Antigen presenting cells
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Antigen presenting cells. Antigen presenting cells - (1) dendritic cells (2) macrophages (3) B cells. Morphology of Dendritic Cells. Paradigm: Immunity is the result of co-evolution of microorganisms and the immune system. Gram-. Gram+. Fungi. Parasites. Virus. INFECTIOUS NON SELF.

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Antigen presenting cells

Antigen presenting cells

  • Antigen presenting cells -

    (1) dendritic cells

    (2) macrophages

    (3) B cells


Antigen presenting cells

Morphology of Dendritic Cells


Antigen presenting cells

Paradigm: Immunity is the result of co-evolution of

microorganisms and the immune system

Gram-

Gram+

Fungi

Parasites

Virus

INFECTIOUS NON SELF

Dendritic cells (DC):

the sentinels of the immune system


Antigen presenting cells

DC orchestrate both innate and adaptive immunity

  • Phagocytosing microorganisms

  • Participating to inflammatory responses

  • Activating appropriate T cell responses


Antigen presenting cells

Myeloid

precursor

Monocytes

MOUSE DCs

CD8a- and CD8a+

DC

Common

DC

precursor

Myeloid-

Lymphoid

Precursor

HSC

Langerhans

cells

Lymphoid

precursor

?

Plasmacytoid

cells

Monocytes

HUMAN DCs

Myeloid

precursor

CD11b+CD33+

DC

Myeloid-

Lymphoid

Precursor

Common

DC

precursor

HSC

Langerhans

cells

Lymphoid

precursor

Plasmacytoid

cells


Antigen presenting cells

DCs progenitors are generated in the bone marrow.

They give rise to circulating DCs precursors.

Circulating DCs precursors enter nonlymphoid tissues as immature DCs. DC are scattered

throughout all non lymphoid tissues where they reside in a resting, (so-called immature) state.

In the absence of ongoing inflammatory and immune responses, they constantly migrate at

low rate to draining lymph nodes.

DCs precursors

DCs progenitors

Immature DCs

In inflammatory conditions, immature DC migrate

to draining lymph nodes where after maturation

(mature DC), they prime the rare circulating

naïve antigen-specific lymphocytes.

Mature DCs


Antigen presenting cells

There are no lineage-specific surface markers that are expressed on all DC.

Moreover, DC are a heterogenous cell population.

However, it has been clearly demonstrated that at least 3 distinct types of DC can be

generated.

Mice:

myeloid DC CD11b+CD11c+,

myeloid DC CD11b+ CD11c+/-, CD4+,

lymphoid DC CD11b- CD8a+

The existence of different subsets of DC has lead to possibility that they can perform

unique functions.


Antigen presenting cells

Dendritic Cell Subpopulations


Antigen presenting cells

Plasmacytoid Dendritic Cells

  • A specialized subpopulation of DC.

  • Resemble plasma cells in morphology – also express some B cell surface markers (e.g. CD45R [B220]).

  • In response to some viral infections, pDC synthesize high levels of type I IFNs – cytokines have direct antiviral effects and activate NK cells.

  • Plasmacytoid DC also are a significant source of IL-12 during the early response – enhance IFN-g production by NK cells, and, subsequently, CD4 and CD8 T cells.


Antigen presenting cells

Process of Dendritic Cell

Migration to Lymph Nodes


Antigen presenting cells

Growth factor-dependent DC

conditioned medium

GM-CSF

or

GM-CSF

IL-4

bone marrow

or

peripheral blood

Maturation

stimulus

Immature DC

Mature DC

Apoptotic DC


Antigen presenting cells

Phagocytosis by DC

triggered

membrane-

ruffling

conventional

phagocytosis

macro

pinocytosis

forced

endocytosis

coiling

phagocytosis

phagosomes

macro-

pinosomes

spacious

vacuoles

phagosomes

phagosomes/

cytosol/replicative

vacuoles

  • Binding of bacteria to the cell surface

  • Actin polymerizationpseudopod extension around the cell surface

  • Engulpment

  • Actin depolymerization

  • Phagosome maturation transport event


Antigen presenting cells

The innate repertoire: the specificity of DC recognition

is mediated by an extended family of receptors binding

a variety of ligands

Gram- bacteria

Gram+ bacteria

Fungi

viruses

Poli I:C

CpG

LPS/LTA

FcR

CR3

(Mac-1)

FcR

FcR

CD11c

TLR 5: flagellinR

Immature DC

ScavengerR

TLR 2: LTA/ Gram+

MARCO R

TLR 4: LPS/ Gram-

TLR 9: CpG

CD14

Collectins

DEC205

PAMP‘s : Pathogen-Associated Molecular Patterns

conserved structures produced only by microorganisms but not by the host


Antigen presenting cells

Toll-like receptors

bacterial glycolipids &

triacylated bacterial lipopeptides

zymosan, peptidoglycan &

diacylated bacterial lipopeptides

E. coli LPS,

hsp60,

RSV F protein

CpG DNA,

chromatin

dsRNA

Flagellin

Imiquimod

CD14

?

?

TLR2/1

TLR2/6

TLR3

TLR4

TLR5

TLR7

TLR8

TLR9

TLR10


Antigen presenting cells

Toll-like receptor

signaling

pathways


Antigen presenting cells

Toll-like receptor signaling pathways


Antigen presenting cells

Gram-

Gram+

Fungi

Virus

Regulated expression

of key molecules for the

Initiation of adaptive

immune responses

INFECTIOUS NON-SELF

PAMP’s

Mature DC

Immature DC

LPS, LTA, CpG, PGN

cytokines

Maturation process

T cell activation

cytokines

NK

CD8

CD4

Adaptive responses

Innate responses


Antigen presenting cells

MATURATION PROCESS

Migratory activity

Cytoskeleton rearrangements

MHC and costimulatory molecules upregulation


Antigen presenting cells

  • Migration

  • Maturing DCs migrate into the T cell area of lymphoid organs.

Immature Dcs express a variety of chemokine receptors (CCR1, CCR5, CXCR1 and CCR6)

that participate in their recruitment to inflammed tissue and/or to allow their residency into

non-lymphoid tissue.

Exemple: Imm. Dcs express CCR6 the receptor for MIP-3a that is constitutively express

in liver and lungs.

Upon maturation, there is a downregulation of receptors for chemokines produced at the

site of inflammation and upregulation of CCR7. SLC is a ligand for CCR7 and is expressed

at high levels by HEVs in LNs and by stromal cells in T cell areas of many secondary

lymphoid organs. ELC (other ligand of CCR7) made in T cell areas of lymphoid tissue.

SLC (6Ckine) and ELC (MIP-3b) act

together to direct DC migration to T cell areas

Of lymphoid tissue and to promote encounter

with T cells.

Adhesion molecules

Banchereau J. et al. Immunobiology of dendritic cells

Ann. Rev. Immunol. 18:767-811, 2000.


Antigen presenting cells

Immature DC

CCR1

CCR2

CCR4

CCR5

CCR6

CXCR1

CXCR4

Mature DC

CCR7

MIP-1a, RANTES, MCP-3, MIP-5

MCPs

TARC, MDC

MIP-1a, MIP-1b, RANTES

MIP-3a

IL-8

SDF-1

MIP-3b, SLC (6Ckine)

Expression of Chemokine Receptors

on Dendritic Cells

Receptor

Ligand

Abbreviations: DC, dendritic cell; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T cell expressed and secreted; MCP, monocyte chemoattractant protein; TARC, thymus and activation-regulated chemokine; MDC, monophage derived chemokine; SDF, stromal derived factor; IL, interleukin; SLC, secondary lymphoid-tissue chemokine.


Antigen presenting cells

Functional downregulation of chemokine receptor expression

during DC maturation

Control

LPS 0.5 h

LPS 1h

RNAse protection assay

Time

0

30’

1 h

2 h

3 h

24 h

LPS

-

+

+

+

+

+

CCR1

[Ca2+] : nM

L32

CLPS 2 h

LPS 3 h

LPS 24h

GAPDH

20 sec

FURA-2 loaded D1 cells stimulated with MIP-1


Antigen presenting cells

Developmental Stages of

Dendritic Cells

Developmental stages of dendritic cells (DCs) in vivo. The generation of DC precursors in the bone marrow, the recruitment of immature DCs in peripheral tissues, and the migration of DCs into the lymphoid organs are illustrated. The maturation of DCs into potent antigen-presenting cells in case of infection or inflammation and their migration have been amply documented (right), but there is also evidence that in the "steady state“ – that is, in the absence of a "danger signal“ – these immature DCs may migrate into the lymphoid organs while remaining at the immature stage (left). The phenotype of the DC migrating in baseline conditions is still unclear. The movement of maturing DCs and the "constitutive" migration of immature DCs have been shown to depend on chemokine gradients.


Antigen presenting cells

MATURATION PROCESS

Migratory activity

Cytoskeleton rearrangements

MHC and costimulatory molecules upregulation


Antigen presenting cells

Spleen-derived DCs


Antigen presenting cells

control

cells

MARCO-

positive

cells


Antigen presenting cells

Overview of Dendritic Cell

Maturation


Antigen presenting cells

MATURATION PROCESS

Migratory activity

Cytoskeleton rearrangements

MHC and costimulatory molecule upregulation


Antigen presenting cells

Molecules Involved in

T Cell – DC Interactions


Antigen presenting cells

Dendritic Cells in Association

with T Cells in vivo.


Antigen presenting cells

Innate immunity regulates the expression of key molecules for the

initiation of the adaptive immune response

B71/2CD40MHC II MHC l

S. thyphimurium

E. coli

l. monocytogenes

M.smegmatis

BCG

S. aureus

S. pyogenes

S. pneumococus

S. gordonii

Lactococco

Lactobacillo

Mature

Immature

MHC class I

MHC class II

B7.1

B7.2

CD40

2.4G2


Antigen presenting cells

Peptides bind to MHC molecules through structurally related anchor residues


Antigen presenting cells

Peptides that bind MHC class II molecules are variable in lenght


Antigen presenting cells

The expression of MHC molecules differs between tissues


Antigen presenting cells

MHC class I loading

Antigen

Endogenous proteins

Exogenous proteins

+/- Ubiquitin

Proteasome

+ PA28

Golgi

HLA class I synthesis

Peptides

TAP

Calnexin

2m

ERp57

Calreticulin

Tapasin

Endoplasmic reticulum


Antigen presenting cells

M. Rescigno PNAS 1998


Antigen presenting cells

Cross-Presentation of Antigen by

Dendritic Cells

  • Cross-presentation is the ability of dendritic cells to deliver exogenous antigens to the class I MHC processing and presentation pathway for the activation of CD8 T cells.

  • The process is rapid, occurring within 3 to 4 hours after antigen uptake.

  • The process requires a functional endocytic pathway –also requires a functional TAP complex, proteosome function and normal transport from the ER-Golgi to the cell surface.

  • The process is inhibited if the phagocytic and macropinocytic activities of the DC are inhibited.

  • Suggests that the DC degrades exogenous antigen in a lysosomal compartment, and then releases relatively long-length peptides into the cytoplasm, where they are ubiquinated, further cleaved by the proteosome, and then delivered to the ER by TAP.


Antigen presenting cells

Peptides derived from phagocytosed antigens can be presented to CD8+ T cells on MHC class I. Phenomenon called

CROSS-PRESENTATION

Soon after or during formation, phagosomes fuse with the ER. After antigen export to the cytosol and degradation by the proteasome, peptides are translocated by TAP into the lumen of the same phagosomes, before loading on phagosomal MHC class I molecule. Therefore, cross-presentation in dendritic cells occurs in a specialized, self-sufficient, ER-phagosome mix compartment.


Antigen presenting cells

Dendritic Cells Deliver Exogenous

Antigens to the Class I MHC Pathway


Antigen presenting cells

The Class II MHC Antigen

Processing and Presentation Pathway


Antigen presenting cells

Function of HLA-DM

(H-2M in Mice)


Antigen presenting cells

HLA-DM is Physically Located

In the MIIC Vesicle


Antigen presenting cells

M. Rescigno PNAS 1998


Antigen presenting cells

Phenotypic Changes Associated

with Antigen Presentation


Antigen presenting cells

Immature DC are less efficient in MLR

when compared to mature DC

MLR

cpm x 10-3

120

mature

immature

100

80

60

40

20

0

0,1

1

100

10

APC (x 10-3)


Antigen presenting cells

.

Allogeneic CD4 T cell proliferation

CD4

iDC

No DCs

mDC

48 h

CFSE

72 h

FSC


Antigen presenting cells

Allogeneic CD8 T cell proliferation

CD8

iDC

No DCs

mDC

48 h

CFSE

72 h

FSC


Antigen presenting cells

Comparison of Antigen Presentation

Abilities of DCs and Macrophages


Antigen presenting cells

Functional Features of Dendritic Cells

Potency

Small numbers of DCs pulsed with low doses of antigen stimulate strong T-cell responses.

Primary responses

Naive and quiescent T cells can be activated with antigens on DCs.

Physiology

CD4+ T helpers and CD8+ T killers are primed in vivo.


Antigen presenting cells

DC are involved in the tolerization of peripheral T cells


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