Paradigm: Immunity is the result of co-evolution of
microorganisms and the immune system Gram-Gram+Fungi INFECTIOUS NON
SELF Virus Parasites Dendritic cells (DC): the sentinels of the
immune system
Slide 4
DC orchestrate both innate and adaptive immunity Phagocytosing
microorganisms Participating to inflammatory responses Activating
appropriate T cell responses
Slide 5
HSC Myeloid- Lymphoid Precursor Myeloid precursor Lymphoid
precursor Common DC precursor Monocytes CD8 - and CD8 + DC
Langerhans cells Plasmacytoid cells ? MOUSE DCs HUMAN DCs HSC
Myeloid- Lymphoid Precursor Myeloid precursor Common DC precursor
Lymphoid precursor Monocytes CD11b+CD33+ DC Langerhans cells
Plasmacytoid cells
Slide 6
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 Immature DCs DCs progenitors
Mature DCs In inflammatory conditions, immature DC migrate to
draining lymph nodes where after maturation (mature DC), they prime
the rare circulating nave antigen-specific lymphocytes.
Slide 7
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-
CD8 + The existence of different subsets of DC has lead to
possibility that they can perform unique functions.
Slide 8
Dendritic Cell Subpopulations
Slide 9
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- production by NK cells, and, subsequently, CD4 and CD8
T cells.
Slide 10
Process of Dendritic Cell Migration to Lymph Nodes
Slide 11
Growth factor-dependent DC Immature DCMature DCApoptotic DC
GM-CSF or GM-CSF IL-4 conditioned medium bone marrow or peripheral
blood Maturation stimulus
Slide 12
conventional phagocytosis macro pinocytosis triggered membrane-
ruffling forced endocytosis coiling phagocytosis phagosomesmacro-
pinosomes spacious vacuoles phagosomesphagosomes/
cytosol/replicative vacuoles Binding of bacteria to the cell
surface Actin polymerizationpseudopod extension around the cell
surface Engulpment Actin depolymerization Phagosome maturation
transport event Phagocytosis by DC
Slide 13
Slide 14
The innate repertoire: the specificity of DC recognition is
mediated by an extended family of receptors binding is mediated by
an extended family of receptors binding a variety of ligands
Immature DC Fc R Fc R Fc R CR3 (Mac-1) CD11c ScavengerR TLR 4: LPS/
Gram- DEC205 CD14 Collectins PAMPs PAMPs : Pathogen-Associated
Molecular Patterns TLR 2: LTA/ Gram+ Gram - bacteria Gram +
bacteria Fungi viruses Poli I:C CpG LPS/LTA TLR 9: CpG TLR 5:
flagellinR conserved structures produced only by microorganisms but
not by the host MARCO R
Slide 15
TLR2/1TLR2/6TLR3TLR4TLR7TLR8TLR10TLR9TLR5 zymosan,
peptidoglycan & diacylated bacterial lipopeptides E. coli LPS,
hsp60, RSV F protein bacterial glycolipids & triacylated
bacterial lipopeptides ?? Flagellin CpG DNA, chromatin dsRNA
Imiquimod Toll-like receptors CD14
Slide 16
Toll-like receptor signaling pathways
Slide 17
Toll-like receptor signaling pathways
Slide 18
Maturation process Gram-Gram+Fungi INFECTIOUS NON-SELF PAMPs
cytokines Virus NK Mature DC T cell activation CD4 CD8 Immature DC
Innate responses Adaptive responses LPS, LTA, CpG, PGN Regulated
expression of key molecules for the Initiation of adaptive immune
responses cytokines
Slide 19
MATURATION PROCESS Migratory activity Cytoskeleton
rearrangements MHC and costimulatory molecules upregulation
Slide 20
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.
Slide 21
Expression of Chemokine Receptors on Dendritic Cells
ReceptorLigand Immature DC CCR1 CCR2 CCR4 CCR5 CCR6 CXCR1 CXCR4
Mature DC CCR7 MIP-1 , RANTES, MCP-3, MIP-5 MCPs TARC, MDC MIP-1 ,
MIP-1 , RANTES MIP-3 IL-8 SDF-1 MIP-3 , SLC (6Ckine) 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.
Slide 22
ControlLPS 0.5 hLPS 1h CLPS 2 hLPS 3 hLPS 24h 20 sec [Ca 2+ ] :
nM Functional downregulation of chemokine receptor expression
during DC maturation FURA-2 loaded D1 cells stimulated with MIP-1
L32 GAPDH CCR1 0301 h2 h3 h24 h Time LPS -+++++ RNAse protection
assay
Slide 23
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.
Slide 24
MATURATION PROCESS Migratory activity Cytoskeleton
rearrangements MHC and costimulatory molecules upregulation
Slide 25
Spleen-derived DCs
Slide 26
control cells MARCO- positive cells
Slide 27
Overview of Dendritic Cell Maturation
Slide 28
MATURATION PROCESS Migratory activity Cytoskeleton
rearrangements MHC and costimulatory molecule upregulation
Slide 29
Molecules Involved in T Cell DC Interactions
Slide 30
Dendritic Cells in Association with T Cells in vivo.
Slide 31
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 Immature Mature MHC class II MHC class I B7.1
B7.2CD402.4G2
Slide 32
Slide 33
Peptides bind to MHC molecules through structurally related
anchor residues
Slide 34
Peptides that bind MHC class II molecules are variable in
lenght
Slide 35
The expression of MHC molecules differs between tissues
Slide 36
Golgi Proteasome + PA28 Peptides +/- Ubiquitin Endogenous
proteins Exogenous proteins Antigen HLA class I synthesis 2m ERp57
Calreticulin Tapasin Endoplasmic reticulum Calnexin TAP MHC class I
loading
Slide 37
M. Rescigno PNAS 1998
Slide 38
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.
Slide 39
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.
Peptides derived from phagocytosed antigens can be presented to
CD8+ T cells on MHC class I. Phenomenon called
CROSS-PRESENTATION
Slide 40
Dendritic Cells Deliver Exogenous Antigens to the Class I MHC
Pathway
Slide 41
The Class II MHC Antigen Processing and Presentation
Pathway
Slide 42
Function of HLA-DM (H-2M in Mice)
Slide 43
HLA-DM is Physically Located In the MIIC Vesicle
Slide 44
M. Rescigno PNAS 1998
Slide 45
Phenotypic Changes Associated with Antigen Presentation
Slide 46
Immature DC are less efficient in MLR when compared to mature
DC 20 40 60 80 100 120 100 10 10,1 cpm x 10 -3 0 APC (x 10 -3 ) MLR
mature immature
Slide 47
. mDC iDC No DCs CD4 CFSE 48 h 72 h FSC Allogeneic CD4 T cell
proliferation
Slide 48
iDC mDC CD8 48 h 72 h No DCs FSC CFSE Allogeneic CD8 T cell
proliferation
Slide 49
Comparison of Antigen Presentation Abilities of DCs and
Macrophages
Slide 50
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.
Slide 51
Differentially expressed genes during DC maturation mostly
encode enzymes and protein involved in the control of: 1) cell
cycle 2) cytoskeleton rearrangements 3) antigen processing 4)
inflammation 5) apoptosis
Slide 52
NK cells activation Immature DC MIP-1 MIP-1 MIP-2 Mature DC
Early-activated DC IL- 12 CD4 + T cell priming Apoptotic DC IL-1
TNF- RANTES MIP-1 MCP-1 Immature DC Transitional DC Innate immune
response 0 h2 h4 h6 h8 h18 h Time actin cytoskeleton rearrangments
actin cytoskeleton rearrangments MIAP1 MIAP2 TRAF1 TRAF2 Adaptive
immune response CD4 + T cell priming CTL priming CD40 B71/2
Peptide+MHCI/II increase of DC processing efficiency IL6 IL10
INFECTIOUS NON-SELF
Slide 53
DC T cell CD40L CD40 receptor-mediated endocytosis antigen
processing pathways MHC II TCR CD28 B7-1/2 phagocytosis DC-T
interactions after antigen uptake fluid-phase-endocytosis
macropinocytosis NF B activation MHC I soluble mediators cytokine
receptors CD95 (Fas) FasL Live bacteria Toxins OX40L OX40 IMMUNE
SYNAPSIS IL2
Slide 54
Kinetic of a bacterial-induced DC transcriptome: functional
clustering of differentially expressed genes INFLAMMATION /
CHEMOKINES / CYTOKINES Increasing expression 0 h 4 h 6 h 12 h 18 h
24 h 48 h AMYLOID A TGF Flt3 IL6 MIP-1 RANTES IL12p40 MIP-1 MIP2
IL1 IL1RA TNF EGF-BP C1qC C1qB C1q MCP5 PDGF LT JE mvrf186 0 h 4 h
6 h 12 h 18 h 24 h 48 h TGF 1 C3 IL15 IL10 0 h 4 h 6 h 12 h 18 h 24
h 48 h LT VEGF IP-10 MCSF IL12p35 GM-CSF 0 h 4 h 6 h 12 h 18 h 24 h
48 h IL2 Nature - Imm. 2, 882-888, 2001
Slide 55
0 100 200 300 01020304050 mBM-DC Macrophages hours IL-2 is
produced by BM-derived DC following bacterial activation but NOT by
macrophages pg/ml Nature - Imm. 2, 882-888, 2001
DCs produce IL-2 after contact with microbial stimuli but not
after contact with inflammatory cytokines DCs are able to
discriminate between a cytokine-mediated inflammatory process and
the presence of infections
Slide 58
Microbial-MPs Receptors Chemokines Cytokines NK T cell
activation CD4 CD8 Immature DC INNATE response ADAPTIVE response
Chemokines Cytokines NKT IL2 Trends in Immunol.23(4) 2002 IL-2 is a
T, NK and B cell growth factor DC-derived IL-2 may be relevant for
NK, T and B cell activation IFN
Slide 59
NK cells activation Immature DC MIP-1 MIP-1 MIP-2 Mature DC
Early-activated DC IL- 12 CD4 + T cell priming CTL priming
Apoptotic DC IL-1 TNF- RANTES MIP-1 MCP-1 Immature DC Transitional
DC Innate immune response maturation time IL2 0 h2 h4 h6 h8 h18 h
Time actin cytoskeleton rearrangments actin cytoskeleton
rearrangments MIAP1 MIAP2 TRAF1 TRAF2 Adaptive immune response CD4
+ T cell priming CTL priming CD40 B71/2 Peptide+MHCI/II increase of
DC processing efficiency IL6 IL10 IL2 Gram- Gram+ Parasites
INFECTIOUS NON-SELF IL2 yeast
Slide 60
DC are involved in the tolerization of peripheral T cells
