Category Archives: Immunology

Nimbus Therapeutics Appoints Chief Medical Officer Annie C. Chen, M.D., MPH, to President of the Company’s Tyk2 Subsidiary – Business Wire

CAMBRIDGE, Mass.--(BUSINESS WIRE)--Nimbus Therapeutics, a biotechnology company coupling targets selected based on causal human biology with structure-based drug discovery and development, today announced the promotion of Chief Medical Officer, Annie C. Chen, M.D., MPH, to President of the companys Tyk2 subsidiary, Nimbus Lakshmi, Inc. In this role, Dr. Chen will provide executive leadership for financial, business, and development activities associated with the companys tyrosine kinase 2 (Tyk2) program, in addition to continuing her role as Chief Medical Officer for Nimbus Therapeutics.

Were at a very exciting juncture as Nimbus gears up to once again become a clinical-stage company, and there is no better person to helm that effort than Annie, said Jeb Keiper, M.S., MBA, Chief Executive Officer of Nimbus. Annies extensive background in immunology, her experience leading clinical strategy to bring multiple therapies forward to regulatory approval, and her passionate dedication as a clinician to the well-being of her patients will be of enormous value to our Tyk2 program as it advances into the clinic.

Tyk2 is a genetically validated target for the treatment of many autoimmune and inflammatory disorders, and through Nimbus structure-based drug discovery efforts, we have developed promising allosteric modulators that effectively inhibit this target, said Dr. Chen. Im honored to lead these multidisciplinary efforts for Nimbus as we initiate clinical studies and chart the programs future path.

Dr. Chen, who received her medical training as an adult rheumatologist, has served as Chief Medical Officer of Nimbus since 2015. She provided oversight for the companys acetyl CoA carboxylase clinical program for NASH and supported business development and financing efforts, before its acquisition by Gilead. Prior to joining Nimbus, Dr. Chen was Executive Director of Clinical Research, Section Head of Vaccines at Merck and Co., where she oversaw clinical research activities for a broad portfolio of vaccines, from discovery through registration and life cycle management. Dr. Chen also held the role of Section Head of Immunology, where she oversaw clinical research for small molecule and protein therapeutics. Prior to Merck, Dr. Chen held roles of increasing responsibility at Genentech, and began her career at Celera Genomics.

About Nimbus Therapeutics

Nimbus Therapeutics designs breakthrough medicines. Utilizing its powerful structure-based drug discovery engine, Nimbus designs potent and selective small molecule compounds targeting proteins that are known to be fundamental drivers of pathology in highly prevalent human diseases and that have proven difficult for other drug makers to tackle. The companys LLC/subsidiary architecture enables diverse and synergistic partnerships to deliver breakthrough medicines. Nimbus is headquartered in Cambridge, Mass. For more information, please visit http://www.nimbustx.com.

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Nimbus Therapeutics Appoints Chief Medical Officer Annie C. Chen, M.D., MPH, to President of the Company's Tyk2 Subsidiary - Business Wire

Jeffrey Goldberg Appointed Chief Executive Officer and Director of Immunitas Therapeutics – BioSpace

One of the central challenges of drug development has been bridging the gap between laboratory research in model organisms to meaningful clinical advances in humans, said Jeff Goldberg. Immunitas and our scientific co-founders use single cell genomic sequencing and sophisticated computational biology techniques to look at human biology directly. I believe this innovative approach can help to accelerate the development of new therapies for patients. I am excited to be joining the Immunitas team as we discover and develop these highly targeted new immuno-oncology therapies.

Immunitas identifies novel, promising oncology targets with potential applicability across both solid and liquid tumors. Additionally, as part of the discovery process, Immunitas develops key biomarkers to guide the selection of patients who may benefit from its new drugs. The company leverages its expertise in antibody discovery and engineering to create therapies that modulate these targets. Immunitas is currently advancing a number of programs toward early human studies, including a lead program with fully-human monoclonal antibodies that will be developed as single agents using a clinical biomarker strategy to guide early efficacy studies.

Jeff Goldberg has over 20 years of industry experience driving programs from discovery through all phases of drug development to commercialization in multiple therapeutic areas, including oncology, neurology, renal, and rare diseases, said Lea Hachigian, President and Director, Immunitas Therapeutics. We are fortunate to have his demonstrated ability leading and building teams as we create an oncology company powered by our human biology-focused approach to immunology.

Mr. Goldberg joins the Immunitas Board of Directors, which includes Dr. Laura Brass, Managing Director at Novartis Venture Fund, Dr. Jrgen Eckhardt, Head of Leaps by Bayer, Bayers strategic venture capital unit, Dr. Lea Hachigian, Principal, Longwood Fund, Dr. Lucio Iannone, Director of Venture Investments of Leaps by Bayer, Dr. Christoph Westphal, co-founder and General Partner of Longwood Fund, and Dr. Vincent Xiang, Managing Director at Hillhouse Capital.

Jeff Goldberg is an experienced biotech program and brand leader with over 20 years of industry experience. He has driven programs from discovery and pre-clinical through IND, clinical trials, NDA, and commercialization in multiple therapeutic areas, including oncology, neurology, renal, and other rare and orphan diseases. Mr. Goldberg joins Immunitas from Akcea Therapeutics, where he was Chief Operating Officer from the time of its formation in January 2015. Previously, Mr. Goldberg was VP of Business Operations, leading both program management and business development at Proteostasis Therapeutics, Inc., a biotech company focusing on neurology and rare diseases. He also spent more than 11 years in positions of increasing responsibility with Genzyme and Sanofi, providing brand management for two marketed products within Sanofi Oncology. Prior to joining Sanofi Oncology, Mr. Goldberg served as Global Program Lead for Genzyme's stem cell mobilization agent Mozobil, leading the global launch team and overseeing the program management and marketing functions for the product. He began his career at Genzyme as Director, Program Management overseeing the development and launch of Renvela in patients undergoing dialysis. Mr. Goldberg has both an MBA and a Master's degree in Chemical Engineering from the Massachusetts Institute of Technology, and a B.S. in Chemical Engineering from Cornell University.

About Immunitas Therapeutics

Immunitas Therapeutics, founded by Longwood Fund, employs a single cell genomics platform to dissect the biology of immune cells in human tumors, thereby advancing discoveries directly from the bench into meaningful clinical improvements. Our focus on human data allows us to start with and stay closer to the biology that is most relevant in patients and greatly accelerates the pace of our research. The Immunitas team of scientific pioneers innovates around each step of the drug development process, first identifying novel targets, then designing therapeutic strategies, and developing key biomarkers to guide the selection of patients who may benefit from our new drugs. Lead programs from this platform have demonstrated single agent activity against challenging tumors and fully-human monoclonal antibodies are advancing towards clinical studies. http://www.ImmunitasTx.com.

Immunitas the human approach to oncology

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Jeffrey Goldberg Appointed Chief Executive Officer and Director of Immunitas Therapeutics - BioSpace

Real-World Outcomes & Technology Company OM1 Closes $50 Million Series C Financing To Make Healthcare More Measured, Precise, And Pre-Emptive -…

BOSTON, Dec. 18, 2019 /PRNewswire/ -- OM1, a real-world outcomes and technology company, today announced $50 million in Series C financing led by Scale Venture Partners, with participation from existing investors, including General Catalyst (GC), Polaris Partners, and 7wire Ventures. In conjunction with the funding, Rory O'Driscoll, Partner at Scale Venture Partner, has joined OM1's Board of Directors.

"Clinical outcomes are the most important metric in healthcare," said Dr. Richard Gliklich, CEO and founder of OM1. "With this funding, OM1 will accelerate our work towards delivering rapid access to real-world outcomes and evidence and with helping our customers apply those data in impactful ways."

Increasingly healthcare stakeholders, including regulators, payer and providers, are seeking real-world evidence for supporting outcomes-based decision making. By organizing health information and applying artificial intelligence (AI) technology, OM1 helps customers generate and use real-world evidence more rapidly and effectively to gain regulatory approval, understand the effectiveness, safety and value of treatments, and personalize care.

"AI and data are driving factors in the transformation of many industries," said Driscoll. "OM1 is at the forefront of bridging these two in transformative ways in healthcare, and we are excited to be part of the journey to drive the better development of medicine and delivery of care."

OM1 focuses on specific therapeutic areas, including chronic conditions like immunology, rheumatology, cardiometabolic disorders, musculoskeletal conditions and central nervous system (CNS)/behavioral health. Among the products developed by OM1 are industry-leading therapeutic-focused registries for advancing medical research, such as in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and state-of-the art AI solutions for measuring and predicting outcomes for patients and populations.

The funding comes on the heels of a high-growth year for OM1 in which the company has seen more than 400% growth in year-over-year sales. OM1 will use the funding to continue the buildout of its data-driven solutions for real-world evidence, value-based care, and predictive medicine.

OM1 was founded in 2015 by Dr. Richard Gliklich, an Executive-in-Residence (XIR) at GC and the former founder of Outcome, a technology and services company focused on real world research and health outcomes that was acquired in 2011. Dr. Gliklich is also the principal investigator for a major federal effort focused on outcomes measurement and standardization.

For more information, visitwww.om1.com.

Contact

Renee HurleyHead of Marketing, OM1617-620-9571rhurley@om1.com

About OM1

OM1 is a leading real-world outcomes and technology company leveraging big clinical data and AI to better understand, compare, and predict patient outcomes. OM1's real world evidence platform, clinical registries and AI technologies enable clients to accelerate research, measure and benchmark health outcomes and to personalize patient care. Learn more at http://www.om1.com.

About Scale Venture Partners

Scale (@scalevp) invests in software companies that are building the intelligent connected world. Investments include: Bill.com, Box (BOX), Cloudhealth, Pantheon, Demandbase, DocuSign (DOCU), ExactTarget (ET), HubSpot (HUBS), JFrog, Lever, OneLogin, and WalkMe. Scale partners with entrepreneurs to support accelerated growth from the first customer to market leadership. Founded in 2000, Scale has over $1 billion under management and is located in Silicon Valley. For more information, visit http://www.scalevp.com.

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SOURCE OM1

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Real-World Outcomes & Technology Company OM1 Closes $50 Million Series C Financing To Make Healthcare More Measured, Precise, And Pre-Emptive -...

Study finds differences in energy use by immune cells in ME/CFS – National Institutes of Health

News Release

Thursday, December 12, 2019

NIH-funded research suggests changes in the immune system in myalgic encephalomyelitis/chronic fatigue syndrome.

New findings published in the Journal of Clinical Investigation suggest that specific immune T cells from people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) show disruptions in the way they produce energy. The research was supported by the National Institutes of Health.

This research gives us additional evidence for the role of the immune system in ME/CFS and may provide important clues to help us understand the mechanisms underlying this devastating disease, said Vicky Whittemore, Ph.D., program director at NIHs National Institute of Neurological Disorders and Stroke (NINDS), which partially funded the study.

ME/CFS is a severe, chronic, and debilitating disease that can cause a range of symptoms including pain, severe exhaustion, cognitive impairment, and post-exertional malaise, the worsening of symptoms after physical or mental activity. Estimates suggest that between 836,000 and 2.5 million people in the United States may be affected by ME/CFS. It is unknown what causes the disease and there are no treatments.

Research by Alexandra Mandarano and collaborators in the laboratory of Maureen Hanson, Ph.D., professor of molecular biology and genetics at Cornell University in Ithaca, New York, examined biochemical reactions involved in energy production, or metabolism, in two specific types of immune cells obtained from 45 healthy controls and 53 people with ME/CFS. Investigators focused on CD4 T cells, which alert other immune cells about invading pathogens, and CD8 T cells, which attack infected cells. Dr. Hansons team used state-of-the-art methods to look at energy production by the mitochondria within T cells, when the cells were in a resting state and after they had been activated. Mitochondria are biological powerhouses and create most of the energy that drives cells.

Dr. Hanson and her colleagues did not see significant differences in mitochondrial respiration, the cells primary energy-producing method, between healthy and ME/CFS cells at rest or after activation. However, results suggest that glycolysis, a less efficient method of energy production, may be disrupted in ME/CFS. Compared to healthy cells, CD4 and CD8 cells from people with ME/CFS had decreased levels of glycolysis at rest. In addition, ME/CFS CD8 cells had lower levels of glycolysis after activation.

Our work demonstrates the importance of looking at particular types of immune cells that have different jobs to do, rather than looking at them all mixed together, which can hide problems specific to particular cells, said Dr. Hanson. Additional studies focusing on specific cell types will be important to unravel whats gone wrong with immune defenses in ME/CFS.

Dr. Hansons group also looked at mitochondrial size and membrane potential, which can indicate the health of T cell mitochondria. CD4 cells from healthy controls and people with ME/CFS showed no significant differences in mitochondrial size nor function. CD8 cells from people with ME/CFS showed decreased membrane potential compared to healthy cells during both resting and activated states.

Dr. Hansons team examined associations between cytokines, chemical messengers that send instructions from one cell to another, and T cell metabolism. The findings revealed different, and often opposite, patterns between healthy and ME/CFS cells, suggesting changes in the immune system. In addition, the presence of cytokines that cause inflammation unexpectedly correlated with decreased metabolism in T cells.

This study was supported in part by the NIHs ME/CFS Collaborative Research Network, a consortium supported by multiple institutes and centers at NIH, consisting of three collaborative research centers and a data management coordinating center. The research network was established in 2017 to help advance research on ME/CFS.

In addition to providing valuable insights into the immunology of ME/CFS, we hope that the results coming out of the collaborative research network will inspire more researchers, particularly those in the early stages of their careers, to work on this disease, said Joseph Breen, Ph.D., section chief, Immunoregulation Section, Basic Immunology Branch, National Institute of Allergy and Infectious Diseases (NIAID), which partially funded the study.

Future research studies will examine metabolism in other subsets of immune cells. In addition, researchers will investigate ways in which changes in metabolism affect the activity of T cells.

This study was supported by NINDS grant U54NS105541, NIAID grant R21AI117595, Simmaron Research, and an anonymous private donor.

NINDS (https://www.ninds.nih.gov/) is the nations leading funder of research on the brain and nervous system.The mission of NINDS is to seek fundamental knowledge about the brain and nervous system and to use that knowledge to reduce the burden of neurological disease.

About the National Institutes of Health (NIH):NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www.nih.gov.

NIHTurning Discovery Into Health

Mandarano et al. Myalgic encephalomyelitis/chronic fatigue syndrome patients exhibit altered T cell metabolism and cytokine associations, Journal of Clinical Investigation. December 12, 2019

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Study finds differences in energy use by immune cells in ME/CFS - National Institutes of Health

Society for Immunotherapy of Cancer to Host Unique 2-Day Workshop Focused on Interrogating the Tumor-Specific Surfaceome for Immune Targeting – PR Web

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MILWAUKEE (PRWEB) December 18, 2019

The Society for Immunotherapy of Cancer (SITC) will host an innovative workshop on April 2324, 2020, in San Diego, which will focus on the identification and biology of cancer cell surface molecules, and implications for cancer immunotherapy drug delivery and targeting.

The SITC Surfaceome Workshop is geared toward academic and industrial researchers from a variety of fields including medical oncology; bioinformatics; cancer biology; genetics/epigenetics and immunology, among others. Organized by prominent members of the immuno-oncology community, including Samir M. Hanash, MD, PhD, from The University of Texas MD Anderson Cancer Center and Avery D. Posey Jr., PhD, from University of Pennsylvania School of Medicine, the workshop will include oral presentations by leading experts in the field, including a keynote by Carl H. June, MD, from the University of Pennsylvania.

Immunotherapies targeted to tumor cells or the tumor microenvironment, such as bispecific molecules, antibody-drug conjugates, and genetically-engineered lymphocytes, show great promise. Moreover, the technologies to create and develop these treatments are advancing rapidly, said SITC President Mario Sznol, MD. We initiated this conference to address a potential limitation for application of these novel approaches to a broad group of patients, which is the identification and understanding of tumor-specific cell surface targets.

The program will aim to define the cancer cell surfaceome, describe techniques used to investigate it, and summarize methods to evaluate the normal tissue expression of identified tumor cell surface targets. Discussions will also focus on the application and development of immunotherapies and other cancer therapies for cancer cell surface targets.

This workshop will also provide an intimate opportunity for attendees to discuss their work with experts in the field, develop collaborations and learn about novel studies of the tumor cell surfaceome. Starting in January, individuals are encouraged to submit an abstract for an opportunity to present their research; a select number of oral abstract presentation slots will be available. Those abstracts not selected for oral presentation will also have the opportunity to present as a poster. Abstract submission is open to anyone working in this field. Encore presentations are welcome. Abstract submissions are due by February 28, 2020, at 5:00 p.m. PST.

The SITC Surfaceome Workshop will take place on April 2324, 2020, at the Hotel Republic San Diego. Registration rates, criteria for abstract submissions and program schedule are available on SITC Cancer Immunotherapy CONNECT.

About SITCEstablished in 1984, the Society for Immunotherapy of Cancer (SITC) is a nonprofit organization of medical professionals dedicated to improving cancer patient outcomes by advancing the development, science and application of cancer immunotherapy and tumor immunology. SITC is comprised of influential basic and translational scientists, practitioners, health care professionals, government leaders and industry professionals around the globe. Through educational initiatives that foster scientific exchange and collaboration among leaders in the field, SITC aims to one day make the word cure a reality for cancer patients everywhere. Learn more about SITC, our educational offerings and other resources at sitcancer.org and follow us on Twitter, LinkedIn, Facebook and YouTube.

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Society for Immunotherapy of Cancer to Host Unique 2-Day Workshop Focused on Interrogating the Tumor-Specific Surfaceome for Immune Targeting - PR Web

Eli Lilly Stock Rises as Earnings Guidance Beats Analyst Expectations – Barron’s

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Shares of the drugmaker Eli Lilly jumped 1.1% in premarket trading on Tuesday as the company announced 2020 financial guidance that is higher than current Wall Street estimates. The guidance comes a day after the company increased its quarterly dividend by 15% and helps extend a breakout that began in November.

Eli Lilly (ticker: LLY) projected that its operating margin would be 31% on a non-GAAP basis next year. This is better than what investors we spoke with were expecting and represents a step-up from the 28.6% operating margin in 3Q19, wrote Cantor Fitzgerald analyst Louise Chen in a note out Tuesday.

Lilly said it expected revenue in 2020 of between $23.6 billion and $24.1 billion. As of Tuesday morning, the Wall Street consensus estimate was $21.1 billion, according to FactSet.

The company said it expected non-GAAP earnings per share of between $6.70 and $6.80 in 2020, higher than the Wall Street consensus estimate of $5.95, according to FactSet.

We expect 2020 to be a year of strong operating and financial performance for Lilly, characterized by revenue growth for our key medicines both in the U.S. and in international markets, ongoing productivity initiatives leading to further margin expansion, continued progress in our clinical pipeline of new medicines, and solid cash flow, said Josh Smiley, the companys chief financial officer, in a statement.

The back story. Shares of Lilly are up 6.2% so far this year. The stock is trailing the S&P 500, which is up 27.3% this year, the S&P 500 Health Care sector index, up 17.5% this year, and the S&P 500 Pharmaceuticals industry group, up 9.5% this year.

Whats new. In its announcement Tuesday, Lilly said that it expected its 2020 revenue growth to be driven by sales of products including the diabetes drug Trulicity, the psoriasis drug Taltz, the migraine drug Emgality, and Reyvow, another migraine drug recently approved by the Food and Drug Administration.

Lilly said that if it meets the revenue forecast, it will hit the 7% revenue compound annual growth rate it had previously projected for the 2015-2020 time frame.

The company also increased its dividend on Monday, announcing that the first quarter dividend in 2020 will be 74 cents per share, up from 64.5 cents per share.

Lilly is in the early phase of an exciting period of prolonged growth for the company, driven by an expanding portfolio of new medicines focused on diabetes, oncology, immunology, and neuroscience, said the companys chairman and CEO, David Ricks,

Looking forward. The company will discuss the new financial guidance on a conference call set to begin at 9 a.m.

Write to Josh Nathan-Kazis at josh.nathan-kazis@barrons.com

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Eli Lilly Stock Rises as Earnings Guidance Beats Analyst Expectations - Barron's

Sanofi Says Its $2.5 Billion Biotech Takeover Is Just the Beginning – The Motley Fool

As the year comes to a close, Sanofi (NASDAQ:SNY) has a holiday gift for investors in the form of a new strategy. The French drugmaker announced a $2.5 billion biotech takeover in the growing immuno-oncology field earlier this week, then a day later said it is dropping research in the diabetes and cardiovascular fields. This is big news because Sanofi's top-selling drug is diabetes drug Lantus. The problem is that with pricing pressure from competitors, Lantus' sales have been sliding -- and fast.

IMAGE SOURCE: GETTY IMAGES.

Lantus brought in more than $1.2 billion in the third quarter of 2017, and by the same period last year, the figure dropped to less than $1 billion. Sanofi reported a 17.5% decline in Lantus sales to $837 million in the third quarter of this year. To make matters worse, the rest of the diabetes and cardiovascular business has followed, weighing down earnings, while areas including oncology and immunology grew.

That's why the stock market applauded new Chief Executive Officer Paul Hudson's plan to refocus the business. Sanofi shares gained 6.2% on Tuesday after Hudson's comments.

Hudson, in his quest to focus on products and areas that are growing, targets $11 billion in sales for eczema treatment Dupixent. Sales of the drug soared 142% in the third quarter to reach $635 million. The company also will prioritize the development of six innovative investigational products in the areas of hemophilia, lysosomal storage disorders, respiratory syncytial virus, breast cancer, and multiple sclerosis.

Halting research in diabetes and cardiovascular, along with other efforts, is meant to help Sanofi reach $2.2 billion in savings by 2022. In other financial news, the company plans on expanding its business operating income margin to 30% by that year and to 32% by 2025. Business operating income is a non-GAAP measure of financial performance in which Sanofi eliminates elements such as acquisition-related effects and adds items like share of profits or losses from certain investments. The company also aims to increase annual free cash flow 50% by 2022.

Sanofi is reorganizing its operations into three business units: specialty care, vaccines, and general medicine. Consumer healthcare, which includes products like over-the-counter painkillers, will be a stand-alone business with its own R&D and manufacturing processes. Reutersreported that Sanofi might sell the unit or look for a joint venture. Consumer healthcare generated $5.2 billion in sales for Sanofi in 2018, a 3% increase from the previous year. That was about half of the figure generated by the specialty care unit, which grew 29% year over year.

Sanofi said cash from its businesses will be spent on further investment internally, acquisitions, and -- good news here, investors -- increasing the annual dividend. The last payment, in May, was $3.42, increasing for the 25th straight year.

Considering all of the good news, Sanofi isn't looking expensive. According to Zacks research, it trades at 14.16 times earnings, slightly cheaper than the large-cap pharmaceutical industry average of 14.85. The stock has gained 17% so far this year to about $49, but Wall Street predicts at least a bit more upside, with the average analyst price target at $52. Investors should also bear in mind that analysts might adjust their estimates and outlooks in the wake of Hudson's presentation.

With total net sales down 1.1% in the third quarter and the former big businesses of diabetes and cardiovascular slowing, Sanofi didn't present the best investment case a few weeks ago. This week's news, however, changed the landscape. The company is acquiring immuno-oncology company Synthorx (NASDAQ:THOR) to boost a part of its own business that is growing. It is halting the spending on struggling units and reallocating resources to stronger ones. And it continues to think of investors with the goal of boosting dividends.

For those looking to add to pharmaceutical holdings, Sanofi looks like a promising candidate going into 2020 and beyond.

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Sanofi Says Its $2.5 Billion Biotech Takeover Is Just the Beginning - The Motley Fool

University of Iowa professor honored for her work in immunology – UI The Daily Iowan

University of Iowa professor of microbiology and immunology Gail Bishop has been named a fellow of the American Association for the Advancement of Science for her work in the field of immunology.

After a long career in science, University of Iowa professor of microbiology and immunology Gail Bishop was named a 2019 fellow of the American Association for the Advancement of Science after nomination from her peers.

Bishop has worked at the UI for 30 years and studied T and B lymphocytes, which are white blood cells that moderate the bodys defense against pathogens. Through her work, she discovered one of the molecules she was studying was important in preventing a certain type of lymphocyte from turning into a tumor.

Bishop first came to the UI in 1989 as an assistant professor and became active in the cancer center. She was then named the Associate Director for Basic Science Research in the Holden Comprehensive Cancer Center.

The lymphocytes she studies are the microbes in the body that remember what immunizing factors they encounter through vaccinations or natural infection, Bishop said. She is interested in signals the lymphocytes get from other cells and environmental cues that regulate an immune response, she said.

Her research on B lymphocytes led her to her work in the cancer center, Bishop said. Using mouse models, she and the research team removed the molecule that regulates the amount of B lymphocytes in the mouses system, she said.

The mice had large lymph nodes and developed a type of tumor called cell lymphoma, which is the most common type of white blood cell cancer in humans, Bishop said.

Related: UI professor receives grant to train medical field in collaboration with language interpreters

I think you have to really enjoy the science itself and not be in it for prizes or fame or anything like that, Bishop said, because although those things do occasionally come, theyre so intermittent and so unpredictable that if that was your goal, youd be miserable all the time.

Through her time as a researcher, a number of people have worked in her lab. Bruce Hostager, a current researcher in her lab, started working with Bishop when he was a postdoctoral student and has been working with her continuously for 25 years.

Through his time working with Bishop, Hostager has seen the way they conduct research change, he said. The technology used in editing the genetics in mice models has evolved to make their work easier, he added.

Her research is one thing that shes being recognized for, but also for some of her service to the scientific community, both nationally and here at the university, Hostager said.

Stanley Perlman, one of Bishops colleagues at the UI, nominated her to be honored as a fellow in the American Association for the Advancement of Science because of her contributes to immunology.

There is not a large number of women working in biology, Perlman said. More and more women are working in biology at the college level, but as you go up the hierarchy there are a lot of men, he added.

Seeing Bishop be honored as a fellow of the American Association for the Advancement of Science may allow her to serve as a role model for women in science, Perlman said.

I just think recognizing Gails talents and contributions is important, even without it being a role model for anyone or affecting anyone else, Perlman said.

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University of Iowa professor honored for her work in immunology - UI The Daily Iowan

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2 – Science

Interferon Insight

Uncontrolled type I IFN activity has been linked to several human pathologies, but evidence implicating this cytokine response directly in disease has been limited. Here, Duncan et al. identified a homozygous missense mutation in STAT2 in siblings with severe early-onset autoinflammatory disease and elevated IFN activity. STAT2 is a transcription factor that functions downstream of IFN, and this STAT2R148W variant was associated with elevated responses to IFN/ and prolonged JAK-STAT signaling. Unlike wild-type STAT2, the STAT2R148W variant could not interact with ubiquitin-specific protease 18, which prevented STAT2-dependent negative regulation of IFN/ signaling. These findings provide insight into the role of STAT2 in regulating IFN/ signaling in humans.

Excessive type I interferon (IFN/) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2, a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2R148W in homozygosity (but not heterozygosity) were hypersensitive to IFN/, which manifest as prolonged Janus kinasesignal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFN/ signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFN/ signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFN/ activity and supporting efforts to target this pathway therapeutically in IFN-associated disease.

Type I interferons (including IFN/) are antiviral cytokines with pleiotropic functions in the regulation of cellular proliferation, death, and activation. Reflecting their medical importance, type I IFNs have been shown to be essential to antiviral immunity in humans (1), whereas their potent immunomodulatory effects have been exploited to treat both cancer and multiple sclerosis (2, 3).

IFN/ also demonstrates considerable potential for toxicity, which became apparent in initial studies in rodents (4) and subsequent clinical experience in patients (5, 6). Thus, the production of and response to type I IFNs must be tightly controlled (7). Transcriptional biomarker studies increasingly implicate dysregulated IFN/ activity in a diverse spectrum of pathologies ranging from autoimmune to neurological, infectious and vascular diseases (811).

The immunopathogenic potential of IFN/ is exemplified by a group of monogenic inborn errors of immunity termed type 1 interferonopathies, wherein enhanced IFN/ production is hypothesized to be directly causal (12). Neurological disease is typical of these disorders, which manifest as defects of neurodevelopment in association with intracranial calcification and white matter changes on neuroimaging, suggesting that the brain is particularly vulnerable to the effects of excessive type I IFN activity (9). A spectrum of clinical severity is recognized, from prenatal-onset neuroinflammatory disease that mimics in utero viral infectionAicardi-Goutires syndrome (13)to a clinically silent elevation of IFN activity (14).

However, the central tenet of the type I interferonopathy hypothesis, namely, the critical pathogenic role of type I IFNs (12), has yet to be formally established (15). Evidence for an IFN-independent component to disease includes (i) recognition that other proinflammatory cytokines are also induced by nucleic acid sensing, which might contribute to pathogenesis (16); (ii) imperfect correlations between IFN biomarker status and disease penetrance (14); (iii) the absence of neuropathology in mouse models of Aicardi-Goutires syndrome despite signatures of increased IFN activity (17); and (iv) the observation that crossing to a type I IFN receptor deficient background does not rescue the phenotype in certain genotypes (e.g., STING and ADAR1) (18, 19), although it does in others (e.g., TREX1 or USP18) (20, 21). Here, we provide concrete evidence of the pathogenicity of type I IFNs in humans, shedding new light on the critical importance of signal transducer and activator of transcription 2 (STAT2) in the negative regulation of this pathway.

We evaluated two male siblings, born in the United Kingdom to second cousin Pakistani parents. Briefly, patient II:3, born at 34 weeks + 6 days with transient neonatal thrombocytopenia, was investigated for neurodevelopmental delay at 6 months (which was attributed to compensated hypothyroidism). Aged 8 months, he presented with the first of three episodes of marked neuroinflammatory disease, associated with progressive intracranial calcification, white matter disease, and, by 18 months, intracranial hemorrhage (Fig. 1A). These episodes were associated with systemic inflammation and multiorgan dysfunction, including recurrent fever, hepatosplenomegaly, cytopenia with marked thrombocytopenia, raised ferritin, and elevated liver enzymes. Latterly, acute kidney injury with hypertension and nephrotic range proteinuria developed (see Table 1, Supplementary case summary, and table S1).

(A) Neuroimaging demonstrating calcifications [brainstem/hypothalamus (proband II:3, top), cerebral white matter/basal ganglia/midbrain/optic tract (sibling II:4, top and middle)], hemorrhages [occipital/subdural/subarachnoid (proband II:3, middle)], and cerebral white matter and cerebellar signal abnormality with parenchymal volume loss (both, bottom), accompanied by focal cystic change and cerebellar atrophy (sibling II:4). (B) Whole blood RNA-seq ISG profiles: controls (n = 5); proband II:3 (n = 4); and patients with mutations in: TREX1 (n = 6), RNASEH2A (n = 3), RNASEH2B (n = 7), RNASEH2C (n = 5), SAMHD1 (n = 5), ADAR1 (n = 4), IFIH1 (n = 2), ACP5 (n = 3), TMEM173 (n = 3), and DNASE2 (n = 3). (C) IFN scores (RT-PCR) of patients, parents, and n = 29 healthy controls. ****P < 0.001, ANOVA with Dunnetts posttest. (D) Renal histopathology in proband (400 magnification) showing TMA with extensive double contouring of capillary walls (silver stain, top left); endothelial swelling, mesangiolysis, and red cell fragmentation (top right); arteriolar fibrinoid necrosis (bottom left); and myxoid intimal thickening of an interlobular artery (bottom right, all hematoxylin and eosin). (E) Transcriptional response to JAK inhibitor (JAKi) ruxolitinib in both patients (RT-PCR).

HLH, hemophagocytic lymphohistiocytosis; EEG, electroencephalogram.

This clinical phenotype was reminiscent of a particularly severe form of type I interferonopathy. In keeping with this observation, IFN-stimulated gene (ISG) transcripts in whole blood, measured by RNA sequencing (RNA-seq) and reverse transcription polymerase chain reaction (RT-PCR), were substantially elevated over multiple time points at similar magnitudes to recognized type I interferonopathies (Fig. 1, B and C), notably without evidence of concomitant induction of IFN-independent inflammatory pathways (fig. S1). Disease in the proband, which not only met the diagnostic criteria for hemophagocytosis but also included features of a thrombotic microangiopathy (TMA) (Fig. 1D), was partially responsive to dexamethasone and stabilized with the addition of the Janus kinase (JAK) inhibitor ruxolitinib (Fig. 1E and fig. S2). Sadly, however, this child succumbed to overwhelming Gram-negative bacterial sepsis during hematopoietic stem cell transplantation.

Patient II:4, his infant brother, presented with abnormal neurodevelopment and neuroimaging in the neonatal period, characterized by apneic episodes from 3 weeks of age in conjunction with parenchymal calcifications and hemorrhage, abnormal cerebral white matter, and brainstem and cerebellar atrophy (Fig. 1A). Blood tests revealed an elevated ISG score (Fig. 1, B and C), anemia, elevation of D-dimers, and red cell fragmentation on blood film, together with proteinuria and borderline elevations of ferritin and lactate dehydrogenase; renal function was normal, and blood pressure was on the upper limit of the normal range for gestational age. Introduction of ruxolitinib led to prompt suppression of ISG expression in whole blood (Fig. 1E) and an initial reduction in apneic episodes, but neurological damage was irretrievable, and he succumbed to disease at 3 months of age. Mothers pregnancy with patient II:4 had been complicated by influenza B at 23 weeks gestation.

Whole-exome sequencing analysis of genomic DNA from the kindred, confirmed by Sanger sequencing (Fig. 2, A and B), identified an extremely rare variant in STAT2 (c.442C>T), which substituted tryptophan for arginine at position 148 in the coiled-coil domain (CCD) of STAT2 (p.Arg148Trp, Fig. 2C). The Arg148Trp variant was present in the homozygous state in both affected children and was heterozygous in each parent and one healthy sibling, consistent with segregation of an autosomal recessive trait (table S2). This variant was found in the heterozygous state at extremely low frequency in publicly available databases of genomic variation [frequency < 0.00001 in Genome Aggregation Database (22)], and no homozygotes were reported. A basic amino acid, particularly arginine, at position 148 is highly conserved (fig. S3). In silico tools predicted that this missense substitution was probably deleterious to protein function (table S2). STAT2 protein expression in patient cells was unaffected by the Arg148Trp variant, in contrast to the situation for pathogenic loss-of-expression STAT2 variants, which resulted in a distinct phenotype of heightened viral susceptibility (Fig. 2D) (23, 24). Filtering of exome data identified an additional recessive variant in CFH (c.2336A>G and p.Tyr779Cys; fig. S4) present in the homozygous state in II:3 but absent from II:4. We considered the possibility that this contributed to TMA in the proband, but functional studies of this variant showed negligible impact on factor H function (fig. S5).

(A) Pedigree, (B) capillary sequencing verification, (C) protein map, and (D) immunoblot (fibroblasts) showing normal expression of STAT2 protein. DBD, DNA binding domain; LD, linker domain; SH2, Src homology 2 domain; TAD, trans-activation domain.

The transcription factor STAT2 is essential for transcriptional activation downstream of the receptors for the innate IFN-/ (IFNAR) and IFN- and their associated JAK adaptor proteins. In the current paradigm (25), STAT2 is activated by tyrosine phosphorylation, associated with IFN regulatory factor 9 (IRF9) and phosphorylated STAT1 (pSTAT1) to form the IFN-stimulated gene factor 3 (ISGF3) to effect gene transcription by binding to IFN-stimulated response elements in the promoters of ISGs. Although loss-of-function variants in STAT2 increase susceptibility to viral disease (23, 24), evidence here suggested pathological activation. Germline gain-of-function variants have been reported in STAT1 (26, 27) and STAT3 (28, 29) but not hitherto STAT2. Consistent with the apparent gain of IFN activity associated with mutant STAT2R148W, we observed in patient fibroblasts (Fig. 3, A and B) and peripheral blood mononuclear cells (PBMCs; fig. S6) the enhanced expression of ISG protein products across a range of IFN concentrations. However, basal and induced production of IFNB mRNA by fibroblasts was indistinguishable from controls (Fig. 3C); nor was IFN protein substantially elevated in patient samples of cerebrospinal fluid (II:3) or plasma (II:4) as measured by a highly sensitive digital enzyme-linked immunosorbent assay (ELISA) assay (30), albeit samples were acquired during treatment (table S3). Thus, the response to type I IFNs, but not their synthesis, was exaggerated. This heightened IFN sensitivity was accompanied by enhancement of key effector functions, as revealed by assays of IFN-mediated viral protection (Fig. 3D) and cytotoxicity (Fig. 3E). Collectively, these data indicated that STAT2R148W was not constitutively active but rather resulted in an exaggerated response upon IFN exposure. To confirm that the Arg148Trp variant was responsible for this cellular phenotype, we transduced STAT2-null U6A cells (31) and STAT2-deficient primary fibroblasts (23) with lentiviruses encoding either wild type (WT) or STAT2R148W, recapitulating the heightened sensitivity of cells expressing the latter to IFN (Fig. 3, F and G, and fig. S7).

Unless stated, all data are from patient II:3 and control fibroblasts. (A) ISG expression (immunoblot, IFN for 24 hours) and (B) densitometry analysis (n = 3, t test). MX1, MX dynamin like GTPase 1; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; RSAD2, radical S-adenosyl methionine domain containing 2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) IFNB mRNA (RT-PCR) external polyinosinic:polycytidylic acid (poly I:C) treatment (25 g/ml for 4 hours; n = 3, t test). US, unstimulated. (D) Antiviral protection assay (mCherry-PIV5). Twofold dilutions from IFN (16 IU/ml), IFN (160 IU/ml) n = 7 replicates, representative of n = 2 experiments (two-way ANOVA with Sidaks posttest). (E) Cytopathicity assay (IFN for 72 hours; n = 3, t test). (F) As in (A), ISG expression in STAT2/ U6A cells reconstituted with STAT2WT or STAT2R148W (immunoblot, IFN for 24 hours). (G) As in (B), n = 3 to 4, t test. Data are presented as means SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., nonsignificant.

To explore the underlying mechanism for heightened type I IFN sensitivity, we first probed STAT2 activation in IFN-stimulated fibroblasts. In control lysates, levels of pSTAT2 had almost returned to baseline between 6 and 24 hours of treatment despite the continued presence of IFN (Fig. 4, A and B). In contrast, pSTAT2 persisted for up to 48 hours in patient cells. This abnormally prolonged pSTAT2 response to IFN was also observed in PBMCs of both patients (fig. S8). Consistent with immunoblot data, immunofluorescence analysis showed persistent ( 6 hours) nuclear localization of STAT2 in patient fibroblasts after IFN treatment, at times when STAT2 staining was predominantly cytoplasmic in control cells (Fig. 4, C and D, and fig. S9). This was accompanied by continued expression of ISG transcripts for 36 hours after the washout of IFN in patient cells as measured by RNA-seq and RT-PCR (Fig. 4, E and F). Thus, the type I IFN hypersensitivity of patient cells was linked to prolonged IFNAR signaling.

All data are from patient II:3 and control fibroblasts. (A) pSTAT2 time course [immunoblot, IFN (1000 IU/ml)] and (B) densitometry analysis (n = 5 experiments, two-way ANOVA with Sidaks posttest). (C) Immunofluorescence analysis [IFN (1000 IU/ml); scale bar, 100 m; representative of n = 3 experiments] with (D) image analysis of STAT2 nuclear translocation (n = 100 cells per condition, ANOVA with Sidaks posttest). A.U., arbitrary units. (E) RNA-seq analysis of IFN-regulated genes (n = 3 controls) with (F) validation by RT-PCR (n = 3, two-way ANOVA with Sidaks posttest). CPM, read counts per million. (G) pSTAT2 decay (immunoblot). IFN (1000 IU/ml; 30 min) followed by extensive washing and treatment with 500 nM staurosporine (STAU). Times relative to STAU treatment. (H) No significant differences by densitometry analysis (n = 3, t test). Data are presented as means SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The IFNAR signaling pathway is subject to multiple layers of negative regulation that target STAT phosphorylation directlythrough the action of tyrosine phosphatasesor indirectly by disrupting upstream signal transduction (7). Prolonged tyrosine phosphorylation is reported with gain-of-function mutations in STAT1, in association with impaired sensitivity to phosphatase activity (27). By contrast, we observed no impairment of dephosphorylation of STAT2R148W in pulse-chase assays with the kinase inhibitor staurosporine (Fig. 4, G and H), implying instead a failure of negative feedback upon the proximal signaling events that generate pSTAT2.

To localize this defect, we analyzed by phosflow and immunoblot the successive activation steps downstream of IFNAR ligand binding in Epstein-Barr virus (EBV)transformed B cells from the proband (II:3) and a heterozygous parent (I:2). As was the case for STAT2 phosphorylation, we also observed prolonged phosphorylation of both JAK1 and STAT1 after IFN treatment (Fig. 5, A to D). This points to a defect in regulation of the most proximal IFNAR signaling events, upstream of STAT2 (7). We observed no evidence of this phenotype in cells bearing STAT2R148W in the heterozygous state, consistent with autosomal recessive inheritance and the lack of clinical disease or up-regulation of IFN activity in heterozygous carriers. This genetic architecture provides a notable contrast to gain-of-function mutations affecting other STAT proteins, all of which are manifest in the heterozygous state (2629).

Time course of IFN stimulation (1000 IU/ml) in EBV B cells from patient II:3 [homozygous (hom)], parent I:2 [heterozygous (het)], and n = 3 controls. (A) Immunoblot and (B) densitometry analyses. (C) Representative histograms (flow cytometry) and (D) mean fluorescence intensity (MFI). Data are means SEM of three repeat experiments (*P < 0.05, **P < 0.01, t test).

Known negative regulators of IFNAR signaling are suppressor of cytokine signaling (SOCS) 1 and SOCS3 (32) and the ubiquitin-specific protease 18 (USP18) (33). SOCS1 and SOCS3 participate in regulation of additional JAK-STAT signaling pathways, such as those activated by IFN and interleukin 6 (IL-6) (34, 35), whereas USP18 acts specifically upon IFNAR signaling (33). To better localize the molecular defect in patient cells, we examined the signaling responses to IFN (STAT1 phosphorylation) and IL-6 (STAT3 phosphorylation), based on the prediction that defects of SOCS1 or SOCS3 regulation would manifest under these conditions. These experiments revealed that regulation of STAT1 and STAT3 phosphorylation was normal in patient fibroblasts (fig. S10). Together with the absence of evidence of up-regulation of the IFN and IL-6 pathways in the analysis of whole blood RNA-seq data (fig. S1), these observations effectively ruled out the involvement of SOCS1 and SOCS3 in the clinical phenotype, leading us to suspect a defect of USP18 regulation.

To investigate this possibility, we primed patient and control cells with IFN for 12 hours, washed them extensively, and rested and restimulated them with IFN or IFN after 48 hours. In these experiments, IFN-induced pSTAT2 and pSTAT1 were strongly inhibited by priming in control cells, consistent with desensitization, a well-established phenomenon of type I IFN biology (Fig. 6, A and B) (36). In marked contrast, the response to IFN restimulation in patient cells was minimally suppressed, indicating a failure of desensitization. Desensitization has been shown to be exclusively mediated by USP18, an IFN-induced isopeptidase (37), through its displacement of JAK1 from the receptor subunit IFNAR2 (38, 39)a function that is independent of its isopeptidase activity toward the ubiquitin-like protein ISG15 (33). STAT2 plays a critical role as an adaptor protein by supporting binding of USP18 to IFNAR2 (Fig. 6C) (40). Both the clinical and cellular effects of STAT2R148W resemble homozygous USP18 deficiency, which was recently described as the molecular cause of a severe pseudo-TORCH syndrome associated with elevated type I IFN expression (table S4) (41). Although this STAT2:USP18 interaction has been shown to be essential for negative regulation of type I IFN signaling in vitro (40), its significance in vivo has not previously been examined. Furthermore, the precise residue(s) of STAT2 that bind USP18 were unresolved, although this interaction had been localized to a region including the CCD and/or DNA binding domain(s) of STAT2 (40).

(A) Desensitization assay (immunoblot, fibroblasts) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 4, ANOVA with Sidaks posttest). (C) Schematic of USP18 mechanism of action and proposed model of STAT2R148W pathomechanism. (D) Modeling of exposed WT (R148)/mutant (W148) residue, demonstrating charge-change (blue, positive; red, negative) and possible steric restriction. (E) Coimmunoprecipitation of USP18 by STAT2 in U6A cells expressing STAT2WT or STAT2R148W with (F) densitometry analysis (USP18/STAT2, ratio to WT; one-sample t test). Data are means SEM (**P < 0.01, ****P < 0.0001). IB, immunoblot.

Because USP18 was induced normally in patient cells (Fig. 6, A and B) and in vivo (Fig. 1B), our data implied that STAT2R148W impedes the proper interaction of STAT2 with USP18, compromising its regulatory function (Fig. 6C). Molecular modeling of STAT2R148W placed the substituted bulky aromatic tryptophan, and resulting charge change, at an exposed site within the CCD (Fig. 6D). Consistent with our suspicion that this might impair the STAT2:USP18 interaction through electrostatic or steric hindrance, coimmunoprecipitation experiments in U6A cells stably expressing WT or STAT2R148W demonstrated a statistical significance reduction of USP18 pull down STAT2R148W compared with WT (Fig. 6, E and F), providing a molecular mechanism for the USP18 insensitivity of patient cells.

Although disruption to the STAT2R148W:USP18 interaction was the most plausible explanation for the clinical and molecular phenotype, we also considered the contribution of alternative regulatory functions of STAT2. Beyond the role of tyrosine phosphorylated STAT2 in innate IFN signal transduction, the unphosphorylated form of STAT2 (uSTAT2) has additional, recently described functions in the regulation of other cytokine signaling pathways. For example, uSTAT2 negatively regulates the activity of IFN (and other inflammatory cytokines that signal via STAT1 homodimers) by binding to uSTAT1 via its CCD (42). This interaction appears to limit the pool of STAT1 available for incorporation into transcriptionally active (tyrosine phosphorylated) STAT1 homodimers. Conversely, uSTAT2, induced by type I IFN signaling, has been reported to promote the transcriptional induction of IL6 through an interaction with the nuclear factor B subunit p65 (43). To investigate the potential relevance of these regulatory functions of STAT2, we first examined the induction of IL6 by RT-PCR analysis of RNA isolated from whole blood of patients, their heterozygous parents, and healthy controls. We found no evidence of increased expression of IL6 or its target gene SOCS3 (fig. S11, A and B), consistent with our previous pathway analysis of RNA-seq data (fig. S1) and implying that STAT2R148W does not influence IL-6 induction. Next, to explore any impact on STAT2s negative regulatory activity toward STAT1, we examined the transcriptional responses to IFN in patient fibroblasts and in U6A cells expressing STAT2R148W. Although we were able to reproduce the previously reported findings of heightened transcription of the IFN-regulated gene CXCL10 in U6A cells lacking STAT2, alongside a nonsignificant trend for IRF1 (fig. S12, A and B) (42), STAT2R148W did not enhance transcript levels of either CXCL10 or IRF1 above WT, in agreement with the data showing the preserved ability of STAT2R148W to bind STAT1 in a coimmunoprecipitation assay (fig. S12, C and D). Together, these studies effectively exclude a contribution of the USP18-independent regulatory functions of STAT2 to the disease phenotype.

To conclusively demonstrate the impairment of STAT2:USP18-mediated negative regulation in patient cells, we tested the impact of overexpression or knockdown of USP18. First, we probed IFNAR responses in fibroblasts stably expressing USP18. As predicted, USP18 was significantly impaired in its ability to suppress IFN signaling in patient cells, relative to controls, both in terms of STAT phosphorylation (Fig. 7, A and B) and STAT2 nuclear translocation (Fig. 7, C and D), recapitulating our prior observations with IFN priming (Fig. 6A). The reciprocal experiment, in which USP18 expression was stably knocked down using short hairpin RNA (shRNA), revealed significantly prolonged STAT2 phosphorylation in control cells at 24 hours, recapitulating the phenotype of patient cells (Fig. 7, E and F). In contrast, there was no effect of USP18 knockdown in patient cells, demonstrating that they are USP18 insensitive. Incidentally, we noted that the early peak (1 hour) of STAT2 phosphorylation in USP18-knockdown control fibroblasts was marginally reduced (Fig. 7E). This subtle reduction was also apparent in STAT2R148W patient fibroblasts (Fig. 4B), although not in EBV B cells (Fig. 5). We speculate that the cell typespecific induction of other negative regulator(s) of IFNAR signaling at early times after IFN treatment, such as SOCS1, might be responsible for this observation. RT-PCR analysis confirmed the increased expression of SOCS1 mRNA in whole blood of patients (fig. S11C), whereas examination of RNA-seq data from IFN-treated fibroblasts revealed an eightfold enhancement of SOCS1 expression at 6 hours in patient cells as compared with controls (Padj = 0.0001; Fig 4E). Together, these data provide preliminary support for the hypothesis that alternative negative regulator(s) of IFNAR signaling may be up-regulated in patient cells. Nevertheless, such attempts at compensation are clearly insufficient to restrain IFNAR responses in the context of STAT2R148W, reflecting the nonredundant role of STAT2/USP18 in this process (39). Collectively, these data support a model in which the homozygous presence of the Arg148Trp STAT2 variant compromises an essential adaptor function of STAT2 toward USP18, rendering cells USP18 insensitive and culminating in unrestrained, immunopathogenic IFNAR signaling.

All data are from patient II:3 and control fibroblasts. (A) STAT phosphorylation in USP18 and vector expressing fibroblasts (immunoblot) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 3, ANOVA with Sidaks posttest). (C) Immunofluorescence analysis of STAT2 nuclear translocation [IFN (1000 IU/ml 30 min); representative of n = 3 experiments] with (D) image analysis (n = 100 cells per condition, ANOVA with Sidaks posttest). (E) Time course of STAT phosphorylation upon IFN stimulation (1000 IU/ml for 0, 1, 6, and 24 hours) of cells transduced with USP18 shRNA or nontargeting (NT) shRNA with (F) densitometry analysis of pSTAT2 (n = 3, t test). Data are means SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001).

We report a type I interferonopathy, caused by a homozygous missense mutation in STAT2, and provide detailed studies to delineate the underlying molecular mechanism. Our data indicate the failure of mutant STAT2R148W to support proper negative regulation of IFNAR signaling by USP18revealing an essential regulatory function of human STAT2. This defect in STAT2 regulation results in (i) an inability to properly restrain the response to type I IFNs and (ii) the genesis of a life-threating early-onset inflammatory disease. This situation presents a marked contrast with monogenic STAT2 deficiency, which results in heightened susceptibility to viral infection due to the loss of the transcription factor complex ISGF3 (23, 24). Thus, just as allelic variants of STAT1 and STAT3 are recognized that either impair or enhance activity of the cytokine signaling pathways in which they participate (44), we can now add to this list STAT2. Our findings also highlight an apparently unique property of human STAT2: That it participates directly in both the positive and negative regulation of its own cellular signaling pathway. Whether this is true of STAT2 in other species remains to be determined. Our findings also localize the interaction with USP18 to the CCD of STAT2, indicating a specific residue critical for this interaction. This structural insight may be relevant to efforts to therapeutically interfere with the STAT2:USP18 interaction to promote the antiviral action of IFNs.

This monogenic disease of STAT2 regulation provides incontrovertible evidence of the pathogenic effects of failure to properly restrain IFNAR signaling in humans. The conspicuous phenotypic overlap with existing defects of IFN/ overproduction, particularly with regard to the neurological manifestations, provides compelling support for the type I interferonopathy hypothesis, strengthening the clinical rationale for therapeutic blockade of IFNAR signaling (15). JAK1/2 inhibition with ruxolitinib was highly effective in controlling disease in the proband; however, the damage that already accrued at birth in his younger brother was irreparable, emphasizing the importance of timely IFNAR blockade in prevention of neurological sequelae. A notable aspect of the clinical phenotype in patient II:3 was the occurrence of severe TMA. Our studies did not support a pathogenic contribution of the coinherited complement factor H variant in patient II:3. This evidence, together with clinical hematological and biochemical results suggestive of incipient vasculopathy in patient II:4who did not carry the CFH variantsuggests that type I IFN may have directly contributed to the development of TMA. Although it is not classically associated with type I interferonopathies, TMA is an increasingly recognized complication of both genetic (41, 42) and iatrogenic states of IFN excess (43), consistent with the involvement of vasculopathy in the pathomechanism of IFN-mediated disease. The fact that STAT2R148W is silent in the heterozygous state at first sight offers a confusing contrast with gain-of-function mutations of its sister molecules STAT1 and STAT3, both of which produce autosomal dominant disease with high penetrance (2629). However, the net gain of IFNAR signaling activity results from the isolated loss of STAT2s regulatory function, which evidently behaves as a recessive trait. There are other examples of autosomal recessive loss-of-function disorders of negative regulators, including USP18 itself (41, 45); the unique aspect in the case of STAT2R148W is that the affected molecule is itself a key positive mediator within the regulated pathway.

In light of the intimate relationship between STAT2 and USP18 revealed by these and other recent data (40), it is reasonable to conclude that the clinical manifestations of human USP18 deficiency are dominated by the loss of its negative feedback toward IFNAR rather than the STAT2-independent functions of USP18 including its enzymatic activity (40, 46, 47). In mouse, white matter pathology associated with microglia-specific USP18 deficiency is prevented in the absence of IFNAR (21). There are now three human autosomal recessive disorders that directly compromise the proper negative regulation of IFNAR signaling and thus produce a net gain of signaling function: USP18 deficiency, which leads to embryonic or neonatal lethality with severe multisystem inflammation (41); STAT2R148W, which largely phenocopies USP18 deficiency; and ISG15 deficiency, in which there is a much milder phenotype of neurological disease without systemic inflammation (45). ISG15 stabilizes USP18, and human ISG15 deficiency leads to a partial loss of USP18 protein (41). Thus, a correlation is clearly evident between the extent of USP18 dysfunction and the clinical severity of these disorders, with STAT2R148W closer to USP18 deficiency and ISG15 on the milder end of the spectrum (table S4). Those molecular defects that result in a failure of negative regulation of IFNAR signaling (i.e., STAT2R148W and USP18/) lead to more serious and extensive systemic inflammatory disease than do defects of excessive IFN/ production (41), suggesting that the STAT2:USP18 axis acts to limit an immunopathogenic response toward both physiological (48) and pathological (41) levels of IFN/. Thus, variability in the efficiency of this process of negative regulation might be predicted to influence the clinical expressivity of interferonopathies. Determining the cellular source(s) of physiological type I IFNs and the molecular pathways that regulate their production are important areas for future investigation.

Some limitations of our results should be acknowledged. Although strenuous efforts were made, we were only able to identify a single kindred, which probably reflects the rarity of this variant. As more cases are identified, our understanding of the clinical phenotypic spectrum will inevitably expand. Furthermore, for practical and cultural/ethical reasons, limited amounts of cellular material and tissues were available for analysis. As a result, we were unable to formally evaluate the relevance of STAT2 regulation toward type III IFN signaling; however, existing data suggest that USP18 plays a negligible role in this context (38). Together, our findings confirm an essential regulatory role of STAT2, supporting the hypothesis that type I IFNs play a causal role in a diverse spectrum of human disease, with immediate therapeutic implications.

We investigated a kindred with a severe, early-onset, presumed genetic disease, seeking to determine the underlying pathomechanism by ex vivo and in vitro studies. Written informed consent for these studies was provided, and ethical/institutional approval was granted by the NRES Committee North East-Newcastle and North Tyneside 1 (ref: 16/NE/0002), South Central-Hampshire A (ref: 17/SC/0026), and Leeds (East) (ref: 07/Q1206/7).

Dermal fibroblasts from patient II:3 and healthy controls were obtained by standard methods and cultured in Dulbeccos modified Eagles medium supplemented by 10% fetal calf serum and 1% penicillin/streptomycin (DMEM-10), as were human embryonic kidney 293 T cells and the STAT2-deficient human sarcoma cell line U6A (31). PBMCs and EBV-transformed B cells were cultured in RPMI medium supplemented by 10% fetal calf serum and 1% penicillin/streptomycin (RPMI-10). Unless otherwise stated, cytokines/inhibitors were used at the following concentrations: human recombinant IFN-2b (1000 IU/ml; Intron A, Schering-Plough, USA); IFN- (1000 IU/ml; Immunikin, Boehringer Ingelheim, Germany); IL-6 (25 ng/ml; PeproTech, USA); and 500 nM staurosporine (ALX-380-014-C250, Enzo Life Sciences, NY, USA). Diagnostic histopathology, immunology, and virology studies were conducted in accredited regional diagnostic laboratories to standard protocols.

Whole-exome sequencing analysis was performed on DNA isolated from whole blood from patients I:1, I:2, II:3, and II:4. Capture and library preparation was undertaken using the BGI V4 exome kit (BGI, Beijing, China) according to manufacturers instructions, and sequencing was performed on a BGISEQ (BGI). Bioinformatics analysis and variant confirmation by Sanger sequencing are described in the Supplementary Materials.

RNA was extracted by lysing fibroblasts in TRIzol reagent (Thermo Fisher Scientific) or from whole blood samples collected in PAXgene tubes (PreAnalytix), as described previously (49). Further details, including primer/probe information, are summarized in the Supplementary Materials and table S5.

Whole-blood transcriptome expression analysis was performed using nine whole blood samples, from the proband taken before and during treatment, and five controls. In addition, the four patient II:3 samples taken before treatment and samples from six patients with mutations in TREX1, three with mutations in RNASEH2A, seven with mutations in RNASEH2B, five with mutations in RNASEH2C, five with mutations in SAMHD1, four with mutations in ADAR1, two with mutations in IFIH1, three with mutations in ACP5, three with mutations in TMEM173, and three with mutations in DNASE2 were analyzed, as described in the Supplementary Materials. RNA integrity was analyzed with Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA purification and fragmentation, complementary DNA (cDNA) synthesis, and target amplification were performed using the Illumina TruSeq RNA Sample Preparation Kit (Illumina). Pooled cDNA libraries were sequenced using the HiSeq 4000 Illumina platform (Illumina). Fibroblasts grown in six-well plates were mock-treated or treated with IFN for 6 or 12 hours, followed by extensive washing and 36-hour rest, before RNA extraction. The experiment was performed with patient II:3 and control cells (n = 3) in triplicate per time point. RNA was extracted using the ReliaPrep RNA Miniprep kit (Promega) according to manufacturers instructions and processed as described above, before sequencing on an Illumina NextSeq500 platform. Bioinformatic analysis is described in the Supplementary Materials. PMBC and fibroblast STAT2 patient and control data have been deposited in ArrayExpress (E-MTAB-7275) and Gene Expression Omnibus (GSE119709), respectively.

Details of lentiviral constructs, mutagenesis, and preparation are included in the Supplementary Materials. Cells were spinoculated in six-well plates for 1.5 hours at 2000 rpm, with target or null control viral particles, at various dilutions in a total volume of 0.5 ml of DMEM-10 containing hexadimethrine bromide [polybrene (8 g/ml); Sigma-Aldrich]. Cells were rested in virus-containing medium for 8 hours and then incubated in fresh DMEM-10 until 48 hours, when they were subjected to selection with puromycin (2.0 g/ml) or blastocidin (2.5 g/ml) (Sigma-Aldrich). Antibiotic-containing medium was refreshed every 72 hours.

EBV B cells were seeded at a density of 8 105 cells/ml in serum-free X-VIVO 15 medium (Lonza, Basel, Switzerland) and stimulated with IFN (1000 IU/ml) for the indicated times. After staining with Zombie UV (BioLegend, San Diego, CA, USA), cells were fixed using Cytofix buffer (BD Biosciences, Franklin Lakes, NJ, USA). Permeabilization was achieved by adding ice-cold PermIII buffer (BD Biosciences, Franklin Lakes, NJ, USA), and cells were incubated on ice for 20 min. After repeated washing steps with phosphate-buffered saline (PBS)/2% fetal bovine serum (FBS), cells were stained for 60 min at room temperature with directly conjugated antibodies (table S6). Samples were acquired on a Symphony A5 flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). The gating strategy is shown in fig. S13.

Immunoblotting was carried out as previously described (1) and analyzed using either a G:BOX Chemi (Syngene, Hyarana, India) charge-coupled device camera with GeneSnap software (Syngene) or a LI-COR Odyssey Fc (LI-COR, NE, USA). Densitometry analysis was undertaken using ImageStudio software (version 5.2.5, Li-COR). For complement studies, sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions was performed on patient/parental serum [diluted 1:125 in nonreducing buffer (PBS)] or affinity-purified factor H (diluted to 200 ng in nonreducing buffer), separated by electrophoresis on a 6% SDS-PAGE gel, and transferred to nitrocellulose membranes for immunoblotting (antibodies in table S6). Blots were developed with Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and imaged on a LI-COR Odyssey Fc (LI-COR).

U6A cells were lysed in immunoprecipitation buffer [25 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, and 10 mM sodium fluoride, with complete protease inhibitor (Roche, Basel, Switzerland)]. Lysates were centrifuged at 13,000 rpm at 4C for 10 min. Soluble fractions were precleared for 1 hour at 4C with Protein G Sepharose 4 (Fast Flow, GE Healthcare, Chicago, USA) that had been previously blocked with 1% bovine serum albumin (BSA) IP buffer for 1 hour. Precleared cell lysates were immunoprecipitated overnight with blocked beads that were incubated with anti-STAT2 antibody (A-7) for 1 hour and then washed three times in IP buffer before boiling with 4 lithium dodecyl sulfate buffer at 95C for 10 min to elute the absorbed immunocomplexes. Immunoblot was carried out as described above.

Fibroblasts grown on eight-well chamber slides (Ibidi, Martinsried, Germany) were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature before blocking/permeabilization with 3% BSA/0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were incubated overnight with anti-STAT2 primary antibody (10 g/ml; C20, Santa Cruz Biotechnology, Dallas, USA) at 4C, and cells were washed three times with PBS. Secondary antibody [goat anti-rabbit Alexa Fluor 488 (1 g/ml), Thermo Fisher Scientific] incubation was performed for 1 hour at room temperature, followed by nuclear staining with 4,6-diamidino-2-phenylindole (DAPI; 0.2 g/ml; Thermo Fisher Scientific). Cells were imaged with an EVOS FL fluorescence microscope with a 10 objective (Thermo Fisher Scientific). The use of STAT2-deficient cells (23) demonstrated the specificity and lack of nonspecific background of the staining approach. Image analysis was performed in ImageJ. The DAPI (nuclear) image was converted to binary, and each nucleus (object) was counted. This mask was overlaid onto the STAT2 image, and the mean fluorescence intensity of STAT2 within each nucleus was calculated (see also fig. S9). About n = 100 cells were analyzed per image.

The structure of human STAT2 has not been experimentally determined. We therefore used comparative modeling to predict the structure. The sequences of both the WT and mutant were aligned to mouse STAT2 (Protein Data Bank code 5OEN, chain B). For each sequence, 20 models were built using MODELLER (50), and the one with the lowest discrete optimized protein energy score was chosen. Protein structures and electrostatic surfaces were visualized with PyMOL (Schrodinger, USA).

Fibroblasts grown on 96-well plates were treated with IFN (1000 or 10,000 IU/ml) or DMEM-10 alone for 72 hours. Cells were fixed in PBS containing 5% formaldehyde for 15 min at room temperature and then incubated with crystal violet stain. Plates were washed extensively then allowed to air dry. The remaining cell membrane-bound stain was solubilized with methanol and absorbance at 595 nm measured on a TECAN Sunrise plate reader (Tecan, Switzerland). Background absorbance was subtracted from all samples, and the results were expressed as a percentage of the absorbance values of untreated cells.

Fibroblasts grown on 96-well plates were pretreated in septuplicate for 18 hours with twofold serial dilutions of IFN and IFN, followed by infection with mCherry-expressing parainfluenza virus 5 (PIV5) in DMEM/2% FBS for 24 hours. Monolayers were fixed with PBS containing 5% formaldehyde, and infection was quantified by measuring mean fluorescence intensity of mCherry (excitation, 580/9; emission, 610/20) using a TECAN Infinite M200 Pro plate reader (Tecan, Switzerland). Background fluorescence was subtracted from all samples, and the results were expressed as a percentage of the fluorescence values of untreated, virus-infected cells.

Unless otherwise stated, all experiments were repeated a minimum of three times. Data were normalized/log10-transformed before parametric tests of significance in view of the limitations of ascertaining distribution in small sample sizes and the high type II error rates of nonparametric tests in this context. Comparison of two groups used t test or one-sample t test if data were normalized to control values. Comparisons of more than one group used one-way analysis of variance (ANOVA) or two-way ANOVA as appropriate, with posttest correction for multiple comparisons. Statistical testing was undertaken in GraphPad Prism (v7.0). All tests were two-tailed with 0.05.

immunology.sciencemag.org/cgi/content/full/4/42/eaav7501/DC1

Materials and Methods

Supplementary case summary

Fig. S1. Ingenuity pathway analysis of whole blood RNA-seq data.

Fig. S2. Longitudinal series of laboratory parameters.

Fig. S3. Multiple sequence alignment of STAT2.

Fig. S4. Factor H genotyping and mutant factor H purification strategy.

Fig. S5. Functional analysis of factor H Tyr779Cys variant.

Fig. S6. Immunoblot analysis of MX1 expression in PBMCs.

Fig. S7. Transduction of STAT2-deficient primary fibroblasts.

Fig. S8. Prolonged STAT2 phosphorylation in PBMCs.

Fig. S9. STAT2 immunofluorescence image analysis.

Fig. S10. STAT phosphorylation is not prolonged in patient cells in response to IFN or IL-6.

Fig. S11. RT-PCR analysis of gene expression in whole blood.

Fig. S12. STAT2R148W does not impair regulation of STAT1 signaling.

Fig. S13. Phosflow gating strategy.

Table S1. Laboratory parameters, patients II:3 and II:4.

Table S2. Rare variants segregating with disease.

Table S3. Digital ELISA detection of IFN protein concentration.

Table S4. Phenotypes of monogenic defects of USP18 expression and/or function.

Table S5. RT-PCR primers and probes.

Table S6. Antibodies.

Data file S1. Raw data (Excel).

References (5159)

Acknowledgments: We are grateful to the patients and our thoughts are with their family. Funding: British Infection Association (to C.J.A.D.), Wellcome Trust [211153/Z/18/Z (to C.J.A.D.), 207556/Z/17/Z (S.H.), and 101788/Z/13/Z (to D.F.Y. and R.E.R.)], Sir Jules Thorn Trust [12/JTA (to S.H.)], UK National Institute of Health Research [TRF-2016-09-002 (to T.A.B.)], NIHR Manchester Biomedical Resource Centre (to T.A.B.), Medical Research Foundation (to T.A.B.), Medical Research Council [MRC, MR/N013840/1 (to B.J.T.)], MRC/Kidney Research UK [MR/R000913/1 (to Vicky Brocklebank)], Deutsche Forschungsgemeinschaft [GO 2955/1-1 (to F.G.)], Agence Nationale de la Recherche [ANR-10-IAHU-01 (to Y.J.C.) and CE17001002 (to Y.J.C. and D.D.)], European Research Council [GA 309449 (Y.J.C.); 786142-E-T1IFNs], Newcastle University (to C.J.A.D.), and ImmunoQure for provision of antibodies (Y.J.C. and D.D.). C.L.H. and R.S. were funded by start-up funding from Newcastle University. D.K. has received funding from the Medical Research Council, Wellcome Trust, Kidney Research UK, Macular Society, NCKRF, AMD Society, and Complement UK; honoraria for consultancy work from Alexion Pharmaceuticals, Apellis Pharmaceuticals, Novartis, and Idorsia; and is a director of and scientific advisor to Gyroscope Therapeutics. Author contributions: Conceptualization: C.J.A.D., S.H., and T.A.B. Data curation: C.F., G.I.R., A.J.S., J.C., A.M., R.H., Ronnie Wright, and L.A.H.Z. Statistical analysis: C.J.A.D., B.J.T., R.C., G.I.R., F.G., D.F.Y., S.C.L., V.G.S., A.J.S., L.A.H.Z., C.L.H., D.K., and T.A.B. Funding acquisition: C.J.A.D., D.D., Y.J.C., R.E.R., D.K., S.H., and T.A.B. Investigation: C.J.A.D., B.J.T., R.C., F.G., G.I.R., D.F.Y., Vicky Brocklebank, V.G.S., B.C., Vincent Bondet, D.D., S.C.L., A.G., M.A., B.A.I., R.S., Ronnie Wright, C.L.H., and T.A.B. Methodology: C.J.A.D., B.J.T., R.C., F.G., D.F.Y., A.J.S., D.D., K.R.E., Y.J.C., R.E.R., C.L.H., and D.K. Project administration: C.J.A.D., K.R.E., S.H., and T.A.B. Resources: S.M.H., Robert Wynn, T.A.B., J.H.L., J.P., E.C., S.B., K.W., and D.K. Software: C.F., A.J.S., M.Z., L.A.H.Z., and Ronnie Wright. Supervision: C.J.A.D., K.R.E., Y.J.C., D.D., C.L.H., R.E.R., D.K., S.H., and T.A.B. Validation: B.J.T., R.C., A.J.S., V.G.S., and C.L.H. Visualization: C.J.A.D., B.J.T., R.C., and S.C.L. Writing (original draft): C.J.A.D., with B.J.T., R.C., S.H., and T.A.B. Writing (review and editing): C.J.A.D., G.I.R., A.J.S., S.C.L., M.Z., S.M.H., K.R.E., R.E.R., D.K., S.H., and T.A.B. Competing interests: The authors declare that they have no competing interests. Data and materials availability: GEO accession: GSE119709. ArrayExpress accession: E MTAB-7275. Materials/reagents are available on request from the corresponding author(s). MBI6 is available from Claire Harris under a material agreement with Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the UK Department of Health.

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Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2 - Science

New frontiers in allergy, cancer and the immune system – ABC News

The immune system. Fantastic when it works terrible when it doesn't.

In this episode of the Health Report, we canvas the new frontiers of knowledge of the immune system from infancy through to adulthood and problems like allergy and cancer.

And we begin with a primer on the immune system what is it and how does it work?

This panel discussion was recorded at the World Science Festival Brisbane 2019.

Presenter:

Dr Norman Swan

Guests:

Professor Nigel McMillanCancer biologist, Menzies Health Institute Queensland

Professor Katie Allen

Paediatric allergist; gastroenterologist, Murdoch Children's Research Institute

Professor Mark SmythSenior scientist, immunology coordinator, QIMR Berghofer Research Institute

Producer:

James Bullen

The rest is here:
New frontiers in allergy, cancer and the immune system - ABC News