ArticlePDF AvailableLiterature Review

Cross-priming in health and disease

June 2010
Nature Reviews Immunology 10(6):403-14

June 2010
10(6):403-14

DOI:10.1038/nri2780

Source
PubMed

Authors:

Christian Kurts

University of Bonn

Percy Knolle

Technische Universität München

Cross-priming is an important mechanism to activate cytotoxic T lymphocytes (CTLs) for immune defence against viruses and tumours. Although it was discovered more than 25 years ago, we have only recently gained insight into the underlying cellular and molecular mechanisms, and we are just beginning to understand its physiological importance in health and disease. Here we summarize current concepts on the cross-talk between the immune cells involved in CTL cross-priming and on its role in antimicrobial and antitumour defence, as well as in immune-mediated diseases.

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Content may be subject to copyright.

Immune defence against intracellular pathogens or

tumours is the domain of CD8+ cytotoxic T lymphocytes

(CTLs). These cells recognize antigenic peptides asso-

ciated with MHC class I molecules, which are expressed

on the surface of all cells in the body. When patho-

gen- or tumour-specific effector CTLs detect antigen

being presented by a cell, they destroy the cell to avert

the spread of infection or cancer. To avoid the killing

of healthy bystander cells that have endocytosed viral

or tumour debris, this endocytosed material does not

usually enter the MHC class I-loading machinery in the

endoplasmic reticulum. The endogenous MHC class I

pathway is reserved for peptides derived from intra-

cellularly synthesized proteins. Therefore, CTL cytotoxic-

ity is focused on cells in which microorganisms replicate

or that are malignant.

However, this restriction of MHC class I loading is

insufficient in one situation: naive CTLs need to be acti-

vated by professional antigen-presenting cells (APCs),

usually dendritic cells (DCs), before they can exert their

cytotoxic effector functions. So when an intracellular

pathogen does not infect APCs or compromises their

endogenous MHC class I pathway, or when a tumour is

not APC-derived (which is the case for most tumours),

CTLs can only be activated if APCs present extracellular

antigen on their MHC class I molecules. This process,

termed cross-presentation, was named after the phenom-

enon of cross-priming that was discovered in the 1970s1,

in which antigens from intravenously injected cells

‘crossed’ into the MHC class I pathway of host APCs

for CTL priming. Cross-priming has since been shown

to be required for defence against many viruses and

tumours2,3, and it is essential for vaccinations with pro-

tein antigens, which must be cross-presented to activate

CTLs4. Self antigens are also cross-presented but rather

than cross-priming, this normally results in deletion of

autoreactive CTLs, in a process termed cross-tolerance5.

Technical and conceptual advances in recent years

have provided insights into the underlying mechanisms of

cross-priming, especially the associated intracellular anti-

gen processing mechanisms and DC subsets, which have

being reviewed elsewhere4,6,7. The physiological impor-

tance of cross-priming and cross-tolerance in health

and disease is less well understood. Here, we summarize

recent advances in our understanding of the regulation

of cross-priming and its implications for defence against

infections, CTL-mediated diseases and tumours.

Basic mechanisms of cross-priming

Roles of APC types in cross-priming. In viv o depletion or

functional inhibition of DCs compromises both cross-

priming and cross-tolerance8,9, identifying these cells as

the most relevant cross-presenting APCs in mice6,7,10,11.

Among the DCs, only those expressing the surface mark-

ers CD24, CD8α and CD103 (also known as αE integrin)

usually cross-present antigen6,7,10,11. Cross-presenting DC

subsets are usually identified by ex vivo analysis of DCs

isolated from antigen-injected mice, because selective

in v ivo targeting of DC subsets is difficult. The targeting of

DC subsets has become possible by the discovery that the

transcription factor BATF3 (basic leucine zipper tran-

scriptional factor ATF-like 3) is expressed by CD8α+ and

CD103+ DCs and is necessary for cross-presentation12.

Using BATF3-deficient mice, it has been proposed that

these DCs constitute a unified subset with a common

lineage13. Under inflammatory conditions, in mucosal

tissues and in tumours, DCs that lack expression of

CD24, CD8α and CD103 can also cross-present14–18.

However, the human counterparts of cross-presenting

mouse DC subsets remain to be identified.

Classifying DC subsets on the basis of cell surface

markers is convenient for flow-cytometric analysis, but

does not mechanistically explain why a cell can cross-

present. Recent studies have proposed two mechanistic

*Institutes of Molecular

Medicine and Experimental

Immunology, University Clinic

of the Rheinische Friedrich-

Wilhelms-Universität,

Sigmund-Freud-Strasse 25,

53105 Bonn, Germany.

‡School of Medicine and

Pharmacology, Sir Charles

Gairdner Hospital, University

of Western Australia,

Nedlands, Perth 6009,

Australia.

Correspondence to C.K.

e-mail: ckurts@web.de

doi:10.1038/nri2780

Cross-presentation

The ability of certain

antigen-presenting cells to load

peptides that are derived from

exogenous antigens onto MHC

class I molecules. This property

is atypical, because most cells

exclusively present peptides

from their endogenous

proteins on MHC class I

molecules. Cross-presentation

can lead to cross-priming or

cross-tolerance.

Cross-priming

The initiation of an

immunogenic CD8+ T cell

response to an antigen that is

not synthesized by the

antigen-presenting cell.

Cross-presentation is essential

for the initiation of immune

responses to viruses that do

not infect antigen-presenting

cells.

Cross-priming in health and disease

Christian Kurts*, Bruce W. S. Robinson‡ and Percy A. Knolle*

Abstract | Cross-priming is an important mechanism to activate cytotoxic T lymphocytes

(CTLs) for immune defence against viruses and tumours. Although it was discovered more

than 25 years ago, we have only recently gained insight into the underlying cellular and

molecular mechanisms, and we are just beginning to understand its physiological

importance in health and disease. Here we summarize current concepts on the cross-talk

between the immune cells involved in CTL cross-priming and on its role in antimicrobial

and antitumour defence, as well as in immune-mediated diseases.

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Cross-tolerance

The antigen-specific

tolerization of CD8+ T cells by

antigen-presenting cells that

cross-present self or innocuous

antigens, which results in

BIM-mediated (BCL-2

inhibitable) deletion of

autoreactive CD8+ T cells.

C-type lectin receptors

A large family of receptors that

bind glycosylated ligands and

have several roles, such as in

cell adhesion, endocytosis,

pathogen recognition, natural

killer cell target recognition and

dendritic cell activation.

explanations, which are not mutually exclusive. The first

hypothesis proposed that only cross-presenting DCs pos-

sess the antigen processing machinery that loads endo-

cytosed antigen onto MHC class I molecules19. Therefore,

based on this hypothesis, cross-presentation must depend

on specific antigen processing mechanisms that can be

regulated separately, so that MHC class I-restricted pres-

entation of endogenous antigens remains operational.

Insulin-regulated aminopeptidase (IRAP; also known as

cystinyl aminopeptidase), a trimming peptidase located

in endosomal compartments, has been recently identified

as a processing molecule with such specificity20; how-

ever, its general role in cross-presentation remains to be

established. The second hypothesis proposed that cross-

presentation depends on distinct endocytosis mecha-

nisms that introduce antigen directly into the organelle

(or organelles) in which cross-presentation occurs21

(BOX 1). In support of this, uptake of antigen for cross-

presentation is restricted to distinct endocytosis receptors,

such as Fc receptors and certain members of the C-type

lectin receptor family, such as C-type lectin 9A (CLEC9A;

also known as DNGR1), CLEC7A (also known as

dectin 1), DC-SIGN (also known as CD209), DEC205

(also known as CD205) and mannose receptor 1 (also

known as CD206)19,22–24. These receptors are expressed

by CD8α+ DCs21, providing a possible mechanistic

explanation for why this DC subset can cross-present.

If CTL-mediated immune responses depend on

cross-priming, one might ask why it is restricted to only

a few DC subsets. One explanation is that DCs capable

of cross-presentation automatically become targets for

CTL killing after endocytosis of viral or tumour debris.

Hence, during a systemic infection, all DCs might be

killed if they all could cross-present. Although DCs

possess mechanisms to avoid CTL-mediated killing to

some degree25, it is clear that cross-presenting DCs can

be effectively killed during viral infections26. However,

this killing does not seem to affect the ensuing immune

response as CTLs need less than one day of antigen

presentation by DCs for activation and a little longer for

developing cytolytic functionality27,28, and they can then

carry out their tasks without continual stimulation, at

least for some days. DCs that cannot cross-prime can

maintain other immune responses that depend on CD4+

T helper (TH) cells, such as antibody production and

macrophage stimulation, because they will not be killed

by CTLs, unless they are virally infected themselves.

Consistent with this notion, non-cross-presenting DCs

are superior at activating TH cells19,21.

Cross-priming CTLs specific for peripheral tissue

antigens involves the cooperation between the different

DC types. Tissue DCs transport antigen from tissues to

secondary lymphoid organs and transfer it to resident

CD8α+ DCs, which then cross-prime CTLs29,30. This

antigen transfer mechanism provides several theoretical

advantages7: first, migratory DCs may distribute antigen

to several resident DCs, which together can prime more

CTLs; second, antigen endocytosed by non-cross-present-

ing DCs can also be used for cross-priming; third, non-

cross-presenting DCs carrying viral antigens would not

be killed by viral-specific CTLs before reaching draining

lymph nodes; fourth, viruses may compromise endog-

enous antigen processing pathways in infected migratory

DCs, but not in the antigen-receiving DCs; and fifth, the

CTL will have to inspect only the few antigen-receiving

resident DCs rather than a large number of migratory

DCs, which may allow faster location of their cognate

antigen. However, this concept of antigen transfer also

contains some conceptual problems: even if antigen is

transported by non-cross-presenting DCs, eventually it

must reach a cross-presenting DC, which then might be

killed by CTLs. Furthermore, the information obtained

from direct encounters with microbial molecular patterns

must somehow be transferred to the cross-priming DCs.

Finally, the mechanism of antigen transfer between

DCs is unclear.

In addition to DCs, macrophages, neutrophilic

granulo cytes and mast cells can cross-present antigen but

cannot cross-prime CTLs31. B cells only cross-present

antigen recognized by their immunoglobulin recep-

tors and can cross-prime CTLs when Toll-like receptors

(TLRs) are also engaged32. Resting B cells can tolerize

CTLs when externally loaded with antigenic peptide33,

but evidence for a functional role of these cells in cross-

tolerance in vivo is lacking. By contrast, liver sinusoidal

endothelial cells (LSECs) can efficiently cross-tolerize

CTLs that are specific for food antigens or commensal

bacteria34. In summary, distinct APC types have different

roles in cross-priming and cross-tolerance.

DC activation and DC licensing. Immunogenic responses

require that DCs encounter antigen that is associated

with molecular patterns indicative of the presence of

microorganisms or other ‘danger’ signals35. Sensing such

patterns (for example, by activation of TLRs) leads to the

upregulation of co-stimulatory molecules and enhances

Box 1 | Distinct organelles for cross-presentation in dendritic cells

The cell biological mechanisms involved in cross-presentation have long remained

poorly understood. In particular, it was unclear how antigens may avoid lysosomal

degradation and enter the MHC class I-loading pathway, which is known to start in the

cytoplasm and to occur in the endoplasmic reticulum (ER)4. However, loading in the ER

would imply that the few peptides derived from endocytosed material would have to

compete with the high levels of endogenously produced peptides in the ER for loading

of MHC class I molecules. An elegant solution to this problem came from studies

showing that loading of peptides from particulate antigens occurs in phagosomes and

hence is sequestered from loading of endogenous peptides in the ER144,145. These

organelles were shown to contain the MHC class I-loading machinery and, after

isolation, were sufficient to import and cross-present exogenously added antigenic

peptides. However, it remains controversial how the loading machinery reaches

phagosomes146. During maturation, phagosomes fuse with lysosomes, lower their pH

and become competent for peptide loading onto MHC class II molecules147.

Cross-presentation of soluble antigens can occur in stable early or recycling

endosomes that do not mature to late endosomes, in which peptides are loaded on

MHC class II molecules21,38,148,149. These early endosomes are supplied with antigen by

distinct endocytic mechanisms, such as that mediated by the mannose receptor,

whereas late endosomes received antigen by other means. According to this model,

cross-presentation and MHC class II-restricted presentation of soluble antigens are

physically separated, whereas MHC class I and II loading of particulate antigens is

temporally separated in the same phagosomes. For both antigen classes,

cross-presentation is sequestered from MHC class I loading of endogenous peptides,

avoiding competition of peptides for MHC class I loading.

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MHC class II

MHC

class II

MHC class I

TCR

CD40L

IL-2

CD80 or

CD86 CD28

CD27

↓ PDL1

↓ TRAIL

expression

TLR agonist

CD70

CD4+ TH cell

CD8+ CTL

CCL3, CCL4

and CCL5 CCR5

Cross-presenting

TLR

Antigen

CD40

TLR

CD1d

TCR

α-GalCer

Antigen

CCR4

CCL22

TCR

CCL17

cross-priming; the enhancement of cross-priming being

especially effective after TLR3 and TLR9 stimulation36,37.

Furthermore, TLR signalling enhances peptide loading

onto MHC class I molecules by inducing the relocation

of components of the peptide-loading machinery to the

endosomes in which cross-presentation occurs38. Many

C-type lectin receptors preferentially recognize foreign

glycosylation structures characteristic of microorganisms

and thereby allow some degree of self–foreign discrimi-

nation21,39. Engaging these receptors may synergize with

TLR signals to promote upregulation of co-stimulatory

molecules because distinct signalling pathways are used40,

and may theoretically enhance cross-priming. However,

this cannot be extrapolated from studies targeting anti-

gens to C-type lectin receptors, because such targeting

enhances cross-priming by increasing antigen uptake,

which is difficult to discriminate from C-type lectin recep-

tor signalling. Also, activation of RIG-I-like cytoplasmic

receptors for nucleic acids and the inflammasome boost

CTL responses41,42 and indirect evidence suggests that

these cytoplasmic sensors can stimulate cross-priming43,44,

but this has not yet been formally shown.

Although co-stimulatory signals are necessary for

immunogenic cross-priming, they are insufficient in

some models to break cross-tolerance45,46, unless specific

TH cells are present45,47. This has led to the realization that

DCs require signals from specific TH cells for immuno-

genic cross-priming. CTLs activated without T cell help

(termed ‘helpless’ CTLs) lack expression of specific anti-

apoptotic molecules, have a short life-span and cannot

carry out cytotoxic effector functions5,48–50. The neces-

sity for specific T cell help can be viewed as requesting

a ‘second opinion’ from TH cells before programming

CTLs for cytotoxic functions. Both autoreactive TH cells

and CTLs are present at a low frequency in a normal

repertoire, but the likelihood that clones specific for the

same autoantigen coexist will be much lower. Hence,

autoimmune responses become less likely when antigen

must be recognized by two lymphocyte subsets that had

been negatively selected for self-reactivity. An analogous

safety strategy is implemented in antibody production, in

which specific B cells and TH cells need to both recognize

the foreign antigen. However, in contrast to B cells, CTLs

lack MHC class II molecules and cannot directly receive

specific help from TH cells. Therefore, cross-presenting

DCs act as a temporal bridge between these cells. The

TH cells convert the DCs to a transient state in which they

can programme the CTL for sustained cytotoxic effec-

tor functions and memory differentiation (FIG. 1a)51,52.

When such DCs are adoptively transferred into naive

mice, they retain the ability to cross-prime CTLs, show-

ing that they store the information received from the

TH cells. This has been termed ‘DC licensing’.

DC licensing is usually facilitated by TH cell-expressed

CD40 ligand (also known as CD154), which interacts

with CD40 on DCs31. It remains unclear to what extent

signalling events induced by TH cells or by TLRs over-

lap in cross-priming DCs. Some differences must exist,

because TH cells and pattern-recognition receptors

synergistically activate DCs and both are necessary for

optimal cross-priming4,45.

Figure 1 | Molecular mechanisms of licensing dendritic cells for classical

cross-priming. a | The molecular mechanisms involved in classical cross-priming

are illustrated. Dendritic cells (DCs) take up antigen by distinct endocytosis

mechanisms (not shown) and present it to CD4+ T helper (TH) cells through MHC

class II molecules and cross-present it to CD8+ cytotoxic T lymphocytes (CTLs)

through MHC class I molecules. Activated CD4+ TH cells can stimulate CTLs through

the production of interleukin-2 (IL-2) and license DCs for cross-priming through

CD40 ligand (CD40L)–CD40 interactions. Licensed DCs upregulate expression of

co-stimulatory molecules, such as CD70, CD80 and CD86, and downregulate

inhibitory molecules, such as programmed cell death ligand (PDL1). Toll-like

receptor (TLR) ligands further activate DCs and increase their cross-presentation

activity. Cross-primed CTLs are programmed for survival and cease TNF-related

apoptosis-inducing ligand (TRAIL) production. ‘Helpless’ CTLs activated by

unlicensed DCs die following secondary encounter with antigen in their effector

phase (not shown). b | Chemokine-mediated regulation of cross-priming is

illustrated. CD4+ TH cells, and the DCs they license, produce CC-chemokine ligand 3

(CCL3), CCL4 and CCL5 in the presence of TLR ligands, which recruit naive CTLs for

classical cross-priming. Alternatively, DCs licensed by natural killer T (NKT) cells

produce the CC-chemokine receptor (CCR4) ligand CCL17, and NKT cells

themselves produce the CCR4 ligand CCL22, which recruit naive CCR4+ CTLs for

cross-priming. The CCR4- and CCR5-mediated recruitment pathways are

synergistic. In this figure, dashed arrows indicate antigen routing for cross-

presentation. α-GalCer, α-galactosylceramide; TCR, T cell receptor.

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Inflammasome

A large multiprotein complex

formed by a nucleotide-binding

domain (NBD)-, leucine-rich

repeat (LRR)-containing family

(NLR) protein, the adaptor

protein apoptosis-associated

speck-like protein containing a

caspase recruitment domain

(ASC) and pro-caspase 1. The

assembly of the inflammasome

leads to the activation of

caspase 1, which cleaves

pro-IL-1β and pro-IL-18 to

generate the active

pro-inflammatory cytokines.

DC licensing

A concept that DCs must

be converted by an

antigen-specific T helper cell

into a functional state required

for immunogenic activation of

CTLs, which decreases the

likelihood of autoimmunity, but

requires coordination of cell

encounters by chemokines.

CTL programming

A concept that CTLs receive

additional information during

their activation that affects

their effector functions,

life-span, memory

differentiation or migratory

properties. These signals

determine whether

cross-presentation leads

to cross-priming or

cross-tolerance.

CTL programming. Licensed cross-presenting DCs

provide signals that dictate whether cross-priming or

cross-tolerance ensues, a process termed ‘CTL program-

ming’27,28,53. Understanding the identity and regulation

of these programming signals is important for optimiz-

ing vaccinations and understanding autoimmunity, and

hence they have been intensely studied. As candidate

signals, altered antigen presentation and modified co-

stimulatory and/or co-inhibitory signals have been pro-

posed. The duration of T cell receptor (TCR) signalling

affects whether activated CTL survive or not27,28, whereas

the TCR affinity dictated the level of CTL expansion

and ensured that only high affinity CD8+ T cell clones

develop into memory CTLs54. It is unknown, however,

how these qualities of TCR signalling may be regulated

by the DCs and by licensing TH cells.

CD70 is a candidate co-stimulatory signal for CTL

programming. This activation-induced tumour necro-

sis factor (TNF) receptor family molecule interacts

with CD27, which is expressed by CTLs, and increases

their survival17,55. However, the expression of CD70 by

DCs is not regulated by TH cells56. Downregulation of

programmed cell death ligands (PDLs), which bind to

the co-inhibitory receptor PD1 of the CD28 family and

promote CTL tolerance, might also contribute to CTL

programming57. It is unclear whether these ligands are

regulated by TH cells. The expression of interleukin-12

(IL-12) by DCs is regulated by TH cells and TLR ligands,

and promotes CTL effector functions58; however, its

ability to programme CTLs for survival is unclear.

CTL survival can be programmed by IL-2 during

priming59. However, IL-2 is not thought to be produced

by DCs and therefore cannot be the elusive survival

signal that licensed DCs provide. Theoretically, it may

originate from TH cells, but this would require their pres-

ence during CTL priming, which is inconsistent with

DCs storing the licensing information. Furthermore,

TH cell-derived IL-2 seems to have a distinct, probably

antigen-nonspecific, role in CTL memory maintenance53.

Alternatively, IL-2 may be produced by the CTL them-

selves. Indeed, prolonged TCR signalling during priming

induces autocrine IL-2 production60, and such IL-2 can

sustain CTL survival, at least when the CTLs are present

at high numbers61. Also, CD70–CD27 signals upregulate

autocrine IL-2 secretion by CTLs62, whereas it is down-

regulated by PD1 ligation63, suggesting that these two

candidates for the DC-derived CTL survival signal may

operate indirectly by regulating autocrine IL-2 produc-

tion. A converse effect on CTL survival has been reported

for autocrine TNF-related apoptosis-inducing ligand

(TRAIL), which CTLs produce by default and which is

shut off by specific TH cells64. Thus, helpless CTLs may

use TRAIL to kill themselves or each other if they also

express the TRAIL receptor. However, IL-2 does not

inhibit TRAIL production65, and causal connections to

CD70 and PD1 ligands have not yet been established. In

conclusion, many mechanistic steps important in cross-

priming have been described, but a comprehensive theory

of the molecular mechanisms by which licensed DCs pro-

gramme CTLs for survival, cytotoxic effector function and

memory generation that links these steps is lacking.

The cellular encounters for cross-priming

CTL activation in lymphoid organs. The need for DC

licensing by TH cells has an important limitation: three

rare immune cells — the cross-presenting DC, the anti-

gen-specific TH cell and the CTL — need to all interact.

However, because the licensing information is stored

within DCs, these interactions may not necessarily occur

at the same time. The CTL may theoretically interact

with the licensed DC after the DC has interacted with

the licensing TH cell51. Nevertheless, relying on random

encounters during normal T cell recirculation would

probably be too time-consuming to guarantee timely

defence against rapidly replicating microorganisms.

Recent studies showed that the encounters necessary

for CTL activation are greatly enhanced by chemokines.

In particular, CC-chemokine receptor 5 (CCR5) ligands

are produced following DC–TH cell interactions under

inflammatory conditions, which were mimicked by the

injection of TLR ligands66 (FIG. 1b). These chemokines

allowed CCR5-expressing naive CTLs to be attracted

to licensed DCs, where they could scan for cognate

antigen. Although TH cells and DCs first need to find

each other, the subsequent attraction of the third rare

cell type is thereby greatly enhanced and this decreases

the time spent by naive CTLs scanning unlicensed DCs.

Once activated, CTLs also produce CCR5 ligands67,

which may guide other naive CTLs towards DCs that

have successfully cross-primed. CTLs also produce

XC-chemokine ligand 1 (XCL1; al so known as lympho-

tactin) that binds to XC-chemokine receptor 1 (XCR1),

which is selectively expressed by CD8α+ DCs, and this

interaction supports cross-talk between these cell types

and enhances cross-priming68.

Natural killer T (NKT) cells, which recognize glyco-

lipid antigens presented by CD1d molecules, can also

enhance cross-priming, for example by upregulating

co-stimulatory molecules on DCs69. This process has

been recently shown to involve cognate DC licensing70

and consequently a tripartite immune cell encounter.

Chemokines, specifically CCR4 ligands and espe-

cially CCL17 (also known as TARC), produced by

cross-priming DCs are instrumental in this setting70

(FIG. 1b). Thus, at least two chemokine systems recruit

naive CTLs towards DCs that have been licensed by

TH cells or NKT cells. The CTL chemotaxis mediated

by CCR4 and CCR5 is synergistic, providing a mecha-

nistic rationale for combining ligands for NKT cells

with TLR ligands plus TH cell epitopes in vaccination

strategies. However, it remains unclear how naive

CTLs are induced to express CCR4 or CCR5 before

they interact with DCs that were licensed by NKT or

TH cells, respectively.

Regulation of effector CTLs in tissues. DCs can also

regulate the CTL effector phase in tissues50,71,72. If DCs

are infected, they may do so by using the endogenous

MHC class I pathway, unless it has been compromised by

the infection. If tissue DCs are not infected, they would

need to cross-present the antigen to ensure the antigen

specificity of this regulation. However, tissue DCs are

usually of the CD8α–CD11b+ subset, which has been

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CD8+

CTL

CXCR3

or CCR5

CXCR3 or

CCR5 ligand

Blood vessel

Endothelial

cell

LFA1

ICAM1

Virus-infected

tissue cell

Cytokines

and interferons

Virus

CD8+

CTL

CD4+

T cell

MHC

class I

MHC

class II

TCR

Tissue

shown to be unable of cross-presentation, at least under

steady-state conditions and in v itro6,7. If such DCs could

acquire this ability by inflammation-induced redifferen-

tiation into cross-presenting cells (for example CD8α+

DCs), the ability to cross-present would essentially be

a functional state that can be regulated, rather than a

property of DC subsets as entities with distinct line-

age. Recently, CD8α–CD103+ DCs have been detected

in certain tissues, which are developmentally related

to CD8α+ DCs13, and can cross-present tissue antigens

after migrating into draining lymph nodes11. However,

it is unclear if these cells can cross-present antigen while

in tissues. Thus, it is currently undecided whether DCs

within non-lymphoid tissues can regulate effector CTLs

in an antigen-specific manner.

An alternative mechanism by which tissue DCs could

regulate infiltrating effector CTLs in an antigen-specific,

but non-MHC class I-restricted, manner involves the

presentation of antigen on MHC class II molecules to TH

cells, which results in the production of cytokines and

chemokines that recruit and/or regulate CTLs (FIG. 2). This

has been described in mouse models of mesotheli oma73,

immune-mediated kidney disease50 and genital herpes

simplex virus (HSV) infection72.

There is also an antigen-specific and MHC

class I-restricted CTL recruitment mechanism. This is

not facilitated by DCs, but instead by cross-presenting

endothelial cells. Such cells have been detected in pancre-

atic islets74, the central nervous system75 and the liver76,

and have been shown to establish firm T cell contact

and activation, which in turn will increase lymphocyte

function-associated antigen 1 (LFA1; also known as αLβ2

integrin)–intercellular adhesion molecule 1 (ICAM1)-

mediated transmigration thorough endothelial barriers

into tissues. Subsequently, tissue-resident DCs may then

regulate the incoming CTLs; for example, by cross-talk

wit h TH cells and chemokine production. However, cross-

presenting endothelial cells might be killed by CTLs,

causing unwanted side-effects. This may be avoided by

high levels of co-inhibitory molecule expression, such as

PDL1, by the endothelial cells, which could help prevent

CTL-mediated damage77. Thus, in addition to the classical

paradigm of CTL recruitment into tissues by attachment

to adhesion molecules on inflamed endo thelial cells,

two specific pathways exist that are facilitated by cross-

presenting endothelial cells or by chemokines resulting

from specific TH cell–DC crosstalk (FIG. 2).

Cross-priming during infections

Viral infections. Cross-priming is necessary for CTL-

mediated immune responses against microorgan-

isms that do not target DCs, such as Epstein–Barr

virus (EBV), hepatitis B virus (HBV) or poliovirus

(TABLE 1). Furthermore, cross-priming is important for

DC-infecting viruses that evade the endogenous MHC

class I pathway, such as members of the Herpes virus

family, especially murine cytomegalovirus (MCMV).

However, many viruses infect DCs, suggesting that

avoiding DCs is not a survival advantage for the virus,

perhaps because cross-priming exists.

MCMV infects various cells, including DCs, and

produces immune evasins that inhibit MHC class I-

restricted antigen presentation78. Hence, MCMV-infected

DCs fail to induce a virus-specific CTL-mediated

immune response79. Despite escaping from the

endogenous antigen presentation pathway, MCMV-

specific CTLs eventually develop, demonstrating

that cross-priming by uninfected DCs must occur80.

This pathway also induces CTL-mediated immunity

against other immune evasin-producing viruses, such

as EBV81 and HSV-1 (REF. 7). But even when effective

cross-priming occurs, MCMV-infected cells still

escape CTL-mediated killing because these cells present

viral epitopes that are different from those that the

CTLs are cross-primed against80. Interestingly, ablation

of immune evasins from MCMV results in decreased

cross-priming and antiviral immunity82. This paradoxi-

cal observation can be explained by the rapid control

of viral replication and decreased viral antigen burden

in the absence of these molecules. Other viruses also

directly avoid cross-priming: vaccinia viruses escape DC

cross-priming by restricting late viral antigens to a partic-

ular cellular compartment that is inaccessible for cross-

presentation but accessible for MHC class II-restricted

antigen presentation83.

Figure 2 | Recruitment of cross-primed effector cytotoxic T lymphocytes into

non-lymphoid tissues. Viral infection of tissue cells leads to their secretion of

pro-inflammatory cytokines and interferons that upregulate the expression of adhesion

molecules, such as intercellular adhesion molecule 1 (ICAM1), by endothelial cells.

Effector cytotoxic T lymphocytes (CTLs) attach to these molecules and are

nonspecifically recruited from the bloodstream into non-lymphoid tissues. In addition,

recent studies have revealed two antigen-specific recruitment mechanisms: first,

endothelial cells in certain tissues, such as the liver, pancreatic islets or the brain, can

cross-present microbial antigen, which allows them to selectively recruit antigen-specific

CTLs. Second, when specific CD4+ T helper (TH) cells detect microbial (or self) antigen on

non-cross-presenting tissue DCs, ligands for CC-chemokine receptor 5 (CCR5) or

CXC-chemokine receptor 3 (CXCR3) are produced that recruit CTLs into the infected

tissue. LFA1, lymphocyte function-associated antigen 1; TCR, T cell receptor.

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Table 1 | Cross-priming of CTLs in viral infections, tumours and CTL-mediated diseases

Disease Finding Refs

Viral infections

Influenza virus Cross-priming by distinct DC subsets at different time points after infection 17,55

EBV Cross-priming induces CTLs against latency-associated antigens expressed in B cells 81

Poliovirus Virus-specific immunity requires cross-priming by bone marrow-derived APCs 3

Lymphocytic choriomeningitis virus Type I IFNs induces virus-specific cross-priming 84

HIV Cross-priming of vaccines induces CTLs specific for HIV components 153

Herpes simplex virus 1 Requirement of DC licensing; division of labour between migratory DCs, lymph

node-resident cross-priming CD8α+ DCs and CD103+ DCs that migrate and

cross-present

7,11,29,51

Coxsackie virus Viral evasion of DC cross-priming 154

Murine cytomegalovirus Presentation of distinct epitopes in infected tissue allows for immune escape despite

successful cross-priming

HCV NS3-specific cross-priming following uptake of apoptotic infected cells 155

HBV Cross-priming of circulating viral antigens 86,87

Vaccinia virus Antigen transfer from infected cells enables cross-priming 156

Adenovirus Antigen transfer from infected macrophages to CD8α+ DCs in cross-priming 30

Non-viral infections

Listeria monocytogenes Essential role of cross-priming for bacteria-specific immune responses 9

Plasmodium spp. DCs cross-priming contributes to CTL-mediated immunity; also promoting

experimental cerebral malaria after brain infiltration of cross-primed CTLs

157

Trypanosoma cruzi DC cross-priming confers protection 158

Mycobacteria DC cross-priming after uptake of apoptotic infected cells 94

Chlamydia trachomatis Cross-priming requires antigen from infected cells 159

CTL-mediated diseases

Type 1 diabetes Central and peripheral cross-tolerance; cross-priming of diabetogenic CTLs 5,97,103,104

Multiple sclerosis Central and peripheral cross-tolerance; cross-priming of encephalitogenic CTLs; role of

cross-presenting endothelial cells

75,111,112,160

Glomerulonephritis and interstitial

nephritis

Activation of nephritogenic CTLs 50,108

Psoriasis vulgaris Activation of CTLs specific for skin autoantigens 109

Ankylosing spondylitis Epidemiological link to EBV infection 114

Autoimmune hepatitis Potential mimicry of EBV and HCV epitopes that are cross-primed; potential role of

cross-presenting endothelial cells

34,115,116

Thyroiditis Vaccination of mice with thyroglobulin caused CTL-mediated thyroiditis 161

Transplant rejection Cross-priming of host CTL specific for graft antigens 117

Tumours

Mesothelioma Weak cross-priming of localized CTLs 124

Melanoma and thymoma Cross-priming restricted by poor TH cell response 123

Leukaemia and lymphoma Cross-tolerance induction 2,162

Transformed embryo cells Cross-presenting DCs can be activated to cross-prime 52

Melanoma Cross-priming is autophagy dependent 163

Hepatocellular carcinoma Cross-priming in an autochthonous mouse model 122

Lung cancer Efficient cross-priming but no tumour eradication 125

Glioma of the brain Cross-primed CTLs enter and are retained in brain tumours 75

Insulinoma Weak cross-priming increasing with tumour size 127

APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; EBV, Epstein–Barr virus; HBV, hepatitis B virus; HCV, hepatitis C virus; IFN, interferon;

TH cell, T helper cell.

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Central tolerance

Self-tolerance that is created at

the level of the central

lymphoid organs. Developing

T cells in the thymus and

B cells in the bone marrow that

strongly recognize self antigen

face deletion or marked

suppression.

Antigenic mimicry

A mechanism for the induction

of autoimmunity, in which a

pathogen expresses a protein

or peptide that is similar to a

self protein. After the induction

of a pathogen-specific immune

response, a cross-reactive

response to self results in

autoimmune pathology.

Type I interferons (IFNs) are released from virus-

infected cells or sentinel cells such as plasmacytoid DCs,

and they can induce DC maturation and support cross-

priming. This may be important when neither viral nor

microbial-derived signals are available to induce the

maturation of cross-priming DCs84. Type I IFNs are effi-

ciently induced by triggering TLR3, which recognizes

double-stranded viral RNA and effectively stimulates

cross-priming37. TLR3-induced type I IFN expression

is associated with control of viral infection, albeit at the

expense of immune-mediated organ damage85. Despite

these mechanisms, some viruses still do not elicit effi-

cient CTL responses despite abundant antigen produc-

tion. For example, hepatitis C virus (HCV) is recognized

by RIG-I, but blocks the subsequent induction of type I

IFNs, and HBV induces the production of IL-6 but not

type I IFNs by infected cells86,87. The efficacy of type I

IFN treatment in patients with chronic viral hepatitis

may involve improved cross-priming, in addition to any

direct antiviral effect.

Viruses such as HIV, influenza virus or HCV can

escape established CTL responses by mutating their

antigens (TABLE 1). Furthermore, during the course of

an infection, different viral antigens are expressed, such

as the early and late antigens of HSV. Thus, continuous

delivery of viral antigens is necessary to cross-prime CTL

clones specific for new antigens in lymph nodes. Such

antigen delivery can be mediated by migratory DCs29 or

by the endocytosis of viral antigens that drained though

lymph vessels by lymph node-resident DCs88. In HSV

infection of the skin or lung, antigen is transported by

migratory CD103−CD11bhi tissue DCs (or by Langerhans

cells in the skin) to draining lymph nodes and trans-

ferred to cross-presenting CD8α+ DCs17, or is conveyed

by migratory CD103+CD11b− DCs that directly cross-

present in draining lymph nodes7. By contrast, migratory

CD103−CD11bhi DCs transported viral antigen during

influenza virus infection from the lung to the bronchial

lymph nodes and directly cross-primed CTLs without the

need to transfer antigen to CD8α+ DCs 17. The role of these

DC subsets in other viral infections is unclear.

Bacterial infections. The importance of cross-priming

for bacterial infection has been mostly studied using

Listeria monocytogenes. Following infection, L. mono-

cytogenes gains access to the cytoplasm of splenic mac-

rophages and hepatocytes, which subsequently undergo

apoptosis. DCs acquire antigen for cross-priming fol-

lowing uptake of debris of infected cells, which is cru-

cial for defence against L. monocytogenes infection9.

Cross-priming can also occur in the absence of protein

synthesis, on the condition that the innate immune

stimulatory function of L. monocytogenes is preserved89.

Surprisingly, cross-priming DCs are also important for

bacterial dissemination into the spleen, as they take up

circulating bacteria and allow their initial intracellular

survival, thereby promoting subsequent bacterial pro-

liferation and spread to other cell populations90. This

may localize infections to sites where DCs can later

orchestrate innate and adaptive immune responses91.

Shortening the time of bacterial exposure curtailed

specific immunity by decreasing TH cell responses92,

showing the importance of DC licensing, which requires

time-consuming establishment of physical contact with

licensing TH or NKT cells. Terminating CTL immunity

against infections that do not provide stimulation for a

minimum time (that is, are eliminated by innate immune

mechanisms) may focus adaptive immune responses

to those infections requiring CTL immunity but may

also allow pathogen escape mechanisms to establish

persistent infection.

Acquisition of bacterial antigen by DCs has been

most intensively studied during mycobacterial infection,

in which mycobacteria persist in phagosomal vesicles

and dispersion of bacterial antigens into the cytoplasm

does not occur. Here, apoptosis of infected cells, facili-

tating the uptake of bacterial antigens, is indispensable

for cross-priming by DCs and induction of efficient

CTL-mediated immunity93. Similar to viral infections,

transport of bacterial antigens by DC into lymph nodes

was also required for cross-priming94. However, process-

ing of bacterial antigens from apoptotic vesicles in DCs

differs from that of viral antigens by requiring saposins,

which open vesicles and release bacterial antigens into

the cytoplasm for processing94. Taken together, DC

cross-priming supports the induction of CTL-mediated

immunity against pathogens with a narrow host cell

tropism that avoid infecting DCs or possess immune

escape mechanisms.

Cross-priming in immune-mediated diseases

Although central tolerance is an efficient process, some

autoreactive CTLs escape negative selection and enter

the circulation. In secondary lymphoid organs, cross-

tolerance serves as a second checkpoint that can eliminate

such escapees5,8. However, cross-tolerance occurs only

when the autoantigen dose and/or CTL affinity are suffi-

ciently high31,49. If not, then autoreactive CTLs may escape

cross-tolerance and cause disease when cross-primed95.

CTLs are the main effector cells of human type 1

diabetes96. Cross-priming is necessary and sufficient for

islet infiltration and destruction in transgenic diabetes

models31,49 and in non-obese diabetic (NOD) mice97,

which have multigenic disease susceptibility similar to

human illness, including DC defects that compromise

cross-tolerance98. However, the identity of the antigens

that are cross-presented is not clear. Theoretically, these

antigens could be pancreatic self antigens or microbial

antigens mimicking such antigens. The suggestion of a

role for microbial antigens is supported by studies with

transgenic mice expressing viral components in islets,

which developed CTL-mediated diabetes after virus

infection99. However, the lymphocytic choriomenin-

gitis virus used in these studies infects DCs, which

then directly prime CTLs, and therefore cross-priming

becomes unnecessary. Antigenic mimicry was suggested

to be supported by the epidemiological association of

diabetes with coxsackie virus infections, but this is now

thought to result from changes in the micromilieu of

infected islets rather than viral antigens99,100. Thus, the

evidence for antigenic mimicry as a cause of type 1

diabetes is unconvincing101.

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CD8+

CTL

Lymph node

Cross-priming

↑ by CD40

agonist and TLR7

Cross-presentation

↑ by chemotherapy, monoclonal

antibodies and vaccines

Circulation

Antigen

release

Traffic into

tumour

Tumour attack

MHC

class I

Tumour

Blood vessel

Tumour

TCR

Tumour-derived

antigen

Tumour-specific CTL

Nature Reviews | Immunology

Oligodendrocyte

A type of glial cell that creates

the myelin sheath that

insulates axons and improves

the speed and reliability of

signal transmission by neurons.

If so, then diabetogenic CTLs are likely to be cross-

primed by pancreatic islet autoantigens. One candidate

is an MHC class I-binding peptide from the insulin

B chain102, an antigen also relevant in NOD mice103.

Pancreatic autoantigens are cross-presented in the pan-

creatic lymph nodes and this normally leads to cross-tol-

erance5,104, unless specific TH cells are present31. Notably,

TH cells specific for pancreatic autoantigens are also

activated at this site105 and might license DCs for cross-

priming. This may be important in patients with type 1

diabetes, because many of them possess particularities

in MHC class II-restricted antigen presentation and in

the responding TH cells106. Diabetes has long been known

to be closely associated with the MHC class II loci107,

although MHC class I-restricted CTLs are the effectors

of disease. The abnormal TH cells of patients with diabetes

might function by licensing DCs in the pancreatic lymph

node, which then cross-prime diabetogenic CTLs.

Most concepts on the role of CTLs and cross-priming

in autoimmunity have been extrapolated from type 1

diabetes models. However, the rapid destruction of

pancreatic islets has hampered investigating intra-organ

cross-talk between immune cells during the CTL effector

phase. Work in autoimmunity models of the kidney and

skin, which are more resistant to CTL-mediated killing of

tissue cells, has provided new insights. Autoreactive CTLs

specific for kidney glomerular antigens are cross-primed

in the renal lymph node and are recruited by chemo-

kines into the kidney, where they inflict damage50. These

chemokines were produced following cross-talk of auto-

reactive TH cells with tubulointerstitial DCs presenting

renal autoantigen, explaining the long-known ability of

TH cells to facilitate kidney access of autoreactive CTLs;

for example, in interstitial nephritis108. TH cell-dependent

recruitment of autoreactive effector CTLs was also noted

in psoriasis109.

In multiple sclerosis research, CTLs are being redis-

covered as important effectors110. Myelin-specific CTLs

that escape thymic tolerance and cross-tolerance mech-

anisms can damage oligodendrocytes111,112. Recent work

in humanized mice has shown that several classes of

bacteria contain an MHC class I-binding peptide that

mimics myelin basic protein, a candidate autoantigen

in multiple sclerosis113. But as with type 1 diabetes, the

exact autoantigens in multiple sclerosis are unknown,

hampering elucidation of the role of cross-priming and

of antigenic mimicry.

CTL-mediated responses against EBV have been linked

with ankylosing spondylitis114, long known to be associated

with HLA-B27 and with autoimmune hepatitis115. Hepatic

autoantigens are also recognized by HLA-A-restricted

CTLs specific for HCV116. In organ transplantation, host

CTLs cross-primed against graft antigens will be restricted

to the host haplotype, but nevertheless can damage graft

cells, either by alloreactivity or by killing host endothelial

cells growing in the graft117. There are further examples for

associations between disease entities and CTLs (TABLE 1),

but in nearly all cases, a causal role of cross-priming

remains to be formally shown.

Cross-priming and tumours

There are notionally three different types of tumour

antigens: ‘self’ antigens, to which a person is partially

tolerant (for example, normal embryonic or differen-

tiation antigens that also happen to be expressed in

tumours), ‘neo-antigens’ to which the host is not toler-

ant (for example, those due to mutations and viruses)

and ‘potential’ antigens, which are epitopes that may be

unmasked by treatments such as chemotherapy, which

increases antigen delivery into the cross-presentation

pathway118. Tumours contain thousands of potentially

strongly immunogenic neo-antigens caused by muta-

tions119. Several studies have shown that cross-presen-

tation of these antigens is an efficient and continuous

process120; therefore, other reasons as to why the tumour

cells are not rejected must exist. Several experimental

procedures have been used to analyse cross-priming in

tumour models2,120–123, and these studies have confirmed

that cross-presentation of tumour antigens occurs, it per-

sists for the dominate antigen and, interestingly, tends to

remain localized to the tumour-draining lymph node120.

Within these lymph nodes, both CD8α+ and CD8α– DCs

cross-present tumour antigens12,123, although whether

this reflects multiple mechanisms for delivery of tumour

antigen into the cross-presentation pathway, such as

through Fc receptor-mediated uptake of endogenous

antibodies that have opsonized tumour cells, or other

aspects of tumour antigen delivery, is not known.

Figure 3 | Cross-priming and immunotherapy in an effective antitumour immune

response. The first step in any successful antitumour immune attack usually involves

cross-presentation of antigens released from tumours, a process that can be boosted by

chemotherapies, monoclonal antibodies and vaccines (with or without dendritic cells

(DCs) or heat shock proteins). However, this cross-presentation of tumour antigen is not

sufficient to eradicate tumours; DC activation is required to turn cross-presentation into

a cross-priming event, a process boosted by agents such as CD40 agonists and Toll-like

receptor 7 (TLR7). Additional steps are then required; for example, expansion and

circulation of antitumour-specific effector cytotoxic T lymphocytes (CTLs), entry of those

effector cells into the tumour site and attack of the tumour — each step being subject to

regulation. Therefore, boosting cross-presentation and/or cross-priming alone would be

insufficient for tumour eradication: scientifically validated therapeutic combinations will

be required to turn cross-priming into successful anticancer immunotherapy.

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Although in some tumour models cross-tolerance

occurs, in others a weak, ineffective and localized CTL

response is induced124. A strong CTL response rarely

results from tumour antigen cross-priming without

further manipulation125. These weak responses are

understandable, as many tumour tissues lack intense

inflammation and/or danger signals and do not contain

pathogen-associated molecular patterns that usually

drive a strong CTL response126. The efficiency of cross-

priming to tumour antigens probably depends on the

cell line and/or tissue of origin plus other factors, such

as antigen dose127 (TABLE 1).

In summary, tumour antigens are cross-presented

efficiently in draining lymph nodes but fail to elicit a

strong antitumour CTL response.

Cross-priming and cancer treatment

It is in cancer therapy that cross-priming has triggered

the most interest. For example, one of the main mecha-

nisms whereby chemotherapy works is by stimulating

antitumour immunity by increased cross-presentation

and cross-priming of antitumour CTLs128. Importantly,

different cancer chemotherapies kill tumour cells

by different mechanisms, not all of which are immuno-

genic. Certainly it seems that the induction of apoptosis

by cancer chemotherapies can increase the amount of

tumour antigen delivered into the cross-presentation

pathway128. The outcome, however, depends on the

effects of chemotherapy on the host immune response

and importantly, the way in which chemotherapy

kills the tumour cell129–131. Increased cross-priming of

tumour-specific CTLs induced by chemotherapy has

been shown to facilitate long term cures of advanced

solid, non-immunogenic tumours when combined with

appropriate immunotherapies132 (FIG. 3).

Our knowledge of cross-presentation will also affect

cancer surgery. In almost all published studies, the

cross-presentation of tumour antigens is restricted to

the lymph node that drains the tumour. Indeed, this may

generate a level of CTL activity within the local ‘sentinel’

lymph node that represents a first ‘checkpoint’ in stop-

ping tumour spread124. Therefore, it has been proposed

that the removal of tumour-draining lymph nodes, even

if they do not show evidence of tumour spread, may

have a negative effect on antitumour immunity.

It has been shown that stronger antitumour effects

are seen if cross-priming of CTLs to tumour antigens is

increased, for example by using adjuvants such as isco-

matrix133, viral vectors134, heat shock proteins135, endog-

enous danger signals such as high-mobility group box 1

(HMGB1)136 or uric acid crystals43,44, or by using mono-

clonal antibodies. Indeed, it is possible that one way in

which monoclonal antibodies work in cancer immuno-

therapy is by opsonizing tumour cells and increasing their

delivery into the cross-priming pathway137. New strate-

gies are also being developed for linking antigens with

DC-targeting molecules which favour particular uptake

receptors and hence deliver the tumour antigen into the

appropriate cross-presentation pathway19,21,35.

One new concept that evolved from studies of

tumour antigen cross-priming is the use of a tumour

as its own vaccine132. Because the multitude of tumour-

specific mutations cannot be easily identified, strat-

egies that destroy tumours in a way that delivers

increased antigen doses into the cross-presentation

pathway may well increase the loading of these neo-

antigens and boost cross-priming128. This will not gen-

erate a strong and prolonged antitumour response; if

that were the case, then partial responses to chemo-

therapy would lead to immune-based tumour eradica-

tion after chemotherapy has ceased. However, it does

predict that immunotherapies that target DCs follow-

ing chemotherapy are more likely to be effective than

those that do not.

In cases in which tumour antigens or oncogenic

viruses have been identified, various vaccination strat-

egies have been used to induce antitumour responses

through the cross-presentation pathway. One promis-

ing approach is the use of synthetic long peptides. These

seem to be more efficient than full-length proteins at

entering the cross-presentation pathway138. Synthetic

long peptides can induce measurable CTL and TH cell

responses against potentially oncogenic viruses in

humans139,140.

Box 2 | Why was cross-priming so controversial?

Cross-priming has long been controversial, which today may sound surprising given the conceptual advantages it offers.

One reason is that several of the principal experimental models used in this field disfavour cross-presentation: for

example, many viruses — including the widely studied lymphocytic choriomeningitis virus (LCMV) — can infect dendritic

cells (DCs), and the associated antigens are presented by the endogenous MHC class I pathway, bypassing the need for

cross-priming150. Second, antigens that were originally thought not to be cross-presented were found to be so when

more sensitive methods were used127. Third, some antigenic epitopes, such as those located in signal sequences of viral

proteins, are rapidly degraded and therefore are not effectively cross-presented151. Fourth, immortalized cell lines, which

do not mimic all of the functions of bona fide APCs in vivo, were used as surrogate DCs. Fifth, among primary DCs, not all

subsets can efficiently cross-present6,7. Sixth, collagenase-based isolation of primary DCs inhibits endocytosis receptors

that internalize antigen for cross-presentation152.

There were also conceptual problems: some scientists were sceptical about cross-presentation because the underlying

cell biological mechanisms had long remained undefined. Finally, the ability to cross-present implies that DCs that have

taken up viral or tumour components might be killed by effector cytotoxic T lymphocytes (CTLs) (not during priming

because cytotoxic activity requires time to be established), and it was hard to imagine that the immune system should be

targeted against itself. This contradiction has been resolved by the independence of activated CTLs from continual

stimulation by DCs27,28 and by the existence of non-cross-presenting DC subsets6,7 that are not killed and hence can

maintain non-CTL-mediated immune responses.

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Signals 1 and 2

T cells require presentation of

antigen (signal 1) and

co-stimulatory signals (signal 2)

for immunogenic activation;

cytokines have been proposed

to be a third signal that

dictates T cell differentiation.

Concluding remarks

Antiviral vaccinations have long been taking advantage

of cross-priming4. Recent findings have clarified some

of the underlying mechanisms, such as the endocytosis

mechanisms in DCs that facilitate cross-presentation,

the organelles in which cross-presentation occurs, DC

licensing, CTL programming, chemokine-mediated

CTL recruitment and the interplay of cross-priming with

chemotherapeutic agents. Translating these advances

into the clinic may allow for the design of more effec-

tive vaccination strategies, for example by optimized

antigen targeting to endocytosis receptors that engage

the cross-presentation machinery in suitable DC sub-

sets. Adjuvants can be chosen that not only augment co-

stimulation, but also improve the cross-presentation of

antigen. Furthermore, the use of adjuvants that induce

different chemokines might further improve cross-prim-

ing. These chemokines may be viewed as a discrete signal

that profoundly affects CTL priming by acting before the

antigen- and co-stimulation-derived signals (also termed

signals 1 and 2, respectively, for T cell activation)141 and

DC-derived cytokines (proposed to be a third signal)58.

Hence, chemokines have been interpreted as ‘signal 0’

in T cell priming142.

Improvements are especially needed in tumour vac-

cination, as recent clinical studies on DC-based tumour

vaccination have been disappointing132,143. Future stud-

ies need to focus on how to harness cross-presenta-

tion to generate chemo-immunotherapy strategies for

therapeutic benefit. Understanding which current

chemotherapies work with, rather than against, the

anticancer immune response may facilitate new cancer

immunotherapies.

We are only beginning to understand in which infec-

tions and in which CTL-mediated diseases cross-prim-

ing is involved or crucial. Certainly, clarifying its role

in human disease is far more difficult than doing so in

mouse disease models. In vitro models have long been

insufficient (BOX 2), but are now improved and supple-

mented by knockdown techniques, and hence will prob-

ably provide further mechanistic insight into the role of

cross-priming in many disease entities.

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mechanism for efficient recruitment of tumor-specific

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Acknowledgments

The authors thank W. Kolanus (LIMES Institute Bonn), J.

Yewdell (NIH), M. von Herrath (LIAI) and R. Slattery (Monash

University Melbourne) for comments and discussions. We

apologize to all colleagues whose work could not be cited

owing to space restrictions. The authors are supported by the

German Research foundation (DFG Sonderforschungsbereiche

704, 670 and 645, Transregio 57, Klinische Forschergruppe

228), the German Academic Exchange service (DAAD) and

the Group of Eight, Australia.

Competing interests statement

The authors declare no competing financial interests.

DATABASES

UniProtKB: http://www.uniprot.org

BATF3 | CCL17 | CCR4 | CCR5 | CD8α | CD24 | CD27 |

XCL1 | XCR1

FURTHER INFORMATION

Christian Kurts’s homepage: http://www.immei.uni-bonn.de

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

REVIEWS

414

JUNE 2010

VOLUME 10 www.nature.com/reviews/immunol

TYK2 signaling promotes the development of autoreactive CD8 cytotoxic T lymphocytes and type 1 diabetes

Article

Full-text available

Feb 2024

Tyrosine kinase 2 (TYK2), a member of the JAK family, has attracted attention as a potential therapeutic target for autoimmune diseases. However, the role of TYK2 in CD8⁺ T cells and autoimmune type 1 diabetes (T1D) is poorly understood. In this study, we generate Tyk2 gene knockout non-obese diabetes (NOD) mice and demonstrate that the loss of Tyk2 inhibits the development of autoreactive CD8⁺ T-BET⁺ cytotoxic T lymphocytes (CTLs) by impairing IL-12 signaling in CD8⁺ T cells and the CD8⁺ resident dendritic cell-driven cross-priming of CTLs in the pancreatic lymph node (PLN). Tyk2-deficient CTLs display reduced cytotoxicity. Increased inflammatory responses in β-cells with aging are dampened by Tyk2 deficiency. Furthermore, treatment with BMS-986165, a selective TYK2 inhibitor, inhibits the expansion of T-BET⁺ CTLs, inflammation in β-cells and the onset of autoimmune T1D in NOD mice. Thus, our study reveals the diverse roles of TYK2 in driving the pathogenesis of T1D.

Identification of Key Genes Associated with Tumor Microenvironment Infiltration and Survival in Gastric Adenocarcinoma via Bioinformatics Analysis

Article

Full-text available

Mar 2024

Simple Summary This study aimed to pinpoint immune-related genes that show heightened activity in cancerous tissue and explore their correlation with cell infiltration in the tumor microenvironment using bioinformatics analysis. By examining gene expression from both stomach cancer and adjacent healthy tissues, we aimed to uncover their significance in cancer development and their impact on the body’s immune response. We identified several genes, including FN1, COL1A2, THBS2, COL3A1, COL5A1, and BGN, which appear to be associated with poorer outcomes for stomach cancer patients. These genes also demonstrate connections to specific immune cells within cancerous tissue. Understanding the role of these genes in the immune response to cancer could facilitate the development of novel treatments and enhance prognostic capabilities for individuals with stomach cancer. Abstract Objective: Gastric carcinoma (GC) is the fifth most commonly diagnosed cancer and the third leading cause of cancer-related deaths globally. The tumor microenvironment plays a significant role in the pathogenesis, prognosis, and response to immunotherapy. However, the immune-related molecular mechanisms underlying GC remain elusive. Bioinformatics analysis of the gene expression of GC and paracancerous healthy tissues from the same patient was performed to identify the key genes and signaling pathways, as well as their correlation to the infiltration of the tumor microenvironment (TME) by various immune cells related to GC development. Methods: We employed GSE19826, a gene expression profile from the Gene Expression Omnibus (GEO), for our analysis. Functional enrichment analysis of Differentially Expressed Genes (DEGs) was conducted using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database. Results: Cytoscape software facilitated the identification of nine hub DEGs, namely, FN1, COL1A1, COL1A2, THBS2, COL3A1, COL5A1, APOE, SPP1, and BGN. Various network analysis algorithms were applied to determine their high connectivity. Among these hub genes, FN1, COL1A2, THBS2, COL3A1, COL5A1, and BGN were found to be associated with a poor prognosis for GC patients. Subsequent analysis using the TIMER database revealed the infiltration status of the TME concerning the overexpression of these six genes. Specifically, the abovementioned genes demonstrated direct correlations with cancer-associated fibroblasts, M1 and M2 macrophages, myeloid-derived suppressor cells, and activated dendritic cells. Conclusion: Our findings suggest that the identified hub genes, particularly BGN, FN1, COL1A2, THBS2, COL3A1, and COL5A1, play crucial roles in GC prognosis and TME cell infiltration. This comprehensive analysis enhances our understanding of the molecular mechanisms underlying GC development and may contribute to the identification of potential therapeutic targets and prognostic markers for GC patients.

Differential centrifugation enhances the anti-tumor immune effect of tumor lysate-pulsed dendritic cell vaccine against glioblastoma*

Article

Full-text available

Oct 2022

Objective This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell (DC) vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma. Methods Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail. The modified tumor lysate was generated by differential centrifugation. The maturity markers of DCs in each group, namely the modified tumor lysate, tumor lysate, and negative and positive control groups, were assessed using flow cytometry. Furthermore, their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE. IFN-γ secretion levels were measured with ELISA. Intracellular reactive oxygen species were measured using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The results were statistically analyzed using an unpaired Student's t-test and were considered significant at P < 0.05. Results Compared with tumor lysate-pulsed DCs, modified tumor lysate-pulsed DCs had a higher expression of maturity markers: CD1a (7.38 ± 0.53% vs. 4.47 ± 0.75%) and CD83 (19.81 ± 4.09% vs. 9.64 ± 1.50%), were better capable of stimulating lymphocyte proliferation [proliferation index (PI): 8.54 ± 0.16 vs. 7.35 ± 0.05], secreting IFN-γ, and inducing stronger in-vitro cytotoxic T lymphocyte (CTL) cytotoxicity against glioblastoma cells. In addition, we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs. Conclusion Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines, and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate. Abstract Glioblastoma is the most common primary malignant tumor of the central nervous system with an annual incidence rate of 3-5/100 000 and a poor prognosis of 14.6 months, accounting for about 50% of all gliomas [1-2]. Although there are numerous studies on glioblastoma, the anatomical location of the central nervous system limits the application of locoregional treatment. In addition, the tumor is characterized by endogenous radio-and chemoresistance, heterogeneity, and an immunosuppressive microenvironment; these factors limit the progress of glioblastoma treatment. The standard treatment is to safely maximize surgical resection with adjuvant radiotherapy and chemotherapy [3-4]. Other adjuvant treatments include tumor treating fields, immunotherapy, gene therapy, and molecular targeted therapy, among which immunotherapy has great potential in the treatment of glioblastoma. As the most effective antigen-presenting cells, dendritic cells (DCs), bridge the innate and adaptive immune system [5-7]. DCs are cultured in vitro for maturation, sensitized with tumor-associated antigens or tumor-specific antigens, and then injected back into Downloaded from

Efferocytosis in dendritic cells: an overlooked immunoregulatory process

Article

Full-text available

May 2024

Efferocytosis, the process of engulfing and removing apoptotic cells, plays an essential role in preserving tissue health and averting undue inflammation. While macrophages are primarily known for this task, dendritic cells (DCs) also play a significant role. This review delves into the unique contributions of various DC subsets to efferocytosis, highlighting the distinctions in how DCs and macrophages recognize and handle apoptotic cells. It further explores how efferocytosis influences DC maturation, thereby affecting immune tolerance. This underscores the pivotal role of DCs in orchestrating immune responses and sustaining immune equilibrium, providing new insights into their function in immune regulation.

Sinusoidal cells and liver immunology

Chapter

Jan 2024

Acute bacterial and viral infections

Chapter

Jan 2024

MHC class I-dressing is mediated via phosphatidylserine recognition and is enhanced by polyI:C

Article

Apr 2024

In addition to cross-presentation, cross-dressing plays an important role in the induction of CD8⁺ T cell immunity. In the process of cross-dressing, conventional dendritic cells (DCs) acquire major histocompatibility complex class I (MHCI) from other cells and subsequently prime CD8⁺ T cells via the pre-formed antigen-MHCI complexes without antigen processing. However, the mechanisms underlying the cross-dressing pathway, as well as the relative contributions of cross-presentation and cross-dressing to CD8⁺ T cell priming are not fully understood. Here, we demonstrate that DCs rapidly acquire MHCI-containing membrane fragments from dead cells via the phosphatidylserine recognition-dependent mechanism for cross-dressing. The MHCI dressing is enhanced by a TLR3 ligand polyinosinic-polycytidylic acid (polyI:C). Further, polyI:C promotes not only cross-presentation but also cross-dressing in vivo. Taken together, these results suggest that cross-dressing as well as cross-presentation is involved in inflammatory diseases associated with cell death and type I IFN production.

Cellular and Epigenetic Aspects of Trained Immunity and Prospects for Creation of Universal Vaccines in the Face of Increasingly Frequent Pandemics

Article

Sep 2023
Genetika

The inevitability of pandemics creates an urgent requirement for emergency action to develop effective technologies to reduce harm to the human population in the period between the onset of an epidemic and the development and production of a vaccine. In this review we discuss the possibility of engineering universal vaccines. Such vaccines would exploit the nonspecific potential of innate immunity, would allow the population to be vaccinated when an unidentified pathogen appears, and would reduce disease severity until pathogen-specific vaccines become available. There are strong evidences that bacterial or viral vaccines such as BCG, measles and polio have heterologous protective effects against unrelated pathogens. This is attributed to the innate immune system’s ability to maintain the memory of past infections and use it to develop immune defenses against new ones. This effect has been called “trained” immunity. The use of trained immunity may also represent an important new approach to improving existing vaccines or to developing new vaccines that combine the induction of classical adaptive immune memory and innate immune memory. Such approaches can be boosted by genetic technology and prove extremely useful against future pandemics.

An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment

Preprint

Full-text available

Mar 2024

Targeting cancer cell mitochondria holds great promise for cancer treatment, yet current strategies for specifically and effectively destroying cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics technology designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM with a mitochondrial leading sequence and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using two strategies: adeno-associated virus (AAV) carrying a cancer-specific promoter (cfos) or cancer-targeted monoclonal antibody-tagged exosome-associated AAV (mAb-Exo-AAV). Induction with ViviRen (an NLuc luciferin) elicits robust endogenous NLuc bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, IMM potential depolarization and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastomas (administrated with AAV) or triple-negative breast cancers (administrated with mAb-Exo-AAV) xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

Adoptive T-Cell Therapy for the Treatment of Lung Cancer

Chapter

Mar 2024

Lung cancer is an imminent health threat and the predominant cause of cancer-related fatalities worldwide. Significant advancements in identifying important mutational variations in the cells have resulted in the creation of molecularly targeted medicines, the efficacy of which has been hampered by the establishment of resistance mechanisms. The current technological revolution in cancer treatment has resulted in substantial medical therapy and cancer research advances. New cancer treatment techniques, such as immunotherapy, are being hailed as “game changers” and “miracles in the making” due to encouraging results in treating patients with advanced diseases. In the present immunotherapy epoch, a growing number of immunotherapeutic medications have been used in the clinical management of several types of cancer. Adoptive cell therapy (ACT), which uses T cells that have been genetically engineered, such as tumor-infiltrating lymphocytes (TIL therapy) and chimeric antigen receptor-modified T (CAR-T) cells, is a revolutionary immunotherapy technique that offers the most promising advanced cancer therapeutics, with unrivaled results in hematological malignancies. This has made it possible to treat solid tumors like lung cancer with immunotherapy in new ways. Nonetheless, due to the tumor’s immunosuppressive microenvironment, its efficacy remains inadequate. As a result, in the current environment, employing adoptive cell therapies to improve efficacy, survival, persistence, and safety in solid tumors remains an important frontier in cancer immunotherapy. A general discussion of the biological milieu of lung tumors is provided in this chapter along with recent developments in adoptive T-cell therapy (T-CT) research, the molecular basis of effective lung cancer management, as well as challenges and potential future directions for the clinical use of TIL and CAR-T cell therapy against lung cancer.

Kidney dendritic cell activation is required for progression of renal disease in a mouse model of glomerular injury

Article

Full-text available

Jul 2009

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy

Article

Full-text available

Aug 2007

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen–specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

TUMOR-ANTIGENS RECOGNIZED BY T-LYMPHOCYTES

Article

Jan 1994

Transplantation experiments have demonstrated that most mouse tumors express antigens that can constitute targets for rejection responses mediated by syngeneic T lymphocytes. For human tumors, autologous cultures mixing tumor cells and blood lymphocytes or tumor-infiltrating lymphocytes have produced CD8(+) and CD4(+) cytolytic T cell (CTL) clones that recognize tumor cells specifically. Attempts to identify the target antigens by biochemical fractionation of tumor cells up to now have failed, with the important exception of the identification of underglycosylated mucins present on breast and pancreatic carcinomas. Gene transfection approaches have proved more successful. A gene family named MAGE codes for antigens recognized by autologous CTL on a melanoma tumor. These genes are not expressed in normal tissues except for testis. They are expressed in many tumors of several histological types. Differentiation antigens coded by genes such as tyrosinase are also recognized on human melanoma by autologous CTL. The identification of human tumor rejection antigens opens new possibilities for systematic approaches to the specific immune therapy of cancer.

Gout-associated uric acid crystals activate the NALP3 inflammasome

Article

Mar 2007

Spatial separation of cross-presentation and endogenous antigen presentation

Article

Aug 2008

ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells

Conference Paper

Oct 2004

P Guermonprez

Tumor Growth Enhances Cross-Presentation Leading to Limited T Cell Activation without Tolerance

Article

Feb 2002

Linh Tra Nguyen

Using a tumor model of spontaneously arising insulinomas expressing a defined tumor-associated antigen, we investigated whether tumor growth promotes cross-presentation and tolerance of tumor-specific T cells. We found that an advanced tumor burden enhanced cross-presentation of tumor-associated antigens to high avidity tumor-specific T cells, inducing T cell proliferation and limited effector function in vivo. However, contrary to other models, tumor-specific T cells were not tolerized despite a high tumor burden. In fact, in tumor-bearing mice, persistence and responsiveness of adoptively transferred tumor-specific T cells were enhanced. Accordingly, a potent T cell–mediated antitumor response could be elicited by intravenous administration of tumor-derived peptide and agonistic anti-CD40 antibody or viral immunization and reimmunization. Thus, in this model, tumor growth promotes activation of high avidity tumor-specific T cells instead of tolerance. Therefore, the host remains responsive to T cell immunotherapy.

tovirus-infected non-haematopoietic cells requires presentation of exogenous antigen

Article

Systemic Antigen Cross-Presented by Liver Sinusoidal Endothelial Cells Induces Liver-Specific CD8 T-Cell Retention and Tolerization

Article

May 2009
HEPATOLOGY

Unlabelled: Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. Conclusion: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

Cross-presentation: A general mechanism for CTL immunity and tolerance

Article

Sep 1998
Immunol Today

MHC class I-restricted presentation is usually associated with the processing of endogenous antigens. However, this restriction element can present antigens that do not originate within the presenting cells. Here, Francis Carbone and colleagues describe how such cross-presentation is critical for cytotoxic T-cell surveillance of peripheral antigens, both self and foreign, located outside the secondary lymphoid compartment.

Cross-priming in health and disease

Abstract

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