› docs › librariesprovider26 › ... · IMMUNOLOGY NETWORK2019-11-12 · 3 Celebrating 10 years Foreword from Prof Laurent Rénia Executive Director Singapore Immunology Network

S I N G A P O R E I M M U N O L O G Y N E T W O R K

Towards Research Excellence in Human Immunology

Celebrating 10 years2006 – 2016

T A B L E O F C O N T E N T

Foreword from Prof Laurent Rénia, SIgN ED ........................................................ 3

Message by Mr Lim Chuan Poh, A*STAR Chairman ........................................ 4

Message by Dr Benjamin Seet, BMRC ED ................................................................. 5

Beginnings. Pioneers, Alumni ............................................................................................ 6

Milestones ............................................................................................................................................. 8

Our Scientists ..................................................................................................................................... 10

Nurturing Talent. Students, Scholars .......................................................................... 14

SIgN Culture and Philosophy .............................................................................................. 16

Outreach ................................................................................................................................................. 18

Scientific Breakthroughs ......................................................................................................... 20

Translational Programs ............................................................................................................ 24

Industry Partnerships ................................................................................................................ 28

Science through Art ...................................................................................................................... 30

Acknowledgements ...................................................................................................................... 32

3

Celebrating 10 years

Foreword from Prof Laurent RéniaExecutive DirectorSingapore Immunology Network

As the Executive Director of SIgN, I am extremely honoured to present this report covering our first 10 years of activities. Since

its conceptualization in 2005 and establishment in 2006 by Prof Philippe Kourilsky and Prof Lam Kong Peng, SIgN has been dedicated to Human Immunology with a mission to improve health and create value for the society.

Moving with the timesSIgN has established itself as an international centre for Immunology focusing on Infection and Immunity, Inflammation and Immunoregulation. Significant developments have been achieved through our team of dedicated researchers and external collaborators from Singapore and abroad. As a dynamic institute, SIgN has undergone changes in its scientific leadership: pioneer researcher leaders, who have played significant roles in establishing SIgN, have moved to new endeavours. Young talented research leaders have joined and will now lead SIgN to new heights.

Translating researchAt SIgN, we have gained recognition over the years for our ability to translate research into innovations. Through close partnerships between SIgN researchers and commercial and public entities, we have developed many projects that have generated new knowledge and innovations to further economic growth and improve lives.

Developing for the futureSIgN firmly believes the importance of nurturing young scientists and is supporting multiple activities to simulate analytical thinking, interdisciplinary collaboration and entrepreneurial spirit. Equipped

with these skill sets, we are confident that our young researchers will be ready to face and solve challenges of the future.

As SIgN matures, its importance as an International Centre of Excellence grows. We are proud to lead the way in bringing integrated cross-disciplinary approaches to problems of both local and international significance. SIgN is committed to maintain scientific excellence and originality, which are alongside A*STAR’s philosophy and mission. I look forward to seeing more contributions from SIgN in the next decade.

Message by Mr Lim Chuan PohChairmanAgency for Science, Technology and Research


T he establishment of SIgN was a critical part of the Biomedical Sciences (BMS) initiative launched in 2000 to develop BMS

as one of the key industry sector. Phase 1 of the BMS initiative, from 2001 to 2005, was focused on developing a critical mass of basic research capabilities. Phase 2 built on the efforts of Phase 1 to develop Translational and Clinical Research (TCR) capabilities to close the gap between bench and bedside. SIgN was therefore born in 2006 as part of this TCR efforts in Singapore, and pertinently, chose to focus on human immunology.

In just one decade, SIgN has built a strong core of research capabilities in human immunology and elevated Singapore’s position within the international research community in this area. SIgN has published close to 650 scientific papers since 2007, with more than half in scientific journals with impact factor above 5. These include several papers in top tier journals such as Nature and Science. With a strong emphasis on TCR, the institute successfully established a network of scientists and clinicians with a shared objective to resolve the complexity of the immune system and translate this knowledge into useful clinical applications. In RIE 2015, SIgN fostered collaborations with over 30 clinicians and clinician-scientists covering important medical and healthcare areas such as infectious diseases, cancer, immunotherapy, aging and allergy. The joint project between SIgN and the National University Health System (NUHS) to study allergy in Singapore is a good example. The study, which identified that house dust mites is the major cause for common airway allergies in Singapore, has deepened our understanding of the local environment, and encouraged further research to improve allergy management in Singapore.

With SIgN’s expertise in immunology and the collaborations with the clinical community, SIgN has also attracted interest in partnerships from

multiple companies on translational and clinical research. In the last five years, SIgN has formed research collaborations with 21 biopharmaceutical, consumer care, and food and nutrition companies including industry giants such as Sanofi, Merck, Janssen and Nestle, as well as local SMEs such as Veredus, Curiox Biosystems and Apta Biosciences on research areas ranging from infectious diseases to aging. More specifically, SIgN is working with Sanofi Pasteur to jointly conduct a three-year clinical trial to understand age-related loss of immunity.

Despite being a young research institute, SIgN has already nurtured several promising young scientific talent for both A*STAR and the wider BMS community. Dr Florent Ginhoux, Senior Principal Investigator at SIgN, for example, has received various accolades for his outstanding research in the area of dendritic cells. He was one of the few scientists outside of Europe to be named an EMBO Young Investigator in 2013, and was also awarded the European Macrophage and Dendritic Cell Society Junior Prize. Another outstanding scientist is Dr Lisa Ng, who is widely recognized for her major contributions in the prevention and treatment of epidemic viral infections such as SARS and influenza. Lisa’s work has also added immensely to SIgN’s advance in the area of infectious diseases particularly in the chikungunya virus.

As we prepare to enter RIE 2020, I am confident that SIgN will continue to do well and contribute to A*STAR’s mission of advancing science and developing innovative technology to further economic growth and improve lives.

On this very meaningful occasion, I would like to congratulate Prof Laurent Renia and his team at SIgN for the success thus far and I look forward to greater contributions from the institute in the next decade.

54

Message by Dr Benjamin SeetExecutive DirectorBiomedical Research Council


Our understanding of the immune system and our ability to better regulate and harness it for therapeutic purposes will have profound

impact on the practice of medicine. The boundaries of immunology as a scientific discipline will overlap and merge with those of other biomedical sciences; whilst the clinical relevance of immunology will become increasingly important in the diagnosis, prognosis, treatment and prevention of acute and chronic diseases, including major health challenges like cancer, diabetes and cardiovascular disease.

The Singapore Immunology Network or SIgN, has in the short decade since its founding, established a strong foundation and expertise in scientific discovery and clinical application in the field of immunology. For example, it has developed a comprehensive immunomonitoring platform that utilises a systems approach for biomarker discovery, identifying novel therapeutic targets and clinical trials monitoring. It also has notable capabilities in the functional studies of immune cell behaviour; in human monoclonal antibody development; in understanding the role of the microbiome in health and disease; as well as in senescence of the immune system as we age.

Together with its strong network of clinical partners in Singapore and internationally, SIgN has translated its research to addressing emerging infectious disease threats like Chikungunya, Zika and Ebola; towards understanding allergy and atopy in Singapore children; and in the contribution of the immune system to the development of cancer, diabetes and heart failure. This has allowed it to establish its place and relevance in the Singapore biomedical research landscape.

I would like to congratulate SIgN for its outstanding work and many collaborative efforts over the decade, and I look forward for more impactful contributions to come.

BeginningsPioneers, Alumni

The SIgN of TimesThe Singapore Immunology Network was conceptualized during the formulation of the nation’s Science & Technology (S&T) 2010 plan. I proposed to set up a collaborative program in immunology research that would involve laboratories in Biopolis, universities and hospitals working together. The idea was endorsed by then A*STAR Chairman, Mr Philip Yeo, A*STAR Deputy Chairman and NUS Provost, Prof Tan Chorh Chuan, and BMRC Chairman, Dr Sydney Brenner. Thus, in 2006, the Singapore Immunology Network was born.

SIgN has come a long way and has done very well in recruiting eminent scientists to Singapore, training a new generation of immunologists and publishing high impact research in top-tier journals. The current Executive Director, Professor Laurent Renia, was one of the earliest scientists to join SIgN and has contributed immensely to the institute’s achievements, especially in the area of infectious diseases. Other early recruits that had contributed to SIgN’s success included Drs Phillipe Kourilsky, Paola Castagnoli, Jean-Pierre Abastado, Ren Ee Chee and many others.

I have no doubt that SIgN is as relevant today as it was 10 years ago. The recent spectacular success of antibody-based checkpoint inhibitor drugs against cancers and the future potential applications of chimeric antigen-receptor (CAR) T cells in immunotherapy illustrated the tremendous translational potential of immunology to human healthcare. And not to forget the ever-increasing needs for new vaccines and therapies to combat infectious diseases such as Dengue, MERS and SARS. The future for SIgN is very bright indeed.

PROF LAM KONG PENG, Founding Executive Director, 2006-2008

VIPs at the official inauguration of SIgN on 15 January 2008.

(From left to right: Prof Paola Castagnoli, then-Scientific Director of SIgN; Mr Philip Yeo, former Chairman of A*STAR; Prof Philippe Kourilsky, then-Chairman of SIgN; Mr S Iswaran, then-Minister of State, Ministry of Trade and Industry (MTI); Mr Lim Chuan Poh, Chairman of A*STAR; Prof Lam Kong Peng, then-Executive Director of SIgN)

WONG SIEW CHENG, Principal Investigator Joined in January 2006

JEAN-PIERRE ABASTADO, Principal Investigator Joined in May 2006 and left in September 2013

LISA NG, Principal Investigator Joined in March 2007

SUBHRA KUMAR BISWAS, Principal Investigator Joined in May 2006

ALESSANDRA NARDIN, Head, Translational Immunology Group Joined in August 2006

REN EE CHEE, Principal Investigator Joined in April 2008

WHAT IS YOUR SWEETEST MEMORY OF SIGN IN THE GOOD OLD DAYS ?

The day we moved into Immunos building. A new home where the SIgN family can embark on our journey together as one entity.

My sweetest memory at SIgN was the first retreat initiated and organized by Jo Eyles-Keeble at Bintan.

I remember getting to know new cultures (more European focus) and build up the infrastructure and science.

One of the oldest memories I have is the exciting time we had when some of us were sharing the ”temporary” SIgN Lab at IMCB level 6

before moving to Immunos.

When I arrived in 2006 we only had some lab space in Proteos…and a PCR machine! There was no cutting-edge technology, only the idea of a project, and little SIgN identity…the people joining one by one all contributed to create the infrastructure and the research from that

nothing – it was very challenging but also very exciting.

I was fascinated by the idea of a special place right here in Singapore where immunology and immunologists can flourish. You can count

the number of similar entities in the world on one hand.

76

Beginnings. Pioneers, Alumni

Milestones

Conceptualization of SIgN

Founding Chairman: Prof Philippe Kourilsky

Founding Executive Director: Prof Lam Kong Peng

2006

2012

2008

2013

2007Immunology labs across Biopolis were grouped under SIgN at the new Immunos building of Biopolis Phase 2

Prof Paola Castagnoli appointed as Scientific Director

Official inauguration of SIgN

Launch of Singaporean Society for Immunology (SgSI) Founding President: Prof Mike Kemeny

First International Singapore Symposium of Immunology by SIgN & SgSI

Dr Lisa Ng received the 8th Asean Young Scientist and Technologist Award

Discovered early immune responses in Chikungunya fever relevant for rational design of Chikungunya virus vaccines and diagnostics development. (Lisa Ng’s Lab. Kam YW et al. EMBO Mol Med. 2012)

Prof Laurent Renia appointed as Acting Executive Director

Launch of first Lab-on-Chip for detection of multiple tropical infectious diseases developed in collaboration with Veredus Laboratories

Hosted Singapore’s inaugural European Molecular Biology Organisation (EMBO) Workshop on Complex Systems in Immunology

Dr Florent Ginhoux received the EMBO Young Investigator Award

Collaboration with Sanofi Pasteur, NUS and NUH to study loss of immunity and reduced responsiveness to vaccination in elderly

Collaboration with L’Oreal to study immune responses in the skin

Inaugural NIF School on Advanced Immunology by iFReC (Japan) and SIgN (Singapore)

9

Milestones

Milestones

2009

2010 2011

2014 2015

Set up of Human Monoclonal Antibodies platform

Set up of 2-Photon imaging facility

Development of a novel immunisation method to induce fast and effective protection in humans against the life-threatening malaria parasite (Laurent Renia’s Lab. Roestenberg M et al. N Engl J Med. 2009)

Discovery of brain immune cells origin that can lead to new strategies to manipulate microglia for treatment of brain disorders (Florent Ginhoux’s Lab. Ginhoux F et al. Science. 2010)

Launch of SIgN-NTU PhD Program in Immunology with 7 students selected for the first intake

Dr Norman Pavelka awarded A*STAR Investigatorship

8

Set up of CyTOF facility

First of several collaborations with Servier to develop immunity-modulating drugs to combat cancer and autoimmune diseases

Launch of SIgN Association of Post-Docs (SIgNAPs)

Elucidation of human skin antigen-presenting cells that may facilitate systemic spread of Dengue virus infection (Katja Fink’s Lab. Cerny D et al. PLoS Pathog. 2014)

Discovery of exposure to house dust mites as the primary case of respiratory allergies in Singapore (Olaf Rotzschke’s Lab. Andiappan AK et al. Allergy 2014)

Consolidation of Clinical Immunomonitoring platform (Flow Cytometry, CyTOF, Genomics, Bioinformatics, Translational Immunology)

Prof Laurent Renia appointed as Executive Director

Development of sophisticated mass cytometry panel for high-dimensional unambiguous and unbiased characterization of the myeloid cell system (Evan Newell’s Lab. Becher B et al. Nat Immunol. 2015)

Collaboration with Chugai for the development of an anti-Dengue therapeutic antibody

Collaboration with Janssen on immunomonitoring of HBV-specific T cell responses

Co-organized with SgSI the 6th Congress of the FIMSA (Federation of Immunological Societies of Asia-Oceania) held for first time in Singapore

LAURENT RÉNIA2007

Knowing your enemies: infectious ideas against pathogens

KATJA FINK2009

Dengue vaccine and immunotherapeutics development

EVAN NEWELL2012

High dimensional analysis of antigen-specific T cells

GENNARO DE LIBERO2010-2016

Tuberculosis Lipid Immunity

LISA NG2007

Immune responses of arthrogenic arboviruses in patients and experimental animal models to develop

rationally-guided immune-based therapies

NORMAN PAVELKA2011

Host-fungal interactions and microbiota

Our ScientistsINFECTION INFLAMMATION IMMUNOREGULATION

1 1

FLORENT GINHOUX2009

Dendritic cell and macrophage ontogeny and differentiation

MARIA LAFAILLE2009-2015

Allergy and inflammation

ALESSANDRA MORTELLARO2008

Inflammasome-mediated innate immune responses to pathogens and danger signals

INFECTION INFLAMMATION IMMUNOREGULATION

Our Scientists

1 0

WONG SIEW CHENG2006

Characterisation of myeloid cells and their subsets in health and disease

SUBHRA BISWAS2006

A dysregulated monocyte/macrophage response underlies the pathogenesis of several human diseases

LUCIA MORI2010-2016

T cell immunity to lipids and metabolites

OLAF RÖTZSCHKE2008

Genetic and functional analysis of allergy and immune-regulatory pathways

ANNA-MARIE FAIRHURST2010

Examination of immune regulators in autoimmune mouse models and clinical samples

INFECTION INFLAMMATION IMMUNOREGULATION

REN EE CHEE2008

Redefining existing paradigms with new perspectives

ANIS LARBI2010

Biology of aging and immunophenotyping: the place of immunity in the biology of aging

NG LAI GUAN2009

Studying how cells move in vivo to allow a better understanding of the immune system

1 3

TECHNOLOGY PLATFORMS

WANG CHENG-I2009

Human Monoclonal Ab Technologies

ZHONG PINGYU2012

Human Monoclonal Ab Technologies

MICHAEL POIDINGER2011

Bioinformatics

Immunomonitoring Platform: Biomarker discovery, identifying novel points of therapeutic intervention, clinical trials monitoring

Translational Immunology Group: Planning and management of translational immunology projects originated from SIgN discovery research

Flow Cytometry: State-of-the-art cell sorting and flow cytometry facility

Deep Immunophenotyping:

High-dimensional cell analysis by CyTOF

Functional Genomics: Transcriptomics and sequencing technologies for immunogenomics research

ALESSANDRA NARDIN2006

Translational Immunology Group

Our Scientists

1 2

FRANCESCA ZOLEZZI2011-2016

Functional Genomics Genomics technologies to support all aspects of immunogenomics research

Bioinformatics: State-of-the-art analytical methods to derive knowledge from generated data

Human Monoclonal Ab Technologies: Generation of novel therapeutic human monoclonal antibodies against various targets

Functional Imaging:

Multi-photon imaging technologies for examining dynamics of immune cell behavior

Mouse Models Of Human Diseases:Modeling human pathologies and developing novel therapeutics

1

2

Nurturing TalentStudents, ScholarsTo date, SIgN has nurtured and trained: 29 returning A*STAR scholars 60 post graduate PhD students 303 internship students

(from universities, polytechnics, junior colleges and secondary schools)

Partners and Programs National University of Singapore (NUS) Nanyang Technological University (NTU) A*STAR Graduate Academy (A*GA) SIgN-NTU Immunology PhD Program A*STAR Graduate Scholarship (AGS) A*STAR Research Attachment Program (ARAP) Singapore International Graduate Award (SINGA)

3

4 5

1 5

1. 2012: Pioneer batch of SIgN Immunotechnologies Training Course (ITTC) students.

2. Sharrada Subramaniam, PhD student from NTU attached to Dr Maria Lafaille’s Lab won Best Poster at SgSI’s 6th Symposium in 2013.

3. PhD student Thomas Champion from University of Manchester attached to Dr Wong Siew Cheng’s lab presenting his poster during FIMSA2015.

4. Choo Zheng Chen from Raffles Institution, attached to Dr Alessandra Mortellaro’s lab in 2014 presented his project and shared his time and experience at SIgN during the A*STAR Student Research Attachment Program 2014 Closing Ceremony.

5. Teo Teck Hui and Lum Fok Moon, NUS PhD Students attached to Prof Laurent Renia and Dr Lisa Ng, at their graduation party after obtaining their PhDs in 2015.

Nurturing Talent. Student, Scholars

1 4

1

3

2

SIgN Culture & PhilosophySIgN Spirit

SIgN organizes off-site annual Scientific Training Retreats since 2009 for all staff to promote skills development in our workforce, strengthen staff interactions and foster team-building.

2011: Turi Beach Batam

4 5 6

7

9 10

11 12

8

1 7

SIgN Culture & Philosophy

1 6

3. 2009: SIgN’s 1st Scientific Retreat - Bintan Lagoon Resort

1. 2011: Turi Beach Batam

2.4.8. 2012: Nirwana Gardens

5.6.7.10.12. 2015: Bintan Lagoon Resort

9.11. 2016: SIgN’s 1st PI Leadership Retreat – Penang Eastern & Oriental Hotel

1

2

Outreach

4

3

1 9

Outreach

1 8

1. 2013 Biopolis Carnivale

For Biopolis’ 10th anniversary celebration, SIgN showcased our research activities and facilitated lab tours to students and members of the public.

2. 2014 inaugural A*STAR Corporate Social Responsibility Day

SIgN participated in the fund-raising bazaar for charity. Donations collected through the sales of home-made baked goods and pre-loved items were donated to Singapore Children Society and the Community Chest.

3. 2015 Sg50 Science Jubilee! A*STAR Open House

SIgN showcased our research to members of the public during A*STAR Open House for Science@50 celebration. Our exhibit focused on arboviruses and allergies and lab tours were conducted for visitors.

4. Singapore Science Festival - X-periment!

Since 2011, SIgNAPs (SIgN Association of Post-Docs) actively participates to X-periment! organized annually by Science Centre Singapore. Themes such as “Allergies and Asthma” and “Immunology in Everyday Life & Health” were showcased with hands-on activities organized for the public.

1

5

2

6

43

7 8

Scientific BreakthroughsSIgN Papers Featured on Journal Covers

1. Haniffa M, Shin A, Bigley V, McGovern N, Teo P, See P, Wasan PS, Wang XN, Malinarich F, Malleret B, Larbi A, Tan P, Zhao H, Poidinger M, Pagan S, Cookson S, Dickinson R, Dimmick I, Jarrett RF, Renia L, Tam J, Song C, Connolly J, Chan JK, Gehring A, Bertoletti A, Collin M, Ginhoux F. Human Tissues Contain CD141(hi) Cross-Presenting Dendritic Cells with Functional Homology to Mouse CD103(+) Nonlymphoid Dendritic Cells. Immunity. 2012 Jul 27;37(1):60-73.

2. Licandro G, Khor HL, Beretta O, Lai J, Derks H, Laudisi F, Conforti-Andreoni C, Qian HL, Teng GG, Ricciardi-Castagnoli P, Mortellaro A. The NLRP3 inflammasome affects DNA damage responses after oxidative and genotoxic stress in dendritic cells. Eur J Immunol. 2013 Aug;43(8):2126-37.

3. Becher B, Schlitzer A, Chen J, Mair F, Sumatoh HR, Teng KW, Low D, Ruedl C, Riccardi-Castagnoli P, Poidinger M, Greter M, Ginhoux F, Newell EW. High-dimensional analysis of the murine myeloid cell system. Nat Immunol. 2014 Dec;15(12):1181-9.

4. Evrard M, Chong SZ, Devi S, Chew WK, Lee B, Poidinger M, Ginhoux F, Tan SM, Ng LG. Visualization of bone marrow monocyte mobilization using Cx3cr1gfp/+Flt3L-/- reporter mouse by multiphoton intravital microscopy. J Leukoc Biol. 2015 Mar;97(3):611-9.

5. Kam YW, Lum FM, Teo TH, Lee WW, Simarmata D, Harjanto S, Chua CL, Chan YF, Wee JK, Chow A, Lin RT, Leo YS, Le Grand R, Sam IC, Tong JC, Roques P, Wiesmüller KH, Rénia L, Rotzschke O, Ng LF. Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein. EMBO Mol Med. 2012 Apr;4(4):330-43.

6. Biswas SK, Mantovani A. Orchestration of metabolism by macrophages. Cell Metab. Apr 4 2012; 15(4):432-437.

7. Ginhoux F, Schultze JL, Murray PJ, Ochando J, Biswas SK. New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol. 2015 Dec 17;17(1):34-40.

8. Newell EW, Davis MM. Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells. Nat Biotechnol. 2014 Feb;32(2):149-57.

Immunity

Previews

Professional Cross-Presenting CD8a-Type CD141hi

Dendritic Cells: We Have Got You in Our Skin!

Marc Dalod1,2,3,*1Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Universite UM2, Campus de Luminy case 906, 13288 Marseille, France2INSERM, UMR1104, 13288 Marseille, France3CNRS, UMR7280, 13288 Marseille, France*Correspondence: [email protected]://dx.doi.org/10.1016/j.immuni.2012.07.008

In this issue of Immunity, Haniffa et al. (2012) identify the presence of professional cross-presenting humandendritic cells in the skin, the liver, and the lung and also presented comparative genomics to align humanand mouse dendritic cell types across tissues.

Mouse dendritic cell (DC) subsets have

been very well characterized in the last

10 years and can be classified in five

major subsets that exist both in lymphoid

and nonlymphoid tissues (Guilliams et al.,

2010). The CD8a-type DC is particularly

efficient at cross-presenting exogenous

antigens for the activation of CD8+

T lymphocytes. Until recently (Segura

et al., 2012), the DCs migrating from

human nonlymphoid tissues into their

draining lymph nodes had not been well

characterized, except for Langerhans

cells (LCs). The delineation of homologies

between human and mouse DC subsets

had been hampered by differences in

surface marker expression and in acces-

sibility of equivalent tissues as a source

for these cells. However, by using an

approach as broad and unbiased as

possible for comparing immune cell types

across species, namely functional geno-

mics, we succeeded in aligning human

blood and mouse spleen DC subsets

(Robbins et al., 2008), thereby revealing

that the human DC subset expressing

high CD141 (BDCA3) is the equivalent to

mouse CD8a-type DCs. This discovery

was confirmed at the functional level as

well as by identifying markers specific

for these cell types and conserved across

species, in particular the endocytic C-

type lectin receptor CLEC9A (Caminschi

et al., 2008; Sancho et al., 2008) and the

chemokine receptor XCR1 (Bachem

et al., 2010; Crozat et al., 2010). A similar

conservation of DC subsets was reported

for sheep skin-draining lymph and skin

(Contreras et al., 2010). Hence, a universal

and simplified classification of DC sub-

sets in fivemajor populations, irrespective

of the tissues and species of origin, was

proposed (Guilliams et al., 2010). It

predicted that CD11chiCD1a+CD141hi

CLEC9A+TLR3hi DCs exist in the tissue

parenchyma of human nonlymphoid

organs, which would be the professional

cross-presenting human DCs, more

generally specialized in the activation of

CD8+ T lymphocytes, and is able to

produce IL-12p70 more efficiently than

the other DC subsets. No experiments

had yet been reported to rigorously test

this hypothesis.

If human professional cross-presenting

DCs exist in epithelia, they would consti-

tute the most functionally and anatomi-

cally relevant population of human DCs

to target clinically to induce efficient

CD8+ T cell responses against intracel-

lular pathogens or tumors. Hence, ad-

dressing this point is of major importance.

However, designing experiments to seek

for human professional cross-presenting

DCs in nonlymphoid tissues is a major

challenge because it requires overcoming

many issues. First, it is difficult to access

human tissues in sufficient quantities

and of high enough quality, knowing that

the frequency of human professional

cross-presenting DCs is expected to be

extremely low in nonlymphoid tissues as

in lymphoid organs. Second, optimized

protocols have to be set up to efficiently

isolate and rigorously phenotype epithe-

lial DCs. Third, appropriate functional

tests must be designed to evaluate the

relative cross-presentation efficiency of

different DC subsets despite their differ-

ence in susceptibility to apoptosis and in

expression of innate immune recognition

receptors.

In this issue of Immunity, Haniffa et al.

(2012) have succeeded in this challenge,

performing a tour-de-force study to seek

for professional cross-presenting DCs in

human skin, skin-draining lymph nodes,

liver, and lung and to characterize in

depth the DC subsets from human skin,

by comparison with human blood. The

authors used (1) CD11c, HLA-DR, CD14,

CD1a, CD1c, CD141, CLEC9A, XCR1,

and other markers for rigorous and

detailed phenotypic identification of DC

subsets in lineage� cells from various

human tissues (Figure 1), (2) a functional

assay for evaluating the cross-presenta-

tion capacity of human blood and skin

DC subsets, (3) allogeneic mixed lympho-

cyte reactions to measure skin DC sub-

set ability to activate CD4+ and CD8+

T cells, (4) different stimuli to probe the

pattern of cytokine production by skin

DC subsets, (5) cell cycle analysis, and

phenotypic characterization of blood

DC subsets upon incubation with digest-

ing skin, to investigate the potential

precursor-progeny relationship between

blood and skin CD141hi DCs, and (6)

comparative genomics to explore the

relationships between human and mouse

DC subsets from different tissues.

Haniffa et al. (2012) show that the

CD141hiCLEC9A+XCR1+ DCs are much

more potent for cross-presentation of

soluble hepatitis B surface antigen

than any of the other four subsets of

human skin antigen-presenting cells

(APCs) examined, including LCs. Indeed,

CD141hiCLEC9A+XCR1+ DCs are at least

as potent for cross-presentation as the

DCs derived in vitro from monocytes,

the gold standard to look at cross-

presentation by human APCs. Previously,

LCs had been proposed to be the skin

APC subset that was the most potent for

Immunity 37, July 27, 2012 ª2012 Elsevier Inc. 3

nature immunology volume 16 number 7 july 2015 683

selectively expressed by cDCs (Zbtb46GFP), and a defined sorting scheme to identify progeni-tors of cDC subtypes. Through the use of chro-matin profiling, they identify the transcription factor IRF8 as a critical factor in the early regulatory circuits that lock cDC fate3.

DCs were first observed in 1973 (ref. 4), but their location among the myeloid and lymphoid branches of the hematopoietic tree has yet to be agreed upon5. Although under-standing of how the DC lineage develops

DC origins by demonstrating how progeni-tor cells commit to the various DC subtypes in mice through distinct intermediate stages. Schlitzer et al. use single-cell mRNA sequenc-ing to analyze the heterogeneity of progenitor DC populations and find, among individual cells, varying levels of commitment to develop into specific conventional DCs (cDCs)2. Grajales-Reyes et al. use mice with expression of green fluorescent protein (GFP) from the locus encoding the transcription factor Zbtb46,

and establish transcription upon induction, these cis elements may be dispensable for the induction of gene expression but instead are essential for the maintenance of stable gene expression in particular biological con-texts that promote alternative cell fate or cell division. It seems reasonable to assume that the emergence of such dedicated cis ele-ments owes to the increasing complexity of transcriptional regulation in multicellular organisms with a more complex body plan. The identification and characterization of additional such elements and the common molecular logic underlying their function will help to better elucidate cell-fate maintenance and offer unique tools with which to probe cellular behaviors.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

1. Holmberg, J. & Perlmann, T. Nat. Rev. Genet. 13, 429–439 (2012).

2. Taniuchi, I. & Ellmeier, w. Adv. Immunol. 110, 71–110 (2011).

3. Sellars, M. et al. Nat. Immunol. 16, 746–754 (2015).4. Smith, Z.D. & Meissner, A. Nat. Rev. Genet. 14,

204–220 (2013).5. Chen, T., Ueda, Y., Dodge, J.E., wang, Z. & Li, E.

Mol. Cell. Biol. 23, 5594–5605 (2003).6. wu, H. & Zhang, Y. Cell 156, 45–68 (2014).7. Rountree, M.R., Bachman, K.E. & Baylin, S.B.

Nat. Genet. 25, 269–277 (2000).8. Lee, P.P. et al. Immunity 15, 763–774 (2001).9. Ragunathan, K., Jih, G. & Moazed, D. Science 348,

1258699 (2014).10. Boucheron, N. et al. Nat. Immunol. 15, 439–448

(2014).11. DuPage, M. et al. Immunity 42, 227–238 (2015).12. Mullen, A.C. et al. Curr. Biol. 11, 1695–1699

(2001).13. Feng, Y. et al. Cell 158, 749–763 (2014).14. Chang, J.T. et al. Science 315, 1687–1691

(2007).15. Kueh, H.Y., Champhekar, A., Nutt, S.L., Elowitz, M.B. &

Rothenberg, E.V. Science 341, 670–673 (2013).

genes other than Cd4. Nonetheless, the distinct roles of HDACs, Ezh2 and DNA methyltrans-ferases indicate that permissive and repressive histone modifications, as well as DNA methy-lation, are used to sustain lineage-specific gene expression in different cellular and bio-logical contexts. More studies will be needed in the future to demonstrate in a cell type–, stage-, locus- and context-specific manner how various epigenetic marks are used to maintain a stable cellular identity.

Cell division has a critical part in cell fate determination12. Along with several other reports10,11,13, the observations by Sellars at al. point to cell division as a critical variable that influences lineage stability3. Although asym-metric cell division can lead to heterogeneity of the daughter cells, along with stochastic dilution of limiting transcription factors or epigenetic marks, how cell division affects T cell lin-eage stability remains largely unknown12–15. Examination of the effect of cell cycle on the distribution of factors that participate in transcriptional regulation and cell-lineage specification, as well as the inheritance of epi-genetic modifications, will provide valuable insights into the mechanisms of the determina-tion and maintenance of the T cell lineages.

Compromised stability of heritable CD4 expression in E4P-deficient CD4+ T cells, or loss of repression of Cd4 in the absence of the silencer S4 or DNA methyltransferases in CD8+ T cells3, together with the reported role of a cis-regulatory element in sustaining regulatory T cell identity13, suggest that a distinct set of cis-regulatory elements is ded-icated to the maintenance of differentiated cellular states. Unlike traditional enhancers that act together with promoters to initiate

at given sites may enable selective manipulation of the methylation states of the DMR to assess their effects on CD4 expression. Likewise, it will be interesting to explore the mechanisms of S4- and E4P-mediated modulation of the DMR.

Published studies have revealed key roles for lineage-specification factors not only in the establishment of lineage identity in differ-entiated cells but also in its maintenance. Such findings provide a rationale for the hypothesis that epigenetic modifications of specialized cis-regulatory elements may preserve cellular lineage identity and the differentiated state in the presence of fluctuating intracellular and extracellular cues. In particular, during cell division, epigenetic ‘bookmarks’ at some of these sites would ensure the inheritance of transcrip-tional states of key genes that define a given dif-ferentiated cell type1,9. That notion is supported by the observation that proper histone acety-lation controlled by the histone deacetylases HDAC1 and HDAC2 is needed to maintain stability of the CD4+ T cell lineage by repressing the cytotoxic T cell program, including expression of CD8 and Runx3 (ref. 10). In addi-tion, trimethylation of histone H3 at Lys27 by Ezh2, a component of the polycomb repres-sive complex PRC2, seems to help maintain the identity of activated regulatory T cells, a sublineage of helper CD4+ T cells11. The study by Sellars et al. makes substantial contributions to this body of work by demonstrating that the maintenance of DNA methylation or directed active demethylation may have essential roles in solidifying cytotoxic or helper T cell lineages by repressing or stabilizing CD4 expression3. It is conceivable that dynamic regulation of DNA methylation has a broader effect on the transcriptional regulation of lineage-related

deborah R. winter and Ido Amit are in the

department of Immunology, weizmann Institute of

Science, Rehovot, Israel.

e-mail: [email protected] or

[email protected]

dCs are ready to commitDeborah R Winter & Ido Amit

Dendritic cell progenitors commit to a specific conventional dendritic cell fate earlier than previously thought, by initiating transcription-factor regulatory circuits unique to their subtype.

Dendritic cells (DCs) are a critical compart-ment of innate immunity and perform

several specialized immunological functions1. In this issue of Nature Immunology, two studies critically contribute to the understanding of

NE wS AND V IE wS

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.



























[email protected]





NE wS AND V IE wS

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.



























[email protected]





NE wS AND V IE wS

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

The origin of microglia and how their homeostasis is maintained in the brain have been controversial since their discovery as resident macrophages. Since the 1990’s, embryonic and postnatal myeloid progenitors were assumed to contribute to adult microglia in the brain, but how microglia originate was still under debate. A recent study by Florent Ginhoux et al.1 in mice discovered that embryonic macrophages from the yolk sac, formed before embryonic day 8, gave rise to almost the entire population of microglia found in the adult brain. Furthermore, peripheral myeloid cells from fetal and adult hematopoiesis contributed minimally, if at all. Microglia and their yolk sac progenitors also depended on different receptors and ligands compared to other monocytes and tissue macrophages. These findings may bring to an end the long-running dispute about the origin of these multifaceted brain cells.

com m u n i t y co r n e r

JoAnne McLaurinThere are two hypotheses proposed for microglial origin—the neuroectodermal tissue or the myeloid-monocytic lineage, which is most widely accepted. Yet the existence of microglial progenitors that derive embryonically from myeloid or mesenchymal cells that are different from monocytic origin is still controversial.

Parabiosis of two mice and chimeras with and without radiation used to investigate the origin of microglia have, unfortunately, not provided definitive results. Using new mouse models, the tour de force by Ginhoux et al.1 now shows that microglia derive from embryonic primitive myeloid progenitors that are distinct from the postnatal hematopoietic origin of other tissue macrophages and that their numbers are maintained independently of circulating hematopoietic cells in the adult brain. In contrast to monocytes, microglia depended on colony-stimulating factor-1 receptor (CSF-1R)—but not the ligand CSF-1—for development, suggesting ontogenic differences, according to the authors1. But these results may also simply represent differences in environmental signals, such as the

1. Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

2. Lee, J.K. et al. Intracerebral transplantation of bone marrow–derived mes-enchymal stem cells reduces amyloid-β deposition and rescues memory deficits in Alzheimer’s disease mice by modulation of immune responses. Stem Cells 28, 329–343 (2010).

3. Lebson, L. et al. Trafficking CD11b-positive blood cells deliver therapeutic genes to the brain of amyloid-depositing transgenic mice. J. Neurosci. 30, 9651–9658 (2010).

4. Ransohoff, R.M. & Cardona, A.E. The myeloid cells of the central nervous system parenchyma. Nature 468, 253–262 (2010).

5. Lin, H. et al. Discovery of a cytokine and its receptor by functional screen-ing of the extracellular proteome. Science 320, 807–811 (2008).

6. Alliot, F., Godin, I. & Pessac, B. Microglia derive from progenitors, origi-nating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117, 145–152 (1999).

7. Mildner, A. et al. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat. Neurosci. 10, 1544–1553 (2007).

Microglial pilgrimage to the brain

Ginhoux et al.1 may have brought pre-vious disputes on the origin of micro-glia to an end. They used a genetic pulse-labeling strat-egy based on the expression pattern of the transcription factor Runx-1 to label yolk sac mac-rophages between embryonic days 6.5 and 10, bracketing the initial time of microglial progeni-tor entry. Virtually all adult microglia arose from yolk sac macrophages pres-ent between embry-onic days 7.25 and 7.75, which entered the embryo right after vascularization at day 8, before primitive or definitive

hematopoiesis starts in the embryo. These experiments show clearly that

microglia come from a single pop-ulation of extra-embryonic yolk sac macrophages and that periph-eral myeloid cells do not contribute to maintenance of adult microglia afterward.

In the diseased bra in , micro-glia have diverse protective func-tions similar to phagocytic mac-rophages, den-

dritic cells and anti-inflammatory tissue macrophages—such as debris removal, presentation of antigen to

Richard Ransohoff

“These experiments show clearly that microglia come from a single population of extraembryonic yolk sac macrophages and that peripheral myeloid cells do not contribute to maintenance of adult microglia afterward.”

Microglia in the adult brain come from primitive macrophages in the yolk sac.

“This study alleviates some of the skepticism I have had for using blood monocytes to deliver drugs into the brain.”

NIB

SC /

Pho

to R

esea

rche

rs, I

nc.

1380 volume 16 | number 12 | december 2010 nature medicine

© 2

010

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.


















Richard Ransohoff




NIB

SC /

Pho

to R

esea

rche

rs, I

nc.


© 2

010

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.


















Richard Ransohoff




NIB

SC /

Pho

to R

esea

rche

rs, I

nc.


© 2

010

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.

nature immunology volume 15 number 12 december 2014 1095

Jonathan Michael Irish is in the Department of

Cancer Biology and the Department of Pathology,

Microbiology and Immunology, Vanderbilt

University School of Medicine, Nashville,

Tennessee, USA.

e-mail: [email protected]

High-content single-cell biology and machine-learning tools are powering

a new era of ‘systems immunology’. Routine mass-cytometry experiments now measure more than 30 features of each of millions of cells, and comprehensive maps of cell identity can be derived from a single cytometry tube1,2. The application of computational tools has substantially augmented the ability to visual-ize high-dimensional mass-cytometry data and model results3–6. These modern tools have revealed that traditional analyses can overlook cells whose phenotypes are not well under-stood (‘Cyto Incognito’, Fig. 1). In contrast to expert-driven manual analyses, computational approaches aim to comprehensively describe cellular phenotypes with minimal bias. With the increasing capacity of single-cell cytometry, both canonical markers and novel markers can be measured simultaneously and act together to robustly distinguish cell identity in highly heterogeneous tissues. Mass-cytometry panels are designed so that each cell will have multiple well-characterized functional markers that unambiguously confirm its identity. Machine-learning approaches then arrange cells into maps or organize cells along progressions on the basis of similar features (Fig. 1). In this issue of Nature Immunology, Becher and colleagues use mass cytometry to create a modern reference map of the healthy mouse myeloid system across eight tissues and reveal the role of the recep-tor for the growth factor GM-CSF (encoded by Csf2rb) in the development of various cell populations, including tissue-resident natural

Beyond the age of cellular discoveryJonathan Michael Irish

The combination of machine-learning tools and mass-cytometry measurements of more than 30 protein markers per cell comprehensively maps cell identity in the heterogeneous myeloid cell system and reveals the global effect of deletion of the gene encoding the receptor for the growth factor GM-CSF.

killer cells, nonlymphoid dendritic cells (DCs), alveolar macrophages and eosinophils7.

The study by Becher et al. increases the under-standing of known cells and resolves previously obscure populations7. For example, three pre-viously unknown cell populations identified by unsupervised analysis are Siglec-FhiSSChi eosinophils, Ly6CloCD43+ monocytes and a CD11blo stage of Nkp46+NK1.1+ innate lym-phocytes. The authors confirm these cellular identities by traditional fluorescence-activated cell sorting followed by Giemsa staining.

Another striking aspect of the study is the use of eight different mouse tissues: lungs, spleen, bone marrow, thymus, brain, liver, mesenteric lymph nodes, and kidneys. The speed with which the healthy landscape can be defined in detail is impressive. As research moves for-ward to study disease populations or animal models, the question will regularly be “Is this abnormal?” This study provides an outstanding reference point for mapping disease states.

Previous observation have shown that mice lacking Csf2rb have deficits in various myeloid

Figure 1 Machine learning and mass cytometry combine to create comprehensive maps of cellular phenotype. Classically (left), the myeloid cell system has been poorly described due to inherent heterogeneity and disagreement on key markers, nomenclature and population boundaries. Traditional analysis approaches identify major populations using canonical markers and may leave unexplored cell subsets with unexpected phenotypes (‘Cyto Incognito’). In contrast, machine learning (middle) provides a more comprehensive view that automatically resolves a two-dimensional map of cell types from high-dimensional single-cell data, which can be read by humans. Phenotypic distances between cells are measured and cells are arranged so that proximity indicates similarity. Cell groups from various tissues and conditions are characterized by distinct protein features, as in the heat maps used by Becher et al. (right)5. In this heat map, color represents the average expression of one of the 33 proteins (columns) in one of the 28 automatically identified cell populations (rows). For example, the expression profile for plasmacytoid DCs (pDCs) is presented in the top row. Applied to myeloid system cells from eight tissues, this approach reveals previously underappreciated populations of tissue-resident macrophages and identifies phenotypically distinct subpopulations of heterogeneous DC, monocyte, neutrophil and eosinophil (Eo) populations. Unexpected phenotypes that would probably have been overlooked in a focused, classic study are revealed. For example, Csf2rb∙/ ∙ mice lack tissue-resident natural killer cells, a subgroup of the innate lymphoid population. This observed absence of innate lymphoid cells is picked up by a mass cytometry panel designed to study the myeloid compartment.

Classic myeloid system Modern quantitative map of cellular phenotypes

DCspDCs

EoInnate

lymphoidcells

AlveolarMΦ

Microglia

Neutrophils

Spleenkidney

MΦ

RedpulpMΦ

Monocytes

33-dimensional protein-expression profile

N e w S A N D V I e w S

npg

© 2

014

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature biotechnology volume 31 number 7 JulY 2013 609

Christopher J. Harvey and Kai W. Wucherpfennig are in the Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA, and Kai W. Wucherpfennig is also in the Program in Immunology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. e-mail: [email protected]

Newell et al.1 have exploited the advantages of mass cytometry to simultaneously ana-lyze the specificities of many T cells. Because the cytotoxic function of CD8 T cells is trig-gered by T-cell receptor (TCR) recognition of epitopes presented by major histocompat-ibility complex (MHC) class I molecules on target cells, they stained T cells with panels of peptide-MHC tetramers. As shown by Altman and Davis in 1996, virus-specific T cells can be visualized and isolated with fluorescently labeled tetramers of peptide-MHC complexes7. Since then, tetramers have become an essen-tial tool for investigation of T-cell responses in basic and clinical immunology. The Davis and Schumacher laboratories reported that com-binatorial tetramer staining can moderately increase the number of fluorescent tetramers one can use to simultaneously stain a single sample8,9; however, spectral overlap has limited the impact of this approach.

To adapt combinatorial tetramer staining to mass cytometry, Newell et al.1 use ten heavy-metal labels to create a three-metal tetramer barcode (Fig. 1). Tetramers are made by mixing biotinylated peptide-MHC complexes at a 4:1 ratio with streptavidin; the multivalent nature of tetramers compensates for the low affin-ity of peptide-MHC complexes for TCRs. To create the three-metal code, the authors label streptavidin in separate reactions with each of the ten metals. A unique combination of three metals is used for a single epitope. Three tetramers are separately made for each peptide-MHC complex using streptavidin labeled with three different metals. The three differentially labeled tetramers are then combined and used as a single staining reagent.

When stained in this way, any cell expressing a TCR capable of binding a given peptide-MHC tetramer should be coincidentally labeled by all three metals. As a result, specifically labeled T cells should be detected in all three—and only those three—mass spectrometer chan-nels. Cells detected in only one or two of the

they suffer from an inherent technical limita-tion imposed by spectral overlap. The recent development of quantum dots with narrow spectral windows has expanded the num-ber of parameters that researchers can detect simultaneously using flow cytometry, but ‘com-pensating’ for spectral bleed-through becomes increasingly complex as the number of fluores-cent parameters is increased3.

Mass cytometry represents an elegant, highly innovative solution to this problem. This breakthrough technology—first described in 2009 by Scott Tanner and colleagues4 at the University of Toronto—is poised to again transform immunology and other fields of biomedical research. The fundamental idea behind mass cytometry is that fluorophores can be replaced with heavy metals (bound to antibodies through a chelator), which are then quantified with high resolution by mass spec-trometry. The approach thus takes advantage of the high mass accuracy of mass spectrometers, enabling simultaneous analysis of a very large number of metal-tagged antibodies in a CyTOF (cytometry by time of flight) instrument.

Approximately 40 transition-state element isotopes not normally found in biological sys-tems (referred to as ‘metals’) with distinct mass can currently be examined5. Cells stained with metal-conjugated antibodies are atomized in an argon plasma stream, creating ion clouds from single cells. Metal ions falling within a certain mass range pass through a series of cones and deflectors and are detected based on time of flight. Metals do not have the spectral overlap problems associated with fluorophores, therefore eliminating the need for compensa-tion. The use of metals, however, is not without limitations, as certain isotype preparations are impure (contaminated by isotypes with a mass of m + 1). To date, only a small subset of the possibilities afforded by this novel technology has been explored, but it is already evident that mass cytometry has great potential for bio-medical research5,6.

The ability to identify and track cytotoxic T cells capable of recognizing particular epitopes is essential for studying human immune responses in the context of infectious diseases, cancer and autoimmunity. Yet despite the centrality of T cell–epitope interactions to the immune response, our ability to deter-mine which epitopes are important in a given individual is extremely limited. In this issue, Newell et al.1 present an elegant approach for simultaneous detection of T cells specific for a large number of candidate epitopes. The authors apply their novel technique to define rotavirus epitopes recognized by T cells in healthy humans, but it should be applicable to identifying T cells specific for epitopes in any protein of known sequence, whether derived from a pathogen, a tumor or a candidate vac-cine. The solution presented in this report therefore represents a decisive advance in human immunology.

In the realm of immunology, few technologi-cal breakthroughs have led to more massive gains in our understanding than the devel-opment of flow cytometry2. It is difficult to envision how we could examine the complex behavior of the many different immune cell populations without this approach. Whereas early flow cytometers could measure only one or two fluorophores, modern multilaser instruments enable isolation of even rare popu-lations of cells based on detection of a substan-tial number of cellular parameters. However, as flow cytometers rely on fluorescent dyes,

Cracking the code of human T-cell immunityChristopher J Harvey & Kai W Wucherpfennig

Combinatorial tetramer staining coupled with mass cytometry allows simultaneous detection of T cells specific for a wide array of peptide epitopes.

n e W s a n d v i e W s

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature biotechnology volume 31 number 7 JulY 2013 609

Christopher J. Harvey and Kai W. Wucherpfennig are in the Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA, and Kai W. Wucherpfennig is also in the Program in Immunology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. e-mail: [email protected]

Newell et al.1 have exploited the advantages of mass cytometry to simultaneously ana-lyze the specificities of many T cells. Because the cytotoxic function of CD8 T cells is trig-gered by T-cell receptor (TCR) recognition of epitopes presented by major histocompat-ibility complex (MHC) class I molecules on target cells, they stained T cells with panels of peptide-MHC tetramers. As shown by Altman and Davis in 1996, virus-specific T cells can be visualized and isolated with fluorescently labeled tetramers of peptide-MHC complexes7. Since then, tetramers have become an essen-tial tool for investigation of T-cell responses in basic and clinical immunology. The Davis and Schumacher laboratories reported that com-binatorial tetramer staining can moderately increase the number of fluorescent tetramers one can use to simultaneously stain a single sample8,9; however, spectral overlap has limited the impact of this approach.

To adapt combinatorial tetramer staining to mass cytometry, Newell et al.1 use ten heavy-metal labels to create a three-metal tetramer barcode (Fig. 1). Tetramers are made by mixing biotinylated peptide-MHC complexes at a 4:1 ratio with streptavidin; the multivalent nature of tetramers compensates for the low affin-ity of peptide-MHC complexes for TCRs. To create the three-metal code, the authors label streptavidin in separate reactions with each of the ten metals. A unique combination of three metals is used for a single epitope. Three tetramers are separately made for each peptide-MHC complex using streptavidin labeled with three different metals. The three differentially labeled tetramers are then combined and used as a single staining reagent.

When stained in this way, any cell expressing a TCR capable of binding a given peptide-MHC tetramer should be coincidentally labeled by all three metals. As a result, specifically labeled T cells should be detected in all three—and only those three—mass spectrometer chan-nels. Cells detected in only one or two of the

they suffer from an inherent technical limita-tion imposed by spectral overlap. The recent development of quantum dots with narrow spectral windows has expanded the num-ber of parameters that researchers can detect simultaneously using flow cytometry, but ‘com-pensating’ for spectral bleed-through becomes increasingly complex as the number of fluores-cent parameters is increased3.

Mass cytometry represents an elegant, highly innovative solution to this problem. This breakthrough technology—first described in 2009 by Scott Tanner and colleagues4 at the University of Toronto—is poised to again transform immunology and other fields of biomedical research. The fundamental idea behind mass cytometry is that fluorophores can be replaced with heavy metals (bound to antibodies through a chelator), which are then quantified with high resolution by mass spec-trometry. The approach thus takes advantage of the high mass accuracy of mass spectrometers, enabling simultaneous analysis of a very large number of metal-tagged antibodies in a CyTOF (cytometry by time of flight) instrument.

Approximately 40 transition-state element isotopes not normally found in biological sys-tems (referred to as ‘metals’) with distinct mass can currently be examined5. Cells stained with metal-conjugated antibodies are atomized in an argon plasma stream, creating ion clouds from single cells. Metal ions falling within a certain mass range pass through a series of cones and deflectors and are detected based on time of flight. Metals do not have the spectral overlap problems associated with fluorophores, therefore eliminating the need for compensa-tion. The use of metals, however, is not without limitations, as certain isotype preparations are impure (contaminated by isotypes with a mass of m + 1). To date, only a small subset of the possibilities afforded by this novel technology has been explored, but it is already evident that mass cytometry has great potential for bio-medical research5,6.

The ability to identify and track cytotoxic T cells capable of recognizing particular epitopes is essential for studying human immune responses in the context of infectious diseases, cancer and autoimmunity. Yet despite the centrality of T cell–epitope interactions to the immune response, our ability to deter-mine which epitopes are important in a given individual is extremely limited. In this issue, Newell et al.1 present an elegant approach for simultaneous detection of T cells specific for a large number of candidate epitopes. The authors apply their novel technique to define rotavirus epitopes recognized by T cells in healthy humans, but it should be applicable to identifying T cells specific for epitopes in any protein of known sequence, whether derived from a pathogen, a tumor or a candidate vac-cine. The solution presented in this report therefore represents a decisive advance in human immunology.

In the realm of immunology, few technologi-cal breakthroughs have led to more massive gains in our understanding than the devel-opment of flow cytometry2. It is difficult to envision how we could examine the complex behavior of the many different immune cell populations without this approach. Whereas early flow cytometers could measure only one or two fluorophores, modern multilaser instruments enable isolation of even rare popu-lations of cells based on detection of a substan-tial number of cellular parameters. However, as flow cytometers rely on fluorescent dyes,

Cracking the code of human T-cell immunityChristopher J Harvey & Kai W Wucherpfennig

Combinatorial tetramer staining coupled with mass cytometry allows simultaneous detection of T cells specific for a wide array of peptide epitopes.

n e W s a n d v i e W s

npg

© 2

013

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

HIF1a Allows Monocytes to Takea Breather during Sepsis

Derek W. Gilroy1,* and Simon Yona1,*1Centre for Clinical Pharmacology and Therapeutics, Division of Medicine, 5 University Street, University College London,London WC1E 6JJ, UK*Correspondence: [email protected] (D.W.G.), [email protected] (S.Y.)http://dx.doi.org/10.1016/j.immuni.2015.02.016

How the immune system is negatively affected by sepsis is not fully understood. In this issue of Immunity,Shalova et al. (2015) show that during human sepsis monocytes upregulate hypoxia-inducible factor-a(HIF1-a) activity and acquire an immunosuppressive phenotype while retaining anti-bacterial and wound-healing properties.

Inflammation is a good thing. It removes

infections and paves the way for tissue

repair and the re-establishment of ho-

meostasis. It’s a delicate balance be-

tween pro- and anti-inflammatory signals

followed by a sequence of pro-resolution

events. However, inflammation can also

be a very bad thing. During sepsis, for

instance, an overexuberant and pro-

longed activation of the innate immune

system results in a ‘‘cytokine storm’’

causing physiological shock and multiple

organ failure. That notwithstanding, phar-

macological interventions aimed at quel-

ling these unbridled drivers of the innate

response have proven largely ineffective

in treating the critically ill. Instead, a pre-

dominantly anti-inflammatory period—

the compensatory anti-inflammatory

response syndrome (CARS)—is now re-

garded as the primary clinical concern.

This phase is associated with microcircu-

latory dysfunction, coagulopathy, cata-

bolic predominance, and bioenergetic

failure leading to multi-organ failure (Full-

erton and Singer, 2011). However, it is

principally marked by vulnerability to hos-

pital-acquired infection and repeated

episodes of sepsis. Indeed, the immune

component of CARS, described as immu-

noparalysis, anergy, or leukocyte re-pro-

gramming, (Hotchkiss et al., 2009) might

pose an equal if not a greater threat to

the host than the initial ‘‘cytokine storm.’’

The proposed mechanisms involved are

complex. Loss of key effector cells espe-

cially via apoptosis, a shift in cytokines

from a pro-inflammatory to an anti-inflam-

matory profile, alteration in cell-surface

receptor expression, and multiple other

alterations have been reported. Impor-

tantly, gene-expression profiling of the in-

flammatory response in the critically ill has

demonstrated that although there is vari-

ability between different initiating stimuli

(Tang et al., 2010), a consistent picture

emerges regardless of etiology (Xiao

et al., 2011) or pathogen (Tang et al.,

2008). This indicates that a fundamental

human transcriptomic response to severe

inflammatory stress might exist across all

forms of sepsis. Thus, these data indicate

that the etiology of the innate immune

response during sepsis episodes might

not be as heterogeneous as previously

suspected. This renders the regulation of

inflammation an appealing therapeutic

target in the critically ill. In this issue of

Immunity, Shalova et al. (2015) have

examined the impact of Gram negative-

induced sepsis on monocyte-effector

function. The overall aim is understand

how a severe inflammatory insult nega-

tively affects the innate immune system

leading, in turn, to the poor clinical

outcome that is typically associated with

sepsis. These authors found that the

phenotype of monocytes taken from

patients within a few hours of infection

shifted from a pro-inflammatory to

an immune-suppressive phenotype in

monocytes taken from patients who

recovered. It is proposed that elevated

hypoxia-inducible factor-a (HIF1-a) trig-

gers IRAKM, a negative modulator of

TLR signaling, leading to an endotoxin-

tolerant monocyte that interestingly also

acquires anti-bacterial and wound-heal-

ing properties.

Sepsis is a complex response to injury

and infection, and trying to understand

its regulatory pathways is no trivial under-

taking. The pathogenesis that underpins

its impact on organ dysfunction and its

subsequent immune suppression pre-

disposing to nosocomial infection, not

the mention its dire long-term mortality,

cannot be ascribed to one single cell,

soluble mediator, receptor, or signaling

factor. Nonetheless, in an attempt to un-

derstand the role of at least one of the

key protagonists within the pantheon of

sepsis, Shalova et al. carried out tran-

scriptomic analysis on circulating mono-

cyte populations from patients with active

sepsis (within a few hours of a defined

gram-negative infection) and compared

this phenotype to monocytes from the

same patients 4–12 weeks later, in which

they termed ‘‘recovery phase.’’ In cells

taken from patients with active sepsis

the usual pro-inflammatory suspects (nu-

clear factor-kB [NF-kB], interleukin-6 [IL-

6], IL-1b, and chemokines CCL3, CCL5)

emerged alongside elevated IL-10 and

cell surface markers of immune dysfunc-

tion. As expected, a different picture was

painted by monocytes taken from those

patients who recovered from sepsis being

more akin to the transcriptome of mono-

cytes from healthy volunteers. Thereafter,

a comparative transcriptomic profile of

these respective monocyte populations

was obtained after their stimulation with

LPS ex vivo. ‘‘Recovery monocytes’’ dis-

played a range of cytokines and chemo-

kines whereas ‘‘sepsis monocytes’’ were

largely refractory to LPS in this regard.

Moreover, sepsis monocytes displayed

apparent reduced antigen presentation

and downregulation of major histocom-

patibility complex-II (MHC-II) genes and

co-stimulatory molecules; the hallmarks

of cells experiencing endotoxin tolerance.

The authors proposed that elevated

HIF1-a and subsequent upregulation of

Immunity 42, March 17, 2015 ª2015 Elsevier Inc. 397

Immunity

Previews

Our current definition of essential genes is based on whether or not gene knockout cells survive. Rancati and colleagues now reveal a new class of essential genes — called ‘evolvable essential genes’ — that can overcome loss of function with time by evolving alternative, usually unrelated, cellular processes driven by aneuploidy.

To identify evolvable essential genes, the authors used a stringent, three-level screen based on the sur-vival of the progeny of Saccharomyces cerevisiae cells each with a gene deletion of one of ~1,000 genes that are considered to be essential for viability. At every stage, cells were cultured for at least 10 days to allow time for the adaptive evolution to occur, and mutant strains that yielded either no progeny (inacti-vation of a non-evolvable essential gene) or too many progeny (inacti-vation of a non-essential gene) were eliminated. The first screen identified heterozygous diploid strains, of which mutant offspring survived. The second and the third screen involved tetrad analysis, in which the four haploid spores are separated and cultured individually, of the

identified mutants in two genetic backgrounds. This strategy yielded 104 putative evolvable essential genes, 16 of which were disregarded after further validation by microma-nipulation-based pedigree analysis showed maximum viability of the offspring. Thus, 88 bona fide essen-tial genes (~9%) could be deleted without resulting in stereotypical cell lethality, and are thus classified as evolvable-essential genes.

Adaptive evolution probably involves whole-chromosome and segmental aneuploidy, rather than point mutations. Interestingly, specific karyotypic changes seem to recur when functionally similar genes are deleted. For example, several strains that were deleted in genes associated with the nucleo-porin (NUP) complex acquired an extra copy of chromosome VIII. Just introducing an extra copy of this chromosome increased the survival of NUP-associated mutants, indicat-ing that this particular aneuploidy is sufficient to rescue the gene deletion. Further functional analysis found that just one gene — BRL1 — mitigates the effect of chromosome VIII aneuploidy, not simply by

restoring the disrupted function but by altering membrane fluidity. Adaptive evolution thus co-opts seemingly unrelated pathways.

Evolvable genes were enriched for components of subcellular com-partments found only in eukaryotes (nucleus, endoplasmic reticulum and Golgi apparatus) but not for proteins involved in universal key processes such as DNA, RNA and protein synthesis. This finding indicates that evolutionary ‘younger’ essential genes might have retained some degree of adaptability.

Thus, a quantitative redefinition of gene essentiality that incorporates both viability and evolvability is required. Distinguishing non-evolvable from evolvable essential genes should be considered when ranking potential drug targets to minimize drug resistance, if these findings can be validated in patho-genic fungi or in other disease- causing cells such as cancer cells.

Liesbet Lieben, Associate Editor, Nature Reviews Disease Primers

E VO L U T I O N

Redefining gene essentiality

ORIGINAL ARTICLE Liu, G. et al. Gene essentiality is a quantitative property linked to cellular evolvability. Cell http://dx.doi.org/10.1016/ j.cell.2015.10.069 (2015)

a quantitative redefinition of gene essentiality that incorporates both viability and evolvability is required

PhotoDisc/G

etty Images

R E S E A R C H H I G H L I G H T S

NATURE REVIEWS | GENETICS www.nature.com/nrg

Nature Reviews Genetics | Published online 14 December 2015; doi:10.1038/nrg.2015.23

© 2015 Macmillan Publishers Limited. All rights reserved









E VO L U T I O N




PhotoDisc/G

etty Images













E VO L U T I O N




PhotoDisc/G

etty Images





Articles of Significant Interest Selected from This Issue by the Editors

The SIV Accessory Protein Vpx Induces Degradation of Host Restriction Factor SAMHD1 in the Nucleus

SAMHD1, a nuclear protein in dendritic and myeloid cells, restricts human immunodeficiency virus type 1 (HIV-1) by depleting dNTPpools. Simian immunodeficiency virus (SIV) and HIV-2 encode Vpx, a small, virion-packaged accessory protein that targets SAMHD1for proteasomal degradation. Hofmann et al. (p. 12552–12560) identify the nuclear localization signal (NLS) of SAMHD1 and show thatan altered SAMHD1 lacking the NLS antagonizes Vpx and retains antiviral activity. Remarkably, Vpx induces the degradation ofSAMHD1 in the nucleus through nuclear E3 ubiquitin ligase and proteasomes.

Activities of the Adeno-Associated Virus Assembly-Activating Protein AAP

Adeno-associated virus caspid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins(VPs). AAP targets VPs to the nucleolus, where newly formed capsids are detected. Naumer et al. (p. 13038 –13048) show that AAPrequires two hydrophobic stretches for interaction with VPs and the assembly-supporting activity. Binding of AAP at the 2-foldsymmetry axes of VPs induces a conformational change in nonassembled VPs that may facilitate subunit interactions at the 3-fold and5-fold symmetry axes. These data provide strong evidence that AAP acts as a scaffolding protein.

Novel Molecular Mechanisms Regulating Herpes Simplex Virus 1 Latency and Reactivation

Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons as a histone-associated circular episome that can reactivateto cause clinical disease. However, the molecular mechanisms regulating the latency-reactivation transition are poorly defined. Ertel etal. (p. 12741–12759) dissected the epigenetic mechanisms that contribute to maintenance of latency and reactivation of HSV-1 byinvestigating the role of the cellular insulator protein, CTCF, in each process. The results suggest that CTCF binding to the HSV-1genome is essential for maintenance of latency and regulated in a transcript-dependent manner, while the loss of CTCF binding is anintegral step in HSV-1 reactivation.

Circulating HIV-1 Variants Have Novel CD4 Requirements

Human immunodeficiency virus type 1 (HIV-1) replicates poorly in macaques, a reality that has hindered development of relevantnonhuman primate model systems for studies of HIV-1 infection. Humes et al. (p. 12472–12483) demonstrate that most circulating,recently transmitted HIV-1 variants do not mediate entry efficiently using macaque CD4. Species-specific differences in the functionalcapacity of CD4 to permit HIV-1 entry mapped to a single amino acid polymorphism. Correlates of macaque CD4 use by pandemicHIV-1 variants, such as soluble CD4 sensitivity, were identified that could be exploited for development of relevant nonhuman primatemodels of HIV-1 pathogenesis.

Chikungunya Virus-Specific Epitopes for Diagnostics and Vaccine Development

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Although anti-CHIKVantibodies have been reported, the fine specificity of the antibody response against CHIKV is not known. Kam et al. (p. 13005–13015)show that the E2 and E3 glycoproteins, capsid, and nsP3 proteins are targets of anti-CHIKV antibody responses in patient cohorts. Thesefindings identify CHIKV-specific epitopes for use in future seroepidemiological studies that will enhance an understanding of theCHIKV-specific immune response and foster development of CHIKV vaccines.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02706-12

SPOTLIGHT

December 2012 Volume 86 Number 23 Journal of Virology p. 12471 jvi.asm.org 12471

on January 25, 2016 by guesthttp://jvi.asm

.org/D

ownloaded from


The SIV Accessory Protein Vpx Induces Degradation of Host Restriction Factor SAMHD1 in the Nucleus

SAMHD1, a nuclear protein in dendritic and myeloid cells, restricts human immunodeficiency virus type 1 (HIV-1) by depleting dNTPpools. Simian immunodeficiency virus (SIV) and HIV-2 encode Vpx, a small, virion-packaged accessory protein that targets SAMHD1for proteasomal degradation. Hofmann et al. (p. 12552–12560) identify the nuclear localization signal (NLS) of SAMHD1 and show thatan altered SAMHD1 lacking the NLS antagonizes Vpx and retains antiviral activity. Remarkably, Vpx induces the degradation ofSAMHD1 in the nucleus through nuclear E3 ubiquitin ligase and proteasomes.

Activities of the Adeno-Associated Virus Assembly-Activating Protein AAP

Adeno-associated virus caspid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins(VPs). AAP targets VPs to the nucleolus, where newly formed capsids are detected. Naumer et al. (p. 13038 –13048) show that AAPrequires two hydrophobic stretches for interaction with VPs and the assembly-supporting activity. Binding of AAP at the 2-foldsymmetry axes of VPs induces a conformational change in nonassembled VPs that may facilitate subunit interactions at the 3-fold and5-fold symmetry axes. These data provide strong evidence that AAP acts as a scaffolding protein.

Novel Molecular Mechanisms Regulating Herpes Simplex Virus 1 Latency and Reactivation

Herpes simplex virus type 1 (HSV-1) establishes latency in sensory neurons as a histone-associated circular episome that can reactivateto cause clinical disease. However, the molecular mechanisms regulating the latency-reactivation transition are poorly defined. Ertel etal. (p. 12741–12759) dissected the epigenetic mechanisms that contribute to maintenance of latency and reactivation of HSV-1 byinvestigating the role of the cellular insulator protein, CTCF, in each process. The results suggest that CTCF binding to the HSV-1genome is essential for maintenance of latency and regulated in a transcript-dependent manner, while the loss of CTCF binding is anintegral step in HSV-1 reactivation.

Circulating HIV-1 Variants Have Novel CD4 Requirements

Human immunodeficiency virus type 1 (HIV-1) replicates poorly in macaques, a reality that has hindered development of relevantnonhuman primate model systems for studies of HIV-1 infection. Humes et al. (p. 12472–12483) demonstrate that most circulating,recently transmitted HIV-1 variants do not mediate entry efficiently using macaque CD4. Species-specific differences in the functionalcapacity of CD4 to permit HIV-1 entry mapped to a single amino acid polymorphism. Correlates of macaque CD4 use by pandemicHIV-1 variants, such as soluble CD4 sensitivity, were identified that could be exploited for development of relevant nonhuman primatemodels of HIV-1 pathogenesis.

Chikungunya Virus-Specific Epitopes for Diagnostics and Vaccine Development

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Although anti-CHIKVantibodies have been reported, the fine specificity of the antibody response against CHIKV is not known. Kam et al. (p. 13005–13015)show that the E2 and E3 glycoproteins, capsid, and nsP3 proteins are targets of anti-CHIKV antibody responses in patient cohorts. Thesefindings identify CHIKV-specific epitopes for use in future seroepidemiological studies that will enhance an understanding of theCHIKV-specific immune response and foster development of CHIKV vaccines.


doi:10.1128/JVI.02706-12

SPOTLIGHT

December 2012 Volume 86 Number 23 Journal of Virology p. 12471 jvi.asm.org 12471


.org/D

ownloaded from


Diversity and Evolution of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread pathogen that affects immunocompromisedindividuals and the developing fetus. Sijmons et al. (p. 7673–7695) quadruple the availablesequence data for genome-wide analysis of HCMV diversity and evolution and provide the mostdetailed map of HCMV interhost variability to date. The analysis reveals the wide extent ofdisruptive mutations in clinical HCMV strains and highlights the fundamental role of recom-bination in the evolution of this herpesvirus. These data will be an important resource for futurestudies of pathogenicity determinants, virus biology, and vaccine design.

Coopting Autophagic Processes by Dengue Virus Benefits Replication

Autophagy is a catabolic process that degrades cellular components in a lysosome-dependentmanner and can promote or restrict viral replication. Metz et al. (p. 8026 – 8041) established anunbiased, image-based flow cytometry approach to quantify autophagic flux. The results indi-cate that Dengue virus induces initial activation of autophagic flux, followed by inhibition ofgeneral and specific autophagy. Moreover, the autophagy receptor SQSTM1/p62 suppressesviral replication and is degraded by the proteasome at late stages of infection. This work suggeststhat there are time-dependent opposing effects of autophagy on Dengue virus.

HIV-1 Is Restricted prior to Provirus Integration in CD34� Cells

Cells can block HIV-1 at specific stages of the viral life cycle via the activities of well-characterized restriction factors and transcriptional silencing machinery. Infection of mu-rine stem cells (MSCs) by murine leukemia viruses (MLVs) is blocked postintegration bytranscriptional silencing. Griffin and Goff (p. 8096 – 8100) show that the dominant point ofrestriction of HIV-1 in human CD34� stem cells is prior to integration of viral DNA. Therefore,MLV restriction by MSCs and HIV-1 restriction by human CD34� cells are fundamentallydifferent processes.

Regulatory T Cells Limit Chikungunya Virus-Induced Joint Pathology

Persons infected with chikungunya virus (CHIKV) develop incapacitating joint pain that com-promises daily activities. CD4� T cells contribute to joint inflammation during the course ofCHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatoryT cells (Tregs) by forming a complex with interleukin-2 (IL-2). Lee et al. (p. 7893–7904) showthat IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology byinhibiting the infiltration of CD4� T cells. These findings suggest that activation of Tregs couldserve to control CHIKV-mediated disease.

Bile Acids: Novel Therapeutic Candidates for Prion Disease

Prion diseases are fatal, transmissible, neurodegenerative diseases with no available treatments.Pathology is triggered by the misfolding of the prion protein (PrP), which causes misfolding ofmore PrP and spreads throughout the brain, leading to astrogliosis and neuronal loss. Cortez etal. (p. 7660 –7672) provide evidence that the bile acids ursodeoxycholic acid (UDCA) andtauroursodeoxycholic acid (TUDCA) have anti-prion effects by impeding PrP misfolding andmediating neuroprotection. Given that these compounds are in clinical use for other humanconditions, these bile acids are promising therapeutic candidates for future clinical trials inhuman prion disease.


doi:10.1128/JVI.01422-15

Phylogenetic network of 124 HCMVfull-genome sequences shows numer-ous reticulate connections that illus-trate recombination.

Differential effect of dengue virus onautophagic flux during the course ofinfection.

HIV-1 infection of CD34� cells re-sults in dramatically decreased levelsof circular viral DNA intermediatesin the nucleus compared with that inpermissive cells.

Expansion of Tregs abrogatesCHIKV-induced joint pathology.

UDCA treatment reduces astrocyto-sis bioluminescence (blue) in prion-infected Tg(Gfap-luc) mice.

SPOTLIGHT

August 2015 Volume 89 Number 15 jvi.asm.org 7445Journal of Virology


.org/D

ownloaded from


Diversity and Evolution of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread pathogen that affects immunocompromisedindividuals and the developing fetus. Sijmons et al. (p. 7673–7695) quadruple the availablesequence data for genome-wide analysis of HCMV diversity and evolution and provide the mostdetailed map of HCMV interhost variability to date. The analysis reveals the wide extent ofdisruptive mutations in clinical HCMV strains and highlights the fundamental role of recom-bination in the evolution of this herpesvirus. These data will be an important resource for futurestudies of pathogenicity determinants, virus biology, and vaccine design.

Coopting Autophagic Processes by Dengue Virus Benefits Replication

Autophagy is a catabolic process that degrades cellular components in a lysosome-dependentmanner and can promote or restrict viral replication. Metz et al. (p. 8026 – 8041) established anunbiased, image-based flow cytometry approach to quantify autophagic flux. The results indi-cate that Dengue virus induces initial activation of autophagic flux, followed by inhibition ofgeneral and specific autophagy. Moreover, the autophagy receptor SQSTM1/p62 suppressesviral replication and is degraded by the proteasome at late stages of infection. This work suggeststhat there are time-dependent opposing effects of autophagy on Dengue virus.

HIV-1 Is Restricted prior to Provirus Integration in CD34� Cells

Cells can block HIV-1 at specific stages of the viral life cycle via the activities of well-characterized restriction factors and transcriptional silencing machinery. Infection of mu-rine stem cells (MSCs) by murine leukemia viruses (MLVs) is blocked postintegration bytranscriptional silencing. Griffin and Goff (p. 8096 – 8100) show that the dominant point ofrestriction of HIV-1 in human CD34� stem cells is prior to integration of viral DNA. Therefore,MLV restriction by MSCs and HIV-1 restriction by human CD34� cells are fundamentallydifferent processes.

Regulatory T Cells Limit Chikungunya Virus-Induced Joint Pathology

Persons infected with chikungunya virus (CHIKV) develop incapacitating joint pain that com-promises daily activities. CD4� T cells contribute to joint inflammation during the course ofCHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatoryT cells (Tregs) by forming a complex with interleukin-2 (IL-2). Lee et al. (p. 7893–7904) showthat IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology byinhibiting the infiltration of CD4� T cells. These findings suggest that activation of Tregs couldserve to control CHIKV-mediated disease.

Bile Acids: Novel Therapeutic Candidates for Prion Disease

Prion diseases are fatal, transmissible, neurodegenerative diseases with no available treatments.Pathology is triggered by the misfolding of the prion protein (PrP), which causes misfolding ofmore PrP and spreads throughout the brain, leading to astrogliosis and neuronal loss. Cortez etal. (p. 7660 –7672) provide evidence that the bile acids ursodeoxycholic acid (UDCA) andtauroursodeoxycholic acid (TUDCA) have anti-prion effects by impeding PrP misfolding andmediating neuroprotection. Given that these compounds are in clinical use for other humanconditions, these bile acids are promising therapeutic candidates for future clinical trials inhuman prion disease.


doi:10.1128/JVI.01422-15

Phylogenetic network of 124 HCMVfull-genome sequences shows numer-ous reticulate connections that illus-trate recombination.

Differential effect of dengue virus onautophagic flux during the course ofinfection.

HIV-1 infection of CD34� cells re-sults in dramatically decreased levelsof circular viral DNA intermediatesin the nucleus compared with that inpermissive cells.

Expansion of Tregs abrogatesCHIKV-induced joint pathology.

UDCA treatment reduces astrocyto-sis bioluminescence (blue) in prion-infected Tg(Gfap-luc) mice.

SPOTLIGHT

August 2015 Volume 89 Number 15 jvi.asm.org 7445Journal of Virology


.org/D

ownloaded from

2 1

Scientific Breakthroughs

2 0

Special Editorial Mentions of SIgN Papers

8

20

48 52

66

110

126

119

96

Publications

645 scientific publications from 2007-2015

Impact Factor

>20: 42

10-20: 86

5-10: 195

N.A. or < 5: 322

140

120

100

80

60

40

20

0

2007 2009 2011 20132008 2010 2012 2014 2015

Publication HightlightsAndiappan AK, Melchiotti R, Poh TY, Nah M, Puan KJ, Vigano E, Haase D, Yusof N, San Luis B, Lum J, Kumar D, Foo S, Zhuang L, Vasudev A, Irwanto A, Lee B, Nardin A, Liu H, Zhang F, Connolly J, Liu J, Mortellaro A, Wang de Y, Poidinger M, Larbi A, Zolezzi F, Rotzschke O. Genome-wide analysis of the genetic regulation of gene expression in human neutrophils. Nat Commun. 2015 Aug 10;6:7971.

Andiappan AK, Puan KJ, Lee B, Nardin A, Poidinger M, Connolly J, Chew FT, Wang DY, Rotzschke O. Allergic airway diseases in a tropical urban environment are driven by dominant mono-specific sensitization against house dust mites. Allergy. 2014 Apr;69(4):501-9.

Becher B, Schlitzer A, Chen J, Mair F, Sumatoh HR, Teng KW, Low D, Ruedl C, Riccardi-Castagnoli P, Poidinger M, Greter M, Ginhoux F, Newell EW. High-dimensional analysis of the murine myeloid cell system. Nat Immunol. 2014 Dec;15(12):1181-9.

Chew V, Chen J, Lee D, Loh E, Lee J, Lim KH, Weber A, Slankamenac K, Poon RT, Yang H, Ooi LL, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP. Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma. Gut. Mar 2012; 61(3):427-438.

2 3

Scientific Breakthroughs

2 2

Chittezhath M, Dhillon MK, Lim JY, Laoui D, Shalova IN, Teo YL, Chen J, Kamaraj R, Raman L, Lum J, Thamboo TP, Chiong E, Zolezzi F, Yang H, Van Ginderachter JA, Poidinger M, Wong AS, Biswas SK. Molecular profiling reveals a tumor-promoting phenotype of monocytes and macrophages in human cancer progression. Immunity. 2014 Nov 20;41(5):815-29.

Eyles J, Puaux AL, Wang X, Toh B, Prakash C, Hong M, Tan TG, Zheng L, Ong LC, Jin Y,Kato M, Prévost-Blondel A, Chow P, Yang H and Abastado JP. Tumor cells disseminate early but immunosurveillance limits metastatic outgrowth in a mouse model of melanoma. J Clin Invest. 2010 May 24. 2010 Jun;120(6):2030-9.

Facciotti F, Ramanjaneyulu GS, Lepore M, Sansano S, Cavallari M, Kistowska M, Forss-Petter S, Ni G, Colone A, Singhal A, Berger J, Xia C, Mori L, De Libero G. Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus. Nat Immunol. 2012 Mar 18;13(5):474-80.

Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010 Nov 5;330(6005):841-5.

Hoeffel G, Wang Y, Greter M, See P, Teo P, Malleret B, Leboeuf M, Low D, Oller G, Almeida F, Choy SH, Grisotto M, Renia L, Conway SJ, Stanley ER, Chan JK, Ng LG, Samokhvalov IM, Merad M, Ginhoux F. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac–derived macrophages. J Exp Med. 2012 Jun 4;209(6):1167-81.

Howland SW, Poh CM, Rénia L. Activated Brain Endothelial Cells Cross-Present Malaria Antigen. PLoS Pathog. 2015 Jun 5;11(6):e1004963.

Kam YW, Lum FM, Teo TH, Lee WW, Simarmata D, Harjanto S, Chua CL, Chan YF, Wee JK, Chow A, Lin RT, Leo YS, Le Grand R, Sam IC, Tong JC, Roques P, Wiesmüller KH, Rénia L, Rotzschke O, Ng LF. Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein. EMBO Mol Med. 2012 Apr;4(4):330-43.

Lee Y, Chittezhath M, Andre V, Zhao H, Poidinger M, Biondi A, D’Amico G, Biswas SK. Protumoral role of monocytes in human B-cell precursor acute lymphoblastic leukemia: involvement of the chemokine CXCL10. Blood. Jan 5 2012; 119(1):227-237.

Li P, Wong JJ, Sum C, Sin WX, Ng KQ, Koh MB, Chin KC. IRF8 and IRF3 cooperatively regulate rapid interferon-beta induction in human blood monocytes. Blood. Mar 10 2011; 117(10):2847-2854.

Liu G, Yong MY, Yurieva M, Srinivasan KG, Liu J, Lim JS, Poidinger M, Wright GD, Zolezzi F, Choi H, Pavelka N, Rancati R. Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability. Cell. 2015 Dec 3;163(6):1388-99.

Russell B, Suwanarusk R, Borlon C, Costa FT, Chu CS, Rijken MJ, Sriprawat K, Warter L, Koh EG, Malleret B, Colin Y, Bertrand O, Adams JH, D’Alessandro U, Snounou G, Nosten F, Renia L. A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax. Blood. Sep 29 2011; 118(13):e74-81.

Schlitzer A, McGovern N, Teo P, Zelante T, Atarashi K, Low D, Ho AW, See P, Shin A, Wasan PS, Hoeffel G, Malleret B, Heiseke A, Chew S, Jardine L, Purvis HA, Hilkens CM, John Tam J, Poidinger M, Stanley RE, Krug AB, Renia L, Sivasankar B, Ng LG, Collin M, Ricciardi-Castagnoli P, Honda K, Haniffa M, Ginhoux F. IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity. 2013 May 23;38(5):970-983.

Shalova IN, Lim JY, Chittezhath M, Zinkernagel AS, Beasley F, Hernández-Jiménez E, Toledano V, Cubillos-Zapata C, Rapisarda A, Chen J, Duan K, Yang H, Poidinger M, Melillo G, Nizet V, Arnalich F, López-Collazo E, Biswas SK. Human Monocytes Undergo Functional Re-programming during Sepsis Mediated by Hypoxia-Inducible Factor-1α. Immunity. 2015 Mar 17;42(3):484-98.

Teng TS, Foo SS, Simamarta D, Lum FM, Teo TH, Lulla A, Yeo NK, Koh EG, Chow A, Leo YS, Merits A, Chin KC, Ng LF. Viperin restricts chikungunya virus replication and pathology. J Clin Invest. 2012 Dec 3;122(12):4447-60.

Wong KL, Tai JJ, Wong WC, Han H, Sem X, Yeap WH, Kourilsky P, Wong SC. Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood. Aug 4 2011; 118(5):e16-31.

Translational ProgramsHuman Immunology has great potential for translation into clinical applications. Translational research with cohorts of patients and healthy volunteers is facilitated in SIgN by cutting-edge translational platforms (Clinical Immunomonitoring and Human Therapeutic Antibodies) and clinical project management. Below are some of the areas in which SIgN investigators have engaged the Singapore clinical community.

Area Program and clinical collaborators

INFECTIONS

Immune responses in dengue patients, biomarkers of dengue severity, functions of skin immune cells after dengue infection (TTSH)

Immunity studies of Chikungunya virus, influenza virus and other respiratory pathogens in Singapore (TTSH)

New biomarkers and drugs in tuberculosis (TTSH)

Immunoclinical investigations in malaria (TTSH)

Immune responses in Hepatitis B Virus infection (NUHS)

Gut microbiome and nosocomial infection (NUHS)

CANCER AND IMMUNITY

Metabolomics and microenvironment modelling platform for HCC therapeutics and biomarker development (NCC)

Novel human antibodies against breast cancer cells (NCC)

Novel therapeutic antibodies against acute myeloid leukemia and gastric cancer stem cells (NUH)

Immune microenvironment of hepatocellular carcinoma, gastric and pancreatic cancer (NCC, SGH, NUH)

IBD and colorectal cancer biomarkers (NUH)

Tumor-associated immune cells in human cancers (NUH)

2 5

IMMUNOTHERAPYAdoptive transfer of human regulatory T cell to treat graft-versus-host disease

AGING AND IMMUNITYImmunoaging, immune biomarkers in frailty and response to vaccination in elderly (NUHS)

MATERNAL AND PEDIATRIC IMMUNITY

Immune cells during fetal development (KKH)

CARDIOVASCULARAsian Network for Translational Research and Cardiovascular trials (ATTRaCT) - Biomarkers and Immunology

INFLAMMATION AND AUTOIMMUNITY

HLA associations with hypersensitivity and adverse drug reactions (SGH, CGH, HSA)

Immune pathogenesis of rheumatoid arthritis, gout, systemic lupus erythematosus, idiopathic uveitis (NUH, SERI)

Anti-inflammatory and anti-infectious therapeutic targets (Duke-NUS)

SKIN IMMUNITY

Pathophysiology of itch in Atopic Dermatitis and Psoriasis (NSC)

Skin-specific immunity in aging (NSC)

Immune cell subsets in the skin (SGH)

ALLERGY Studies of atopy and allergic diseases in Chinese Singaporeans (NUS)

Progenitor cells in respiratory diseases (NUH, NUS)

Translational Programs

2 52 4

CLINICAL IMMUNOMONITORING PLATFORMSIgN’s Clinical Immunomonitoring platform is dedicated to the definition of immunomarkers and immunological endpoints with clinically relevant impact via:

The platform comprises 4 cores, namely (i) Flow Cytometry Core, (ii) CyTOF Core, (iii) Functional Genomics Core and (iv) Bioinformatics Core. The integrated workflow of the platform incorporates upstream study design, SOP-driven sample collection and biobanking, as well as high-throughput analysis of samples using the most sophisticated technologies available and bioinformatics analysis downstream.

The platform is engaged in several collaborations with Industry and clinical partners. Some of these studies address diseased populations with unmet clinical needs that may benefit from a better understanding of disease biology, ultimately leading to predictive or diagnostic biomarkers improving patient care or new targets for clinical interventions. Studies in healthy individuals such as vaccine clinical trials potentially contribute to rational vaccine design for long-term protective immunity against infectious diseases. Examples of such studies include a Phase IV clinical trial study with Sanofi Pasteur and NUHS aimed at understanding the loss of immunity and reduced responsiveness to vaccination in the elderly, and the ATTRaCT Study (a collaborative effort between A*STAR, SingHealth and NUHS institutions), which aims to gain deeper insight into the role of inflammation in heart failure, the leading cause of mortality worldwide.

2 7

Translational Programs

2 6

HUMAN THERAPEUTIC ANTIBODIES PLATFORMThe Human Therapeutic Antibodies platform at SIgN utilizes state-of-the-art antibody technologies to discover, engineer and characterize novel fully human antibodies and develop them into therapeutic candidates to treat a wide range of medical conditions, including infection, cancer, inflammation, and autoimmune diseases.

The platform has developed streamlined, high-throughput single B cell PCR cloning technology to isolate natural disease-fighting antibodies directly from virus-infected patients. A large panel of neutralizing antibodies against dengue virus has also been discovered. In addition, the combinatorial phage display technology is employed to discover antibodies against human disease antigens, including cytokines and tumor markers on cancer stem cells, from a proprietary library of more than 30 billion unique clones.

The platform’s advanced antibody discovery/engineering technologies have attracted the interest of various biotech and pharmaceutical companies to co-develop and/or license therapeutic antibody candidates. Much interest has also been generated within Singapore clinical community which sees in our antibody technologies a way to address unmet medical needs in the future.

2 72 6

2014

2010

Vivalis Therapeutic antibodies against Chikungunya virus

Cytos Biotechnology VLP-based influenza vaccine

Siena Biotech Development of anti-DKK-1 therapeutic antibodies

Veredus Laboratories Lab-on-chip diagnostic kit for tropical diseases

2013

2009

Servier Mechanism of action of a candidate therapeutic antibody

Servier Identification of HLA alleles associated with drug-related adverse events

BD Dissecting phenotype and function of myeloid immune cell subsets

Sanofi Pasteur Frailty, immunosenescence and vaccination in the elderly

Menarini Biomarkers Singapore Novel procedure for selection of rare fetal cells from maternal blood

Curiox Biosystems Pte Ltd Miniaturization of Luminex assay using DropArray technology

Bioo Scientific Development of a new technology for the sequencing of rare transcripts

Industry Partnerships

2 9

2015

2011 2012

Galderma R&D Immunopathogenesis of acne

Servier Antibodies to breast cancer

L’Oréal Singapore Advanced Research Immune responses in the skin

Servier Natural compounds for dendritic cell immunomodulation

Novartis In vitro and ex vivo evaluation of antimalarial drugs

Sengenics International Pte Ltd Aging, Biology and Computing: the ABC of Health-Span

Janssen Sciences Ireland UC Immunomonitoring of HBV-specific T cells

Mead Johnson & Company, LLC Effect of nutritional components on modulation of allergic asthma

MSD Development of imaging probes using fibronectin protein scaffolds

MSD Deep immunophenotyping of tumor-specific T cells in a mouse model

Nestec and Sanofi Pasteur Aging, nutrition and immunity in Singapore

Servier Novel targets and antibodies in autoimmune diseases

Chugai Pharmaceutical Co Ltd Optimization of a potential therapeutic antibody candidate

Apta Biosciences Functional characterization of Seligo targeted at chemokine receptor

MSD Study on mechanisms of neurodegeneration using in vitro differentiated microglia

Industry Partnerships

2 8

1

Science through ArtWinner of the 2011 Competition

1. A Fusion to Great Perfection Weng Keong Chew, Yilin Wang, Jackson Li, Samantha Chew, Chi Ching Goh, Vanessa Manoharan,

Nadja Bakocevic and Jo Keeble

Add together different experiences and expertise, blend with teamwork, and let the magic begin! Images from our laboratory swirl together to show the beauty of using different scientific techniques and tools to understand the immune system.

2. Perspective Deming Lee, Benjamin Toh, Chrissie Lim, Muly Tham, Michelle Hong and Lu-En Wai

3. Poly-maze Chaotic Reaction Fabien Decaillot

4. Divided Victor de Vries

5. The Angiogenic Tree Irina Shalova, Manesh Chittezhath and Subhra Biswas

6. Look At Me Jia Shee Hee

7. The Stuff Dreams Are Made Of Joe Yeong

8. Breakthrough Benjamin Toh, Deming Lee, Xilei Dai and Jean-Pierre Abastado

9. The Immune Odyssey Preston Teng, Christelle Gabriel and Jason Kam

2 3 4

5

6

8

9

7

3 1

Science through Art

3 0

Finalists of the 2011 Competition

Acknowledgements

Acknowledgements

This commemorative book would not have been possible without the contributions of many people. We would like to take this opportunity to thank all SIgN staff, alumni and partners who have shared their valuable inputs for the realization of this book.

COMMEMORATIVE BOOK COMMITTEE

Cheryl Lee

Kim Ng

Lai Guan Ng

Lisa Ng

Norman Pavelka

Francesca Zolezzi

Copyright © 2016

All rights reserved. No part of this publication may be reproduced or used, in any form or by any means - electronic, mechanical, photocopying, recording or otherwise - without prior written permission of SIgN.

S I N G A P O R E I M M U N O LO G Y N E T W O R K ( S I G N ) 8A Biomedical Grove, Immunos Building, Level 4 Singapore 138648

sign.a-star.edu.sg

Documents

› docs › librariesprovider26 › ... · IMMUNOLOGY NETWORK2019-11-12 · 3 Celebrating 10 years Foreword from Prof Laurent Rénia Executive Director Singapore Immunology Network