic mice co-expressing both a single TCR and its respective ligand generate a significant percentage of Foxp3+ Tregs (Apostolou et al., 2002; Jordan et al., 2001). These results suggest that agonist ligand is required to induce Treg differentiation. In contrast, work by van Santen et al using mice in which the amount of agonist ligand could be carefully titrated found that increasing amounts of agonist ligand did not result in an increase in Foxp3+ Treg numbers. Rather, they found that Foxp3+ Tregs were more resistant than non-Tregs to negative selection and that this resulted in an increase in the percentage of Foxp3+ Tregs, but not an actual increase in their numbers (van Santen et al., 2004). Their conclusion was that enhanced survival rather than induced differentiation accounted for the results in the previous studies. We suggest that our model may account for these latter findings. Notably, in the studies by van Santen et al, the agonist ligand was expressed in both the thymic cortex and medulla. Based on our findings that IL2Rβ-induced STAT5 activation is required to complete Foxp3+ Treg differentiation, we would predict that increased agonist ligand in the cortex would result primarily in negative selection due to the limited amounts of IL2 available there. In contrast, we predict that forced expression of IL2 in the cortex would rescue these negatively selected cells by converting them into Foxp3+ Tregs.
A related question concerns the dual role that TCR/CD28 signals play in both negative selection and Foxp3+ Treg development. Work from many labs have established that TCR/CD28 signals are required for both of these processes (Aschenbrenner et al., 2007; Bensinger et al., 2001; Tai et al., 2005; Tang et al., 2003). However, it is unclear how this common signaling mechanism can lead to such dramatically different cell fates. One possibility is that this simply reflects the overall strength of signal with the strongest TCR/CD28-dependent signals leading to cell death while weaker TCR/CD28-dependent signals promote Foxp3+ Treg differentiation. Although this is an attractive explanation, formal proof for such a model is lacking. We propose an alternative model in which high-affinity TCR stimulation induces a program of negative selection that includes enhanced ability to respond to IL2Rβ-dependent ligands. Whether these thymocytes die, or emerge as Foxp3+ Tregs, depends on whether they subsequently encounter STAT5-inducing ligands in the thymus (Sup Fig. 6). Intriguingly, this model would potentially allow for enrichment of Tregs specific for Aire-induced self-antigens which are localized in the medulla (Anderson et al., 2002; Aschenbrenner et al., 2007), and thus have an increased probability of encountering IL2. In contrast, abundant self-antigens, which would be predicted to induce negative selection at a high frequency in the cortex, would preferentially promote negative selection-induced cell death due to the absence of IL2. Such a mechanism would result in greater numbers of Foxp3+ Tregs directed to low abundance self-antigens presented in the thymic medulla, and thus the very cells that might be expected to escape negative selection at higher frequency.
STAT5b-CA transgenic mice were previously described (Burchill et al., 2003). C57BL/6 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Foxp3-GFP x TCR TCli Tg x TCRα+/− mice were provided by Dr. Alexander Rudensky (University of Washington) (Hsieh et al., 2006). Mice used for analysis were 4–6 weeks old, unless otherwise noted. The University of Minnesota Institutional Animal Care and Use Committee approved all animal experiments.
Thymii and peripheral lymphoid organs were isolated from STAT5b-CA and C57BL/6 mice between the ages of 4–6 wks of age and analyzed for 2W1S-specific T cell populations (Moon et al., 2007). Pooled spleen and lymph nodes were incubated with PE or APC labeled 2W1S:I-Ab tetramers followed by incubation with anti-PE or anti-APC microbeads (Millteni Biotech, Auburn, CA). Tetramer specific T cells were then enriched using magnetic LS-columns (Millteni Biotech) and enumerated using flow cytometric counting beads (Caltag, Carlsbad, CA). Enriched T cell populations were stained with CD3, CD4, CD8, CD11b, CD11c, B220, F4/80, GR1 and NK1.1 and intracellular Foxp3 as previously described (Burchill et al., 2007).
Foxp3+ and Foxp3− populations were isolated from Foxp3-GFP x TCRβ6 Tg x TCRα+/− STAT5b-CA and C57BL/6 mice using a Facs Aria cell sorter. TCR Vα2 sequences were cloned and analyzed as previously described (Hsieh et al., 2006).
Thymi from either Foxp3-GFP or STAT5b-CA x Foxp3-GFP male mice were disrupted with ground glass slides. Cells were subsequently stained with the cell surface antibodies CD8 for enrichment of CD4 T cells using magnetic bead columns (Miltenyi Biotech). Purified CD4+CD8-Foxp3−CD25+ and CD4+CD8-Foxp3−CD25+ thymocytes were purified using a Facs Aria cell sorter (Becton Dickinson). Isolated thymocyte subsets were resuspended in DMEM containing 10% FCS (Atlas Biologicals), 50μM β-mercaptoethanol (Sigma), 1% L-glutamine (Mediatech Cellgrow), 1mM sodium pyruvate (Mediatech Cellgrow), 10 mM Hepes (Mediatech Cellgrow), and 1% streptomycin/penicillin (Mediatech Cellgrow). Cells were either seeded into 96-well round bottom plates in the presence of media alone, IL7 (5nM), or IL2 (10u/ml) plus IL7 (5nM). Some cultures were treated with either 10μg/mL anti-TGFβ (R&D systems, St. Paul, MN), 10 μg/mL isotype control IgG1 antibody (GIR-208; kindly provided by Dr. Robert Schreiber, Washington University), 100 nM Trichostatin A (Sigma, St. Louis, MO), 800 nM Apicidin (Sigma, St. Louis, MO) or DMSO (0.2%). IL2 and IL7 were obtained from the Biologicals Resource Branch, National Cancer Institute Preclinical Repository. Cells were incubated for 24 hours at 37°C in a 5% CO2 incubator. Foxp3 expression was analyzed using an LSR II (Becton Dickinson) and Flow Jo Software (Treestar).
Sorted CD4+ CD25− Foxp3-GFP− thymocytes were stimulated for 24 hours with 10 U/mL IL2 and 5 ng/mL IL7 (PeproTech). Cells were then harvested and stained for phospho-STAT5 as previously described (Will et al, 2006). For acetyl-H3 and H4 western blots CD4+ T cells were stimulated overnight with IL2 plus IL7 and either TSA or DMSO. Nuclear lysates were run on a 12% SDS-polyacrylamide, transferred to a nitrocellulose membrane and blocked for one hour with 5% non fat dry milk in 1 X PBS and 0.1% Tween. Blots were incubated with primary anti-acetyl H3 and H4 antibodies (Upstate Biology) and secondary goat anti-Rabbit IgG followed by visualization with an Odyssey Infrared Imaging System.
Supplemental Fig. 1: Foxp3+ Tregs in STAT5b-CA x γc−/− mice are functional suppressor cells. Shown are gated CD8 T cells from littermate control, γc−/− mice and STAT5b-CA x γc−/− mice stained for CD69 (left panel) and CD62L (right panel) expression. CD8+ T cells from γc−/− mice all exhibit an activated phenotype as illustrated by high CD69 and low CD62L expression. CD69 expression is reduced to background levels while CD62L remains high on CD8+ T cells from STAT5b-CAx γc−/− mice indicating that the Foxp3+ Tregs generated in those mice are capable of blocking T cell activation.
Supplemental Fig. 2: Illustration of the 2W1S Tetramer purification procedure.
Supplemental Fig. 3: Gating Strategy used to analyze 2W1S-specific T cells. Following magnetic bead enrichment samples were stained with antibodies to CD11b, CD11c, F4/80, NK1.1, B220, CD4, CD8, CD25, and intracellular Foxp3. Cell populations were fractionated by flow cytometry first using cell size and granularity followed by elimination of doublet events using FSC-A and FSC-W. T cells were then identified by high levels of CD3 expression and low levels of Dump (CD11b, CD11c, F4/80, NK1.1, B220) staining. The T cell population was then fractionated into CD4 and CD8 populations and the 2W1S-positive gate was set at a level such that gating on CD8 T cells resulted in <0.1% 2W1S-positive events. Magnetic bead enrichment results in the specific pull down of 2W1S positive cells. Neither TEa x Rag−/− nor SM1 x Rag−/− TCR transgenic T cells which recognize Ea:I-Ab and FliC:I-Ab respectively bind to the 2W1S:I-Ab tetramer complex.
Supplemental Fig. 4: Conversion of thymic Foxp3+ Tregs in the presence of IL-2 alone. Purified CD4+Foxp3-GFP−CD25− thymocytes from STAT5b-CA mice were stimulated in vitro with media alone, or increasing doses of IL-2 (10U/ml, 100U/ml and 1000U/ml). After twenty-four of stimulation, cells were stained for CD4 expression and the percentage of Foxp3-GFP+ Tregs determined by flow cytometry. Shown is a representative example of two experiments.
Supplemental Fig. 5: Trichostatin A induces a general increase in histone H3 and H4 acetylation. CD4+CD8− thymocytes from STAT5b-CA mice were stimulated overnight with IL2 plus IL7 and either DMSO or TSA. Nuclear lysates were blotted with antibodies to acetylated histone H3 and H4. Blots were reprobed with an antibody to β-actin; twice as much lysate was loaded from DMSO treated cells.
Supplemental Fig. 6: Model of IL2R/STAT5-dependent Foxp3+ Treg development.
We thank Rachel Agneberg, Jessica Oehrlein and Jared Liebelt for assistance with animal husbandry, Paul Champoux for assistance with flow cytometry, and Dan Mueller and Laura Ramsey for review of the manuscript. This work was supported by a Pew Scholar Award, a Cancer Investigator Award, a Leukemia and Lymphoma Society Scholar award, and an NIH grant (AI061165) to M.A.F. The authors declare that they have no financial conflicts of interest. Correspondence and requests for materials should be addressed to MAF at ude.nmu@500arraf.
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Care home costs: Essential tips for home care fundingPosted on: 15 June 2010 by Mark O'haire
The issue of long-term care costs is never far from the headlines. Our guide highlights 10 points to consider if you or a family member face the prospect of moving into a care home.
The prospect of moving into residential care or having a carer in our home is something we'd all prefer to ignore for as long as possible.
But with National Dementia Week highlighting the problems of old age during 4 July – 10 July, with the help of chartered financial planner Ruth Dolan, we guide you through your options which may help tackle the challenges ahead.
Ruth, a member of the care sector team at leading regional law firm Furley Page, says: "The first thing to remember is that your local authority should provide an assessment of your needs so that the most appropriate type of care can be arranged for you. Depending on your means, you may find you have to contribute some or all of the care costs. If that's the case make sure you are aware of all your options."
Ruth's check list includes:
- State benefits: claim all the benefits available including Attendance Allowance, which is non-means tested and paid free of tax for over 65s, and welfare benefits which change continually
- Investments: these should be reviewed regularly. Check on the type of returns (income or capital growth); the risk involved and the tax liabilities
- Annuities: if your income won't meet your care home costs you may need to invest to provide additional income. An annuity with a lump sum is an option
- Property: if you don't want to sell your home to fund your care home costs you could apply to your local authority for a deferred payment scheme. Other options are renting your property or releasing some equity - but seek advice on the various schemes as they can have significant implications.
1) Pay for care in your home
Increasing the level of care at home can delay the need to go into a nursing home. You can expect to pay about £10 an hour to have a qualified carer – provided by your local social services department - come to your home to help with tasks such as preparing meals or dressing and undressing. It is also possible to have a carer stay overnight or even to arrange full-time care at home. Before you receive this service, your level of need will have to be assessed.
You may also be entitled to 'direct payments'. These are local council payments for anyone who has been assessed as needing help from social services and who would like to buy in services privately. The cost of care at home can also be offset by claiming Attendance Allowance (see below).
2) Claim your allowances
Attendance Allowance is a tax-free benefit for people aged 65 or over who need help with personal care because they are physically or mentally impaired. There are two rates: £47.10 for help during the day or during the night (but not both) and £70.35 for help during both the day and night. There is also a 'low' rate of £18.65. It is paid regardless of your level of income or savings. Claim by calling the official Benefit Enquiry Line 0800 882 200 or online at www.dwp.gov.uk/eservice.
3) Speak to the charity professionals
Age UK's telephone advice service provides welfare rights advice for older people and their carers, call 0800 169 6565. Events that lead up to the need for a family member to go into long-term care are often very stressful. The Alzheimer's Society is a valuable source of help and advice about dementia in all its forms. Call 0845 300 0336 or log on to www.alzheimers.org.uk.
4) Buy an 'instant annuity'
Some or all of the cost of full-time care may be financed by buying a type of annuity known as an immediate needs care plan. These pay much more than a standard annuity because typically people go into care when their health is poor and life expectancy is comparatively short. Only a handful of companies offer these plans at present and their rates vary. It is advisable to speak to an IFA before signing up for one of these plans.
5) Keep your savings separate
Local authorities no longer have the right to raid the savings of the husband or wife of someone who has to go into care and has asked for help paying the fees. However, to be sure of avoiding problems with joint accounts, married couples should keep their savings in separate names.
6) Ask for regular reviews
Many of those going into a home will be eligible for a Government payment to cover the nursing element of their care, known as the NHS nursing contribution. There are three levels - low £40 a week, moderate £83 a week, and high £133 a week. With time, the extent of nursing care required may increase, so be sure to ask for a review if you think this is the case. See the Department of Health website for more details.
7) Consider equity release
An alternative to an immediate needs care plan (see above) may be equity release. This can provide you with a lump sum or a regular monthly income by signing away some or all the value of your home.
8) Take power of attorney now
Don't wait until a family member or relative is incapable of managing their own affairs before considering power of attorney. It can make the whole process much more complicated and you may end up with the Court of Protection looking over your shoulder.
9) Beware if you want to avoid paying
Many people believe the state should pay for long-term care and some will be tempted to arrange their affairs so that they can claim they do not have enough money or assets (such as their home) to cover the cost. The authorities will come after you and your family members if they have reason to think you have deliberately set out to evade self-funding.
10) Think about inheritance tax
Legitimate IHT planning may enable you to hand some of your assets to family and friends without falling foul of the rules on disposal of assets.
For more information, please visit www.furleypage.co.uk.
Do you have any tips for care home funding?
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Mark O'haire
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