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Secondary bacterial pneumonia is a

common complication of influenzavirus infection in humans. But whataccounts for the increased morbidity

that is associated with viralbacterialco-infection? Now, Medzhitov andcolleagues describe a mouse model of

co-infection with influenza virus andLegionella pneumophila that can beused to discriminate between the abil-

ity of the host to detect and eliminatea pathogen (resistance) and the abilityto maintain homeostasis with a given

level of pathogen burden (tolerance).All mice that had been infected

intranasally with a sublethal

dose of influenza virus followed3 days later by a sublethal dose ofL. pneumophila died within a week

of co-infection, whereas all micesurvived a single infection with the

virus or the bacteria. Importantly,

there was no significant differencein the viral or bacterial burden after

single infection or co-infection, andco-infection was not associated withsystemic dissemination of either

pathogen. Furthermore, an attenu-ated strain ofL. pneumophila that isunable to secrete virulence factors

still resulted in 100% mortalityin co-infected mice. So, bacterialgrowth or virulence is not required

for the effects of co-infection, which

confirms that this is not a failure of

immune resistance.Using various mouse strains

with genetic deletions of key immune

molecules, as well as antibody-mediated depletion of neutrophilsor natural killer cells, the authors

showed that co-infection still resultsin mortality in the absence of allmajor immune and inflammatory

pathways that might be triggeredby the virus or bacteria. They sug-gest that these results show that an

excessive inflammatory responseleading to immunopathology is notthe cause of death after co-infection.

Even in the absence of all controllableimmunostimulatory signals whenimmunodeficient (Toll-like receptor 2

(Tlr2)/Tlr4/) mice were infectedwith influenza virus and a severely

attenuated L. pneumophila strain that

lacked flagellin and was unable toreplicate or secrete effectors most

mice died following co-infection butnot after a single infection with eitherthe virus or the bacteria.

So, if resistance is not involved inco-infection-associated mortality in terms of the failure of the immune

response to control pathogen growthand spread, increased pathogen

virulence or the effects of immuno-

pathology the alternative scenario

is that the host is unable to tolerate

the tissue damage that results fromco-infection. The lungs of co-infectedmice had significantly increased

necrosis of airway epithelial cellscompared with singly infected mice,and genes involved in tissue protec-

tion and repair were downregulatedin co-infected mice compared withsingly infected mice. Administration

of amphiregulin an epidermalgrowth factor family member that hasa role in maintaining lung homeosta-

sis during influenza virus infection to Tlr2/Tlr4/ mice that had beeninfected with influenza virus and the

severely attenuated L. pneumophilastrain significantly decreased lungdamage and mortality but had no

effect on viral or bacterial burden.Together, the results show that

tolerance can determine the outcome

of an infection independently ofresistance and is, thus, a bona fide

host defence strategy that could betargeted for therapeutic purposes.

Kirsty Minton

I N F E C T I O N

Resistance is futile

ORIGINAL RESEARCH PAPER Jamieson, A. M. et al.

Role of tissue protection in lethal respiratory viral-

bacterial coinfection. Science 25 Apr 2013

(doi:10.1126/science.1233632)

FURTHER READING Schneider, D. S. & Ayres, J. S.

Two ways to survive infection: what resistance and

tolerance can teach us about treating infectious

diseases. Nature Rev. Immunol.8, 889895 (2008)

tolerance can

determine the

outcome ofan infection

independently

of resistance

and is, thus,

a bona fide

host defence

strategy

NPG

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

NATURE REVIEWS |IMMUNOLOGY VOLUME 13 | JUNE 2013

Nature Reviews Immunology| AOP, published online 7 May 2013; doi:10.1038/nri3462

2013 Macmillan Publishers Limited. All rights reserved


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During an adaptive immuneresponse, T follicular helper (T

FH)

cells enter germinal centres andsupport the affinity maturation,isotype class-switching and memory

responses of antigen-specific B cells.Previous studies have focused on howcognate interactions shape germinal

centre responses; Qi and colleaguesnow report that costimulatory signalsdelivered by bystander B cells are

essential for TFH

cell recruitment tothe germinal centre.

Deficiency of inducible T cell

costimulator (ICOS) leads to defec-tive germinal centre responses.This was thought to be owing to the

inability of ICOS-deficient T cells toupregulate CXC-chemokine recep-tor 5 (CXCR5), which is required for

migration into B cell follicles and,as such, for recruitment to germinal

centres. The authors transduced

ICOS-deficient ovalbumin (OVA)-specific CD4+ T cells (OT-II cells)

with CXCR5 and found that theywere still unable to migrate into thefollicles after immunization with

haptenated OVA. Instead, these cellsaccumulated at the T cellB cell(TB) border at the follicular edge.

ICOS-deficient OT-II cells that weretransduced with the transcriptionfactor B cell lymphoma 6 (BCL-6; a

master regulator of TFH

cell develop-ment) also failed to enter the follicles.

Therefore, ICOS signalling promotesT

FHcell recruitment by a mechanism

that is distinct from CXCR5 orBCL-6 upregulation.

Surprisingly, recruitment of wild-

type OT-II cells was not impairedif OVA-presenting dendritic cellsor OVA-presenting B cells were

defective in ICOS ligand (ICOSL)expression. This suggested that ICOSsignalling promotes T cell entry into

the follicles in an antigen-independentmanner. In support of this theory,T cells that had been activated in vitro

could migrate deep into follicles,even if they were not specific for theimmunizing antigen; however, their

entry was strictly dependent on ICOSexpression. Therefore, the authorsexamined whether ICOS signalling

provided by bystander B cells pro-motes T cell entry into the follicles. In

mice with a B cell-specific deficiency

in ICOSL, activated OT-II cellswere unable to enter the follicles. By

contrast, these T cells were recruitedinto follicles in control mice inwhich B cells expressed ICOSL,

even if the B cells were deficient inMHC class II expression.

Closer analyses showed that

ICOSL-expressing bystanderB cells do not alter chemokinegradient sensing by T cells;

instead, they promote persistentrandom motility in T cells in a

phosphoinositide 3-kinase (PI3K)-dependent manner. Two-photon

intravital imaging showed thatactivated T cells at the TBborder extended frequent pseudo-

pods and maintained a polarizedstate, which is crucial for directionalcell migration. By contrast, in

animals with ICOS-deficient T cellsor ICOSL-deficient B cells, T cellswere depolarized and showed

decreased motility at the follicularborder. Finally, the authors showedthat the lack of expression of ICOSL

by bystander B cells led to defectivegerminal centre responses followingimmunization, even when antigen-

specific T cells and B cells werepresent and competent for ICOS andICOSL expression, respectively.

The authors conclude thatbystander follicular B cells at the TB

border provide an ICOS-dependent

signal to activated CD4+ T cells,thereby promoting their eventual

recruitment to germinal centres.Notably, this study shows thatstimulatory signals that are delivered

to T cells by B cells in an antigen-independent manner also supportgerminal centre responses.

Yvonne Bordon

LYMPHOCYTE RESPONSES

Stand by for action!

ORIGINAL RESEARCH PAPER Xu, H. et al.

Follicular T-helper cell recruitment governed by

bystander B cells and ICOS-driven motility.Nature

25 Apr 2013 (doi: 10.1038/nature12058)

bystander

B cells are

essential

for TFH

cell

recruitment to

the germinal

centre






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As well as detecting pathogenicorganisms, Toll-like receptors (TLRs)

coordinate homeostatic responsesto commensal bacteria. Chambonand colleagues now report that TLR

expression by intestinal epithelialcells (IECs) is under circadian con-trol, leading to oscillatory immune

signalling in response to the micro-biota. Notably, in the absence of themicrobiota the circadian clock is

disrupted in IECs and mice developmetabolic disturbances.

To examine whether commensal

bacteria are involved in circadianresponses, the authors compared tem-poral gene expression profiles in IECs

from control and microbiota-depletedmice. IECs from microbiota-depletedmice showed disrupted circadian

control of several nuclear receptors,including PPAR (peroxisomeproliferator-activated receptor-),

REV-ERB and ROR (retinoic acidreceptor-related orphan receptor-),and of components of the circadian

clock machinery. Notably, depletionof the microbiota also led to systemic

metabolic effects, with animals show-ing increased blood levels of glucose,triglycerides and free fatty acids, and

decreased production of insulin.Further analyses showed that

these metabolic disturbances resulted

from increased corticosterone syn-thesis by ileal IECs, which was causedby increased PPAR expression

in these cells. Indeed, microbiota-depleted mice with an IEC-specificdeletion of PPAR showed normal

regulation of circadian clockcomponents and did not show anymetabolic disturbances. Microbial

products promoted the activationof JUN N-terminal kinase (JNK)and the activator protein 1 (AP-1)

transcription factor JUN in IECs, andJUN was shown to repress PPAR.However, although JNK and JUN

showed a circadian mode of activa-tion, their levels of gene and proteinexpression did not change over time.

Instead, the authors found thatthe expression of TLR1 to TLR5

and also TLR9 (but not TLR6 andTLR7) is under circadian control inIECs. This explains how microbiota-

derived products can activate JNKand JUN and repress PPAR in acircadian manner. The temporal

transcription of TLRs in IECs wasshown to be activated and repressedby the alternate binding of ROR or

REV-ERB, respectively, to a con-served site in the TLR gene promot-ers. Using a bioinformatics approach,

the authors found that a largenumber of genes (>2,000) that areexpressed by IECs contain the ROR

and REV-ERB DNA-binding site.This suggests that many of the genesthat are involved in homeostatic IEC

responses are controlled in a micro-biota-dependent circadian manner.Indeed, the authors confirmed that

several immune mediators that areimportant for maintaining epithelialbarrier integrity are regulated in this

way in IECs.Taken together, the data show

a key role for the microbiota and

TLRs in controlling circadianresponses in IECs. Such circadiancontrol of IEC responses could be

important for coordinating homeo-static IEC functions with behavioural

activities in mice.

Yvonne Bordon

MUCOSAL IMMUNOLOGY

TLRs get rhythm

ORIGINAL RESEARCH PAPER Mukherji, A. et al.

Homeostasis in intestinal epithelium is

orchestrated by the circadian clock and

microbiota cues transduced by TLRs. Cell153,

812827 (2013)

FURTHER READING Sheiermann, C., Kunisaki, Y.

& Frenette, P. S. Circadian control of the immune

system.Nature Rev. Immunol.13, 190198 (2013)

TLR

expression

by intestinal

epithelial cells

... is undercircadian

control

COMSTOCKIMAGES






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It has been previously suggested that

thymus-derived regulatory T (TReg

)cells primarily recognize self anti-gens, whereas peripherally derived

TReg

cells respond to foreign antigens.Indeed, conversion of forkhead boxP3 (FOXP3)CD4+ T cells to FOXP3+

TReg

cells is known to be favoured bythe tolerogenic microenvironmentof the gut, where there is an abun-

dance of innocuous foreign antigens,including food- and microbiota-derived components. However,

Cebula et al. now suggest that mostcolonic T

Regcells, including T

Regcells

that recognize intestinal commensal

bacteria, develop in the thymus.The authors used transgenic mice

that express a limited but diverse

T cell receptor (TCR) repertoire(TCRmini mice) to investigate the

origin of colonic TReg

cells onthe basis of high-throughput TCRsequencing analyses. T cells from

TCRmini mice express a single TCR

-chain combined with a variety of

TCR -chains, and their develop-ment and function was found to becomparable to that of non-transgenic

T cells.Sorted individual FOXP3+ T cells

and FOXP3CD4+ T cells from the

thymus, peripheral lymph nodesand intestines of TCRmini mice wereanalysed in terms of the comple-

mentarity determining region 3(CDR3) sequence of the TCR-chain that they expressed. It was

found that the most abundant TCRsdid not overlap between the FOXP3+and FOXP3CD4+ T cell subsets,

irrespective of the organ of origin.Moreover, 86% of the TCRs from

colonic TReg

cells were identical to

50% of the TCRs from FOXP3+CD4+thymocytes. This suggests that in all

organs that were analysed, includingthe intestines, peripheral conversionof FOXP3CD4+ T cells to FOXP3+

TReg

cells did not account for the

majority of TReg

cells. A similar result

was observed in transgenic mice thathad an even broader TCR repertoire.So, colonic T

Regcells seem to derive

predominantly from FOXP3+CD4+thymocytes.

But do these thymus-derived

intestinal TReg

cells respond to foreignantigens such as those derived fromcommensal bacteria? Treatment of

TCRmini mice with antibiotics alteredthe frequency of the most dominantcolonic T

Regcell TCRs, which prob-

ably reflects the antigen-specificexpansion or contraction of colonicT

Regcell populations. However, the

diversity of the colonic TReg

cell TCRrepertoire was not reduced followingthe changes in the composition

of colonic microbiota. Moreover,most colonic T

Regcell TCRs were

shared with FOXP3+CD4+ thymocytes

even after treatment with antibiotics.This indicates that changes in thecomposition of the population of

intestinal commensal bacteria didnot promote substantial recruitmentof peripherally derived T

Regcells.

Finally, hybridomas that weregenerated from colonic T

Regcells

showed responsiveness to sterile

filtrates of caecal contents and tosonicates of individual commensalbacterial species. As 90% of the

TCRs of hybridomas that respondedto caecal contents were expressedby FOXP3+CD4+ thymocytes, the

authors conclude that thymus-derived T

Regcells have a leading

role in establishing intestinal T cell

tolerance against microbiota.Maria Papatriantafyllou

TOLERANCE

The origins of colonic TReg

cells

ORIGINAL RESEARCH PAPER Cebula, A. et al.

Thymus-derived regulatory T cells contribute to

tolerance to commensal microbiota. Nature497,

258262 (2013)

colonic TReg

cells seem

to derive

predominantly

from

FOXP3+CD4+

thymocytes

Ph

oto

dis

c/G

etty

Im

ages






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A major role of the immune system is to maintainhomeostatic tissue function. For example, sterile tis-sue damage that occurs after trauma needs to bedetected and repaired. Pathogens that can invade andcause harm to tissues should be eliminated while ourcommensal microbiome must be tolerated, as it fulfilsfunctions that are required for host survival. The innateimmune system has a number of signalling receptorsthat recognize foreign molecular structures as well asself molecules that are altered, that have become tooabundant or that emerge in areas normally devoid ofthese molecules1,2.

Innate immune signalling receptors monitor theextracellular space as well as many subcellular com-partments for signs of infection, damage or othercellular stressors. The inflammasomes are a groupof multimeric protein complexes that consist of aninflammasome sensor molecule, the adaptor proteinASC and caspase 1. Inflammasome formation is trig-gered by a range of substances that emerge duringinfections, tissue damage or metabolic imbalances.Once the protein complexes have formed, the inflam-

masomes activate caspase 1, which proteolytically acti-vates the pro-inflammatory cytokines interleukin-1(IL-1)3 and IL-18. In addition, inflammasome acti-

vation causes a rapid, pro-inflammatory form of celldeath called pyroptosis4.

With the discovery of pattern recognition receptors(PRRs), such as inflammasome sensor molecules, theirsignalling pathways and their ability to programme cel-lular immune responses, we are beginning to understandhow the immune system protects the host at a molecularlevel. At the same time, we have learnt that immune dys-regulation contributes to prevalent diseases in Westernsocieties such as atherosclerosis, type 2 diabetes, cancer

and neurodegenerative diseases. Thus, a fine balancemust be maintained between the activation and inhi-bition of inflammation to allow the immune systemto remove any sources of danger without causing harm tothe host.

In this Review, we present an overview of the currentunderstanding of inflammasome activation and regu-lation, and we discuss the recent findings about non-canonical processing of IL-1. We also compare thestructures and the regulation of inflammasomes withthat of the apoptosome.

The inflammasomes and their coreceptors

Inflammasome components. At first glance, the inflam-masomes are organized in a very simple manner: inflam-masome sensor molecules (see below) connect to caspase 1

via ASC, which is an adaptor protein encoded byPYCARDthat is common to all inflammasomes. ASC consists of twodeath-fold domains: one pyrin domain and one caspase acti-

vation and recruitment domain (CARD). ASC interactswith the upstream inflammasome sensor molecules viathe pyrin domain5. This interaction triggers the assem-

bly of ASC into a large protein speck consisting mainlyof multimers of ASC dimers6,7. Using its CARD, ASCbrings monomers of pro-caspase 1 into close proximity,which initiates caspase 1 self-cleavage and the formationof the active heterotetrameric caspase 1. Active caspase 1proteolytically activates a number of proteins8, includingpro-IL-1 and pro-IL-18 (REFS 9,10), and induces theirrelease via a non-classical secretion pathway11.

The transcription of pro-IL-1 is induced by theactivation of the transcription factor nuclear factor-B(NF-B), whereas pro-IL-18 is constitutively expressedand its expression is increased after cellular activation.Therefore, these potent pro-inflammatory cytokines are

1Institute of Innate Immunity,

University Hospital,

University of Bonn,

Bonn 53127, Germany.2Department of Medicine

and Division of Infectious

Diseases and Immunology,

University of Massachusetts

Medical School, Worcester

01655, Massachusetts, USA.3Deutsches Zentrum fr

Neurodegenerative

Erkrankungen (DZNE),

Bonn 53175, Germany.4Centre of Molecular

Inflammation Research,Norwegian University of

Science and Technology,

Trondheim NO-7491,

Norway.5Structural Immunobiology

Unit, Laboratory of

Immunology, National

Institute of Allergy and

Infectious Diseases,

National Institutes of Health,

Bethesda, Bethesda 20892

Maryland, USA.

Correspondence to E.L.

e-mail: [email protected]

doi:10.1038/nri3452

ASC

An adaptor protein that was

originally found to form protein

precipitates in apoptotic cells

that are termed protein specks.

Activation and regulation of theinflammasomesEicke Latz14, T. Sam Xiao5 and Andrea Stutz1

Abstract | Inflammasomes are key signalling platforms that detect pathogenic microorganisms

and sterile stressors, and that activate the highly pro-inflammatory cytokines interleukin-1

(IL-1) and IL-18. In this Review, we discuss the complex regulatory mechanisms that facilitate

a balanced but effective inflammasome-mediated immune response, and we highlight the

similarities to another molecular signalling platform the apoptosome that monitorscellular health. Extracellular regulatory mechanisms are discussed, as well as the intracellular

control of inflammasome assembly, for example, via ion fluxes, free radicals and autophagy.

REVIEWS

NATURE REVIEWS |IMMUNOLOGY VOLUME 13 | JUNE 2013 |397

mailto:eicke.latz%40uni-bonn.de?subject=mailto:eicke.latz%40uni-bonn.de?subject=


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IFNAR

Type I IFN

TRIF

TNFR familymembersuch as CD95

Dectin 1

Mitochondrion

Cytoplasm

TLR4

Bacterium

BacterialmRNA

Caspase 11

?

Caspase 1

Pro-IL-1

NLRP3

ASC

IL-1

Caspase 8

Ripoptosome

Fusion

?

?

?

Non-canonical NLRP3inflammasome in responseto Gram-negative bacteria

Non-canonicalcaspase 8inflammasome

Auto-processing?

Phagosome

Lysosome

Actinmobilization

Negativefeedback

Degradation Sequestration

FADD

RIP1

XIAP

IAP1

RIP3

Canonical NLRP3inflammasome

Ripoptosome-induced IL-1processing

TNFR-inducedIL-1 processing

IL-1 secretion Pyroptosis

Pro-IL-18 IL-18

Pro-caspase 11

Type IIFN

IFN-induciblefactor

Nucleus

SYK

ASCMALT1

IAP2

PRR

?

?

?

?

?

formed, under which circumstances caspase 11 activa-tion will lead to NLRP3 activation and when it will causecell death independently of the canonical inflamma-somes. A recent review discusses caspase 11 activationin more detail48.

Noninflammasome processing of IL1The inflammasomes and IL1 processingin vitro andin vivo. Studies that have been carried out in vitro usingcells that are deficient in inflammasome componentshave undoubtedly been instrumental in identifying

several inflammatory triggers as activators of inflam-masomes. However, the contribution of inflammas-omes to IL-1 activation in vivo has not, at times, beennearly as convincing. The reduction in inflammationin mice that are deficient in the inflammasome compo-nents ASC or caspase 1 can be much less pronouncedthan that in mice lacking the IL-1 receptor (IL-1R)or IL-1. It is probable that inflammasome-independentIL-1 activation mechanisms are involved in vivo thatcould account for the observed differences betweenthe in vitro and in vivo studies. For example, the in vivo

Figure 1 | Canonical and non-canonical activation of IL-1.NLRP3 (NOD-, LRR- and pyrin domain-containing 3) needsadditional cofactors for the processing of interleukin-1 (IL-1)in response to Gram-negative bacteria; this pathway has been

termed the non-canonical NLRP3 inflammasome. Toll-like receptor 4 (TLR4) signalling via TIR domain-containing adaptorprotein inducing IFN (TRIF) induces the secretion of type I interferons (IFNs), which lead to the activation of caspase 11 viaautocrine signalling through the IFN/ receptor (IFNAR), possibly involving additional (not yet known) IFN-inducible factors.Caspase 11 is necessary for the activation of caspase 1 and has an independent role in IL-1 secretion and pyroptosis inresponse to Gram-negative bacteria. A possible mechanism for caspase 11 action might be its role in actin mobilization via

cofilin, which might lead to increased fusion of lysosomes to phagosomes and, potentially, to increased leakage of bacterial

mRNA (also termed vita-PAMPs) to the cytoplasm to activate NLRP3 (not shown). A platform consisting of mucosa-associated

lymphoid tissue lymphoma translocation protein 1 (MALT1), caspase 8 and the adaptor protein ASC (termed the

non-canonical caspase 8 inflammasome) is formed in response to stimulation of dectin 1. Caspase 8 might also be activated

by TLRs using the signalling adaptor TRIF in the presence of cycloheximide. In addition, the formation of the ripoptosome is

triggered by the loss of inhibitor of apoptosis proteins (IAPs) with concurrent TLR stimulation. The ripoptosome consisting

of FAS-associated death domain protein (FADD) and receptor-interacting protein 1 (RIP1) activates caspase 8 via RIP3.

However, caspase 8 can also limit ripoptosome action on NLRP3. Members of the tumour necrosis factor receptor (TNFR)

family might also induce pro-IL-1 cleavage, as has been shown for CD95 (also known as FAS). Question marks showpathways that are still speculative. PRR, pattern recognition receptor; XIAP, X-linked inhibitor of apoptosis protein.

R E V I E W S

400 | JUNE 2013 | VOLUME 13 www.nature.com/reviews/immunol



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Cytoplasm

Nucleus

Activatory Inhibitory

IFNGR IFNAR

Type I IFN IL-1R PRR PRR

?

?

CD40

CD40L

TNFR

IFNAR

Type I IFNIFN

IFNGR

IFN

Priming viaexpressionlevels

Degradation

? ?

?

iNOS

PKC

NLRP3inflammasome

Nitrosylation

Nitrosylation

NLRP3 mRNA

miR-223

NLRP3

pro-IL-1

AIM2

NLRC4 NO

P

Caspase 1

T cell

Priming viadeubiquitylation

ROS

Ub

NLRP3

BRCC3

Pro-IL-1 IL-1

Amyloid-

An endogenous peptide that is

generated by proteases in the

brain. It is prone to aggregation

and plaque formation.

Amyloid- plaques are a

hallmark of Alzheimers disease

and can activate the NLRP3

(NOD-, LRR- and pyrindomain-containing 3)

inflammasome.

Cryopyrin-associated

periodic syndrome

(CAPS). Characterized by

NLRP3 (NOD-, LRR- and pyrin

domain-containing 3)

inflammasome hyperactivity

and the excessive release of

interleukin-1, which leads to

an autoinflammatory disease

phenotype with periodic fever

episodes, urticaria and often

severe arthritis.

byPRKCD), which depends on the recognition of bothflagellin and the type III secretion system. This indi-cates that PKC might be activated by PRR signalling67.Therefore, at least some inflammasomes are only fullycapable of responding to danger signals in situations in

which either pro-inflammatory cytokines are present orinnate signalling molecules sense noxious signals.

A few molecules, such as amyloid-, can induce bothNLRP3 priming through TLR activation and NLRP3inflammasome activation68. However, the response toaggregated amyloid- can be dramatically augmentedwhen innate sensors or cytokine receptors become acti-

vated by additional stimuli. These priming mechanismsmight be of great relevance in determining the magnitudeof an inflammatory response to danger signals.

Conversely, genetic differences that influence thethreshold of inflammasome activation might con-tribute to the development of chronic inflammatory

or autoinflammatory diseases. It is known that about60% of patients with cryopyrin-associated periodicsyndrome (CAPS) carry activating mutations in thecoding sequence ofNLRP3 itself or in other inflamma-some components69. This indicates that inflammasome

hyperactivity may be influenced by additional mecha-nisms. Indeed, single nucleotide polymorphisms in thepromoter region ofNLRP3 were found in patients withCAPS69. In these patients, an increase in NLRP3 expres-sion as opposed to the presence of a more active pro-tein (as a result of mutations in the coding sequence)could conceivably result in a lower threshold of NLRP3inflammasome activation in response to danger sig-nals and this could trigger the development of CAPS.Therefore, several mechanisms ensure that inflammas-omes are not accidentally triggered and that an appropri-ately balanced immune response to danger signals canoccur in a regulated manner.

Figure 2 | Extracellular signals regulate the inflammasomes. Pro-interleukin-1 (pro-IL-1) and NLRP3 (NOD-, LRR-and pyrin domain-containing 3) expression are induced by transcriptionally active pattern recognition receptors (PRRs)

or by cytokine receptors. Furthermore, NLRP3 deubiquitylation by the K63-specific deubiquitinase BRCC3 is crucial for its

activation. Direct contact with mature or memory T cells inhibits the inflammasomes, probably via tumour necrosis factorreceptor (TNFR) superfamily interactions. Type I interferons (IFNs) inhibit the transcription of proIL1, but also upregulatethe expression of absent in melanoma 2 (AIM2). Both type I IFNs and IFN inhibit NLRP3 through the induction of nitricoxide (NO) via inducible nitric oxide synthase (iNOS), possibly with the requirement of concomitant priming by PRRs.

Ub shows ubiquitylated proteins. Question marks show pathways that are still speculative. CD40L, CD40 ligand; IFNAR,

interferon-/receptor; IFNGR, interferon- receptor; IL-1R, IL-1 receptor; miR-223, microRNA-223; NLRC4, NOD-,LRR- and CARD-containing 4; ROS, reactive oxygen species; PKC, protein kinase C.

R E V I E W S




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through the hemichannel pannexin 1 (REFS 8284); how-ever, pannexin 1-deficient mice do not show diminishedcaspase 1 activation85. Thus, the mechanisms by whichseveral inflammasome triggers can induce K+ effluxremain unclear.

In addition to the requirement for decreased K+ lev-els, osmotic pressure regulates NLRP3 inflammasomeactivation86. Cells that have been subjected to hypotonicsolutions undergo cellular swelling concomitant witha decrease in intracellular K+ and Cl concentrations.Interestingly, cell swelling induces a regulatory volumedecrease response through transient receptor poten-tial cation channels (TRPM7 and TRPV2) that triggersintracellular Ca2+ mobilization. The mobilized Ca2+has many molecular targets, including TGF-activatedkinase 1 (TAK1; also known as MAP3K7) (REF. 86).Although TAK1 might have a role in a PRR-dependentnon-transcriptional priming pathway87 that is, in thesignalling pathway that leads to the deubiquitylation, forexample, of NLRP3 further investigation into its roleis required. Another transient receptor potential channel

TRPM2 has also been implicated in NLRP3 activa-tion in response to crystalline substances88. This channelsenses intracellular ROS and responds by opening itselfto facilitate Ca2+ influx into the cell; this is intriguing con-sidering that both ion fluxes and the oxidative state (seebelow) have important roles in NLRP3 inflammasomeactivation.

Another regulator of Ca2+, namely C/EPB-homologousprotein (CHOP; also known as DDIT3), has been impli-cated in NLRP3 inflammasome activation89. However,the activation of CHOP following the induction of theunfolded protein response via inhibition of translation isinsufficient for NLRP3 inflammasome activation90 and,therefore, the exact role of CHOP in NLRP3 inflam-masome activation is unclear. Two recent studies haveimplicated calcium-sensing G-protein coupled receptors(GPCRs) in the activation of NLRP3: calcium-sensingreceptor (CASR) and GPRC6A signal via G

iand G

q,

which inhibit adenylyl cyclase and activate phospho-lipase C, respectively91,92. Thus, sensing of extracellularCa2+ leads to reduced cyclic AMP (cAMP) levels throughadenylyl cyclase inhibition and to increased cytoplasmicCa2+ concentrations via the activation of phospholipase C.It is worth noting that the GPCR platelet activating factorreceptor can also stimulate inflammasome formation92,which indicates that other GPCR pathways could controlinflammasome activation. The role of cAMP in inflamma-

some activation remains unclear as one study has reportedthat cAMP can directly inhibit NLRP3 (REF. 91), whereasanother study reported that cAMP levels had no directinfluence on inflammasome activation92. Taken together,these studies show that NLRP3 activation is regulated bythe ion content of the cell (FIG. 3); however, the moleculartargets and the mechanisms by which ion fluxes regulateNLRP3 remain to be fully elucidated.

The redox state of the cell is another important indi-cator of the viability of cells, and many signalling path-ways are influenced by changes in the redox state. Inparticular, ROS facilitate the assembly of the apoptosomein several ways. For example, oxidative modifications to

a b

c90

90

90

Cytochrome c

dsDNA

Pro-caspase 1

Pro-caspase 9

ASC

CARDof APAF1

APAF1

AIM2

Apoptosome

Ligand-binding domain WD40 repeats HIN

dsDNA

PYD

NACHT

CARD

Caspase 9 Caspase 1

Oligomerization domain

Signalling domain

Catalytic domain

AIM2 inflammasome

Apoptosome AIM2 inflammasome

Box 2 | Oligomerization of deathinducing molecular platforms

Receptors of the death-inducing molecular platforms are activated by ligands such

as cytochrome c for the apoptosome component apoptotic protease-activating

factor 1 (APAF1), and double-stranded DNA (dsDNA) for the absent in melanoma 2

(AIM2) inflammasome. By contrast, the NLRP1 (NOD-, LRR- and pyrin

domain-containing 1) inflammasome can be activated by self-proteolysis in its

function-to-find (FIIND) domain131133, which might be relevant to its proteolytic

activation by the anthrax lethal toxin136

. The cleaved fragments remain associatedwith each other but the proteolytic event enables NLRP1 to recruit the adaptor

protein ASC and caspase 1. For the other receptors, ligand binding is thought to

induce conformational changes, promoting oligomerization of the receptors into

signalling platforms that recruit adaptors and activate effector caspases.

Oligomerization of APAF1 and NOD-like receptors (NLRs) is mediated by their

NACHT domains, which bind and hydrolyse deoxyadenosine triphosphate (dATP)

or ATP146,147. However, AIM2 seems to use the multivalent ligand dsDNA as its

oligomerization platform130. Both receptor oligomerization and ligand binding

might be modulated by the regulatory domains of the receptors, including the

WD40 repeats of APAF1, the HIN domain of AIM2 and the leucine-rich repeats

(LRRs) of the NLRs. Two examples of the death-inducing platforms are shown

in the box figure.

The apoptosome consists of seven APAF1 molecules that are activated by

cytochrome c; this then recruits pro-caspase 9 molecules137,148,149 (see figure, part a).

Six- or eight-fold symmetry of the apoptosomes has also been observed, whichsuggests that there are variable oligomerization states137,148,149. Two hypothetical

models of the AIM2 inflammasome highlight the positioning of the activating DNA

either at the centre (see figure, part b) or the periphery (see figure, part c) of the

complex. It is possible that the physiological AIM2 inflammasome may contain

characteristics of both models, and the oligomerization states can be variable owing

to the sequence-independent nature of the DNA-binding. For clarity, only one

caspase dimer is shown for each oligomeric platform. According to a recent model,

the pyrin domain (PYD) of ASC can interact with itself in a helical manner to form

filamentous structures5, which might mediate ASC pyroptosome formation 6.

CARD, caspase activation and recruitment domain.

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Regulation by interacting host proteins. In addition togeneral cellular conditions such as ion concentrationsand the nutritional state, specific regulatory proteinshave evolved that regulate inf lammasome formation(TABLE 1). The formation of the inflammasome can

be controlled both at the level of inflammasome sen-sor molecules and, further downstream, at the level ofASC and caspase 1 interaction. As mentioned above,inflammasomes form large protein aggregates andthere are two levels at which death-fold domains areused to recruit effector inflammasome components.First, ASC is recruited to the inflammasome receptorsby homotypic pyrin domain interactions. Next, in a sec-ond death-fold domain interaction, ASC binds to andactivates pro-caspase 1 via its CARD. Studies have eluci-dated inflammasome regulation at the level of death-folddomain interactions and regulatory proteins.

Caspase activity during cell death is regulated byCARD-containing proteins such as CARD8, whichhas also been shown to be a binding partner of NLRP3(REF. 108). CARD8 has multiple functions in regulat-ing apoptosis, one of which is to directly bind to pro-caspase 9 and to suppress its activation109. Simi larly,mouse caspase 12, which is a paralogue of caspase 1,interacts with caspase 1 to reduce its activity110. A poly-morphism in the caspase 12 (CASP12) gene in humansleads to either a truncated protein or to a full-lengthprotein (caspase 12L)111. Similarly to mouse caspase 12,caspase 12L reduces cytokine production in responseto LPS111, which at high doses can activate theNLRP3 inflammasome and thus caspase 1 without theneed for a second signal. Therefore, caspase 12L and

mouse caspase 12 probably function as decoy proteinsfor caspase 1, thereby limiting its activation. CARD-only proteins (COPs) can also inhibit caspase 1 acti-

vation112. CARD18 (also known as ICEBERG) andCARD16 (also known as pseudo-ICE and COP1)were the first decoy COPs to be described113. Throughtheir CARDCARD interaction, these two inhibitorssequester pro-caspase 1 and inhibit its activation by theinflammasome. CARD17 (also known as INCA), whichis another decoy protein, is upregulated by IFN to sup-press IL-1 generation114. Thus, proteins containing aCARD can sequester caspase 1, thereby blocking theformation of a functional inflammasome complex.

An additional level of fine-tuning and regulationof the inflammasomes can be achieved by interferingwith the pyrinpyrin interaction in the inflamma-somes. Several pyrin domain-only proteins (POPs)and other pyrin-containing proteins have been char-

acterized. POP1 (also known as PYDC1 and ASC2)and POP2 (also known as PYDC2) inhibit the inter-actions between the inflammasome sensor moleculesand ASC115117. However, COPs and POPs have not beenidentified in the mouse genome, which indicates thata more complex regulatory system might be presentin humans. The fact that COPs and POPs have notbeen identified in the mouse genome makes the studyof these proteins difficult, and many of the COPs andPOPs have only been investigated using overexpres-sion studies and not using genetic deletion or RNAinterference.

Alternative splicing of ASC can provide an additionallevel of regulation by giving rise to ASC variants thatnegatively regulate the assembly of the inflamma-somes118. Furthermore, NLRP10 (also known as PYNOD)was suggested to negatively regulate inflammasomes bysequestering ASC119,120, but deletion ofNlrp10 in micedid not confirm this hypothesis121,122. NLRP7 has beensuggested to be a negative regulator of inflammasomes;however, more recent findings indicate that NLRP7might assemble an inflammasome in response tomicrobial acylated lipopeptides28,123.

The role of pyrin, which is a protein that is encodedbyMEFV (Mediterranean fever gene), in inflam-masome regulation also remains unclear. One studyfound that Mefv deletion in mice lead to increased

IL-1 release without influencing caspase 1 activityor inflammasome assembly. These findings suggestedthat pyrin can inhibit IL-1 release downstream of theinflammasomes124. However, another study found thatshort-term IL-1 release was not impaired after Mefvgene deletion and that IL-1 was only inhibited afterseveral days of stimulation125. In addition, mice carry-ingMefvmutations that have been identified in patientswith familial Mediterranean fever (FMF) showed ASC-dependent but NLRP3-independent release of IL-1125.Therefore, whether pyrin is an inhibitor at the level ofIL-1 release or whether it forms a pyrin inflammasomeremains to be determined.

Table 1 (cont.) | Regulatory proteins of the inflammasomes

Regulatoryprotein

Inflammasomecomponenttargeted

Mechanism of action Source of experimental evidence Refs

Other regulators (cont.)

HSP90 NLRP3 Necessary for activation, possibly owing to its chaperonefunction

Biochemical evidence and inhibitor ofHSP90

129

SGT1 NLRP3 Necessary for activation, possibly owing to its chaperonefunction

RNAi and biochemical evidence 129

AIM2, absent in melanoma 2; BCL, B cell lymphoma; CARD, caspase activation and recruitment domain; FMF, familial Mediterranean fever; GBP5,guanylate-binding protein 5; HSP90, heat shock protein 90; IAP, inhibitor of apoptosis protein; IL-1, interleukin-1; NAIP, NLR family, apoptosis inhibitory protein;NLR, NOD-like receptor; NLRC4, NOD-, LRR- and CARD-containing 4; NLRP, NOD-, LRR- and pyrin domain-containing; NOD2, nucleotide-binding oligomerizationdomain-containing protein 2; PKC, protein kinase C; PKR, protein kinase R; POP, pyrin domain-only protein; PYD, pyrin domain; RNAi, RNA interference; XIAP,X-linked IAP. *Expressed only in mice. Expressed only in humans. Two contradictory studies have been published about this interactor.

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149. Qi, S. et al. Crystal structure of the Caenorhabditis

elegans apoptosome reveals an octameric assembly

of CED-4. Cell141, 446457 (2010).

150. Jha, S. et al. The inflammasome sensor, NLRP3,

regulates CNS inflammation and demyelination via

caspase-1 and interleukin-18.J. Neurosci. 30,

1581115820 (2010).

151. Inoue, M. et al. Interferon- therapy against EAE iseffective only when development of the disease

depends on the NLRP3 inflammasome. Sci. Signal.

5,ra38 (2012).152. Lamkanfi, M. et al. Glyburide inhibits the cryopyrin/

Nalp3 inflammasome.J. Cell Biol. 187, 6170 (2009).

153. Juliana, C. et al. Anti-inflammatory compounds

parthenolide and Bay 117082 are direct inhibitors of

the inflammasome.J. Biol. Chem. 285, 97929802

(2010).

AcknowledgementsThe authors would like to thank C. M. De Nardo and B. G.

Monks for critical reading of the manuscript. This work was

supported by grants from the US National Institutes of

Health (NIH) and the Deutsche Forschungsgemeinschaft

(DFG), Germany (to E.L.), and by the Division of Intramural

Research, National Institute of Allergy and Infectious

Diseases, NIH, USA (to T.S.X.). E.L. is a member of the

excellence cluster ImmunoSensation in Bonn, Germany.

Competing interests statementThe authors declare no competing financial interests.

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on the basis of their surface phenotype. Consequently, inboth humans and mice810,1417, T

FHcells are considered to

be CD4+ T cells that express the highest levels of CXCR5,together with the surface receptors inducible T cell costimulator (ICOS) and programmed cell death protein 1(PD1; also known as PDCD1), the transcriptional repressor B cell lymphoma 6 (BCL6) and the cytokine IL21(BOX 3), and that have downregulated the T cell zonehoming receptor CCchemokine receptor 7 (CCR7) andIL7 receptor (IL7R) (TABLE 1).

This phenotypic approach to defining TFH

cells hasfacilitated their molecular and cellular characterization.However, it needs to be appreciated that, just as B cellsundergo important differentiation events at the T cellB cell border (such as the formation of extrafollicular

plasmablasts) and in GCs (such as the formation ofmemory and plasma cells), CD4+ T cells can differentiate

into TFH cell subsets that are strategically located at theseregions to facilitate distinct phases of a T celldependentB cell response6,7,18,19. These T

FHcell subsets probably pro

vide early B cell help at the T cellB cell border, and/orthey represent precursor cells that differentiate into GCT

FHcells following receipt of appropriate signals from

inside the active B cell follicle and that guide the differentiation of GC B cells into memory or plasma cells.In this Review, we discuss recent developments in theinvestigation of the mechanisms that underlie T

FHcell

development and function, the discovery of specializedsubsets of T

FHcells and how perturbations to T

FHcells

potentially contribute to numerous human diseases.

Table 1 | Effector subsets of CD4+ T cells: ontogenic and functional requirements, and roles in disease

CD4+T cell

subset

Inducingcytokines

ActivatedSTATs

Transcriptionfactors

Suppressingcytokines

Canonicalcytokinesproduced

Roles in hostprotection

Associatedpathologies

Refs

TH1 cells IL-12

IFNSTAT4STAT1

T-bet IL-4 and IL-10 IFN Antiviral andantimicrobialimmunity

Cell-mediatedimmunity

Mendeliansusceptibility tomycobacterialdisease (decrease inT

H1 cells)

Multiple sclerosis(increase in T

H1 cells)

1,121,122

TH2 cells IL-4 STAT6 GATA3 and

MAFIFN IL-4, IL-5

and IL-13Immunity to

extracellular parasitesAllergy, asthma or

eczema (increase inT

H2 cells)

1,123

TH17 cells IL-23 and

IL-1IL-6 and

IL-1TGF

STAT3 RORt andROR

IL-4, IFN,IL-27 andIL-2

TGF(suppressesIL-22expression)

IL-17A,IL-17F,IL-21, IL-22and IL-26*

Protection atmucocutaneous sites

Antimicrobialimmunity (forexample, againstCandida spp. andStaphylococcusspp.)

Inflammatory boweldisease

Inflammatory boweldisease (increase inT

H17 cells)

Susceptibility tofungal infections(decrease in T

H17

cells)

1,121,122,124

TH9 cells TGF

IL-4STAT6 PU-1 and

IRF4IFN andIL-27

IL-9 Protection againsthelminth infections

Allergy (atopicdermatitis) andasthma (increase inT

H9 cells)

125

TH22 cells TNF

IL-6STAT1STAT3STAT5

RORt andAHR

High doses ofTGF

IL-22 Barrier immunity(skin, intestines andairways)

Enhancement ofinnate immunity

Tissue regeneration

Allergy (atopicdermatitis) (increasein T

H22 cells)

Inflammation atjoints and barriers(increase in T

H22

cells in mice)

126

TReg

cells TGF andIL-2

STAT5 FOXP3 IL-6 TGF andIL-10

Immune suppression IPEX syndrome(decrease in T

Reg

cells)

75

TFHcells IL-6, IL-21and/or IL-27IL-12

STAT3STAT4STAT1

BCL-6,IRF4, MAF andBATF

IL-2 and IL-10 IL-21, IL-4and IL-10 Help for B cellactivation ordifferentiation

Generation oflong-lived antibodyresponses

Humoralimmunodeficiency(decrease in T

FHcells)

Autoimmunity(increase in T

FHcells)

T cell lymphoma

(increase in TFH

cells)(see TABLE 2)

2325,45

AHR, aryl hydrocarbon receptor; BATF, basic leukine zipper transcriptional factor ATF-like; BCL-6, B cell lymphoma 6; FOXP3, forkhead box P3; GATA3,GATA-binding protein 3; IFN, interferon-; IL, interleukin; IPEX, immunodysregulation, polyendocrinopathy and enteropathy X-linked; IRF4, interferon-regulatoryfactor 4; ROR, retinoid-related orphan receptor; STAT, signal transducer and activator of transcription; TGF, transforming growth factor-; T

FH, T follicular helper;

TH, T helper; TNF, tumour necrosis factor; T

Reg, T regulatory. *Human-specific cytokine. Reported in mice.

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B cell lymphoma 6

(BCL-6). A transcriptional

repressor identified as beingcrucial for the formation of

T follicular helper (TFH

) cells.

Several mechanisms have been

proposed for the role of BCL-6

in TFH

cell commitment,

including suppression of the

expression of transcription

factors that are required for the

generation of alternative TH

fates, suppression of

microRNAs and cooperation

with other transcriptional

regulators to induce the

expression of important TFH

cell-related genes.

Requirements for TFH

cell formation

TFH

cell differentiation requires input from several surfacereceptors (including CD28, ICOS, CD40 ligand (CD40L)and signalling lymphocytic activation molecule (SLAM)family members), as well as from cytokines and theirassociated signalling pathways (for example, signal transducer and activator of transcription 3 (STAT3) or SLAMassociated protein (SAP; also known as SH2D1A)), whichall culminate in the induction of BCL6, an important

regulator of the TFH cell lineage2022. However, additionaltranscription factors (including interferonregulatoryfactor 4 (IRF4), basic leucine zipper transcriptional factorATFlike (BATF) and MAF) and microRNAs also haveimportant regulatory functions during T

FHcell develop

ment2225 (FIG. 1; TABLE 1; Supplementary information S1(table)). The requirements for T

FHcell formation have

been extensively reviewed in the literature2326 and aresummarized in Supplementary information S1 (table). Inthis Review, we discuss the most recent studies that haveidentified pathways that positively and negatively influence T

FHcell generation and function, and that clarify

previous inconsistencies.

The TNF receptor superfamily. B cellactivating factor(BAFF; also known as TNFSF13B) and a proliferationinducing ligand (APRIL; also known as TNFSF13) areligands of the tumour necrosis factor (TNF) superfamilythat regulate B cell survival and differentiation11. Bothligands bind to the common receptors transmembraneactivator and CAML interactor (TACI; also known asTNFRSF13B) and B cell maturation antigen (BCMA;also known as TNFRSF17), and BAFF also binds to the

BAFF receptor (BAFFR; also known as TNFRSF13C);receptors which are predominantly expressed onB cells11. NFBinducing kinase (NIK; also known asMAP3K14) is a component of the BAFFR signallingpathway11. Expression of NIK and BAFFR by B cells wasfound to be required for their constitutive expression ofICOS ligand (ICOSL). The significance of this is thatNIK deficiency in B cells compromises T

FHcell induc

tion27. Thus, sustained BAFFBAFFRNIK signalling inB cells maintains ICOSL expression, thereby facilitatingcognate ICOSICOSL interactions between activatedCD4+ T cells and B cells, which result in optimal T

FHcell

differentiation.

Box 1 | TFH

cells and affinity maturation of GC B cells

Affinitymaturationoftheantibody

responseisbasedontheselective

perpetuation(knownaspositive

selection)ofgerminalcentre(GC)B cells

thathaveacquiredincreasedaffinityfor

foreignantigensthroughsomatic

hypermutation(SHM)oftheirimmunoglobulinvariable-regiongenes.

WhereasSHMitselftakesplaceinB cells

thatoccupytheGCdarkzone,

high-affinityGCB cellsonlyundergo

positiveselectionafterreturningtothe

lightzonewheretheygainpreferential

accesstoforeignantigensthatare

expressedonthesurfaceoffollicular

dendriticcells(seethefigure).However,

theprecisemechanismsbywhich

preferentialaccesstoantigenstranslates

intopositiveselectionofhigh-affinityGC

B cellshavenotbeenclearlyestablished.

ThespecificprovisionofTfollicular

helper(TFH)cell-derivedhelpersignalstohigh-affinityGCB cellsisproposedtobe

oneof,ifnottheonly,majordriversof

antibodyaffinitymaturation12,13.In

theory,thegreaterpropensityof

high-affinityGCB cellstoaccessforeign

antigensshouldaugmenttheirabilityto

internalize,processandpresentforeignpeptidestotheTFH

cellsthatresideinthelightzoneofGCs(seethefigure).

However,althoughitisclearthatTFH

cellsarerequiredtosupporttheGCresponse,ithasbeendifficulttoestablishtheir

preciseroleindrivingaffinitymaturation.Inarecentstudy,aGCresponsewasmanipulatedinmicesuchthatantigen

presentationbyGCB cellstoTFH

cellswasdecoupledfromB cellreceptor(BCR)-mediatedantigenrecognition.This

resultedinthedeliveryofTFH

cellhelptoallGCB cellsandtheproliferativeexpansionofthepopulationofTFH

cells

regardlessoftheiraffinityforantigen12.ThisresultdoesnotprovethatpreferentialdeliveryofTFH

cellhelpisthe

fundamentaldriverofaffinitymaturationbutitclearlydemonstratesthatTFH

cellshavethepotentialtoperformthis

roleifhigh-affinityGCB cellsareindeedsuperioratpresentingantigentoTFH

cells.

CD40L,CD40ligand;CR2,complementreceptor2;FcRII,lowaffinityFcreceptorforimmunoglobulin;Ig,immunoglobulin;

TCR,Tcellreceptor.

Folliculardendritic cell

Germinal centre

BCR

Cell death

Affinity-maturedantibodies

High affinity

B cell TFH cell

BCR

CR2 or FcRII

Ig

Foreign antigen

CD40LCD40

Adhesionmolecules

TCR

MHC class IIpeptide complex

B cell

Low affinity

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SH2 domaincontaining

protein tyrosine

phosphatase 1

(SHP1). A protein tyrosine

phosphatase that is involved in

suppressing intracellular

signals delivered via numerous

activating receptors, including

T cell and B cell antigen

receptors, as well as members

of the signalling lymphocytic

activation molecule (SLAM)

family of surface receptors.

One proposed mechanism of

action is the direct or indirect

dephosphorylation of

components of the T cell

receptor signalling pathway,

such as CD3, LCK,

-chain-associated protein

kinase of 70 kDa (ZAP70) and

phosphoinositide 3-kinase.

Follicular T regulatory cells

A subset of T regulatory (TReg

)

cells that co-opts the

transcriptional machinery of

T follicular helper (TFH

) cells

to facilitate their migration to

germinal centres, where they

can appropriately restrain

humoral immune responses,thereby potentially preventing

overzealous antibody

responses. Follicular TReg

cells

can be identified by the

expression of typical

TFH

cell surface markers

(CXC-chemokine receptor 5

(CXCR5), inducible T cell

co-stimulator (ICOS) and

programmed cell death

protein 1 (PD1)) along with

the TReg

transcription factor

forkhead box P3. Their

mechanism of action remains

to be completely elucidated.

SAP) have poor humoral immunity31. A role for CD84 inSAPdependent T

FHcell generation following immuniza

tion with protein antigen was recently demonstrated33.However, the T

FHcell deficiency in Cd84/ mice was

less severe than in SAPdeficient mice33, and TFH

cellformation following viral infection was unaffected bythe absence of CD84 (REF. 35).

Analysis of genetargeted mice has failed to showa requirement for SLAM family receptors other thanCD84 in T

FHcell formation (reviewed in REF. 23),

although SLAM has been shown to be required for IL4expression by GC T

FHcells18. One interpretation of these

observations is that the severe effect of SAP deficiency inT

FHcells reflects a requirement for numerous SLAM

receptors during the differentiation of TFH

cells.Alternatively, as SLAM receptors can also recruitinhibitors of signalling (such as lipid and tyrosine phosphatases), SAP deficiency might exacerbate negativesignals that are delivered through one or more of theSLAM receptors. Consistent with this idea, loss of LY108reversed the inability of SAPdeficient CD4+ T cells toform T

FHcells and to support B cell responses35. This

was due to a reduced recruitment ofSH2 domain-containingprotein tyrosine phosphatase 1 (SHP1; also known asPTPN6) to the immune synapse by LY108 (REF. 35). Thus,LY108 functions as a rheostat that is capable of deliveringpositive SAPdependent and negative SHP1dependent

signals that dynamically regulate TFH cells (FIG. 1).

The role of PD1. An important phenotypic determinant of T

FHcells is their high expression of PD1

(REF. 14). PD1 has an inhibitory role in TFH

cell differentiation, as mice with impaired PD1 function have moreT

FHcells (CCR7lowICOShi cells in Pd1/ mice) as a result

of increased proliferation and reduced apoptosis3639.Expression of PD1 ligand 1 (PDL1; also known asCD274), rather than PDL2, on B cells constrains T

FHcell

formation via the PD1 pathway38(FIG. 1). Although thesestudies showed that ablating PD1 signalling increasedT

FHcell numbers, they yielded conflicting results about

how this affected the outcome of the GC response3639.For instance, some groups reported increased antigenspecific antibody responses in PDL1deficient mice38or in Plasmodiuminfected mice that had been treatedwith a PDL1specific mAb39. These findings in miceare consistent with data demonstrating that ligation ofPD1 suppresses the proliferation, activation and function of human T

FHcells in vitro40. However, other groups

have reported impaired plasma cell and GC responsesin the absence of PD1 signalling 36,37. These differencesmight reflect nuances in the experimental systems thathave been used, but they might also result from distinct functions of PD1 in the development and functionof not only T

FHcells but also follicular T regulatory cells

(follicular TReg

cells), which express higher levels of PD1than T

FHcells41.

Cytokines. Numerous cytokines have been shownto be important for T

FHcell generation in vivo and

in vitro. The initial cytokines that were identified toinduce T

FHcelllike features in cultured CD4+ T cells

were IL6 and IL21 (TABLE 1). However, subsequentanalyses of IL6 and IL21 or IL21Rdeficient miceyielded conflicting results regarding the necessity ofthese cytokines in regulating T

FHcell formation in vivo

(reviewed in REFS 23,24). Furthermore, recent studieshave provided greater insights into the roles of these

cytokines in TFH cell commitment. Using several mousemodels of viral infection, investigators found varying and transient degrees of impairment in T

FHcell

numbers in the absence of IL6, but they observedconsistently reduced levels of virusspecific IgG4245.A more severe decrease in T

FHcell formation and pro

tective IgG production occurred in the absence of bothIL6 and IL21 (REFS 42,44)(FIG. 1). This requirementfor IL6 and IL21 is consistent with reductions in thenumber of T

FHcells in the absence of functional STAT3

(REFS 30,46), which acts downstream of both cytokines(TABLE 1). Interestingly, the early reduction in the number of mouse T

FHcells in the absence of STAT3 was

Box 3 | IL21 is a potent differentiation factor for human B cells

Interleukin-21(IL-21)wasdiscoveredin2000asapleiotropiccytokinethatiscapableofactivatingmostlymphocyte

populations116.TheinitialdescriptionreportedthatIL-21stronglyinducedtheproliferationofCD40-stimulatedhuman

B cells,butthatitinhibitedIL-4-inducedBcellproliferation116.Sincethen,manystudieshaveestablishedthepotency

ofIL-21asagrowthanddifferentiationfactorforhumanB cells.WhenhumanB cellsareprimedwithTcellhelpinthe

formofaCD40-specificmonoclonalantibodyorCD40ligand(CD40L),IL-21inducesrobustBcellproliferationaswell

astheexpressionofactivation-inducedcytidinedeaminase( AICDA;requiredforimmunoglobulinclassswitching),

Blymphocyte-inducedmaturationprotein1( BLIMP1;alsoknownasPRDM1)andXbox-bindingprotein1(XBP1)83,117,118

,allofwhichmediatethedifferentiationofB cellsintoplasmacells.Consequently,atleastin vitro,IL-21efficiently

inducessubsetsofactivatedB cellstoundergoclassswitchingeithertobecomeIgG-orIgA-expressingcells,orto

becomeplasmablastssecretingIgM,IgG,IgAorIgE83,117120.IL-21predominantlyinducesswitchingtoIgG3,IgG1

andIgA1subclasses117,119,120,whereasIL-21-stimulatednaive,GCormemoryB cellsproducelargequantitiesof

immunoglobulins83,117,119.IL-21canalsoinducetheexpressionofBcelllymphoma6(BCL6)inhumannaiveB cells117,

whichisconsistentwithIL-21havingaroleinestablishingGCs.BeforethediscoveryofIL-21,itwaswellrecognizedthat

classswitchingbyhumanB cellswasregulatedbyIL-4(whichpromotesclassswitchingtoIgG4andIgE),IL-10(which

promotesclassswitchingtoIgG1andIgG3),IL-13(whichpromotesclassswitchingtoIgG4andIgE)andtransforming

growthfactor-(TGF; whichpromotesclassswitchingtoIgA),andIL-10wasalsoconsideredtobeastronginducerofimmunoglobulinsecretion.Furthermore,B cellsurvivalwasshowntobepositivelyregulatedbyIL-4orIL-10(reviewed

inREF. 82).StudiesoverthepastdecadehavehighlightedtheimportanceofIL-21inhumoralimmunityinhumansby

demonstratingthatithastheremarkableabilitytoexertallofthesefunctionsonhumanB cells.

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T cells

T cells that express the T cell

receptor. These T cells are

present in the skin, vagina

and intestinal epithelium as

intraepithelial lymphocytes.

Although the exact function of

T cells is unknown, it has

been suggested that mucosal

T cells are involved in innate

immune responses.

Recent studies have reassessed the nature of NKTcells that provide B cell help. It was shown that following immunization with GalCerantigen conjugates,a small proportion of mouse NKT cells acquired aT

FHlike phenotype62,63. These NKT follicular helper

(NKTFH

) cells were detected in human tonsils62 andtheir development depended on the same factors thatare important for conventional T

FHcells62. By produc

ing IL21, NKTFH

cells supported the rapid formation ofGCs, yielding detectable levels of antigenspecific IgG,with some evidence of affinity maturation6264(FIG. 2).

Although NKTFH

cells resembled TFH

cells, themost striking difference was their inability to invokelonglived memory responses to lipid antigens (FIG. 2).Furthermore, the magnitude of the NKT

FHcellinduced

antibody responses to lipid antigens was inferior tothose driven by conventional T

FHcells6264. Despite this,

administering GalCer as an adjuvant or as a conjugated component of immunizing antigens increasedthe production of antigenspecific antibodies. Thisprobably reflects the direct actions of NKT

FHcells on

antigenspecific CD1d+ B cells, as well as the indirectactions of NKT

FHcells, such as cytokine production, on

other cell types5861. It might also indicate that there issynergy between NKT

FHand T

FHcells. Taken together,

these findings support the inclusion of GalCer invaccine adjuvants.

TFH

cells.The finding that mice and humans lacking the conventional TCR have T

Hcells that elicit

humoral responses against T celldependent antigensled to the realization that T cells can provide helpto B cells to generate GCs65. Similar to conventionalT

FHcells, some human T

FHcells express CXCR5

and localize to follicles and GCs6668. T cells expressa semiinvariant TCR repertoire and recognize nonpeptidic phosphoantigens that are derived from microbial metabolites65. These antigens rapidly activate T cells, which can subsequently acquire T

FHcell fea

tures66,68,69; the differentiation of T cells to TFH

likecells is increased by exogenous IL21 (REFS 68,69) (FIG. 2).As T cells do not produce IL21 (REFS 68,69), theyare dependent on extrinsic sources of this cytokine todifferentiate into T

FHlike cells.

As conventional CD4+ T cells, T cells and NKTcells recognize different repertoires of microbial antigens, their ability to differentiate into T

FHlike effec

tor cells provides a mechanism whereby humoral

immunity can be generated against a broad range ofpathogenassociated antigens (FIG. 2). This expandsthe number of molecular targets that initiate protective immunity, but it might also contribute to postinfection autoimmunity by generating crossreactiveautoantibodies13,70(BOX 2).

Follicular TReg

cells. Recent studies have proposed thatT

FHcells are controlled by follicular T

Regcells a spe

cialized subset of TReg

cells that colocalize within B cellfollicles. Follicular T

Reglike cells were first described

in human tonsils in 2004 (REF. 71), but it took another78 years for them to be examined in greater detail.

Follicular TReg

cells comprise approximately 1015%of the T

FHcell population in human and murine lym

phoid tissues7274. They show characteristics of bothT

FHcells and T

Regcells, but they lack expression of CD40L,

IL4 and IL21 (REF. 73). Abrogating either follicular TReg

cell development or their follicular localizati

in mun ology

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