Immunologia – Cellule Dendritiche

Cellule dendritiche e cellule natural killer: le fasi iniziali della risposta immune

Prof. Guido Ferlazzo
Università degli Studi di Messina
Corso di Laurea in Medicina e Chirurgia
6 Aprile, 2006

Fasi iniziali della risposta immune

Innate

Adherence to epithelium
Macrophages
Dendritic Cells

Local infection, penetration of epithelium

Macrophages
Dendritic Cells
Natural killer cells

Adaptive

Local infection of tissues
T cells
B cells
Protection against infections

Antigen capture

• Epithelial border
• Immature DC
• DC precursors
• MF
• NK
• Eosinophils
• Cytokines

Antigen presentation

• Migration maturation
• Lymphoid organ
• Lymphocyte selection
• Mature DC
• DC apoptosis
• Lymphocyte activation

CTL helper and regulatory T cells

Il secondo segnale (costimolo)

MHC + antigens
APC
IL-2R
Costimulus
T lymphocytes
IL-2

Molecole coinvolte nella sinapsi DC/T

• Adhesion Molecules: ICAM-1, DC-SIGN, LFA-1, ICAM-3
• Semaphorins: Neuropilin-1, SEMA-4A
• B7 Family: B7-1, B7-2, B7-H1, B7-DC, B7RP-1, B7-H3, B7x
• CD28 Family: CD28, CTLA-4, PD-1, ICOS, BTLA
• TNF Family: 4-1BBL, CD27, LOX40L, LIGHT
• TNFR Family: 4-1BB, CD27, OX40, HVEM
• CD40
• Other Mediators: TXA₂, TP

Antigene e costimolo sono necessari per l'attivazione dei T linfociti.

Malignant tumor cells

Expressing TRA but no co-stimulatory molecules

• Naive CD8 T cells specific for TRA cannot be activated by the tumor cells and may be rendered anergic
• Tumor grows progressively
• Transfect tumor cell with B7
• Tumor cells expressing B7 can activate TRA-specific CD8 T cells
• Activated CD8 T cells eliminate tumor
• Mouse can now reject parental B7-negative or GM-CSF-negative tumor
• Transfect tumor cell with GM-CSF
• GM-CSF recruits dendritic cells, which can present tumor antigens to T cells

Processazione dell'antigene

• MHC di classe I
• Peptidi
• Complesso del Golgi
• Peptide
• Calreticolina
• Proteasoma
• Proteina
• TAP
• Tapasina
• Antigene esogeno
• Comparti endocitici
• CLIP
• Catena invariante digerita
• Reticolo endoplasmatico rugoso
• Calnessina
• MHC di classe II
• Nucleo
• Catena invariante
• Via endogena (MHC di classe I)
• Via esogena (MHC di classe II)

Processazione dell'antigene e presentazione mediante molecole MHC-simili

• Peptide antigenico
• Lipide antigenico
• CD1
• MHC di classe I
• MHC di classe II
• Compartimenti endocitici
• Reticolo endoplasmatico rugoso
• TAP
• Peptidi
• Batteri intracellulari
• Lipidi
• Endosoma
• Batteri

Il fenomeno del "Cross-priming"

Un'eccezione delle cellule dendritiche alle vie classiche di presentazione

• Apoptotic (or necrotic?) cells in peripheral tissues
• DC migration to draining lymph node
• Mature DC stimulate Virus/tumor-specific CTLs

Progenitori e precursori ematopoietici

PROGENITOR
BLOOD PRECURSOR
CD34+ myeloid
CD34+ lymphoid
CD14+ CD11c+ CD1-Monocytes
GM-CSF IL-4
M-CSF
CD14- CD11c+ CD1+
GM-CSF IL-4 TGFβ
M-CSF
IL-3

Fattori di induzione

• GM-CSF
• IL-3
• IL-4
• Viruses

Proprietà delle cellule dendritiche immature

• ANTIGEN CAPTURE
• High intracellular MHC II
• Endocytosis
• Phagocytosis
• High CCR1, CCR5, CCR6
• Low CCR7
• Low CD54, 58, 80, 86
• Low CD40
• Low CD83, No DC-LAMP

Proprietà delle cellule dendritiche mature

• ANTIGEN PRESENTATION
• High surface MHC II
• Low endocytosis
• Low phagocytosis
• Low CCr1, CCR5, CCR6
• High CCR7
• High CD54, 58, 80, 86
• High CD40
• High CD83, high DC-LAMP, high p55

Struttura di un linfonodo

• Secondary lymphoid follicle (with germinal center)
• Afferent lymphatic vessel
• Paracortical area (mostly T cells)
• Germinal center
• Primary lymphoid follicle (mostly B cells)
• Medullary cords (macrophages and plasma cells)
• Medullary sinus
• Artery
• Vein
• Efferent lymphatic vessel
• Senescent germinal center
• Marginal sinus

Percorso delle cellule dendritiche

• Mature DCs travel to lymph nodes
• Dendritic cells in peripheral tissues
• Dendritic cells in the lymphatic circulation
• Dendritic cells in lymphoid tissues

T cells enter lymph node across high endothelial venules in the cortex.
T cells monitor antigen presented by macrophages and dendritic cells.
T cells that do not encounter specific antigen leave lymph node through lymphatics.
T cells that encounter specific antigen proliferate and differentiate to effector cells.

Immunofluorescenza

Per cellule dendritiche e per macrofagi

NK cells and DCs colocalize in T cell areas of normal human lymph node

CD20, DEC205, CD3, CD56

Chemokine receptors on dendritic cells

CD34+ progenitors

• BONE MARROW
• BLOOD
• CCR6+ CCR7-TISSUES
• MIP-3_α
• PATHOGEN ENTRY
• Immature DC
• 6Ckine
• CCR6- CCR7+DR. LYMPH NODES
• CCR6- CCR7++mature DC
• MIP-3_β

Cellule dendritiche

1. Catturano gli antigeni nei tessuti periferici
2. Migrano negli organi linfoidi secondari
3. Presentano gli antigeni ai linfociti T

Cellule natural killer

1. Sono la terza popolazione linfocitaria
2. Riconoscono i loro bersagli mediante la somma di segnali ricevuti attraverso recettori attivatori ed inibitori
3. Contengono le infezioni virali e la progressione tumorale finché l'immunità acquisita (adaptive immunity) non è effettiva

NK limit viral load and tumor burden prior to the initiation of the adaptive immune response.

• Production of IFNα, IFNβ, TNFα and IL-12
• NK cell mediated killing of infected cells
• T cell mediated killing of infected cells

Virus titer

Time after viral infection (days)

NK burst precedes T cell activation during viral infections

B cell receptor

Membrane-bound IgM (mIgM) recognition
Light chain
Heavy chain
Igß Igα signaling

T cell receptor

TCR recognition
CD3
CD3α βε δγ ε
ITAMsζ ζsignaling

I linfociti B riconoscono

Determinanti conformazionali ed esterni dell'antigene

• Epitopi B: formati da aa localizzati in punti diversi della catena amino acidica ed avvicinati dal ripiegamento della molecola
• Epitopi T: aa in sequenza

Recettori inibitori delle cellule NK

• KIR: killer immunoglobulin-like receptor (killer inhibitory receptors)
• LIR-1/ILT2
• NKG2A

Ligands: HLA-A3, -A11, HLA-Bw4, HLA-C, all MHC class I, HLA-E

Recettori attivatori delle cellule NK

• Ligands: MICA/B, ULBPs, CD48, PVR CD155, Nectin-2 CD112, NKp46, NKp30, NKp44, NKG2D, 2B4, NTBA, DNAM-1, NKp80
• Receptors: IgV IgC Lectin domain ITAM YxxM (PI3-K binding motif) Tyr-based motif SAP SHP-1 SHP-2

Mechanisms of NK cell functions

Regulated by a balance between activating and inhibitory signals

• Inhibitory receptor
• Activating receptor
• SHP1
• ZAP 70

Physiological conditions
Pathological conditions

• Class I MHC IR No lysis Normal cell AR ligand AR NK cell IR Lysis Neoplastic cell Virus infected cell Class I MHC AR ligand AR NK cell

Sinapsi linfocita-cellula bersaglio

• Nucleo
• Monomeri di perforina
• Granulo
• CTL
• Poro
• Perforina polimerizzata
• Cellula bersaglio

Receptors

• NCRs (NKp30, p44, p46) +
• KIRs, CD94, ILT2 -
• 2B4, NTBA +
• NKG2D +

Ligands: ?, HLA, CD48, ?, MICA, MICB, ULPBs

NK cell and target cell interactions

NK STOP CD16 KIR Class I MHC Natural Killing ADCC Target ADCC Antibody Dependent Cell Cytotoxicity Citotossicità anticorpo-dipendente

La cellula citotossica che effettua ADCC

Deve esprimere sulla membrana un recettore Fc per le Ig.
Le cellule NK hanno il CD16 e lisano cellule “aberranti” anche via ADCC.

Meccanismi di citotossicità dell'immunità innata

• Enzimi litici
• Perforina
• TNF
• Granzimi
• Neutrofilo
• Eosinofilo
• Recettore per Fc
• Macrofago
• Cellula NK
• Cellula bersaglio
• Antigene di superficie (Ag)
• Ag legato all'Ab legato al recettore per l' Fc

Subsets of natural killer cells

Division of labor

• CD56^brightCD16^- NK cells: Cytokine producers: IFNγ, TNF, GM-CSF, CD56_hi c-KIT CD25 KIR CCR7 CD94
• CD56^dimCD16⁺ NK cells: Killers: CD56_low KIR CD16

Colucci et al., Nat Rev Immunol 2003
Natural killer cell content in secondary lymphoid organs
Peripheral Blood
Lymph Node
Spleen
Tonsil

Inflamed!
CD3
CD56

NK% of total cells in the organ:
20 %
5 %
17 %
0.4 %
(7-25%)
(1-7%)
(7-50%)
(0.2-0.7%)

Ferlazzo et al, J.Immunol. 2004

Distribution of lymphocytes estimated in a normal young adult man

• 2.2 % Blood
• 41.3 % Lymph Nodes
• 15.2 % Spleen
• 4.3 % Gut lymphoid tissue
• 10.9 % Thymus
• 10.9 % Bone Marrow
• 15.2 % Other Tissues

F.Trepel, 1974

Natural killer cell content in secondary lymphoid organs

• Peripheral Blood
• Lymph Node
• Spleen
• Tonsil

CD3
CD56
95 : 5
25 : 75
85 : 15
25 : 75

Inflamed!
NK% of total cells in the organ: 20 % (7-25%), 5 % (1-7%), 17 % (7-50%), 0.4 % (0.2-0.7%)

Estimated number of total NK cells resident in this organ(s):
(Westermann and Papst, Clin Investig 1992)
2 x 10⁹
10 x 10⁹
13 x 10⁹
< 10⁹

Ferlazzo et al, J.Immunol. 2004

NK limit viral load and tumor burden prior to the initiation of the adaptive immune response

• Production of IFNα, IFNβ, TNFα and IL-12
• NK cell mediated killing of infected cells
• T cell mediated killing of infected cells

Virus titer
Janeway and Travers, 1994

NK burst precedes T cell activation during viral infections

La cellula dendritica è responsabile dell'attivazione precoce delle cellule NK.

Le cellule dendritiche inducono le cellule NK a:

• Proliferare
• Secernere IFN^γ
• Potenziare la citotossicità

Ferlazzo et al. J.Exp.Med., 2002
Fernandez et al. Nat.Immunol., 2000
Piccioli et al. J.Exp.Med., 2002
Ferlazzo et al. Eur.J.Immunol., 2003

IFN_γ secretion
Activation of cytotoxicity
Proliferation and survival

TNF_α Other soluble molecules?
mbIL-15
NK activating receptor-ligand interaction?
IL-12
TNF_α
Maturation
IFN_α Other soluble molecules?

Subsets di linfociti T

• CD8 T cells: peptide + MHC class I
• CD4 T cells: peptide + MHC class II

Cytotoxic (killer) T cells

cytotoxins
virus-infected cell

TH₁ cells

cytokines
macrophage

TH₂ cells

cytokines
antigen-specific B cell

Cytotoxic effector molecules

• Perforin
• Granzymes
• Fas ligand
• Others: IFN-γ, TNF-β, TNF-α

Macrophage-activating effector molecules

• IFN-γ
• GM-CSF
• TNF-α
• CD40 ligand
• Fas ligand
• Others: IL-3, TNF-β (IL-2)

B-cell-activating effector molecules

• IL-4
• IL-5
• CD40 ligand
• Others: IL-3, GM-CSF, IL-10, TGF-β, Eotaxin