38% of Tottenham's conceded goals occurred after the 75th minute in Premier League.
Everton have scored in 100% of their home matches in Premier League.
62% of Tottenham's points have been earned away in Premier League.
Tottenham were leading at halftime in 65% of their matches in Premier League.
Tottenham have scored in each of their last 7 matches in Premier League.
75% of Everton's points have been earned at home in Premier League.
Tottenham have won 76% of their matches in Premier League.
Tottenham have won 80% of their away matches in Premier League.
Tottenham have scored 61% of their goals away in Premier League.
Tottenham have scored in each of their last 10 away matches in Premier League.
Huesca have failed to win in their last 15 matches in La Liga.
62% of Valencia's matches ended up in a draw in La Liga.
Leganes have been undefeated in their last 6 matches in La Liga.
50% of FC Sevilla's conceded goals occurred after the 75th minute in La Liga.
Rayo Vallecano conceded at least 1 goal in 88% of their home matches in La Liga.
88% of Valencia's matches had under 2.5 goals in La Liga.
Huesca have lost 75% of their away matches in La Liga.
Leganes conceded at least 1 goal in 71% of their home matches in La Liga.
FC Sevilla have scored in 75% of their away matches in La Liga.
FC Sevilla have drawn their last 3 away matches in La Liga.
Rayo Vallecano have scored 73% of their goals at home in La Liga.
61% of Rayo Vallecano's conceded goals were conceded at home in La Liga.
67% of Leganes's points have been earned at home in La Liga.
FC Sevilla have been undefeated in their last 7 matches in La Liga.
Rayo Vallecano have conceded the opening goal in 75% of their matches in La Liga.
75% of Levante's matches had over 2.5 goals scored in La Liga.
Levante have scored in 88% of their away matches in La Liga.
Huesca have lost their last 6 away matches in La Liga.
69% of Leganes's matches had under 2.5 goals in La Liga.
63% of Levante's conceded goals were conceded at home in La Liga.
Levante have drawn their last 3 away matches in La Liga.
Valencia conceded at least 1 goal in 75% of their home matches in La Liga.
Huesca conceded at least 1 goal in 100% of their away matches in La Liga.
Levante have been undefeated in their last 5 away matches in La Liga.
Valencia have scored 31% of their goals after the 75th minute in La Liga.
Huesca conceded at least 1 goal in each of their last 16 matches in La Liga.
Levante have scored in each of their last 6 away matches in La Liga.
FC Augsburg conceded at least 1 goal in 100% of their away matches in Bundesliga.
Wolfsburg have scored in 71% of their away matches in Bundesliga.
FC Augsburg conceded at least 1 goal in each of their last 8 matches in Bundesliga.
FC Augsburg conceded at least 1 goal in 86% of their home matches in Bundesliga.
Aston Villa have been undefeated in their last 7 matches in Championship.
28% of Leeds Utd's conceded goals occurred in the first 15 in Championship.
Aston Villa have scored 2 goals or more in each of their last 7 matches in Championship.
Aston Villa have scored in each of their last 7 matches in Championship.
Aston Villa conceded at least 1 goal in 73% of their home matches in Championship.
Leeds Utd have scored in each of their last 11 away matches in Championship.
Leeds Utd have scored in each of their last 16 matches in Championship.
Aston Villa have scored in each of their last 11 home matches in Championship.
Aston Villa have conceded 2 goals or more in each of their last 3 home matches in Championship.
Leeds Utd have won their last 5 matches in Championship.
Aston Villa have scored 2 goals or more in each of their last 4 home matches in Championship.
Padova conceded at least 1 goal in 100% of their away matches in Serie B.
35% of Cosenza's conceded goals occurred after the 75th minute in Serie B.
80% of Lecce's conceded goals occured in the second half in Serie B.
Venezia have scored in each of their last 9 matches in Serie B.
Lecce have scored 65% of their goals away in Serie B.
Padova have lost 75% of their away matches in Serie B.
70% of Venezia's points have been earned at home in Serie B.
67% of Cosenza's matches had under 2.5 goals in Serie B.
Cosenza have scored 31% of their goals after the 75th minute in Serie B.
45% of Lecce's conceded goals occurred after the 75th minute in Serie B.
Padova have scored in 88% of their away matches in Serie B.
Lecce were leading at halftime in 56% of their matches in Serie B.
Padova have scored 67% of their goals away in Serie B.
31% of Padova's conceded goals occurred after the 75th minute in Serie B.
73% of Venezia's matches had under 2.5 goals in Serie B.
Padova have conceded 2 goals or more in each of their last 7 away matches in Serie B.
Venezia have scored in 100% of their home matches in Serie B.
Padova have scored in each of their last 6 away matches in Serie B.
Ajax Amsterdam have won their last 10 matches in Eredivisie.
FC Utrecht have scored in each of their last 10 matches in Eredivisie.
De Graafschap have failed to win in their last 6 matches in Eredivisie.
Vitesse Arnhem conceded at least 1 goal in each of their last 8 matches in Eredivisie.
FC Utrecht have scored in 100% of their home matches in Eredivisie.
De Graafschap have scored 33% of their goals after the 75th minute in Eredivisie.
68% of Vitesse Arnhem's points have been earned at home in Eredivisie.
FC Utrecht were leading at halftime in 56% of their matches in Eredivisie.
Ajax Amsterdam have scored 2 goals or more in each of their last 10 matches in Eredivisie.
De Graafschap have failed to score in their last 4 matches in Eredivisie.
Ajax Amsterdam have scored in each of their last 5 away matches in Eredivisie.
De Graafschap conceded at least 1 goal in 88% of their home matches in Eredivisie.
75% of Ajax Amsterdam's matches had over 2.5 goals scored in Eredivisie.
82% of De Graafschap's points have been earned at home in Eredivisie.
Ajax Amsterdam have scored in each of their last 10 matches in Eredivisie.
De Graafschap have scored 83% of their goals at home in Eredivisie.
Ajax Amsterdam have been undefeated in their last 10 matches in Eredivisie.
De Graafschap have conceded the opening goal in 75% of their matches in Eredivisie.
68% of FC Utrecht's points have been earned at home in Eredivisie.
FC Utrecht have scored 2 goals or more in each of their last 5 home matches in Eredivisie.
Ajax Amsterdam have won 88% of their matches in Eredivisie.
De Graafschap were losing at halftime in 62% of their matches in Eredivisie.
FC Utrecht have scored 2 goals or more in each of their last 4 matches in Eredivisie.
De Graafschap conceded at least 1 goal in each of their last 7 home matches in Eredivisie.
FC Utrecht have won their last 5 home matches in Eredivisie.


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I have sold a property at 1341 CARMEL PLACE in NANOOSE BAY.
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Charming and immaculate are the words of choice to describe this property. This rancher is set on a 0.25 acre lot with views of snow capped mountains and within a short stroll to Beachcomber Park and marina. The lovely backyard is fully landscaped, fenced and has southern exposure. This home offers low maintenance and outstanding location.


May 2019 Vogalonga - A spectacular event attracting worldwide interest from spectators and participants alike.Coop Supermarket Ponte dei Meloni (quite near San Aponal at San Polo 1338a) Convenient if staying at Ca' San Polo - Conad Supermarket - near the Church of the Frari (Rio tera dei Frari - San Polo 2605/A 2605/B) Open Mon-Sat.00 -.00.
Fondaco Dei Tedeschi on the Grand Canal at Rialto, historically used from the 13th Century by German merchants and found just around the corner from the apartment Ca Giulia, and the Fondaco Dei Turchi (now Venices Natural History Museum) also on the Grand Canal and close. Also available as an iPhone app (Tapvenice Eating) to download from iTunes/Apple Store - Venice Botteghe antiques, bijouterie, coffee, cakes, carpets, glassa handbook for self-assured shoppers - by Michela Scibilia - Time Out - Venice: Verona, Treviso and the Veneto - Venice by Jan Morris. Perhaps visit The Church and Campo of San Giacomo Dell Orio Ca Pesaro (International Gallery of Modern Art) - click here (Museums) for a 'Virtual Map/Tour' of the Gallery, or view it here on YouTube Palazzo Mocenigo at San Stae (Costume Museum) - view it here. Perhaps visit The Church of Santi Giovanni e Paolo (Zanipolo) The Scuola Grande di San Marco Museum of History and Medicine (including displays of ancient texts and medical instruments) set in spectacular surroundings. There is a ceremony of the " Wedding with the Sea " in front of the Church. Join in and practice your Italian while getting to know more about some of the superb local delicacies on offer. The area is close to many of the citys major attractions and only a short walk to the district of San Marco via the Accademia Bridge with its spectacular views of the Grand Canal. The tide in the Venetian Lagoon rises and falls twice every day and sometimes many factors overlap and the tide overflows onto some of the streets. 93 minutes long, is reformatted.85/1 ratio. (Open mornings only - the earlier the better -Tuesday to Saturday).
If it does occur while youre in Venice. Venice for the disabled from the popular Tripadvisor travel website which is full of useful information venice venice and tips based on actual traveller experience What is" Yes for further details take a look. And a valid photo ID is required.
A collage of Venice: at the top left is the Piazza San Marco, followed by a view of the city, then the Grand Canal, and (smaller) the interior of La Fenice and, finally, the Island of San Giorgio Maggiore.About Venice Click on a question below to open and read our ick it again to close.
The Secret Itineraries Tour of the Doges Palace. Often decorated in grand and impressive fashion to showcase the wealth and power exerted by its original owners. Convenient if staying at, giudecca and Ca siti casino dove sei iscritto Dei Fiori, a trip down the length of the Grand Canal on the water bus vaporetto. Sporting or musical events taking place in the city each year or maybe youll simply discover that. The Mayor of Venice, degli Armeni, ca Della Bottega Grocery Shop on the Fondamenta Sebastiano Venier at San Vio Dorsoduro 735 santa croce Coop Supermarket Campo San Giacomo dell musica da casinò Orio Santa Croce.
Perhaps, as a family, you would like to take a course in mask decoration at Ca Macana, one of a number of mask making workshops to be found in the Dorsoduro area of the city.A must-see for any children who are fans of the fictional adventurer Indiana Jones, this church exterior is the setting of the library featured in the film Indiana Jones and the Last Crusade.The tour, which needs to be booked in advance and lasts around 1 hour 15 minutes, also visits the Piombi or Leads in which the legendary Giacomo Casanova was imprisoned and from which he famously escaped.
A great tip to keep your child's interest levels up when visiting a museum is to make the museum shop your first point of call.
In this shorter version, many scene have been cut, like the car chase between David Niven and the girls.In fact, the word casino is Italian in origin.Which museums and galleries are considered the most important in Venice?


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You are here: Home / All posts / #BeBoldForChange: Taking stock of gender inequalities in the UK in the face of Brexit
#BeBoldForChange: Taking stock of gender inequalities in the UK in the face of Brexit
By Colette Fagan, Helen Norman and Nina Teasdale Filed Under: All posts, Brexit, British Politics, Growth and Inclusion Posted: March 9, 2017
As the 2017 International Women's Day global theme calls on us to 'be bold for change', here Professor Colette Fagan, Dr Nina Teasdale and Dr Helen Norman take stock of the UK's gender-related policy measures.
Progress towards gender equality has been uneven and often too slow
Since 1957, equality between women and men has been a founding value of the EU and adherence to this principle is a membership condition
The gender aspect of the European Employment Strategy introduced targets to promote gender equality in the labour market, which member states were held accountable against
The World Economic Forum predicts that the gender gap won't close entirely until 2186
It is essential that the existing EU provisions are written into UK law, and built upon
Gender budgeting and gender impact analysis should become a routine tool for policy design as the government navigates the economic challenges
According to Hilary Clinton in one of her first Presidential candidacy speeches in April 2015, 'there has never been a better time in history to be born female'; and without doubt progress has been made. Women are better represented in political office – global figures rising from 11.3% in 1995 to 22.7% in 2015. Record numbers of women are in employment, and they hold a growing proportion of the senior professional and managerial positions, including on corporate boards. Global female participation in tertiary education has improved and has overtaken male participation rates in almost all developed countries and in half of developing countries. And there has been a global commitment to tackle violence against women and girls as part of the UN's Sustainable Development Goals (2015).
But progress towards gender equality has been uneven and often too slow. Looking forward in the UK we now face the uncertainties of Brexit while still grappling with the effects of the economic austerity measures introduced since the 2008 recession.
A step closer to parity?
European Union (EU) membership, as well as initiatives from supra-national bodies including the United Nations, added weight to the efforts of feminists, union members and others who have campaigned to secure progress towards gender equality in the UK. Since 1957, equality between women and men has been a founding value of the EU and adherence to this principle is a membership condition that the UK government signed up to when it joined in 1973.
Gender equality legislation was introduced with the Equal Pay Act (1970), the Sex Discrimination Act (1975) and statutory maternity leave (1975). Further legal reforms, triggered by EU Directives, followed (e.g. Part-time Workers Regulations 2000 and Unpaid Parental Leave), supplemented with 'soft law' recommendations, and the gender mainstreaming aspect of the European Employment Strategy (1997) introduced targets to promote gender equality in the labour market. National governments' progress against the strategy was subject to scrutiny via annual reports, and the country specific recommendations for policy priorities included gender equality measures.
Steered by the EU's employment strategy, between 1997-2010, significant policy reform was made by the Labour government which initiated fundamental change and modernisation, providing 'new opportunity and new structural spaces' to pursue gender equality. Focusing primarily on increasing women's employment, this included the introduction of targets for pre-school childcare provision (the 2002 Barcelona Objectives), and the 1998 National Childcare Strategy delivered significant expansion. While most of the free pre-school provision has been part-time and term-time (for 3 to 4 year olds and eligble two year olds) it has grown from an entitlement to 12 hours per week in the late 1990s to 15 hours in 2010, with plans to increase to 30 hours from September 2017.
Childcare expansion was complemented by a suite of other reforms: improvements to statutory maternity and paternity leave; the 'right to request' reduced or flexible working hours (initially for parents and then extended); and parental leave was introduced and reformed – most recently, with the launch of Shared Parental Leave (2015). There were also targeted measures to help women in low-income households, including the New Deal for Lone Parents, and the introduction of Working Tax Credits and the National Minimum Wage (1998) – women accounting for two thirds of the recipients of the latter. This group of women, however, has been hit hard by the welfare cuts triggered by the government's austerity measures, and most exposed to increased job insecurity and deteriorating working conditions (Budget Gender Audit).
In contrast, and in the context of widening economic inequalities in the UK, women's representation in managerial positions has improved, including at board level. The latter was accelerated by the Davies Review (2011) which set a voluntary target for gender equality in FTSE 100 boardrooms and against the backdrop of the European Parliament debating whether to introduce a gender quota for corporate boards (Fagan et al. 2012). The UK voluntary target of having 25 per cent women on FTSE 100 boards by December 2015 was exceeded – more than doubling the number in 2011 when the target was set.
Falling back?
After a protracted period of austerity following the 2008 financial crisis, and now Brexit, there is a very real risk that the long-established UK gender rights and gender equality machinery will be weakened or stripped away. According to the World Economic Forum (2016) the UK has slipped from the ninth most gender-equal country in 2006 to 20th place.
Indicators of the magnitude of gender inequalities in the UK include:
Senior decision making positions remain male-dominated. In 2016, only 29 % of MPs and 26.8% of FTSE 100 board members were women (concentrated in non-executive rather than executive director roles).
The UK has a persistent problem of skill shortages in STEM (science, technology, engineering and maths), recognised most recently in the government's proposed new Industrial Strategy. Yet girls and young women remain under-represented in the relevant education and training pathways, compounded by a leaky pipeline whereby employers fail to recruit and retain women with STEM qualifications.
The gendered pay gap persists and becomes wider after motherhood. Recent estimations show that at the current rate of progress equal pay will only be achieved 99 years after the 1970 Equal Pay Act was enacted; even though the current government vowed to close the gap within a generation.
Following the Equality Act (2010), mandatory gender pay gap reporting is to be finally introduced from April 2017 for larger employers, although they have until 2018 to publish this data.
While Shared Parental Leave is a step in the right direction to support fathers' involvement in childcare, it is low paid and reliant on the mother giving up a portion of her maternity leave, so only a minority of fathers have taken it up (what makes dads involved in childcare). Men are also not pursing flexible working; there continues to be a penalty for working part-time and pregnancy discrimination remains prevalent.
The government plan to increase free pre-school childcare to 30 hours from September 2017 is unlikely to roll-out smoothly because only one third of local authorities in England expect there to be enough childcare places. Families that are unable to secure a free pre-school place or need to purchase additional childcare contend with childcare costs which are much higher than in most other European countries.
More of women's employment than men's is concentrated in the public sector, in education, health and other social care and social service roles – exposing women to the pressures and deteriorating employment conditions stemming from public expenditure cuts.
With both the NHS and social care in crisis, the government is pushing families to take responsibility for the care of elderly parents and relatives. However, it is women who will feel the brunt given they assume most responsibility for unpaid care.
Violence against women and girls remains prevalent, with an average of two women a week killed by a violent partner or ex-partner in England and Wales.
So where does this leave us, over a 100 years after IWD was first celebrated?
The World Economic Forum predicts that the gender gap won't close entirely until 2186. There is risk of:
Complacency and lost momentum – the 'policies are in place, action is being taken, so what's the problem' type of complacency.
Measures continuing to treat women as the problem rather than tackling the tacit bias that restricts women's progress; moreover, both women and men need to be involved in the process.
The economic crisis continuing to thwart political vision and limit concrete measures to support women's rights, representation and resources.
When the international community took stock of the 20th anniversary of the Beijing Platform for Action, a post-2015 framework – the sustainable development goals – was adopted, with women's rights and equality between women and men a standalone goal. According to Maycock, (2015) this provides 'a renewed opportunity for global, European and national feminist action'.
Yet, successful action in the UK will become more difficult without the platform of the EU's legal framework, policy initiatives and alliances. It is too early to determine the gendered effects of Brexit, but adherence to two principles are required to prevent backsliding. First, it is essential that the existing EU provisions are written into UK law, and built upon. Second, that gender budgeting and gender impact analysis should become a routine tool for policy design as the government navigates the economic challenges which are widely predicted. Otherwise women will continue to bear the brunt of the economic costs, as they have done under the austerity measures. Being bold for change thus couldn't be more apt or more urgent.
Tagged With: Brexit, employment, EU, family policy, gender inequalities, international women's day
About Colette Fagan
Professor Colette Fagan's research focus is on work and employment, with particular interests in gender relations and inequalities in the workplace and domestic life. She is also a coordinator of the European Commission's Expert Group on Gender, Social Inclusion and Employment.
About Helen Norman
Helen was a Research Fellow in Sociology at the University of Manchester. Her research focuses on fathers and fatherhood, the gendered division of labour and gender inequalities in work and family life. She now works at Leeds University Business School.
About Nina Teasdale
Nina is a Fellow at the Centre for Economic Justice, WiSE, Glasgow Caledonian University. Gender and equality related issues are central to Nina's research interests, with a particular focus on work, employment and organisations.
What makes dads involved in childcare?
Tackling the 'childcare crunch'

redpajama_set_name: RedPajamaCommonCrawl


CHICAGO -- Power hitting Kyle Schwarber had quite the week as far as plate appearances go. They were about as up and down as his season has been up to this point. Schwarber struck out eight straight times. Then he followed that by reaching base eight straight times before he struck out in the 7th inning of the August 16th game against the Reds. The question we try and answer, is Schwarber's high strikeout total cause concern?
It's been noted quite consistently that Schwarber does not exactly have the season we all thought he would. Schwarber burst on the scene in 2015 and in 69 games had a slash line of .246/.355/.487 We all had high hopes in 2016 for Schwarber. However, his season was cut short, and he was able to work back from an intensive knee injury for the World Series.
Fast forward to this year and Schwarber has a line of .204/.321/.442 numbers that leave quite a bit to be desired. He has struck out more than any other Cub this year with a strikeout percentage of 31.2. He's followed closely by Ian Haap and Javier Baez who have struck out 29.4% and 28.9% respectively. However, I do not think that number should be a cause for concern quite yet. This is still the first full season for Schwarber in the big leagues. So to compare his strikeout percentages to others in their first full season, we can look to the likely AL Rookie of the Year and maybe even the MVP in Aaron Judge who has a strikeout rate of 31.7%. Kris Bryant in his NL Rookie of the year campaign had a 30.6% strikeout percentage. That being said since the All-Star break Schwarber has struck out 40% of the time and all season he has been making weaker contact than a player like Schwarber should. However, when he does make solid contact, he has produced with 20 homers to go along with 40 RBIs.
Since his return from the minors in 33 games, he is batting .278 with .388 on-base percentage, .588 slugging with 17 runs, eight homers, and 12 RBIs. His career stat line through 554 at-bats is a .220 average with 36 homers, 83 RBI, 100 runs, 87 walks, and 194 strikeouts.
His .321 season on-base percentage this season is only eight points below the league average. So as much as Schwarber has struggled, it hasn't been as dire as everyone thinks. He will continue to make adjustments, and the eight strikeouts followed by eight trips to the bases shouldn't be a regular thing. There is no doubt in my mind Schwarber either this season or next will find his consistency as far as getting hits and become that huge piece in the Cub's lineup for the next several years.


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Land of the wanderers
Caviar for the masses? China certainly thinks so
The largest caviar maker in the world wants "normal people" to try the expensive delicacy.
Caviar was the traditional delicacy of the Caspian Sea but these days production has a new headquarters: China.
Qiandao (Thousand Island) lake lies 220 miles south of Shanghai. Fringed with mountains, it is a popular holiday spot for Chinese residents fleeing the summer heat of the city.
But it is also home to Kaluga Queen, the biggest caviar maker in the world, responsible for a third of global production.
The sturgeon are kept in floating pens in a corner of Qiandao lake in eastern China
Their caviar ends up everywhere. The company says their eggs have graced the tables of both Queen Elizabeth and Kim Jong Un.
Kaluga Queen plans to go public next year at a valuation of around £600m, the company told Sky News.
"In the beginning, people were surprised because: caviar, from China?" Han Lei, vice general manager of Kaluga Queen, said.
"They never heard about caviar from China. They had many doubts about the quality. About the safety.
"Today, we supply 25 countries and we work with around 100 distributors around the world."
The company was set up by officials from the Chinese ministry of agriculture, who started building their first farm in 2003.
Some of the sturgeon are worth as much as a Ferrari
It was good timing. In 2005, the US banned imports of beluga caviar from sturgeon caught in the Caspian sea because of fears overfishing would wipe out the species.
Farmed caviar is not subject to those restrictions.
"In 2006, we produced the first tins of farmed caviar from China," Mr Han said. "And after that we grew every year."
In a corner of the waters of Qiandao, floating metal and net structures pen in more than 1,000 sturgeon.
Some are up to two metres long and worth as much as a Ferrari, Mr Han said, because of the value of the eggs they carry.
After about four years in the lake, the sturgeon are taken to these inland pools where they live for at least another four years
Different breeds of sturgeon spend four or so years in the lake before they are transported to pools on land.
"We feed them and watch them growing from baby fish to a mature fish," Deny Yun, who manages the farm, told Sky News.
"We feel heart-broken when sending them to factory. But as a company, we have to do so. We must suffer this."
Once they are mature - at least eight years old but for some breeds as old as 15 - the sturgeon are taken to the processing factory.
The eggs are taken out of the sturgeon when they are at least eight years old
They are cut open still alive and the eggs are extracted before the sturgeon is killed and the rest of its body used for meat.
The eggs are cleaned, sorted and salted before they are tinned - a process that takes 15 minutes from start to finish.
Most of Kaluga Queen's caviar is sold under different brands abroad - in the UK it is sold through King's Fine Food, which is on sale at Fortnum and Mason's and Harrods.
But it also supplies customers in China using its own branding. And China is getting an appetite for the stuff.
The eggs are cleaned, sorted and salted then tinned
Prices start at 500 RMB (around £57) for 50g, going up to 9,000 RMB (£1,024) for a 50g tin of beluga caviar.
"We did a lot of promotion to introduce the caviar culture to Chinese people," Mr Han said.
"Now, more and more people start to consume caviar, to like caviar, to be caviar fans.
"The domestic market is growing very fast. After the virus is well controlled by the government, they're going to restaurants more.
"And more and more people like to enjoy the caviar."
That is helping to make up the shortfall in international orders as a result of COVID-19.
Deng Yun manages the farm which has more than 1,000 sturgeon
Kaluga produced 20% less caviar this year, although it aims to make up that deficit in 2021.
And there is a lot of room for growth. Current global production of caviar, from all producers, is around 300 tonnes.
In the heyday of the free-spending, status-flaunting 1980s, production peaked at 1,000 tonnes.
Mr Han said Kaluga Queen wants "normal people to have the chance to try caviar".
"In this way we can meet the demand all over the world," he added.
You'll never be old and wise if you weren't young and crazy.
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CBLB Constrains Inactivated Vaccine–Induced CD8+ T Cell Responses and Immunity against Lethal Fungal Pneumonia
Som G. Nanjappa, Srinivasu Mudalagiriyappa, J. Scott Fites, M. Suresh and Bruce S. Klein
J Immunol September 15, 2018, 201 (6) 1717-1726; DOI: https://doi.org/10.4049/jimmunol.1701241
Som G. Nanjappa
*Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802;
ORCID record for Som G. Nanjappa
Srinivasu Mudalagiriyappa
ORCID record for Srinivasu Mudalagiriyappa
J. Scott Fites
†Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792;
ORCID record for J. Scott Fites
M. Suresh
‡Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706;
Bruce S. Klein
§Department of Internal Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792; and
¶Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792
Fungal infections in CD4+ T cell immunocompromised patients have risen sharply in recent years. Although vaccines offer a rational avenue to prevent infections, there are no licensed fungal vaccines available. Inactivated vaccines are safer but less efficacious and require adjuvants that may undesirably bias toward poor protective immune responses. We hypothesized that reducing the TCR signaling threshold could potentiate antifungal CD8+ T cell responses and immunity to inactivated vaccine in the absence of CD4+ T cells. In this study, we show that CBLB, a negative regulator of TCR signaling, suppresses CD8+ T cells in response to inactivated fungal vaccination in a mouse model of CD4+ T cell lymphopenia. Conversely, Cblb deficiency enhanced both the type 1 (e.g., IFN-γ) and type 17 (IL-17A) CD8+ T cell responses to inactivated fungal vaccines and augmented vaccine immunity to lethal fungal pneumonia. Furthermore, we show that immunization with live or inactivated vaccine yeast did not cause detectable pathologic condition in Cblb−/− mice. Augmented CD8+ T cell responses in the absence of CBLB also did not lead to terminal differentiation or adversely affect the expression of transcription factors T-bet, Eomes, and RORγt. Additionally, our adoptive transfer experiments showed that CBLB impedes the effector CD8+ T cell responses in a cell-intrinsic manner. Finally, we showed that ablation of Cblb overcomes the requirement of HIF-1α for expansion of CD8+ T cells upon vaccination. Thus, adjuvants that target CBLB may augment inactivated vaccines and immunity against systemic fungal infections in vulnerable patients.
This article is featured in In This Issue, p.1603
Fungal infections in immunocompromised patients, such as those with AIDS and CD4+ T cell lymphopenia, have sharply risen in recent years (1). Substantial evidence indicates that CD4+ T cells are necessary to control the fungal infections, but these cells are lacking in CD4+ lymphopenic individuals. We have previously shown that vaccine-induced antifungal cytokine-producing CD8+ T cells, both type 1 (e.g., IFN-γ) and type 17 (IL-17A) cells, can compensate to provide sterilizing vaccine immunity in the absence of CD4+ T cells (2, 3). Although no licensed fungal vaccines are currently available, vaccines targeting CD8+ T cells in vulnerable immunocompromised patients would require an additional layer of safety. Subunits vaccines are highly desirable, but protective fungal epitopes have not been determined for CD8+ T cells. Inactivated vaccines are safe but need adjuvants that may undesirably bias T cell responses that support Ab production. Adjuvants for fungal vaccines tailored to CD4+ lymphopenic individuals should rather induce type 1 (IFN-γ, TNF-α, and GM-CSF) and type 17 (IL-17A) CD8+ T cell cytokine responses that are important for vaccine immunity against pathogenic fungi.
CD8+ T cells elicited by vaccination differentiate and clonally expand to become a large pool of effectors during the expansion phase of which ∼90% will die by apoptosis during the ensuing contraction phase. The remaining 5–10% of effectors, known as memory precursor effector cells, differentiate to become long-lasting memory cells. A threshold number of such memory cells is essential to rapidly clear infection upon re-exposure (4). Thus, the quantum of effector CD8+ T cells following expansion dictates the threshold number of memory cells, whereas the inflammatory milieu affects the type and quality of the response. The inflammatory cytokines dictate counterregulatory types of CD8+ T cell responses; IL-12 promotes type 1 responses; and a combination of IL-6, TGF-β, and IL-1 promote type 17 responses (5). Moreover, Ag load and inflammatory milieu regulate the quality of memory CD8+ T cells. High Ag load causes the clonal deletion and functional exhaustion of effector CD8+ T cells, whereas an inappropriate inflammatory milieu guides their terminal differentiation and dysfunction (6, 7). Thus, both TCR signaling and inflammatory milieu are key targets for developing rational fungal vaccines.
Productive TCR signaling requires at least two signals: signal 1 from the peptide-MHC-TCR complex and signal 2 from costimulatory signals such as CD28. Upon induction of TCR signaling, a cascade of signaling pathways associated with cell proliferation, differentiation, and survival are activated involving tyrosine kinases, MAPKs, and cytoskeleton remodeling that culminate in NFAT–NF-κB–mediated activation of CD8+ T cells (8). Casitas B–lymphoma-b (CBLB), an E3 ubiquitin ligase, is important for maintenance of peripheral T cell tolerance, and its absence leads to augmented TCR signaling resulting in autoimmunity (9). However, CBLB negatively regulates T cell responses during infection, and its deletion enhances CD8+ T cell–mediated tumor immunity (10–12). CBLB controls TCR signaling pathways at various junctures by downregulating the TCR complex, PI3K, PLCγ1, or Vav1 and is associated with T cell anergy and generation of poor quality effector T cells (11, 13–16). Thus, Cblb-deficient T cells can overcome the requirement of CD28 signaling (signal 2) for T cell activation (17). Thus, CBLB targeting has been shown to be beneficial in controlling chronic viral infections and tumors (11, 18).
Our past work has shown that live attenuated fungal vaccination in the absence of CD4+ T cells induces type 1 and type 17 CD8+ T cells that are required for vaccine immunity and are distinct from antiviral and antibacterial effector CD8+ T cells by portraying phenotypic attributes that portend stem cell–like behavior and are maintained as long-lasting memory cells (19–21). In this study, we evaluated whether targeting CBLB would potentiate inactivated fungal vaccine immunity in a mouse model of CD4+ T cell lymphopenia. We assayed the quantity and quality of CD8+ T cell responses to inactivated Blastomyces and Histoplasma yeasts in comparison with lymphocytic choriomeningitis virus (LCMV). We also delineated the effect of CBLB on resistance following inactivated yeast vaccination. Finally, we established the T cell–intrinsic role of CBLB and present novel data that suggest Cblb deficiency can override the requirement of hypoxia-inducible factor-1α (HIF-1α) for vaccine-induced CD8+ T cell responses.
Cblb−/− mice were provided by P.S. Ohashi (University of Toronto, ON, Canada) with permission from J. Penninger (Institute of Molecular Biotechnology, Austria). The C57BL/6, OT-I transgenic (Tg) (TCRα/TCRβ-specific for OT-I epitope), and B6.PL-Thy1a/Cy/Thy1.1 (Thy1.1) were purchased from The Jackson Laboratory. OT-I mice carrying Thy1.1 allele were generated by backcrossing OT-I Tg mice with Thy1.1 mice, which were then used to generate congenic Thy1.1+ OT-I Tg–Cblb−/− mice by backcrossing with Cblb−/− mice. All mice were maintained under specific pathogen-free conditions at the University of Wisconsin-Madison and at the University of Illinois at Urbana-Champaign and were used in accordance with the guidelines of the institutional animal care committees.
LCMV infection and DNA vaccination
For acute viral infection, LCMV Armstrong strain was given (105 PFU) by the i.p. route. For chronic viral infection, LCMV clone 13 strain was given (106 PFU) by the i.v. route. To cause persistent infection (22), mice were infected with LCMV clone 13 and were depleted of CD4+ cells with GK1.5 Ab (200 μg i.p. twice on days 0 and 4 postinfection). For DNA vaccination, 200 μg of endotoxin-free pCMV-NP plasmid was given once via the i.m. (anterior tibialis) route.
Adoptive transfer experiments
Naive Cblb+/+ and Cblb−/− OT-I cells (both Th1.1+) were purified from both draining lymph nodes (dLNs) and spleens using a CD8+ T cell enrichment kit (BD Biosciences). A total of 106 OT-I cells was adoptively transferred i.v. into naive recipient mice.
Fungi, vaccinations, and challenge infection
Wild-type virulent strains of Blastomyces dermatitidis ATCC 26199 and Histoplasma capsulatum ATCC 26032 (G217B) were obtained from American Type Culture Collection. The isogenic attenuated strain of Blastomyces lacking the virulence factor BAD1 (no. 55) (23) was used for vaccination (105–106 CFU). Blastomyces was maintained as yeast on BD Middlebrook 7H10 agar supplemented with oleic acid–albumin complex (Chem Impex International) at 39°C. To measure OT-I T cell responses, recombinant no. 55 strain engineered to carry the OT-I epitope SIINFEKL was used for vaccination (106 CFUs) (19). H. capsulatum was maintained on Histoplasma macrophage medium slants and used for vaccination (107 CFU). To study vaccine resistance, mice were challenged intratracheally with ∼104 CFUs of virulent strain B. dermatitidis (no. 26199). Vaccinations were given s.c. and divided at two sites, one dorsally and one at the base of tail (3, 24).
CD4+ T cell depletion
Except for the LCMV infection studies (unless indicated), CD4+ T cells were depleted using 200 μg/mouse GK1.5 mAb i.v. twice a week (Bio X Cell) for all experiments. The depletion efficiency was >99% as described previously (19).
To assess vaccine-induced CD8+ T cell responses, dLNs and spleens were harvested on indicated days. To measure recall responses, lung tissues were harvested at day 4 after pulmonary challenge. Tissues were homogenized on BD cell strainers, and RBCs were lysed using 4% ammonium chloride–containing buffer. Cells were restimulated with anti-CD3 (clone 145-2C11; 0.1 μg/ml) and anti-CD28 (clone 37.51; 1 μg/ml) Abs in the presence of GolgiStop (BD Biosciences) for 5 h at 37°C. Following incubation, cells were washed and stained with fluorochrome-conjugated Abs for surface markers. Following fixation and permeabilization (BD Cytofix/Cytoperm buffer), cells were stained with Abs against intracellular cytokine. All Abs were purchased from BD Biosciences or eBioscience except anti-CD43 (clone 1B11; BioLegend). Cells were analyzed by a BD LSR II or Cytek Aurora flow cytometer, and data were analyzed using FlowJo (Treestar). All flow cytometry data shown is after gating on lymphocyte gate followed by CD8α+ T cell gate.
CBLB and HIF-1α staining
Single-cell suspensions were Ab stained for surface markers followed by fixation and permeabilization (BD Cytofix/Cytoperm). Cells were stained with anti-mouse CBLB Abs (no. SC-1435 or no. SC-8006; Santa Cruz Biotechnology) followed by staining with fluorochrome-conjugated rabbit anti-goat secondary Ab for SC-1435 clone (13) and rabbit anti-mouse HIF-1α (D1S7W; Cell Signaling Technology). In some experiments, blocking peptide (SC-1435P; Santa Cruz Biotechnology) was used to block primary Ab binding sites before using for staining (13).
Cytokine bead array
Blood was collected from mice when they were moribund, and serum was separated. Serum cytokine concentrations were measured by flow cytometry using CBA Kit (BD Biosciences) as per the manufacturer's protocol.
Transcription factor staining
Abs against T-bet, RORγt, and Eomes were obtained from eBioscience, and cells were stained using a FoxP3 Staining Kit (eBioscience) along with cytokines staining.
HIF-1α inhibitor treatment
Echinomycin was purchased from Cayman Chemical, and stocks were made in methanol (1 mg/ml). Stocks were diluted in sterile PBS and administered to mice i.p. every other day (∼3 μg/mouse) starting from day 4 postvaccination.
Statistical analysis of fungal load was measured by the nonparametric Mann–Whitney U test, and all other analyses were performed using a two-tailed unpaired Student t test. A two-tailed p value of ≤ 0.05 was considered statistically significant.
Antigenic load dictates CBLB-dependent CD8+ T cell responses and immunopathologic condition
We previously reported that Cblb deficiency augments TCR signaling in CD8+ T cells and enhances IFN-γ production during an acute infection with the Armstrong strain of LCMV (resolved in 8–10 d) (13, 22). In contrast to an acute infection in immunocompetent mice, the clone 13 strain of LCMV replicates to very high levels, induces varying levels of CD8+ T cell dysfunction, and results in a chronic viral infection lasting 3–6 mo. Depletion of CD4+ T cells during a chronic LCMV infection leads to severe functional exhaustion of CD8+ T cells and lifelong viral persistence (25). The expression levels of CBLB in CD8+ T cells in this setting were directly proportional to the viral Ag load (Fig. 1A). Thus, it was of interest to determine the role of CBLB in regulating CD8+ T cell responses under conditions of disparate antigenic loads that may be seen in immunocompromised hosts during systemic viral infection. To assess the effect of high viral load on immune regulation by CBLB, we evaluated CD8+ T cell responses in Cblb+/+ and Cblb−/− mice during chronic and persistent viral infections. Absence of CBLB significantly enhanced LCMV-specific (both NP396 and GP33 dominant epitopes) CD8+ T cell responses during both chronic and persistent viral infections (Fig. 1B). Cblb deficiency enhanced CD8+ T cell responses during chronic viral infection (18), but Cblb−/− mice succumbed within 8–9 d after LCMV infection (Supplemental Fig. 1A). Remarkably, circulating levels of proinflammatory cytokines were sharply elevated following LCMV clone 13 infection in Cblb−/− versus Cblb+/+ mice (Supplemental Fig. 1B), which suggested that cytokine storm might underlie the lethality of Cblb−/− mice. Thus, under conditions of high viral load during a chronic LCMV infection, CBLB protects against immunopathologic condition by downregulating antiviral T cell responses.
Role of CBLB during viral infections. Naive Cblb+/+ and Cblb−/− mice were inoculated with LCMV strains to produce acute, chronic (clone 13), or persistent (clone 13, CD4-depleted) infections. (A) Levels of CBLB expression. Values are mean fluorescence intensity (MFI) ± SD. (B) LCMV-specific CD8+ T cell responses. Values are percent ± SD and MFI of IFN-γ (parentheses). (C and D) Epitope-specific CD8+ T cell responses following recombinant DNA (pCMV-NP) vaccination. Percent (C) and IFN-γ expression in MFI (D) of CD8+ T cell responses. n = 4–5 mice/group. *p ≤ 0.05, **p ≤ 0.01.
We next assessed the role of CBLB in regulating T cell responses under conditions of relatively low antigenic stimulation induced by a DNA vaccine. As shown in Fig 1C, following DNA immunization, the percentage and IFN-γ–producing ability of Ag-specific CD8+ T cells were significantly augmented in Cblb−/− mice compared with Cblb+/+ mice. Together, these data suggest that CBLB augments the activation threshold of CD8+ T cells. By doing so, CBLB limits CD8+ T cell responses to weak antigenic stimuli but protects against CD8+ T cell–mediated immunopathologic condition induced by a high Ag load.
Ablation of Cblb enhances antifungal CD8+ T cell responses following live yeast vaccination
As illustrated above, sustained high antigenic load during a chronic LCMV infection led to fatal immunopathologic condition in Cblb−/− mice, and we have shown previously that fungal Ag persists for an extended period of time (∼7 wk) following vaccination with live attenuated Blastomyces yeast (19). First, we asked if CBLB is induced following fungal vaccination. As shown in Fig. 2A, fungal vaccination induced high levels of CBLB in activated CD8+ T cells in both dLNs and spleens that waned in the ensuing weeks of rest (∼8 wk postvaccination), coinciding with the depletion of vaccine Ag (19). Next, we analyzed the response of Cblb−/− mice to vaccination with live attenuated yeast. We vaccinated Cblb+/+, Cblb+/−, and Cblb−/− mice with live attenuated yeast and monitored their morbidity and survival. We did not observe overt signs of distress or mortality over 8 wk postvaccination in any of the groups. Lastly, we assessed the role of CBLB on antifungal CD8+ T cell responses. We have previously shown that both type 1 (IFN-γ, GM-CSF, and TNF-α) and type 17 (IL-17A) responses were important for fungal vaccine immunity mediated by CD8+ T cells (2, 3). As shown in Fig. 2B, there was a significant increase in the frequency of both type 1 and type 17 cytokine–producing CD8+ T cells in Cblb-deficient mice compared with Cblb+/+ mice. Similarly, the number of type 1 and type 17 but not type 2 cytokine–producing CD8 T cells in dLNs and spleens were significantly higher in Cblb−/− mice compared with Cblb+/− and Cblb+/+ controls (Fig. 2C, Supplemental Fig. 2A). Notably, even a single copy of Cblb was enough to suppress the CD8+ T cell responses. Collectively, these data suggest that Cblb deficiency enhances antifungal CD8+ T cell responses to live fungal vaccination without causing lethality or obvious adverse effects.
CBLB constrains antifungal CD8+ T cell responses. Naive Cblb+/+, Cblb+/−, and Cblb−/− mice were vaccinated s.c. with the live attenuated strain no. 55 of B. dermatitidis (105 CFU). On day 16, dLNs and spleens were harvested, and cytokine-producing CD8+ T cells were analyzed by flow cytometry. (A) CBLB expression (MFI) in naive (CD44lo) and activated (CD44hi) CD8+ T cells on indicated days postvaccination. Frequency (B) and numbers (C) of cytokine-producing cells gated on activated (CD44hi) CD8+ T cells. Values are mean ± SD. n = 4 mice per group. Data is representative of two independent experiments. CD4+ T cells were depleted throughout the experiment. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
CBLB constrains antifungal CD8+ T cell responses to an inactivated vaccine
Inactivated vaccines are safer for immunocompromised individuals, but they often fail to elicit protective T cell responses. After the findings above that persistent fungal vaccine Ag does not cause lethality or aberrant gross pathologic condition in Cblb−/− mice, we investigated whether 1) CBLB constrains CD8+ T cell responses to inactivated vaccine and whether 2) CBLB deficiency bolsters both type 1 and type 17 responses to inactivated vaccine. As shown in Fig. 3A, vaccination with inactivated yeast induced stronger Tc1 and Tc17 responses in Cblb−/− mice compared with Cblb+/+ and unvaccinated controls, and responses were augmented with a higher vaccine dose. Similarly, total numbers of both IFN-γ+ (Tc1) and IL-17A+ (Tc17) CD8+ T cells in Cblb−/− mice vaccinated with inactivated yeast were significantly higher than in Cblb+/+ controls in both dLNs and spleens (Fig. 3B, Supplemental Fig. 2B). CD43 expression (26) on cytokine-producing cells in Cblb+/+ and Cblb−/− mice were similar (Supplemental Fig. 2C), suggesting that CBLB does not influence CD43-mediated effector cell functions (26). Collectively, these data suggest that CBLB downregulates antifungal Tc1 and Tc17 cell responses to inactivated fungal vaccine.
CBLB limits CD8+ T cell responses to inactivated fungal vaccine. Naive Cblb+/+ and Cblb−/− were vaccinated with either live or heat-killed strain no. 55 yeast. On day 18, dLNs were harvested to analyze CD8+ T cell responses by flow cytometry. Frequency (A) and numbers (B) of cytokine-producing CD8+ T cells. Values are mean ± SD. n = 4–5 mice/group. CD4+ T cells were depleted throughout the experiment. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
CBLB constrains inactivated vaccine–induced immunity against lethal fungal pneumonia
We next investigated whether enhanced CD8+ T cell responses to inactivated fungal vaccine in Cblb−/− mice translate to better resistance against lethal fungal pneumonia. Vaccination with heat-killed Blastomyces poorly protects Cblb+/+ mice from lethal pulmonary challenge (27). In line with this finding, Cblb+/+ mice vaccinated with inactivated yeast had only 5-fold fewer CFUs than unvaccinated controls after challenge. In sharp contrast, Cblb−/− mice that received inactivated vaccine had ∼4 logs lower (1,295-fold) CFUs compared with their unvaccinated controls (Fig. 4A). Interestingly, unvaccinated Cblb−/− mice had ∼2 logs lower (169-fold) CFUs compared with unvaccinated Cblb+/+ mice, suggesting an innate, vaccine-independent role for CBLB. In concordance with CFU data, recall responses of cytokine-producing CD8+ T cells in infected lungs were significantly higher in Cblb−/− mice compared with Cblb+/+ mice (Fig. 4B). Thus, our data suggest that CBLB constrains antifungal immunity after vaccination with inactivated yeast, and this finding correlates with poor induction and recall responses of CD8+ T cells.
CBLB regulates vaccine immunity to inactivated fungal yeast. Naive Cblb+/+ and Cblb−/− were vaccinated with 105 CFU of heat-killed (h.k.) strain no. 55 yeast. (A) After a month rest, mice were challenged intratracheally with a lethal dose (104 CFU) of virulent strain no. 26199. When unvaccinated mice were moribund (day 16 postinfection), lungs were harvested to quantify fungal burdens. CFUs are shown in whisker plots. n = 4–14 mice/group. (B) Mice were vaccinated with killed yeast as above. Mice were challenged after 2 mo rest, and lungs were harvested (day 4 postinfection) to enumerate cytokine-producing CD8+ T cells. n = 2–4 mice/group. Data is representative of two independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
CBLB downregulates antifungal CD8+ T cell responses to inactivated Histoplasma yeast
To extend our observations to other pathogenic fungi, we evaluated the antifungal CD8+ T cell responses in Cblb+/+ and Cblb−/− mice following vaccination with inactivated Histoplasma yeast. Of note, Histoplasma induces strong type 1 (IFN-γ) responses that are correlated with resistance (28–30). Fig. 5A and 5B show the frequency of cytokine-producing CD8+ T cells in Cblb+/+ and Cblb−/− mice vaccinated with either inactivated yeast or live yeast. As we observed with Blastomyces vaccination, the percentage of cytokine-producing CD8+ T cells (especially type I cells) was significantly higher in Cblb−/− mice compared with Cblb+/+ and unvaccinated mice (Fig. 5, Supplemental Fig. 3). Similar findings were present in live yeast vaccine groups, except that Cblb−/− mice had significantly higher IL-17A+ CD8+ T cells compared with Cblb+/+ controls (Fig. 5B, Supplemental Fig. 3B). In sum, these findings together suggest that CBLB negatively regulates CD8+ T cell responses to vaccination with multiple fungi.
CBLB limits CD8+ T cell responses following vaccination with live or inactivated Histoplasma yeast. Naive Cblb+/+ and Cblb−/− were vaccinated with either heat-inactivated (A) or live (B) Histoplasma yeast (107 CFU). On day 26, dLNs were harvested to analyze CD8+ T cell responses by flow cytometry. Dot plots show the frequency of cytokine-producing CD8+ T cells. Values are mean ± SD. n = 4–5 mice. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Ablation of Cblb bolsters cytokine-producing ability of CD8+ T cells without causing terminal differentiation
We next investigated whether robust activation of Cblb-deficient CD8+ T cells causes their terminal differentiation or functional exhaustion, thereby potentially undermining long-term, vaccine-induced immunity. Fig. 6A shows the expression of transcription factors in cytokine-producing cells. Expression levels of prototypic transcription factors in Tc1 and Tc17 cells were similar in both Cblb+/+ and Cblb−/− mice (Fig. 6A). The ratio of T-bet/Eomes, a prognostic feature of terminal differentiation and memory development (31, 32), was also similar in both Cblb+/+ and Cblb−/− CD8+ T cells (Fig. 6B). Also, the expression levels of CD127 (prosurvival) and KLRG-1 (terminal differentiation) (33) were similar in Cblb+/+ and Cblb−/− mice (Supplemental Fig. 4A). We also analyzed the quality of effector CD8+ T cells by comparing their cytokine-producing ability on a per-cell basis. The mean fluorescence intensity (MFIs) of IFN-γ and IL-17A expression were significantly higher in Cblb−/− mice compared with Cblb+/+ mice following vaccination with inactivated yeast (Fig. 6C), suggesting a better qualitative signature cytokine imprinted on these cells in the absence of CBLB. These data suggest that CBLB deficiency promotes CD8+ T cell responses following vaccination with inactivated yeast without adversely causing their terminal differentiation.
Ablation of CBLB improves the quality of effector CD8+ T cells. Naive Cblb+/+ and Cblb−/− mice were vaccinated with either live or heat-killed strain no. 55 yeast. On day 18, dLNs were harvested to analyze CD8+ T cell responses by flow cytometry. (A) Histograms show transcription factor profile of cytokine-producing CD8+ T cells. (B) Relative proportions of expression of transcription factors in cytokine-producing CD8+ T cells. Relative expression was calculated by dividing MFI of each transcription factor by the sum of both MFIs. (C) Histograms show MFI of cytokines gated on cytokine-positive CD8+ T cells. Values are mean ± SD. n = 4–5 mice. *p ≤ 0.05, **p ≤ 0.01.
CBLB downregulates antifungal CD8+ T cell responses in a T cell–intrinsic mechanism
CBLB is expressed not only in lymphoid cells but also in nonlymphoid cells and affects their activation and functions (34). Although CBLB is a known negative regulator of TCR signaling, it is possible that CBLB expression in non–T cells affects the differentiation of antifungal CD8+ T cells in a cell-extrinsic manner in our models. To address this possibility, we did criss-cross adoptive transfer experiments involving vaccination. As shown in Fig. 7A, irrespective of the background of recipient mice, OT-I cells lacking CBLB showed a significantly higher frequency of cytokine responses (1.5–2-fold) than OT-I cells expressing CBLB, suggesting a stronger intrinsic role of CBLB. Likewise, the total numbers of cytokine-producing CD8+ T cells was significantly higher for Cblb−/−OT-I cells versus Cblb+/+ OT-I cells in either Cblb+/+ and Cblb−/− recipients (Fig. 7B). Of note, although OT-I cells are known to resist becoming IL-17A+ cells, the total number of IL-17A+ Cblb−/− OT-I cells was significantly higher than Cblb+/+OT-I cells (Fig. 7B), although the frequencies were similar between the groups. Thus, our findings indicate that CBLB has a prominent role in constraining antifungal CD8+ T cell responses in a cell-intrinsic manner.
Intrinsic role of CBLB for antifungal CD8+ T cell responses. Enriched Thy1.1+ OT-I T cells (∼106) from naive Cblb+/+ or Cblb−/− mice were adoptively transferred into naive Thy1.2+ Cblb+/+ or Cblb−/− mice. On the following day, recipients were vaccinated with strain no. 55 yeast engineered to express the OT-I epitope (106 CFUs). On day 22, dLNs were harvested to enumerate cytokine-producing CD8+ T cells. Frequency (A) and total numbers (B) of cytokine-producing OT-I cells. Values are mean ± SD. n = 5–6 mice per group. Data are representative of two independent experiments. A t test comparing each cytokine-positive population between Cblb+/+ and Cblb−/− cells in the same recipient genotype. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Cblb deficiency overcomes the requirement of HIF-1α for expansion of vaccine-induced CD8+ T cell responses
HIF-1α is critical for survival of cells under the hypoxic conditions that can prevail during inflammation or tumor growth (35). We have recently shown that HIF-1α is necessary for the expansion of antifungal IFN-γ+ but not IL-17A+ CD8+ T cell responses (21). In this study, we investigated whether Cblb-deficient CD8+ T cells resistant to apoptosis (36) require HIF-1α for their response following fungal vaccination. We tested this hypothesis using the HIF-1α–specific inhibitor, Echinomycin. In concert with prior findings (21), Echinomycin treatment did not affect the expansion of IL-17A+ (Tc17) CD8+ T cells in Cblb+/+ mice but did significantly reduce IFN-γ+ (Tc1) CD8+ T cell responses in these mice in both dLNs and spleens (Fig. 8A, Supplemental Fig. 4B). In contrast, Echinomycin treatment did not affect Tc1 (or Tc17) cells in Cblb−/− mice, suggesting a dispensable role for HIF-1α in the absence of CBLB. Next, we asked whether CBLB affects the protein levels of HIF-1α. Our direct ex vivo staining showed increased levels of HIF-1α in activated (CD44hi) CD8+ T cells over naive (CD44lo) T cells in Cblb+/+ group (Fig. 8B, 8C), suggesting the induction of HIF-1α following vaccination. Direct ex vivo staining showed the similar levels of HIF-1α in both Cblb+/+- and Cblb−/−-activated (CD44hi) CD8+ T cells, suggesting a negligible role of CBLB on HIF-1α levels. Next, we evaluated the role of CBLB on the fate of HIF-1α following restimulation (upon engagement of TCR signaling mimicking infection) in the presence of cyclohexamide (protein synthesis blocker; new HIF-1α synthesis) or MG132 (proteasome inhibitor; polyUb-mediated degradation of HIF-1α). We found higher levels of HIF-1α with MG132 and relatively lower levels with cycloheximide in both Cblb+/+ and Cblb−/− CD8+ T cells, proportionately (Fig. 8C), suggesting a minimal role of CBLB directly on HIF-1α protein levels. Thus, HIF-1α is redundant for expansion of CD8+ T cells in the absence of CBLB following fungal vaccination.
HIF-1α requirement for antifungal CD8+ T cell responses. Naive Cblb+/+ and Cblb−/− mice were vaccinated with attenuated strain no. 55 yeast. Cohorts of mice received either vehicle or Echinomycin (3 μg/mouse) every other day starting from day 4 postvaccination (A). On day 14–16, dLNs were harvested to analyze cytokine-producing CD8+ T cells by flow cytometry. Scatter diagrams show frequency of cytokine-producing cells among CD8+ T cells. Each individual marker represents the value from a single mouse. Data are pooled from two independent experiments. (B and C) On day 16, dLNs were harvested to analyze HIF-1α levels in CD8+ T cells in the presence of cyclohexamide (CHX, 10 μM; EMD Millipore) or MG132 (50 μM; EMD Millipore). Data are representative of two independent experiments. Values are mean ± SD. n = 4–5 mice/group. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
Despite the use of antifungals, individuals with deficiencies in CD4+ T cells, such as AIDS patients, remain susceptible to life-threatening fungal infections. Inactivated vaccines would be desirable for such patients if effective, but such vaccines have limited effectiveness because of weaker T cell responses. In this study, we show that modulating signaling downstream of the TCR is a strategy for developing efficacious fungal vaccines tailored to vulnerable individuals. Knocking out a negative regulator of TCR signaling (e.g., Cblb) augments type 1 and type 17 cytokine–producing CD8+ T cell responses required for effective antifungal vaccine immunity. Our data support the premise that adjuvants targeting such a pathway can bolster vaccine immunity elicited by inactivated fungi. CD8+ T cell epitopes have not been identified for fungi; thus, the use of whole cell–based vaccines may be a rational method for inducing protective CD8+ T cell immunity against such eukaryotic pathogens (37, 38).
Systemic fungal infections can be deadly in AIDS or CD4+ lymphopenic individuals. Our study showed that chronic viral infections are lethal in Cblb−/− mice. However, inactivated fungal vaccination did not have this effect. Hence, perhaps one can vaccinate CD4+ T cell lymphopenic patients by targeting CBLB without undesirable effects. Our idea is to use such adjuvants in a spatial (s.c.) and temporal (limited timeframe) manner. Perhaps this method may potentiate a pool of virus-specific T cell responses and boost their ability to clear virus (39, 40).
Vaccine-induced CD8+ T cells compensate CD4+ T cells for mediating immunity against lethal fungal pneumonia. Antifungal CD8+ T cell immunity is correlated with the expression of both type 1 and type 17 cytokines (2, 3) and activation of neutrophils (3), an important leukocyte essential for killing yeast. Our study shows that CBLB deficiency fosters superior cytokine-producing CD8+ T cells (Fig. 3) that correlate with vaccine immunity (Fig. 4). Interestingly, unvaccinated Cblb−/− mice showed increased resistance compared with unvaccinated Cblb+/+ mice, suggesting a role for innate (nonvaccine) immunity. Recent studies also showed that loss of CBLB promoted the activation of innate immune cells, leading to superior Candida resistance (41–43). Nevertheless, our data indicate that vaccinated Cblb−/− mice are able to mediate much better resistance than vaccinated Cblb+/+ mice. Thus, adjuvants targeting CBLB may bolster the efficacy of inactivated fungal vaccines to enhance CD8+ T cell immune correlates that govern vaccine immunity. Our data also show that Cblb regulates vaccine-induced CD8+ T cell responses in a cell-intrinsic manner, with little effect from non–T cells.
Qualitatively superior memory CD8+ T cells are generated with optimal activation of TCR signaling (44). Overstimulation of TCR signaling, however, leads to CD8+ T cell clonal deletion or functional exhaustion, marked by increased T-bet, decreased Eomes, and poor cytokine production (45). In the absence of Cblb, TCR downregulation is inhibited, leading to overactivation of TCR signaling and enhanced ERK phosphorylation (13). However, our data showed that vaccination with inactivated fungi did not cause terminal differentiation of Cblb-deficient CD8+ T cells but enhanced their cytokine-producing ability on a per-cell basis.
HIF-1α is an important factor that facilitates survival, proliferation, and tumor growth (35). Not surprisingly, tumor cells can thrive better in hypoxic niches by overexpressing HIF-1α, and it serves as a target for treatment against certain cancers (46). T cell–specific ablation of HIF-1α inhibits differentiation of Th17 cells by a RORγt-dependent mechanism (47), and Vhl (negative regulator of HIF-1α)-deficiency in CD8+ T cells enhances their effector functions and causes immunopathologic condition during persistent infection (48). We showed previously that antifungal Tc17 cells require HIF-1α during their differentiation following fungal vaccination but not for their expansion, whereas the converse was true for Tc1 cell responses (21). In this paper, our data showed that Cblb deficiency overcame the requirement of HIF-1α for expansion of antifungal Tc1 cells. Perhaps this may be one reason that Cblb−/− CD8+ T cells function better at tumor sites, a niche known to have hypoxia. Regardless, in sum, we show in this study that CBLB can be a potential target for enhancing antifungal CD8+ T cell responses following vaccination with inactivated yeast, thereby bolstering resistance against lethal fungal pneumonia.
The authors have no financial conflicts of interest.
We thank Dr. Howard Steinberg, Department of Pathobiological Sciences at the University of Wisconsin-Madison for histopathological studies. We thank Kevin Galles, Department of Pediatrics at the UW-Madison and Mauricio Villamar, Department of Pathobiology at the University of Illinois at Urbana-Champaign for genotyping mouse strains. We also thank animal care staff at UW-Madison and in the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign for excellent animal care and management.
This work was supported by National Institutes of Health (NIH) Grants AI119945 (to S.G.N.), AI124299 and AI118326 (to M.S.), and AI035681 and AI040996 (to B.S.K.). J.S.F. received support from T32 AI055397 (NIH–National Institute of Allergy and Infectious Diseases).
The online version of this article contains supplemental material.
Abbreviations used in this article:
CBLB
Casitas B–lymphoma-b
dLN
draining lymph node
HIF-1α
hypoxia-inducible factor-1α
LCMV
lymphocytic choriomeningitis virus
mean fluorescence intensity
transgenic.
Accepted July 12, 2018.
Copyright © 2018 by The American Association of Immunologists, Inc.
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Som G. Nanjappa, Srinivasu Mudalagiriyappa, J. Scott Fites, M. Suresh, Bruce S. Klein
The Journal of Immunology September 15, 2018, 201 (6) 1717-1726; DOI: 10.4049/jimmunol.1701241
Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-β1–Induced OCT4/Nanog Pathway
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The first item I restored on the Straight 8 is the power supply. I removed it from the computer to allow disassembly, testing, and cleaning. The first step I do is a visual inspection to see if any obvious problems exist and to plan what needs to be done. I also take pictures of everything and notes as needed so I can put it back together when I'm done.
The visual inspection only showed some corrosion, dirt, and foreign objects. I found 3 screws from the missing bracket that held the top of the backplanes, 1 bolt with nut attached, a piece of the broken cover, a nail, and some of the white washers that the backplanes pivot on.
I then removed all the capacitors that weren't soldered in. The dielectric in electrolytic capacitors deteriorates when stored. If full voltage is applied without current limit (such as would happen if you just turned on the computer) they can be damaged. The dielectric can be normally be reformed by proper control of the current as they are brought up to their rated voltage. There are many different opinions on the Internet on how this should be done. Use your favorite search engine to search for capacitor reforming for more information.
For the capacitors I used bench power supplies that you can set the output voltage and current limit. I set the current limit to around 20 mA. In working on this writeup I found this manufacturers page that said the reforming current limit should be no larger than the capacitor's maximum leakage current specification. This one said to apply rated voltage through a 1k resistor to reform. My current limit was between these. The capacitors were connected through 4k resistors to the power supply so I could monitor how each was reforming and to make the reforming current balance better between them. The supply was set to the capacitor rated voltage. Make sure you won't exceed the power rating of the resistor. In the past the capacitors I had reformed dropped the leakage current to less than a mA reasonably quickly. These took much longer for the leakage to drop. After most of the capacitors' leakage was below a mA I lowered the series resistor on the leaky capacitors to below 1k to see if that would improve their reforming. It didn't. In the end the capacitor leakage varied from almost none for the best to 500uA, 1.4 mA, 3 mA, 3.3 mA, and 3.4 mA for the worst ones after a long time reforming.
I don't have data on the leakage current specification of the particular capacitors I was reforming. Current production capacitors of similar value are specified as 4 to 6 mA maximum leakage current after 5 minutes. The typical leakage after 1 hour is 20% of the 5 minute leakage. The leaky capacitors were 6-8 mA after 5 minutes and 1.4-3.4 mA after hours at rated voltage. I was thinking the capacitors were usable until I could find some that electrically and visually matched but I ended up leaving them at rated voltage for two days then noticed that the vents were starting to bulge. I was able to find some reasonable replacements for all the capacitors with leakage above 1 mA.
While I had the original capacitors out I cleaned them and used a wire brush in a Dremel to clean the corrosion off the terminals. On my PDP-8/I I had problems with poor connections on the .25" quick disconnect (Faston) connections. They had corroded some so the resistance was enough to cause excessive voltage drop under load. For this restoration I cleaned them all and made sure they fit snugly. I loaded the supply as shown in the table below. For connections the power supply current ran through I used a volt meter to look for voltage drops which are larger than others. On the filter capacitor bank for connections that weren't carrying the supply output current I used an oscilloscope to look at the ripple voltage. I didn't find any really bad connections. On one of the capacitors the ripple level at the rivet holding the terminal on was less than at the terminal. The terminal was slightly loose and rotating it improved the connection. I also found at one of the quick disconnect wires the ripple was larger and and varied when the wire was wiggled.
For inhibit the first numbers are after turn on with machine cold, the second is after running for a while, and the third with test leads from ground to INH-. It appears capacitance to ground significantly shifts the output. More investigation is needed on why this occurs. The load regulation is pretty poor. The G808 card was significantly modified in the later rev K schematic I have from the board in my machine. The poor regulation may be why. The front panel draws from .15A to 1.6 A from the -15 supply depending on how many bulbs are lit. The memory + supply is 42.1 stopped, 41.2 running.
I did a line regulation test, the results are shown with the scope photo in the pictures below. I didn't run a load regulation test though probably should have.
Several of the capacitors were soldered in with bleed resistors across the terminals. For these I reformed them installed in the power supply with the current limit 5-10 mA above the expected current through the bleed resistor. The power supply control cards were removed and from the schematics applying power to the capacitors shouldn't cause problems. Since I don't know the exact value of the bleed resistor and the value changed as they heated up I can't measure the leakage current accurately. One of the 75 volt capacitors seemed to be leakier though it got better after a few hours. They all seem acceptable to use.
Due to the corrosion on the screws I wanted to check the socketed relay. After some careful prying I was able to remove it and a bunch of corrosion bits fell out. Further inspection showed one of the contacts was also damaged. I purchased a new relay. I was trying to get a period relay and found a new old stock one in an old looking box. I overshot since the "new" relay was made in 1962. I cleaned the socket with contact cleaner, alcohol, and a small plastic tube brush. I also used some Caig DeoxIT/PreservIT before installing the new relay.
Since the relay pins were corroded, I pulled the two chassis mounted transistors which were socketed. They were gold plated and didn't have noticeable corrosion on the pins so I reinstalled them with new heat sink compound. During later testing I found one of the transistors was bad and replaced it with a 1981 date code of the same DEC labeled transistor.
I also cleaned the rusted chrome-plated hardware using Kano Exrust. Don't use it on parts that have any gold-colored cadmium plating. The rust had caused a noticeable amount of the chrome plating to flake off on some of the parts. They don't look too bad with just the cleaning. I'll see how long it takes them to start rusting again. Most of the screws are stainless steel so didn't rust but the nuts are chrome plated.
I then cleaned everything and put it back together. One of the capacitor wires broke during the process so that was re-soldered, and a quick disconnect needed replacing.
I loaded the supply with resistors and used pots to simulate the thermistors in the memory stack. The manual gave the rated currents for the supply except the -15 supply is listed as 15 mA. It could be 15A but I haven't found a correct value in a manual. I targeted about half load for testing. I then applied power. I monitored the input line current and output voltages. Using a scope I noticed that the output ripple was not symmetric on the positive and negative cycle of the AC line. I tracked that down to mismatched taps being used for the memory supply. The transformer has 40, 42, and 45V taps available for the memory power supply. The supply is center tapped with 2 diodes making a full wave rectifier. One of the diodes was connected to the 40V tap and the other to the 45V tap. This caused all the current to be pulled from the higher voltage tap. When I connected both diodes to the 40V tap the ripple waveform became symmetric. There was no evidence that the wiring was changed any time recently so either it was made wrong or somebody moved only one wire. Looking at another PDP-8 it looks like the wire from the transformer to the terminal block were incorrectly connected. I have moved the wires to match the other computer.
When I adjusted the pot that was simulating the stack thermistor the memory read/write output voltage didn't change. I found the main pass transistor was shorted. When I replaced it and an open resistor on a G808 power supply control card the output worked properly.
The next fault I fixed is the chattering of the relay with the bad contact discussed above when the supply turns on or off.
The chattering may be why the contact was damaged. The chatter is due to a design error in the G809 rev B design. The schematic in the manual has a later version with the problem fixed. Due to the difficulty modifying the board to the later revision I made a different modification to fix the problem. I also found a wiring difference in the power supply which I though may have been an error but was a intentional change.
Another fault I fixed is the meter on the output of the supply read about 15% high. I assume that the meter read correctly when the machine was new. Analog meters measure current so have a resistor in series internal to the meter to set the full scale voltage. The resistor value likely has decreased with age. I hoped to open the meter and replace the resistor but the case is closed with a crimped metal band which can't be safely removed. Instead I added another resistor in series.
The last fault (as of this writing) is the neon indicator on the back of the power supply doesn't light reliably. The voltage they turn on with increases with usage so they tend to flicker or not come on all the time as they age. I didn't find a source for the Industrial Devices Inc Omni-vision 1020C55 neon indicator so it will stay as it is.


text:
Rangers likely miss chance at acquiring Justin Upton, now need Josh Hamilton
With Justin Upton now off the trade market, the Rangers need to re-sign Josh Hamilton if they're going to seriously contend for a World Series title in 2013.
By JP Starkey Dec 12, 2012, 7:02am CST
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Thearon W. Henderson
On Tuesday night, a three-team trade finally went down in Major League Baseball, involving the Arizona Diamondbacks. Unfortunately for the Rangers, it wasn't the rumored three-team trade from a week ago that would have brought Justin Upton to Texas.
Instead, the Diamondbacks found their shortstop of the future in Didi Gregorious, and sacrificed Trevor Bauer, not Upton, to get it done. Cleveland also held onto to Asdrubal Cabrera, and instead send Shin-Soo Choo to the Cincinnati Reds -- and picked up Bauer in exchange for one year of Choo.
Not bad Cleveland, not bad.
Unfortunately, that takes Upton off the market -- even Kevin Towers has admitted as much.
As you know, the Rangers were actively attempting to acquire Upton, but Texas did not want to give up Elvis Andrus to do so -- a wise move, for the Rangers.
With Upton now off the market, it's essentially imperative for the Rangers to re-sign Josh Hamilton. Though the market hasn't developed like Hamilton probably thought it would for his services, he's still the top bat in this year's free agent class, and he's going to get paid as such.
Texas, though, will face competition from Seattle, and possibly Philadelphia. Further, the Rangers lost a bit of leverage with Upton now off the market. Hamilton knows the Rangers need him if they're going to seriously contend for a World Series title in 2013, and surely, his agent will make Daniels well aware of that fact.
Last week, it was reported that the Rangers were willing to offer Hamilton four years -- and that a deal could likely be hashed out, with options for a fifth and possibly sixth year. It's no guarantee that the Rangers go beyond four years, but if the Mariners offer five, or even six, then Texas might be forced to offer five guaranteed years for Hamilton in order to retain him -- otherwise, they'll run the risk of losing Hamilton and Mike Napoli -- and not even having Upton to show for it.
It's not that Texas will be bad in 2013 even if Hamilton leaves. As currently constructed, the Rangers would have a lineup with Geovany Soto at catcher, Ian Kinsler at first, Jurickson Profar at second, Andrus at short, Adrian Beltre at third, David Murphy in left, either Craig Gentry or Leonys Martin in center and Nelson Cruz in right -- with Mitch Moreland getting at bats somewhere. It's not a terrible lineup, but it's certainly lacking a good bit of thunder.
No, this isn't the dream offseason for the Rangers, and you can argue that it's been a poor offseason. Daniels and the Rangers inexplicably failed to make Napoli a qualifying offer, costing the Rangers a draft pick. Texas whiffed on signing Zack Greinke, though, for the money involved, that might not be the worst thing for the Rangers going forward.
With Upton now off the board, it seems like the Rangers are left with their fall back option -- Josh Hamilton. And hey, that's really not too bad of a fall back option.
Still, this hasn't been a great offseason for the Rangers. Just last week, there was speculation that the Rangers could sign Greinke, Hamilton and trade for Upton. While wholly unrealistic, the idea was planted in Texas' fans minds that it could happen.
It's apparent that the Rangers now need Hamilton -- and that's not so bad. In a sense, Hamilton needs the Rangers too. Hopefully, the two sides can come to a reasonable agreement, and hopefully the Rangers can get away with four guaranteed years.
Be sure to follow JP Starkey on Twitter.
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