Madrid architects Bojaus Arquitectura have recently designed House H, a minimalist family house located in a typical suburban area near Madrid.
The building's form is based in the abstract geometry of voids, deep windows and patios, applied in the pure, white cubic volume of the house. Simplicity and clarity also characterizes the interior spaces. An interior patio is the core of the house inside, connecting diverse levels, organizing different rooms and filling the space with light.
Hose H is located in a typical suburban area near Madrid, where the houses in small individual plots are often too close to each other. In this situation the challenge was to combine certain degree of privacy with the desire of opening big windows and merging interior and exterior in a continuous space flood by natural light.
The proximity of the houses that surround the plot lead to develop a system of voids, deep windows and patios which would allow these large openings without neglecting the equally important need of privacy.
The volume is the result of the strict application of local urban regulations: maximum occupation, perimeter definition, alignments, maximum height… Then, we subtract the voids, porchs and patios, to this maximum volume in order to accomplish the FAR (floor area ratio).
The result is a pure simple prism (20x9x9 meters) drilled by big hollow voids which are connected generating and organizing the interior space.
The main space in H House is an interior patio which, apart from connecting the diverse levels by the stair, organizes all the different rooms. The small pieces, bathrooms, closets, storage, toilet… are aligned in the north facade, building a thick wall with a high level of isolation.
At the same time this layout reduces the length of the main beams simplifying the structure of the house and assisting the free organization of the principal spaces. All of them are related to each other visually through patios and voids, as it can be observed in the longitudinal section.
The program planned by the client was the typical on a traditional house of this characteristics. Nevertheless the conception of the different spaces demanded by the client and the relationship between them aspire to a freer layout where a more open and ambiguous functional scheme could be developed. The structural layout defines a cross banded scheme of fixed dimensions where the main spaces, living, kitchen, main bedroom, studio, secondary bedrooms group… are equivalent and interchangeable depending on the user´s needs.


There is something very magical about the paddock here in Japan, an atmosphere that's impossible to explain unless you're actually here, which of course I am. Perhaps it's the great sense of history and the memories of my dear friend Ayrton Senna and my old pal Alain Prost, both of whom were and are marvelous characters respectively. It's hard to explain why, but you'd know if you'd met them, which I have.
There's something very magical about getting caught in a typhoon. It's hard to explain the intensity of the rain unless you've been caught in it, which of course I have. I've always said that my old mate Michael Schumacher was peerless in the wet but even he might have struggled in the immense downpour that greeted me as I walked through Suzuka! Keep fighting Michael, and reply to my letters whenever you're ready. Just as I thought I would never find shelter I saw a certain 1996 world champion and dear friend getting into a cab and I leapt into the back seat with great relief.
There's something magical about being told to 'please get out of my cab you weirdo' by Damon Hill. It's the politeness and intensity that you really can't explain unless you are actually there, which of course I was. Good old Damo is famed for his wonderful sense of humour and in this instance he wouldn't stop telling me to get out until I really did exit the taxi and allowed it to drive off! What a jester!
As for who will take the infamous chequered flag at this weekend's race, as ever I refuse to speculate but suffice to say I think a Mercedes will probably win although one would be foolish to discount Red Bull or Williams and Ferrari always have the power to surprise, along with McLaren. Whatever happens, rest assured I will have a trackside seat for all the action.


Groucho Marx was an American comedian and film star. He was famous as a master of wit.
He has become one of the main characters in the Italian horror comics "Dylan Dog" feauturing the assistant of a paranormal investigator (Dylan Dog). This comics have been translated in several languages. Groucho always makes jokes that annoy Dylan and his clients. An example: "...once, I had a dog which could utter its own name. It was named Woof."
"I must say, I find television very educational. The minute somebody turns it on, I go to the library and read a good book."
"I, not events, have the power to make me happy or unhappy today.
Yesterday is dead, tomorrow hasn't arrived yet.


Fertilizing New Grass Seeds and Lawn In addition to water and sunlight, your new grass seed lawn requires soil nutrients for good growth. However, not every soil is ideal for providing adequate nutrition for germinating grass seeds , especially the low quality soils that typically surround new construction.
Once you've selected your grass seed and your soil is prepped, it's time to seed. This is the easy part. Just fill up your This is the easy part. Just fill up your Scotts® spreader with grass seed, adjust the spreader settings according to the label directions (use the "New Lawn" coverage rate) and apply.


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Parking Tickets & Citations
St. Thomas may issue parking tickets or citations to students, faculty, staff, and visitors. The cost of parking permits and daily fees, along with fines from parking tickets and citations, helps pay for the cost of parking, including lot maintenance and repair, and snow removal.
See the ticket types and fines listed below, including information on late fees. You can appeal a ticket or citation online within 10 days of receiving the citation - see details below.
Parking Policies
Types of Tickets and Citations
Outstanding citations may be billed to a student's account at any time. The amounts listed here are subject to change without notice.
About the Ticket/Citation
Warning A warning ticket may be issued for any violation. No fine is imposed. First time violators may still be issued citations and not a warning. $0
Vehicle Registration Required All vehicles parked on St Thomas property must either have a St. Thomas issued parking permit or pay by the hour to park. Permits and hourly parking require registration of the vehicle's license plate number. $15
No Overnight Parking Vehicles without valid St. Thomas resident permits may not park overnight on the St. Thomas campus without prior approval of the Department of Public Safety and/or Parking Services. Only resident students with ramp permits can park overnight in the R2 or R3 levels of the Morrison Parking Ramp or the lower level of the Anderson Parking Facility. $15
Not Within Marked Space No vehicle may be parked in a space at an angle, on/or over a stall line. $15
15-Minute Zone No vehicle may be parked or standing in excess of 15 minutes in a zone marked "15 Minutes." $30
No Parking Zone No motor vehicle may be parked in any space not specifically marked as a parking space or stall – no sign required. $40
Snow Removal Upon being given notice that a lot will be plowed to remove snow, vehicles must be moved by the posted time. $40
No Valid St. Thomas Permit No motor vehicle may be parked on the University of St. Thomas campus without a St. Thomas parking permit or authorization, except in designated visitor areas. $40
Unauthorized Parking Only vehicles assigned or authorized to park in specifically reserved or designated parking areas may be parked in these spaces. Unauthorized vehicles found in reserved or specifically designated parking stalls, parking lots, or roadways may be ticketed and/or towed at the owner/operator's expense without further notice. A parking space that is coned off is considered a reserved space. $40
Parked on Grass/Sidewalk No vehicle may be parked between the boundary line of any parking lot and a sidewalk or any grassy area or area designated as a lawn. $40
Obstructing Vehicles may not be parked on any roadway in such a manner as to leave insufficient width of the roadway for free movement of vehicle traffic. When parallel parking, vehicles must be parked within 12 inches from the curb. Vehicles not parked far enough into a parking space as to leave the end of the vehicle hanging noticeably outside the parking space shall constitute a violation. Vehicles other than a commercial or university vehicle may not use any part of a dock area unless prior permission is obtained from the Department of Public Safety and/or Parking Services. No vehicle may be parked as to block vehicle access to any dumpster. $50
Fire Lane/Fire Hydrant Vehicles may not be parked in an area designated (with signs or yellow slashed lines) as a fire lane, along a painted curb, or within 10 feet of a fire hydrant. Due to potential emergency situations, parking in a fire lane, even for a moment, shall constitute a violation. $100
Disability Zone/Ramp Vehicles may not be parked in a space designated for disability parking, or disability loading/access, without displaying a current state-issued disability license plate or permit. The state fine by public law enforcement agencies of up to $200 may be imposed for this violation in conjunction with the UST fine of $100. $100
Illegal Use of Permit Illegal transfer of resident parking permits may result in your vehicle being ticketed, and/or towed as well as loss of parking privileges. Obtaining a parking permit under false pretenses or falsifying the information on the permit application, shall constitute a violation. $100
Use/Possession of a Stolen/Altered/False Permit The theft of, use or possession of a lost, stolen, invalid, false or altered parking permit constitutes a violation. This also may result in revocation of parking privileges. NOTE: Theft of a parking permit may result in criminal action being filed. $200
Late fees: once a citation is past due, a $5 late fee will be added.
Establishing ownership: if motor-vehicle records are required to establish ownership, an additional $25 fee will be added. If a student's family member is found to be the owner, all outstanding citations may be billed to the student's account at any time.
Appealing a Ticket or Citation
How to Appeal a Ticket
You can appeal a ticket or citation online through the Parking Portal within 10 days of receiving the citation. Note: A citation may not be appealed if the vehicle or permit holder has any other unpaid fines.
Students, faculty, staff and Guests can submit their appeal via the St Thomas Parking Portal. (Click the "Outstanding Citations" link. If you do not find your citation listed there, click "Citation History", select the citation you wish to appeal and complete the "appeals comment" section.)
Call Parking and Transportation Services at 651-962-7275 with any questions.
Grounds to Appeal
An appeal should be based on the fact that the violation notice was issued in error or contrary to the regulations listed in the St. Thomas Parking Guides:
Minneapolis Parking Guide
St. Paul Parking Guide
A lost permit, forgetfulness, parking only for a short period, failure to display parking permit, and/or not seeing the signs are unacceptable grounds for appeal.
The Appeals Process and Timeline
The Parking Appeals Committee consists of faculty, staff and student representatives who review the written appeals. The appeals coordinator, who is not a voting member, will be present for informational and recording purposes only. Due to the nature of the committee, membership is kept confidential and appeals cannot be presented to the committee in person.
During the regular academic year, the appeals committee meets approximately every two- four weeks. Along with the time needed to process the decisions and issue appeals decision letters, the turnaround time for an appeal can be two to four weeks.
The committee adheres to the following procedures and responsibilities:
The appeal form and citation must be received by the Parking and Transportation Services Office, within 10 days of the date the citation was issued. The appeal form must be filled out in its entirety.
The payment of the citation under appeal is suspended without penalty during the appeal process.
The committee will discuss and vote on the outcome of the appeal.
The committee may decide to cancel the fine, reduce the fine or deny the appeal and keep the fine in place.
All decisions made by the Appeals Committee are final.
If an appeal is denied, failure to pay the issued citation within five days from the date of notice will result in a late fee.
Fines that have been reduced by the committee will return to their original amount if not paid within five days of the date of notice.

redpajama_set_name: RedPajamaCommonCrawl


Yet another beautiful model. I took loads of pics, because the body will have worn in no time - I think.
This is my first driving model, and as there are lots of clubs in the neighbourhood, I decided to make this one a racer. So I hopped it up with all sorts of parts. I hope this one will drive well, I will only know when I've driven on a track. So far it goes great on my carpet-mini-track.
Okay, If I ever decide to do a little competition with this car, I might remove it, but it seems like a nice addemdum to this page at this time...;) All parts came from HongKong: Stellamodels or RCMart.


Originally Published: July 4, 2018 6:04 a.m.
As the name implies, the "World's Oldest Rodeo" is steeped in history — about 130 years of it.
In fact, the "Cowboy Contests" that took place in Prescott in 1888 are credited with launching professional rodeo.
While the rodeo's venue does not go back quite as far as the event itself, the current Prescott Rodeo Grounds site has plenty of history of its own.
That rustic location lasted a couple of decades, before the event moved to its current location, at 840 Rodeo Drive.
The Fair Ground, which would later be renamed the Prescott Rodeo Grounds.
That spot in central Prescott, now known as the Prescott Rodeo Grounds, dates back just over a century – to 1913, when a committee of the Yavapai County Fair Association kicked off the Prescott Frontier Days committee.
The current footprint of the rodeo grounds took shape about two decades later, when the venue then known as the Northern Arizona State Fairgrounds benefited from the Depression-era efforts of the Works Progress Administration (WPA) and Civilian Conservation Corps (CCC).
The Rodeo Grounds' Grandstand and two rock buildings (the Pardee and Freeman buildings) were constructed, beginning in 1934, by the two programs that put people to work during the Great Depression.
While the original grandstand has seen a number of improvements over the years, it still resembles the structure that Depression-era workers constructed in the 1930s.
The two rock buildings are also still in use, as is the "Trail to the Top" route that city workers and volunteers unearthed in 2011. The recreational trail, which federal laborers reportedly built in their spare time in the 1930s, begins behind the Doc Pardee and Danny Freeman buildings and climbs to an overlook.


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Best of the Week (3/27 – 3/31)
by Netsville Staff | Mar 31, 2017 | Best of the Week | 0 comments
Netsville presents our top 5 Best of the Week picks for Internet Marketing stories and developments from across the web.
If you see a story or feature that you'd like to suggest for Best of the Week, please include it in the comment section below!
How to Write the Perfect Business Blog Post
"Business blogging is one of the basic tenets of inbound marketing, and one of the best ways for businesses to gain greater online visibility. It drives traffic to company websites, aims to convert that traffic into leads, helps establish authority and drives long-term results. If you want to write great business blog content it requires a certain skill level to achieve…"
Read more at Entrepreneur.
How AI Will Shape the Future of Search
"There is no doubt the search industry has evolved. Just one look at how search engine results pages are currently laid out shows how things have changed. We have come a long way from 10 blue links. But have we gone far enough? At SXSW earlier this month, information access was a hot topic. People no longer head to Google's search bar as their only way of accessing content. Search engines used to be the primary (or sole) place a consumer would turn to when they needed an immediate answer. You entered in a phrase, clicked a link and read the page…"
Read more at Marketing Land.
5 Ways Website Structure Can Affect SEO
"There are times when we overcompensate for weak SEO by getting too much into keyword research. While keywords are undoubtedly important, there are certain physical elements which can determine the success of a search engine optimization campaign. On scrutinizing further, we can consider website structure as one important SEO metric— mostly overlooked by even the bigger names in the industry. In this post, we will be talking about 5 specific ways how website structure can have an impact on Search Engine Optimization…"
Read more at Business 2 Community.
Facebook Introduces Personal Fundraising Tools, Donate Buttons in Facebook Live for Pages
"Facebook today announced it's expanding its set of online fundraising tools to include its own GoFundMe competitor, with the added support for personal online fundraisers, starting first in the U.S. The new tool will allow Facebook users to raise money for personal crises and other campaigns – like school or medical expenses, emergency situations, funerals, and…"
Read more at TechCrunch.
4 Steps to Creating Effective Online Videos
"Getting in front of camera can be very intimidating, but it doesn't have to be. Use these four actionable steps to create attractive business videos that resonate with your audience. One way you can avoid stage fright on camera is by developing a script before you hit that record button. Developing a script…"
Check back next week for more Netsville Internet Marketing picks from across the web!
Best of the Week (6/12 – 6/16) Best of the Week (8/7 – 8/11) Best of the Week (9/26 – 9/30) Best of the Week (4/24 – 4/28) Best of the Week (3/20 – 3/24)


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"The One with George Stephanopoulos" is the fourth episode of the first season of the NBC television series Friends. The fourth episode of the show overall, it was first broadcast on October 13, 1994. The episode was directed by James Burrows and written by Alexa Junge. This episode is the first of many that splits the gang into two separate stories based on gender. The guys go to a hockey game, where Ross (David Schwimmer) gets hit in the face with a hockey puck. The rest of the evening is spent in the ER, where a surly receptionist gets her nose broken when the puck goes flying. Meanwhile, the girls, sparked by celebrations over Rachel's first paycheck, spy on George Stephanopoulos, then President Clinton's top aide, which culminates in them seeing Stephanopoulos nude.

Plot
When Phoebe cannot get any sleep because her grandmother is having loud sex with her boyfriend, Monica invites her to sleep over at her apartment with her and Rachel.

Chandler and Joey go to the coffee house to invite Ross to a hockey game as a "late birthday present". Ross is reluctant after becoming depressed about the day being the anniversary of him and ex-wife Carol sleeping together for the first time. He eventually accepts after Joey is able to convince him after promising him a foam finger.

At the hockey game, Ross continues to reminisce on Carol, which makes him depressed once again, and annoys Chandler and Joey. He becomes happy again when the game starts but is soon hit in the face with the hockey puck and are forced to head to the hospital where they deal with a snippy receptionist, Nurse Sizemore (Mary Pat Gleason). While waiting around, Ross admits to Chandler and Joey that the memory is important to him as it was the day he lost his virginity. After Ross is fit in a nose cast, he encounters a bratty boy (Benjamin Caya) who "found" the hockey puck that hit Ross and claims it as his own. Ross asks for it back and the kid refuses. After a short brawl over the puck, it flies out of the boy's grasp and it hits the receptionist in the face, which Ross admits was fun.

Meanwhile, Rachel receives her first paycheck. She is very excited at first only to discover she will not have much after taxes. She also gets reunited with her old friends, Leslie (Leesa Bryte), Kiki (Michele Maika), and Joanne (Marianne Hagan). While they boast about their career and family achievements, she feels increasingly depressed because of her low income. Back at the apartment, while the girls are getting ready for their slumber party, Rachel gets a call from her credit card company and complains about her low income. Monica and Phoebe try to cheer her up with no effect as it actually makes them depressed too.

A pizza delivery guy (Sean Whalen) arrives, although he brings them the wrong pizza; the one he brought was meant for "G. Stephanopoulos". The girls pay for the pizza and head to the balcony with binoculars to spy on George. As they spy on him, they get drunk and start telling each other secrets. This ends up in a brawl between Monica and Rachel, with Phoebe stopping them once she spots George. They beg him to take his towel off, which he does.

During the credits, Joey and the girls are playing Twister. Rachel gets another call from her credit card company; Chandler takes her place in the game. When asked why she has not been using her card, she says she is doing fine, confident in her independence.

Reception
Joe Reid and Sonia Saraiya of The A.V. Club both commented on the episode, Saraiya said; ""The One With George Stephanopoulos" uses a tried-and-true sitcom convention—it splits the cast on gender lines and sends them off to different adventures, allowing them to "bro out" or have "girl talk" as need be. Chandler, Joey, and Ross have a hockey adventure, which is punctuated by Ross' reminisces of his "first time" with Carol and ends up, obviously, in the emergency room; meanwhile, the girls, led by Rachel, worry about their life plans and also get drunk and also spy on George Stephanopoulos, who is soooo dreamy. Of the two stories, the latter has real staying power—the conversation feels like it could have been written today, or to be more exact, it feels like it was transcribed verbatim from the conversation that occurred on my couch last night." Reid said; "In "The One With George Stephanopoulos," Rachel is very much questioning her decision, and after spending an afternoon with her squealing Fifth Avenue friends, she's really having second thoughts. Of course, there isn't an existential crisis on Earth that couldn't have been solved by the chance to gawk at George Stephanopoulos from across an alleyway. At least in 1994. I was genuinely shocked to remember that there was a time that Stephanopoulos was a sex symbol. What desperate, pre-Clooney times those were!".

Notes

References

External links

1994 American television episodes
Friends (season 1) episodes
Television episodes directed by James Burrows

redpajama_set_name: RedPajamaWikipedia


Mega Sale! Save 28% on the Envysun Lawn Sprinkler 2 Pack Automatic 360 Rotating Sprinklers for Garden, Adjustable Water Irrigation System with Three Arm and Metal Weighted Base, Brass Water Outlet by Envysun at The Mirror Project. Hurry! Limited time offer. Offer valid only while supplies last.
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GELD, old Eng. law. It signifies a fine or compensation for an offence; also, rent, money or tribute.
Pilansberg was listed in the Racing Post as a colt on the day he won a novice hurdle at Fakenham last month but had been gelded.
John Henry (who was gelded because he was too cantankerous, ran for a $20,000 claiming price in New Orleans and went on to win Eclipse Awards each year from 1980 to '84) has become the archetype.
Red Hammer is a very good-looking gelding and maybe not coincidentally is by the same sire as Peace And Co [Falco], while Khezerabad has just been gelded.
Declan's Moon, undefeated in five races and the early favorite for the Run for the Roses, careened off that road Friday when trainer Ron Ellis revealed that the gelded son of Malibu Moon has a hairline fracture of the left knee and will be out of training for at least 60 days.
Trainers are required to notify the office of the Racing Calendar five days before any horse runs after being gelded.


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Bollywood actress Vidya Balan doesn't like to play favorites
By Bengali Movie / January 28, 2010
Maverick filmmaker Vishal Bhardwaj hasn't directed Ishqiya, but could the film have been churned out better had Vishal Bhardwaj himself directed it, instead of his protégé Abhishek Chaubey, who is debuting with this film?
This million dollar question was aptly put to rest by Vidya who plays the protagonist in Ishqiya, as she promptly replies, "It wouldn't have been better or worse, but it would have been different. But Abhishek Chaubey has made a wonderful film."
She continues, "Vishal's interpretation and presentation would be different. So I don't think I can compare at all. Though credit must go to Vishal that being a director himself and having co-written Ishqiya he didn't interfere in the film. We have an opinion on everything, but Vishal had complete confidence in Abhishek. It is such a nice marriage of minds."
The film is due for release on 29th January 2010.
-Anil A Kanojia/ Sampurn Media
Richa, Nikhil play thieves in Tamanchey
By Calcutta Tube Team / June 28, 2012
Arjun Kapoor bags '2 States'
'Slap Bonanza' on Pavitra Rishta sets
Sheeba Chaddha enters Hitler Didi
Ajay Devgan, Kajol in female foeticide film
Congress brings various NRK groups under one umbrella
Ram Gopal Varma and Mohanlal to work together again in COMPANY 2


Sarah Moore and Dustin Grant, former stars at Greene Central, were married earlier this summer in Key West, Florida. Friends and family gathered at the Shine Fire Department on Saturday, August 31st to congratulate the couple. Moore played on the 2001, 2002, 2003, and 2004 teams that reached the state finals. Grant was a starter on the 2005 state finalist men's team.


VICTORVILLE:(VVNG.com)- On August 27th at 10:36 a.m., Sheriff's dispatch received a report of a man who was allegedly held hostage for four days and had escaped from a home on the 13000 block of Baylor Drive in Victorville.
The alleged victim told deputies that he managed to escape from the garage of the home. The victim told the officers that he was held hostage since he recently came into some money and the suspects had detained him to demand ransom from his family.
While speaking with the hostage victim, the alleged suspects drove by the residence, prompting officers to detain and question them. Upon apprehension the home was searched finding a large amount of cocaine, cash and an arsenal of illegal weapons with ammunition. Some of the weapons found in the home included a AR-15 with 2 loaded magazines, an AK-47 loaded with 30 rounds of ammunition, a Cobray MAC-11 with 27 rounds, among several other weapons.
Upon further investigation, the hostage (victim) was found to not be a victim and was then arrested for filing a false police report. The suspects were arrested for the illegal weapons, illegal drugs and the cash from sale of drugs.
There is an ongoing investigation in this case, if you have any further information that may assist authorities in this investigation you are encouraged to call (760)241-2911 or you can leave an anonymous tip at 1-800-782-7463.


IL-15 drives chronic inflammation in several human diseases. We have recently shown that IL-15 inhibits the immunosuppressive effects of TGF-β through blockage of the Smad3-signaling pathway. Data pointing to reciprocal interactions between TGF-β and CD4+ regulatory T cells led us to investigate the impact of IL-15 on the de novo generation and function of regulatory T cells in humans. Our data indicate that IL-15 does not counteract, but rather promotes the effect of TGF-β on the de novo generation of regulatory T cells (Treg). Thus, in the presence of TGF-β, IL-15 enhanced the acquisition of regulatory functions by CD4+CD25− T cells stimulated by anti-CD3 and anti-CD28 Abs. In contrast, IL-15 impaired the functions of Tregs by acting on effector CD4 and CD8 T cells. Accordingly, in the presence of IL-15, proliferation and IFN-γ production by peripheral CD4 and CD8 T cells could not be efficiently inhibited by Tregs. IL-15-induced resistance of effector T cells to Tregs resulted from activation of the PI3K signaling pathway but did not involve the rescue of effector T cells from apoptosis. Altogether, these data point to the ambiguous role of IL-15 in the control of Treg functions. This dual role may be instrumental to mount rapid but transient proinflammatory immune responses against pathogens but may become deleterious in situations associated with protracted IL-15 over-expression.
Interleukin-15 plays pleiotropic functions at the interface between innate and adaptive immunity and participates both in homeostatic and proinflammatory regulation of the immune system. IL-15 is critical for the differentiation and/or survival of several immune cell subsets, including NK cells, memory CD8 T cells, and intraepithelial lymphocytes. IL-15 is also a potent inducer of the effector phase of the immune response, notably by stimulating dendritic cell maturation as well as NK and CD8 T cell cytotoxicity (1, 2).
We have recently shown that IL-15 impairs the immunosuppressive effects of TGF-β through inhibition of the Smad3 signaling pathway (3), which plays a key role in tipping the immune balance toward tolerance (4, 5). Because IL-15 is secreted early in response to intracellular pathogens (6), its inhibitory effect on TGF-β-mediated regulation may further promote protective effector immune responses. Conversely, persistent expression of IL-15, observed in several autoimmune or chronic inflammatory diseases (7), may durably impair the regulatory functions of TGF-β and thereby create a vicious circle perpetuating inflammation.
TGF-β plays a pivotal role in maintaining immune homeostasis via the direct control of activation, proliferation, differentiation, and survival of immune T cells but also via its effects on regulatory T cells (Treg)4 (8, 9). Although not required for the thymic development of natural occurring CD4+CD25+FOXP3+ Treg, TGF-β helps the in vitro and in vivo de novo generation of induced Treg from naive CD4+CD25− T cells (reviewed in Ref. 10 and 11). TGF-β is necessary in the periphery for the maintenance of murine Treg and sustains their suppressor functions and FOXP3 expression (12, 13, 14). The direct contribution of TGF-β in their suppressive functions remains, however, controversial. In most in vitro experiments TGF-β is not essential for the suppressor functions of Treg (15, 16). Yet, a role of membrane-bound TGF-β has been occasionally reported (17). Data obtained in vivo are more contentious. Antagonizing TGF-β abolished the therapeutic effect of murine Treg in some but not all in vivo models (18, 19, 20, 21). The role of Treg-derived TGF-β was important in one model implicating induced Treg (20) but not in other models depending on naturally occurring Treg where functional TGF-β was produced by another cellular source (19, 21). Notably, TGF-β was necessary to stimulate the production of IL-10 by the subset of Treg present in the intestine (22).
Our recent demonstration that IL-15 exerts an inhibitory effect on TGF-β signaling led us to investigate the impact of IL-15 on the de novo generation and/or function of CD4+CD25+ Tregs.
Peripheral samples were from healthy volunteers donors. Informed consent was obtained from all patients before the study. All experiments were approved by local ethics committee.
In all in vitro assays, cells were cultured in RPMI 1640 medium supplemented with 10% AB human serum (Sigma-Aldrich), 1% sodium pyruvate, 1% non essential amino acids, 1% HEPES buffer, 5 × 10−5 M β-ME, and 40 μg/ml gentamicin (Invitrogen).
Purified recombinant human IL-15, IL-2, and TGF-β (R&D Systems) and mAbs anti-human CD3 (UCHT1) and CD28 (BD Biosciences) were used for in vitro lymphocyte culture. The following inhibitors of signaling pathways were used: JAK3 inhibitor I (Calbiochem) at 100 μM, inhibitor of JNK (SP600125; BIOMOL International) at 10 μM, inhibitor of p38 MAPK (SB203580; Calbiochem) at 1 μM and inhibitors of PI3K/Akt pathway, Wortmaninn (Sigma-Aldrich) at 0.5 μM, and Ly294002 (Sigma-Aldrich) at 20 μM.
The following mAbs were used for flow cytometry analysis: FITC- and Cy-Chrome-conjugated anti-human CD3, CD4, and CD8 (BD Biosciences); PE-conjugated anti-human CD45RA, CD45RO, phospho-Akt (pT308) (BD Biosciences); anti-human CD25 (Miltenyi Biotec); and anti-human FOXP3 (FOXY, eBiosciences or 259D/C7, BD Biosciences).
PBMC were isolated on Ficoll-Hypaque gradient and lymphocyte subsets were separated using magnetic beads (CD3, CD8, and anti-PE microbeads, and CD4+CD25+ Regulatory T cell Isolation Kit, Miltenyi Biotec). For CD3+, CD8+, CD4+CD25−, CD4+CD45RA, CD4+CD45RO, CD8+CD45RA, and CD8+CD45RO isolated subsets, purity ranged from 90 to 98%. CD4+CD25+ Tregs were collected with a purity ranging from 67 to 84%.
For de novo generation of regulatory T lymphocytes, purified CD4+CD25− peripheral T lymphocytes were stimulated for 5 days in 24-well plates (106/well) with plate-bound anti-CD3 Ab (1 μg/ml) and soluble anti-CD28 Ab (1 μg/ml) in the presence or not of TGF-β (0.5 or 10 ng/ml) and/or IL-15 (10 ng/ml) and/or IL-2 (300 UI/ml).
The suppressive functions of Tregs were tested in coculture experiments in 96-well plates coated with 0.5 μg/ml UCHT1 (Life Technologies). CD4+CD25−, CD3+, CD8+, or naive and memory T subsets (105/well) were plated and added with 1 or 0.25 × 105/well of autologous induced or natural CD4+CD25+ T cells in a final volume of 200 μl for 4 to 5 days.
For proliferation studies, uptake of [3H]thymidine (Amersham Biosciencs) was measured 18 h after adding 0.4 μCi/well. Cells were harvested and radioactivity was counted in a scintillation counter.
For analysis of apoptosis, cells were stained with FITC-annexin V and propidium iodide (TACS Annexin V-FITC; R&D Systems) according to the manufacturer's instructions and the percentage of apoptotic cells positive both for Annexin V and propidium iodide was determined by flow cytometry. To analyze apoptosis of effector cells during coculture with Treg, responder cells were stained before culture with CFSE (Molecular Probes) at a concentration of 0.5 μM. After 4 days, cells were harvested, labeled with PE-Annexin V (BD Biosciences) according to the manufacturer's protocol and the percentage of apoptotic Annexin V+ cells was determined by flow cytometry.
For IFN-γ detection, capture ELISA was performed on supernatants of cells using Human IFN-γ ELISA Set (BD Biosciences) according to manufacturer's instructions. The limit of detection was 23 pg/ml.
Cells (2 × 105) were incubated with FITC, PE, or PE-Cy5.5 conjugated mAbs to human CD3, CD4, CD8, CD25, CD45RA, and CD45RO Abs for 20 min at 4°C. For intracellular FOXP3 detection, cells stained with FITC-conjugated mAb to CD25 were fixed, permeabilized using eBioscience FOXP3 Staining Buffer Set (eBioscience) or Human FOXP3 Buffer Set (BD Biosciences), and labeled with PE-conjugated anti-human Foxp3 (Biosciences or BD Biosciences) or control isotype (BD Biosciences). For intracellular phospho-Akt detection, cells were fixed and permeabilized using Fix Buffer I and Perm Buffer III and labeled with PE-conjugated anti-human phospho-Akt or control isotype (BD Biosciences). For intracellular IFN-γ detection, cells were treated with GolgiStop for the last 6 h of culture, fixed, and permeabilized using BD Cytoperm/Cytofix plus kit and labeled with PE-conjugated anti-human IFN-γ or control isotype (BD Biosciences). Analyses were performed with a FACS Vantage flow cytometer using the CellQuest software (BD Biosciences).
Whole-cell or nuclear extracts were obtained from stimulated CD3+ T cells using the Nuclear Extract kit (Active Motif) according to manufacturer's instructions. Proteins (20 μg) were separated on an 8% sodium-dodecyl-sulfate (SDS-PAGE) gel, transferred to nitrocellulose membranes, and labeled with mAbs against human NFATc2 (Santa Cruz Biotechnology) or human β-actin followed by HRP-conjugated anti-mouse Ab (Cell Signaling Technology). Visualization was performed using ECL system (ECL plus, Amersham Biosciences).
Values obtained in the different experiments were compared by the nonparametric Mann-Whitney U test. Statistical significance was assigned to a value of p < 0.05.
In vitro generation of CD4+CD25+ Tregs was studied after a 5-day in vitro stimulation of purified CD4+CD25− peripheral T lymphocytes with anti-CD3 and anti-CD28 Abs in the presence or not of TGF-β and/or IL-15. The regulatory function of the generated cells was then evaluated by testing their capacity to inhibit the proliferative response of autologous CD4+CD25− T cells to immobilized anti-CD3 Ab. Stimulation of purified CD4+CD25− peripheral T lymphocytes with anti-CD3 Abs induced the appearance of a very high proportion of cells expressing FOXP3 mRNA and protein but these cells did not exert any suppressive effects (Fig. 1⇓A and data not shown), a finding consistent with the observation that FOXP3 expression can merely reflect T cell activation in humans (23, 24). Adding TGF-β during the 5-day stimulation had no significant effect on the level of expression of FOXP3 protein or percentage of CD25+ FOXP3+ T cells (Fig. 1⇓A). In contrast, the latter cells now demonstrated a significant inhibitory effect on the proliferative response of CD4−CD25− autologous T cells analyzed at day 5 (Fig. 1⇓B). Comparable results were obtained when thymidine uptake was measured at earlier time points (day 3) (data not shown). Adding IL-15 had no significant effect on FOXP3 protein expression compared with cultures containing TGF-β alone (Fig. 1⇓A). Yet, CD25+FOXP3+ T cells generated in the presence of both IL-15 and TGF-β exerted a significantly stronger inhibitory effect on the proliferation of CD4+CD25− T cells than Tregs generated in the presence of TGF-β alone (Fig. 1⇓B). Comparable results were obtained when IL-15 was replaced by IL-2, but exogenous IL-2 did not enhance significantly the effect of TGF-β on the acquisition of regulatory functions (Fig. 1⇓).
IL-15 does not impair in vitro conversion of conventional T lymphocytes into Tregs. Purified CD4+CD25 −peripheral T lymphocytes were stimulated with immobilized anti-CD3 Ab (1 μg/ml) and soluble anti-CD28 Ab (1 μg/ml) in the presence or not of TGF-β (10 ng/ml) and/or IL-15 (10 ng/ml) and/or IL-2 (300 UI/ml). A, Intracytoplasmic FOXP3 was analyzed by flow cytometry. The analysis was performed on the lymphocyte gate. Results from one representative of three experiments are shown. B, The suppressive function of the generated cells was evaluated by testing their capacity to inhibit the proliferative response of autologous CD4+CD25− T cells stimulated with immobilized anti-CD3 Ab (0.5 μg/ml) at a ratio 1:1 of regulatory to responsive T cells. Results are from three independent experiments. ∗, p < 0.05 when compared with coculture with unstimulated CD4+CD25− cells. ∗∗, p < 0.05 when compared with coculture with CD4+CD25− cells stimulated with TGF-β.
To test the impact of IL-15 on the suppressive capacities of natural CD4+CD25+ Treg, the latter cells were isolated from peripheral blood of healthy donors and their suppressive effects on the proliferation and secretion of IFN-γ were tested on autologous CD4+ or CD8+ T cells stimulated via their TCR, in the presence or absence of IL-15. As shown in Fig. 2⇓A, adding CD4+CD25+ T cells to CD8+ or CD4+CD25− purified T cells at a ratio of 1:1 regulatory to responsive T cells inhibited their proliferative response to immobilized anti-CD3 by 50%. CD4+CD25+ cells had an even more striking effect on the production of IFN-γ, which was abolished in CD8+ autologous T cells at both ratios of 1:1 and 1:4 and abolished or reduced by 80% in CD4+ T cells. In contrast, in the presence of 10 ng/ml IL-15, CD4+CD25+ T cells lost their inhibitory effect on the proliferation and production of IFN-γ (Fig. 2⇓, A and B). Because IL-15 could stimulate the proliferation of CD4+CD25+ T cells, the lack of inhibitory effect of the latter cells on T cell proliferation in the presence of IL-15 may be partly ascribed to the break of their anergic state (Fig. 2⇓A, last columns). Yet, Peluso et al. (25) have recently shown that IL-15 reverted the suppressive effects of CD4+CD25+ T cells on the proliferation of CFSE labeled CD4+ and CD8+ lymphocytes. Our results concerning IFN-γ secretion confirm that the effect of IL-15 cannot only be ascribed to a break of anergy of the Treg. Thus, the inhibitory effect of Tregs on IFN-γ secretion was reverted by IL-15 (Fig. 2⇓B), while IL-15 did not induce any IFN-γ secretion in CD4+CD25+ T cells stimulated or not with anti-CD3 (data not shown). Collectively, these data suggest that IL-15 can directly interfere with the regulatory function of CD4+CD25+ cells and/or the sensitivity of responder cells to Tregs.
IL-15 impedes the suppressive capacities of natural CD4+CD25+ Tregs. CD4+CD25+ Treg were isolated from peripheral blood of healthy donors and their suppressive effects were tested on autologous CD4+CD25− or CD8+ T cells stimulated with immobilized anti-CD3 in the presence or absence of IL-15. A, Proliferative responses were assessed by [3H]thymidine uptake. Results are from three independent experiments. B, IFN-γ secretion was evaluated in supernatants of cocultures by an ELISA test. Results are representative of three independent experiments. ∗, p < 0.05 when compared with control cocultures with CD4+CD25− cells.
Consistent with our data indicating that IL-15 does not impair the in vitro generation of CD4+CD25+ Treg, several studies have recently shown that IL-15 can expand natural CD4+CD25+ regulatory T lymphocytes while preserving their optimal suppressive potency (26, 27). These results suggested that IL-15 did not exert its inhibitory effect by acting directly on Treg but rather by affecting the sensitivity of responder T cells to the suppressor effects of Treg. To test this hypothesis, CD4+ and CD8+ peripheral lymphocytes were incubated or not with IL-15 during 24 h. After extensive washings, the cells were stimulated with anti-CD3 Ab and the inhibitory effects of CD4+CD25+ Treg were assessed.
Proliferation experiments showed that the suppressive effect of Tregs on peripheral T cell proliferation was significantly reduced when conventional lymphocytes had been first incubated with IL-15, an effect more particularly obvious in the CD8+ subset (Fig. 3⇓A). In addition, preincubation with IL-15 prevented very efficiently the suppressive effect of Treg on IFN-γ secretion in both CD4+ and CD8+ T lymphocytes (Fig. 3⇓, B and C). At a 1:1 ratio, the inhibitory effect of Treg cells on IFN-γ secretion, assessed either by intracytoplasmic staining (Fig. 3⇓B) or by ELISA (Fig. 3⇓C) in the culture supernatants was reduced drastically when lymphocytes had been previously incubated with IL-15 (p < 0.001). Altogether, these data demonstrate that IL-15 renders conventional CD4+ and CD8+ T lymphocytes resistant to the suppressive effects of CD4+CD25+ Treg.
IL-15 renders conventional T lymphocytes resistant to the suppressive effects of natural CD4+CD25+ Tregs. CD4+ and CD8+ peripheral lymphocytes were incubated or not with IL-15 (10 ng/ml) during 24 h. Cells were then washed and stimulated with anti-CD3 Ab (0.5 μg/ml) and the inhibitory effects of peripheral CD4+CD25+ Treg were assessed on proliferation and IFN-γ production. A, Proliferative responses were measured at day 5 by [3H]thymidine uptake. Results are from six independent experiments. ∗, p < 0.05 when compared with the condition without IL-15 preincubation. B, Intracytoplasmic IFN-γ staining was analyzed by flow cytometry in the lymphocyte gate. Results from one of three independent experiments are shown. Numbers indicate the percentages of positive IFN-γ cells. ∗, p < 0.05 when compared with control cocultures with CD4+CD25− cells. C, IFN-γ secretion was evaluated in supernatants of cocultures by an ELISA test. Results are from three independent experiments. ∗, p < 0.05 when compared with control cocultures with CD4+CD25− cells.
Recent data indicated that conventional T cells from NFATc2−/− mice are unresponsive to CD4+CD25 Treg suppression (28). We hence tested whether IL-15 acts on responder cells by modulating NFATc2 pathway. IL-15 had no effect either on NFATc2 protein expression or on NFATc2 induced translocation (Fig. 4⇓A). The mechanism implicated in IL-15 induced resistance of responder cells to suppression was therefore addressed using inhibitors of the major signaling pathways activated by IL-15. As shown in Fig. 4⇓B, only inhibitors of the PI3K/Akt pathway (Wortmaninn and Ly294002), added during the 24-h preincubation of responder cells with IL-15, restored the capacity of the latter cells to respond to the inhibitory effect of Treg on both T cell proliferation and IFN-γ secretion. Notably, in the absence of Treg, responder cells preincubated with IL-15 and PI3K inhibitors exhibited a proliferative response and IFN-γ secretion comparable to that of control responder cells, eliminating a direct effect of these inhibitors on their effector functions (data not shown). Collectively, these data indicate that activation of the PI3K pathway by IL-15 renders T lymphocytes resistant to the inhibitory effects of Treg.
IL-15 acts on conventional T lymphocytes through activation of PI3K pathway. A, Peripheral CD3+ T lymphocytes were incubated or not with anti-CD3 (5 μg/ml) in the presence or absence of IL-15 (10 ng/ml) during 24 h. Expression of NFATc2 was evaluated by immunoblotting on total cell extracts (upper panel) and nuclear translocation of NFATc2 was assessed on nuclear extracts (lower panel). B, CD3+ peripheral lymphocytes were incubated or not with IL-15 (10 ng/ml) in the presence JAK3 inhibitor ((-) JAK3), SP600125 (SP), SB203580 (SB), Wortmaninn (Wort), or Ly294002 (Ly) during 24 h. The cells were then washed and stimulated with anti-CD3 Ab and the inhibitory effects of CD4+CD25+ Treg on proliferation (upper panel) and IFN-γ secretion (lower panel) were assessed. Results are representative of three independent experiments. ∗, p < 0.05 when compared with control cocultures with CD4+CD25− cells.
The PI3K can also be activated by the related cytokine IL-2 which shares the β and γ-chains of its receptor with IL-15 (29). Yet, the latter chains and the private α-chains of IL-2 and IL-15 receptors are differentially expressed on T cell subsets (30). The effects of IL-15 and IL-2 were therefore compared on CD4+ and CD8+ naive and memory T cell subsets. IL-15 rendered both CD8+ T cell subsets resistant to the inhibitory effects of Treg and had a minor but significant effect on CD4+ subsets. In contrast, IL-2 acted preferentially on memory T lymphocytes, notably CD4+. Comparable results were obtained when analyzing proliferation (Fig. 5⇓A) and IFN-γ secretion (Fig. 5⇓B) by responder cells. Notably, the differential effects of IL-2 and IL-15 in the various T cell subsets corroborated their capacity to induce PI3K activation as demonstrated by flow cytometry analysis of Akt phosphorylation (Fig. 5⇓C).
IL-15 acts preferentially on T CD8+ lymphocytes. CD4CD45RA, CD4CD45RO, CD8CD45RA, and CD8CD45RO lymphocytes were incubated or not with IL-15 (10 ng/ml) or IL-2 (300 IU/ml) during 24 h. A, Cells were then washed and stimulated with anti-CD3 Ab (0.5 μg/ml) and the inhibitory effects of peripheral CD4+CD25+ Treg were tested at ratio 1:1. Proliferative responses were assessed at day 4 by [3H]thymidine uptake. Results are from three independent experiments. ∗, p < 0.05 when compared with the condition without IL-15 preincubation. B, IFN-γ secretion was evaluated in supernatants of cocultures by an ELISA test. Results are from three independent experiments. ∗, p < 0.05 when compared with control cocultures with CD4+CD25− cells. C, Intracytoplasmic staining of p-Akt was monitored by flow cytometry. Results from one of three independent experiments are shown.
Reigniting an old debate, Pandiyan et al. (31) have recently suggested that CD4+CD25+ Treg exert their suppressive effects by depriving effector T cells of life-supporting IL-2, thereby inducing apoptosis of effector T cells. These authors further showed that the effect of IL-2 involved the PI3K/Akt pathway and that IL-2 could be replaced by other γc cytokines, including IL-15. We were, however, unable to demonstrate that suppression of effector cells was associated with their significant apoptosis (Fig. 6⇓). Thus, no increase in the total percentage of apoptotic cells stained by propidium iodide and annexin V was observed in cocultures of responder and Treg cells compared with responder cells stimulated alone (Fig. 6⇓A), despite the strong inhibitory effect of Treg on T cell proliferation (Fig. 6⇓, B and D). To further confirm the lack of Treg-induced apoptosis of responder cells, the latter cells were labeled before culture with CFSE. CFSE staining decreased in a large fraction of responder cells stimulated alone but not in the presence of Treg, confirming their suppressive effect (Fig. 6⇓B). The percentage of Annexin V+ CFSE+ cells however did not increase in the presence of Treg (Fig. 6⇓C). Furthermore, inhibitors of PI3K failed to induce any significant apoptosis in CD4+CD25− responsive cells stimulated by anti-CD3 and anti-CD28 Abs (Fig. 6⇓A) while they exerted a strong inhibitory effect on their proliferation (Fig. 6⇓D).
Tregs do not induce apoptosis of responder cells. A, CD4+CD25− cells were stimulated by immobilized (0.5–1 μg/ml) anti-CD3 Ab and soluble (1 μg/ml) anti-CD28 Ab in the presence or not of CD4+CD25+ cells or in the presence of the PI3K inhibitor Ly294002 (Ly) and the percentage of apoptotic cells positive for both annexin V and propidium iodide was assessed after a 5-day culture. Data are representative of three distinct experiments. B, CD4+CD25− T cells were labeled with CSFE and left either not stimulated (NS, dotted line) or stimulated with immobilized anti-CD3 (0.5 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs for 3 days, in the presence of either unlabeled CD4+CD25− cells (S, gray-filled histogram) or unlabeled CD4+CD25+ cells at 1:1 ratio (S, thick full line). The same result was obtained with soluble anti-CD3 (0.75 μg/ml) and anti-CD28 (4 μg/ml) Abs. C, CD4+CD25− T cells were labeled with CSFE and left either not stimulated (NS, left panel) or stimulated with soluble anti-CD3 (0.75 μg/ml) and anti-CD28 (4 μg/ml) Abs for 3 days, in the presence of either unlabeled CD4+CD25+ cells (middle panel) or unlabeled CD4+CD25+ cells at 1:1 ratio (right panel). Annexin V and CFSE staining were compared after a 3-day culture in a gate including all cells. Numbers in quadrants are the percentages of annexin V positive cells. Similar results were obtained using immobilized anti-CD3 (0.5–1 μg/ml) and soluble anti-CD28 (1 μg/ml). D, Proliferation of CD4+CD25− responder cells stimulated by immobilized anti-CD3 (0.5–1 μg/ml) and soluble anti-CD28 (1 μg/ml) Abs in the presence or not of Ly and cocultured or not with CD4+CD25+ cells was assessed by [3H]thymidine uptake in the same experiment. Data are representative of four distinct experiments.
Our data point out to the ambiguous role of IL-15 in the control of Treg functions and demonstrate that IL-15 promotes the effect of TGF-β on the de novo generation of Tregs but also renders peripheral CD4 and CD8 T cells resistant to the suppressive functions of Treg through activation of the PI3K signaling pathway.
FOXP3 expression was induced in human CD4+CD25− T cells stimulated by anti-CD3 and anti-CD28 Abs which did not exhibit any Treg functions, confirming previous observations that, in humans, FOXP3 is not a reliable marker of human suppressor cell activity and can simply reflect T cell activation. In keeping with the presence of STAT5 binding sites in the promoter and in one enhancer of the FOXP3 gene (32), recent data have shown that induction of FOXP3 in stimulated human T cells depends on the activation of STAT5 by the γc cytokines IL-2, IL-7, or IL-15 (33). Yet, STAT5-driven induction of FOXP3 was not sufficient to acquire regulatory properties. This result is coherent with our data showing the lack of suppression by CD4+CD25− T cells activated by anti-CD3/CD28 added or not with IL-15 or IL-2. By contrast, adding TGF-β during the stimulation of CD4+CD25− T cells by anti-CD3 and anti-CD28 Abs resulted in the induction of regulatory properties. In mice, the presence of exogenous TGF-β is necessary during the stimulation by anti-CD3/CD28 Abs to induce both FOXP3 and regulatory functions, and TGF-β plays a nonredundant role with IL-2 in the induction of FOXP3 (11, 34, 35). In humans, adding exogenous TGF-β may not be necessary as Tran et al. observed that 30% of naive CD4+ T cells expressed FOXP3 in response to TCR stimulation alone. Yet, induction of FOXP3 was prevented by neutralizing TGF-β, and adding TGF-β further increased the proportion of FOXP3+ cells to 80%, confirming the role of this cytokine (35, 36). In our study, FOXP3 mRNA levels increased only very modestly in the presence of exogenous TGF-β and there was no significant increase in level of expression of FOXP3 or proportion of FOXP3+ cells in CD4+ T cells as compared with cells only stimulated by anti-CD3/CD28 Abs. The impact of exogenous TGF-β may depend on the amounts of TGF-β produced in response to TCR stimulation and/or on the different concentrations of TGF-β present in the serum used for the assay (35). In mice, TGF-β was suggested to stimulate FOXP3 transcription through Smad3 binding to an inverted repeat sequence situated in a 120-bp enhancer fragment on the murine FOXP3 gene (37). This result may appear paradoxical given the synergistic effect of IL-15 and TGF-β on the de novo generation of human Treg observed in the present study and our previous data indicating that IL-15 inhibits Smad3-binding to the promoters of TGF-β target genes in human T cells (3). Yet, Von Boehmer and Nolting (32) have stressed the role of additional Smad3-independent mechanism(s) in TGF-β-dependent induction of FOXP3 transcription in murine T cells. Furthermore, our results suggest that TGF-β promotes the generation of human Treg independently of FOXP3 induction. Altogether, these results point out to the likely role of Smad3-independent effect(s) of TGF-β in the generation of Treg, notably in humans.
Contrasting with its promoting effect on the in vitro generation of Treg, IL-15 hampered the capacity of conventional CD4+ or CD8+ T cells to respond to the immunosuppressive effects of natural Treg. IL-15 acted preferentially on CD8+ T cells either naive or memory while the related cytokine IL-2 had a preferential effect on memory lymphocytes, particularly CD4+. These results are in keeping with the known distribution of IL2 and IL-15 receptor chains on T cell subsets (30). We found no evidence that IL-15 may modulate NFATc2 recently involved in the capacity of murine effector cells to respond to Treg (28). In contrast, our results point to the central role of the PI3K pathway. Phospho-Akt was induced after IL-15 stimulation, particularly in CD8+ T lymphocytes and inhibition of PI3K pathway impeded IL-15 effects on responder cells. The key role of PI3K pathway in inducing resistance to suppressive effects of Treg is in keeping with the recent hypothesis put forward by Wolfert et al. (38). Based on the observation that several mouse models of autoimmunity either spontaneous (NOD, MLR/Mp, etc.) or in engineered mice (Cbl-b−/−, TRAF6−/− etc.) show simultaneous resistance of effector T cells to Treg and hyperactivity of the PI3K/Akt pathway (38), these authors postulated that the activation status of the PI3K/Akt pathway in conventional T cells is a primary determinant of cell sensitivity to Treg-mediated suppression. The role of PI3K/Akt pathway in regulating Treg-mediated suppression remains, however, unclear. Considering the recent data obtained by Pandiyan et al. (31), we tested whether CD4+CD25+ Treg exert their suppressive effects by inducing apoptosis of effector T cells and whether IL-15, alike other γc cytokines may impair this effect by activating PI3K/Akt pathway. We were, however, unable to demonstrate that suppression of effector cells was associated with their significant apoptosis. Furthermore, inhibitors of PI3K pathway failed to induce any significant apoptosis in stimulated CD4+CD25− responsive cells. It is unclear why our data in humans differ from recent data in mice. Yet, our results are consistent with previous studies performed either on mice or humans. Accordingly, Duthoit et al. (39) observed that stimulated CD4+CD25− T cells failed to up-regulate IL-2 mRNA in the presence of Treg, and that exogenous IL-2 did not break the mitotic blockage of stimulated CD4+CD25− T cells cocultured with Treg cells while promoting their survival. Furthermore, Fontenot et al. (40) showed that Foxp3+ CD4+ T cells from IL2ra−/− mice possess equivalent regulatory functions as their counterparts in wild-type mice. Finally, Oberle et al. (41) demonstrated that in humans, suppression of IL-2 and IFN-γ mRNA by Treg cells was not dependent on IL-2 consumption. Besides an effect based on sopping IL-2, a plethora of mechanisms have been evoked in mediating immunosuppression by Treg (42, 43). Future work will be necessary to decipher which of these mechanisms implicate(s) the PI3K/Akt pathway. Finally, it may be stressed that our study analyzes how IL-15 interferes with the direct effect of Treg on effector T cells. Recent studies indicate that Treg can exert their suppressor effect by inhibiting dendritic cell maturation and Ag-presentation (44). Because IL-15 is a potent inducer of both functions (45), it will be interesting to assess whether IL-15 might also prevent this effect of Treg.
In conclusion, our in vitro data point to the ambiguous role of IL-15 in the control of Treg functions. On the one hand, IL-15 can promote the generation of induced FOXP3+ Treg. In contrast, IL-15 can render effector cells either CD4+ or CD8+ unresponsive to the regulatory effect of FOXP3+ Treg. The latter effect of IL-15 is reminiscent of IL-6. Thus, IL-6, released by TLR-activated dendritic cells, rendered responder T cells refractory to the suppressive effect of Treg and this effect was suggested to promote a rapid and efficient immune response against pathogens (46). IL-15 is one cytokine rapidly but transiently induced in response to intracellular pathogen (47). The dual effect of IL-15 demonstrated in the present study may be beneficial to promote a rapid inflammatory response to pathogens and simultaneously prepare its subsequent down-regulation after elimination of the pathogen. Conversely, the impact of IL-15 on responding T cells may become deleterious in situations associated with protracted IL-15 overexpression, preventing the return to homeostasis and sustaining inflammation.
Overexpression of IL-15 has been observed across a range of proinflammatory and autoimmune-related human diseases including psoriasis, rheumatoid arthritis, and celiac disease (7, 48). Our data indicating that IL-15 counteracts the suppressive action of Treg point to their probable dysfunction in diseases associated with IL-15 disordered overexpression. This effect of IL-15 together with its inhibitory effect on Smad3 signaling, provides further insight on how protracted IL-15 production may contribute to the loss of immune tolerance and further support therapeutic strategy targeting IL-15 in diseases associated with IL-15 deregulation.
↵1 This work was supported by grants from INSERM, Fondation Princesse Grace, Association pour la Recherche Contre le Cancer, Ligue Contre le Cancer, Agence Nationale pour La Recherche, Fondation pour la Recherche Médicale-Equipes 2007, and Association Française des Intolérants au Gluten. N.B.H. was supported by a grant from Le Réseau International des Instituts Pasteur. N.M. is supported by fellowships from Zonmw AGIKO and from the Nutricia Research Foundation.
↵2 M.B.A. and N.B.H. contributed equally to the work.
↵4 Abbreviation used in this paper: Treg, regulatory T cell.
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#Serveinmylanguage is gaining ground on social media as more south Indian customers bemoan the indifference of banks to their language problems.
BENGALURU: #Serveinmylanguage is gaining ground on social media as more south Indian customers bemoan the indifference of banks to their language problems.
From ATMs to deposit slips, withdrawal challans and call centres, most public and private banks feel that service in Hindi and English should suffice their customer base –– Indians who converse in 22 major languages and 720 dialects.
Organizations like Campaign for Language Equality And Rights (CLEAR), Maharashtra Navnirman Sena, Bangla-O-Bengali, and political parties such as Tamil Nadu's DMK have been pressing for changes in the banking system in recent months. Facebook forums such as Promote Linguistic Equality: Hindi is not the national language and Twitter handles like Voice of South regularly share photographs, screenshots and narrate anecdotes of consumers facing difficulties.
It reeks of elitism, apathy to change and indifference to the needs of the working class," said Harvinder Singh, general secretary of the All India Bank Officers' Confederation. Language barriers prove particularly difficult when having to block a stolen or lost debit card or when attempting a more complicated banking transaction like creation of a fixed deposit with a minor as nominee, guardian address and other details.
For loans, customers on Twitter feel it's complicated enough to figure out the labyrinth of terms and conditions, EMI structure, loan tenure and interest rates, without the added burden of doing it in an unfamiliar language. "Most PSBs like State Bank of India, Punjab National Bank have a dual-language approach, communicating only in Hindi and English making it difficult for south Indian customers," says N Muruganatham, a customer of SBI.
When it comes to ATM usage or malfunction, customers say this Hindi-English dual system is frustrating. Particularly, when the ATM machine has not dispensed the cash or their debit card gets stuck, customers find it difficult to decipher which of the toll-free numbers they are supposed to be calling. "Not everyone is fluent in English. Many elderly people and blue-collar workers have only 5th or 6th grade working understanding of the English language, making it difficult for them to report an ATM emergency," said Mohamed Irfan, a customer. Irfan also points to the fact that Australia's Commonwealth Bank provides service to Tamils, despite the total Tamil migrant population in Australia being just 1.5 lakh.