CORSO DI IMMUNOLOGIA
per il corso di Laurea in biotecnologie
a.a. 2005-2006 II semestre
DOCENTE:
Dott.ssa Vladia Monsurrò
Dipartimento di patologia
Sezione di immunologia
Universita’ degli Studi di Verona
045 8074256
[email protected]
ORARIO DELLE LEZIONI:
lunedì:
4:30-6:00
giovedì
4:30-6:00
dal 20 marzo al 25 maggio 2006
aula F Facolta’ di Scienze
DOVE TROVARE LE LEZIONI on
line:
http://www.scienze.univr.it/fol/main?ent=oi&cs=6&id=20061&lang=it
In “materiale didattico”
Per qunato riguarda il programma e l’esame e’ come discusso in classe
PROGRAMMA LEZIONI
Mar 21 Marzo lezione 1
Generalità sul corso
Il sistema immunitario (Introduzione)
Giov 23 Marzo lezione 2
Cellule e tessuti del sistema immunitario
Lun 27 Marzo lezione 3
Riconoscimento dell’antigene:
Molecole del Sistema Immunitario:
Antigeni e anticorpi
Giov 30 Marzo lezione 4
MHC (Major histocompatibility complex)
Lun 3 Aprile lezione 5
MHC-Antigene:
Sua
captazione,
processamento e presentazione.
Giov 6 Aprile lezione 6
TCR
Antigeni tumorali e immunoterapia
preparazione del talk)
(in
Lun 10 Aprile lezione 7
Maturazione, attivazione e regolazione dei
linfociti:
Maturazione dei linfociti ed espressione dei
recettori (genetica Ig e TCR)
Giov 13 Aprile lezione 8
Immunita’ e tumori
Parmiani Talk (ore 17:00)-vedi volantino
Giov 20 Aprile lezione 9
Attivazione
segnale.
linfocitaria,
trasduzione
del
Lun 24 Aprile lezione 10
Attivazione dei linfociti B e produzione di Ab
Giov 27 Aprile lezione 11
Tolleranza immunologica
Giov 4 Maggio lezione 12
Meccanismi effettori
Citochine, Immunita’ innata
Lun 8 Maggio lezione 13
Meccanismi effettori dell’immunita’ cellulomediata, reazioni DTH
Giov 11 Maggio lezione 14
Meccansimi effettori dell’immunita’ umorale
complemento
Lun 15 Maggio lezione 15
Rapporti tra fisiologia e patologia
Ipersensibilità concetti generali
Ipersensibilità di tipo I
Giov 18 Maggio lezione 16
Test prove esami-recupero (metodiche di lab)
Merc 7 giugno e 28 giugno
ESAMI
MHC restriction of cytolytic T lymphocytes. Virus-specific cytolytic T lymphocytes (CTLs) generated from virus-infected
strain A mice kill only syngeneic (strain A) target cells infected with that virus. The CTLs do not kill uninfected strain A targets
(which express self peptides but not viral peptides) or infected strain B targets (which express different MHC alleles than
does strain A). By use of congenic mouse strains that differ only at class I MHC loci, it has been proved that recognition of
antigen by CD8+ CTLs is self class I MHC restricted
Antigen-presenting cells are required for T cell activation. Purified CD4+ T cells do not respond to a protein
antigen by itself but do respond to the antigen in the presence of an antigen-presenting cell (APC). The function of
the APCs is to present a peptide derived from the antigen to the T cell. APCs also express costimulators that are
important for T cell activation; these are not shown.
CAPTAZIONE, PROCESSAMENTO E
PRESENTAZIONE DELL'ANTIGENE
• E‘ il meccanismo attraverso il quale avviene l'attivazione dei
linfociti T da parte di cellule accessorie dette Antigen
Presenting Cells (APC).
• L’attivazione dei linfociti T naive si svolge in un
microambiente complesso, gli organi linfoidi secondari, dove i
movimenti delle APC e dei linfociti T sono governati da
limitazioni anatomiche.
• Le APC professioniste più efficienti nell’attivazione dei linfociti
T naive sono le Cellule Dendritiche (DC).
Functions of different antigen-presenting cells. The three major types of antigen-presenting cells for CD4+ T
cells function to display antigens at different stages and in different types of immune responses. Note that effector T
cells activate macrophages and B lymphocytes by production of cytokines and by expressing surface molecules;
these will be described in later chapters.
INGRESSO
DELL’ANTIGENE:
• pelle
• tratto
gastrointestinale
• tratto
respiratorio
Cellule dendritiche
(DC)
DENDRITIC CELLS (DC)
DC al
microscopio
ottico
DC della cute,
Cellule di
Langerhans
(LC)
DC al
microscopio
elettronico
a scansione
DC nei
LN
Interazione di una DC con un
linfocita T nel linfonodo
DC: rossa
T: verde
INTERAZIONE APC- LINFOCITI T:
presentazione dell’antigene
SINAPSI
IMMUNOLOGICA
Sull’APC, quando il TCR
Riconosce il complesso
MHC-peptide, numerose
proteine di membrana
e intracellulari vengono
mobilizzate rapidamente.
La regione di contatto
fisico tra APC e cellula T
È chiamata SINAPSI
IMMUNOLOGICA o
CLUSTER di ATTIVAZIONE SOPRAMOLECOLARE (SMAC).
TCR, CD3, catene  , i
corecettori CD4 o CD8, e
il CD28 sono mobilizzati
al centro della sinapsi.
Le integrine restano
invece alla sua periferia.
cSMAC= central
Supramolecular
Cluster
pSMAC= periferal
Supramolecular
Cluster
Analisi in “imaging al microscopio confocale ”
della formazione della sinapsi immunologica
a)
b)
c)
d)
Una cell.T specifica per un determinato complesso MHC-peptide lega un’APC.
CD3 (verde) si localizza sullo cSMAC.
L’integrina LFA-1 (rosso) invece si localizza sullo pSMAC.
CD3 è al centro e LFA-1 è alla periferia della sinapsi immunologica.
CAPTAZIONE DELL'ANTIGENE NELL’EPIDERMIDE
Processamento
dell’antigene
captato in periferia
BIOCHIMICA DELLA PROCESSAZIONE
E PRESENTAZIONE DELL'ANTIGENE
each different MHC
molecule expressed can bind a
different peptide produced by the
processing of the same antigen
3 distinct pathways:
1
3
2
From: William E. Paul "Fundamental Immunology", Fourth ed. Lippincott, Williams & Wilkins, 1999.
I compartimenti intracellulari di
endocitosi e secrezione giocano un ruolo
fondamentale nei tre processi
PROCESSAZIONE
E PRESENTAZIONE Ag VIA MHC-II
Antigen processing requires time and cellular metabolism and can be mimicked by in vitro proteolysis. If an antigenpresenting cell (APC) is allowed to process antigen and is then chemically fixed (rendered metabolically inert) 3 hours or
more after antigen internalization, it is capable of presenting antigen to T cells (A). Antigen is not processed or presented if
APCs are fixed less than 3 hours after antigen uptake (B). Fixed APCs bind and present proteolytic fragments of antigens to
specific T cells (C). The artificial proteolysis therefore mimics physiologic antigen processing by APCs. Effective antigen
presentation is assayed by measuring a T cell response, such as cytokine secretion. (Note that this type of experiment is
done with populations of antigen-specific T cells, such as T cell hybridomas, which respond to processed antigens on fixed
APCs, but that normal T cells require costimulators that may be destroyed by fixation. Also, the time required for antigen
processing is 3 hours in this experiment, but it may be different with other antigens and APCs.)
PROCESSAZIONE
E PRESENTAZIONE Ag VIA MHC-I
Pathways of antigen processing and presentation. In the class II MHC pathway (top panel), extracellular protein
antigens are endocytosed into vesicles, where the antigens are processed and the peptides bind to class II MHC
molecules. In the class I MHC pathway (bottom panel), protein antigens in the cytosol are processed by
proteasomes, and peptides are transported into the endoplasmic reticulum (ER), where they bind to class I MHC
molecules. Details of these processing pathways are in Figures 5-10 and 5-14. TAP, transporter associated with
antigen processing.
Presentation of extracellular and cytosolic antigens. When a model protein ovalbumin is added as an extracellular
antigen to an antigen-presenting cell that expresses both class I and class II MHC molecules, ovalbumin-derived peptides
are presented only in association with class II molecules (A). When ovalbumin is synthesized intracellularly as a result of
transfection of its gene (B), or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock
(C), ovalbumin-derived peptides are presented in association with class I MHC molecules. The measured response of
class II-restricted helper T cells is cytokine secretion, and the measured response of class I-restricted CTLs is killing of the
antigen-presenting cells
Presentation of extracellular and cytosolic antigens. When a model protein ovalbumin is added as an extracellular
antigen to an antigen-presenting cell that expresses both class I and class II MHC molecules, ovalbumin-derived peptides
are presented only in association with class II molecules (A). When ovalbumin is synthesized intracellularly as a result of
transfection of its gene (B), or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock
(C), ovalbumin-derived peptides are presented in association with class I MHC molecules. The measured response of
class II-restricted helper T cells is cytokine secretion, and the measured response of class I-restricted CTLs is killing of the
antigen-presenting cells
Presentation of extracellular and cytosolic antigens. When a model protein ovalbumin is added as an extracellular
antigen to an antigen-presenting cell that expresses both class I and class II MHC molecules, ovalbumin-derived peptides
are presented only in association with class II molecules (A). When ovalbumin is synthesized intracellularly as a result of
transfection of its gene (B), or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock
(C), ovalbumin-derived peptides are presented in association with class I MHC molecules. The measured response of
class II-restricted helper T cells is cytokine secretion, and the measured response of class I-restricted CTLs is killing of the
antigen-presenting cells
Presentation of extracellular and cytosolic antigens. When a model protein ovalbumin is added as an extracellular
antigen to an antigen-presenting cell that expresses both class I and class II MHC molecules, ovalbumin-derived peptides
are presented only in association with class II molecules (A). When ovalbumin is synthesized intracellularly as a result of
transfection of its gene (B), or when it is introduced into the cytoplasm through membranes made leaky by osmotic shock
(C), ovalbumin-derived peptides are presented in association with class I MHC molecules. The measured response of
class II-restricted helper T cells is cytokine secretion, and the measured response of class I-restricted CTLs is killing of the
antigen-presenting cells
PROCESSAZIONE DI ANTIGENI ENDOCITATI E
PRESENTATI VIA MHC-II
INVARIANT CHAIN (Ii)
trimero Ii
trimero Ii,  E  MHC
 L‘iniziale assemblaggio nel RE dell‘eterodimero MHC-II è indipendente
dall’associazione con il peptide, perchè il complesso è stabilizzato da Ii.
 Ii contiene il segmento CLIP (MHC-CLassII associated Invariant
chain Peptide), che protegge la tasca del peptide finchè la molecola non
arriva alle vescicole endocitiche.
more acidic
GILT = IFN--Inducible
Lysosomal Thiol reductase,
reduce disulphide bonds
Cathepsins digest Ii
The functions of class II MHC-associated invariant chains and HLA-DM. Class II molecules with bound invariant chain,
or CLIP, are transported into vesicles (the MIIC/CIIV), where the CLIP is removed by the action of DM. Antigenic peptides
generated in the vesicles are then able to bind to the class II molecules. Another class II-like protein, called HLA-DO, may
regulate the DM-catalyzed removal of CLIP. CIIV, class II vesicle; CLIP, class II-associated invariant chain peptide; ER,
endoplasmic reticulum; Ii, invariant chain; MIIC, MHC class II compartment
Morphology of class II MHC-rich endosomal vesicles. A. Immunoelectron micrograph of a B lymphocyte that has
internalized bovine serum albumin into early endosomes (labeled with 5-nm gold particles, arrow) and contains class II MHC
molecules (labeled with 10-nm gold particles) in MIICs (arrowheads). The internalized albumin will reach the MIICs
ultimately. (From Kleijmeer MJ, S Morkowski, JM Griffith, AY Rudensky, and HJ Geuze. Major histocompatibility complex
class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments. The Journal of
Cell Biology 139:639-649, 1997, by copyright permission of The Rockefeller University Press.) B. Immunoelectron
micrograph of a B cell showing location of class II MHC molecules and DM in MIICs (stars) and invariant chain concentrated
in the Golgi (G) complex. In this example, there is virtually no invariant chain detected in the MIIC, presumably because it
has been cleaved to generate CLIP. (Photographs courtesy of Drs. H. J. Geuze and M. Kleijmeer, Department of Cell
Biology, Utrecht University, The Netherlands.)
Associazione dei complessi MHC-II-peptide a microdomini lipidici della
membrana plasmatica (lipid rafts) costitutiti da colesterolo interdigitato a
glicosfingolipidi e sfingomielina
-Rhodamine-CT lega i gangliosidi
dei lipid rafts
-Alexa 488 è FITC-Ab secondario
che lega anti-MHC-II o anti-CD45
PROCESSAZIONE DI ANTIGENI CITOSOLICI
PRESENTATI VIA MHC-I
2a via
CROSSPRESENTAZIONE
1a via
Endocyted protein
La digestione di proteine ubiquitinate nei
proteasomi avviene routinariamente
I TRASPORTATORI DEI PEPTIDI, TAP
(Transporter associated with Antigen Processing) della famiglia
dei trasportatori ABC (ATP-Binding Cassette).
• legano e trasportano peptidi di circa 8-13 AA
• peptidi con residui basici o idrofobici al C terminale sono
trasportati piŭ efficientemente, e sono anche quelli che legano
meglio MHC-I
Role of TAP in class I MHC-associated antigen presentation. In a cell line lacking functional TAP, class I molecules are
not efficiently loaded with peptides and are degraded, mostly in the endoplasmic reticulum (ER). When a functional TAP
gene is transfected into the cell line, normal assembly and expression of peptide-associated class I MHC molecules are
restored. Note that the TAP dimer may be attached to class I molecules by a linker protein called tapasin, which is not
shown in this and other illustrations. TAP, transporter associated with antigen processing.
CROSS-PRESENTATION
1. There is a marked increase of MHC-I synthesis during DC maturation
2. Several proteasome subunits of the immunoproteasome are induced
during DC maturation
3. DC appear very efficient in presenting exogenous internalized antigens in
the context of MHC-I molecules (cross-presentation)
CROSS-PRESENTATION
Solo le CELLULE DENDRITICHE sono in
grado di presentare antigeni endocitati
dall'esterno anche attraverso la
VIA CITOSOLICA,che lega i peptidi a
MHC-I (CROSS-PRESENTATION).
Quindi, la CROSS-PRESENTATION
può portare alla generazione di risposte
T citotossiche contro virus che
non infettano le DC stesse.
CROSS-PRESENTATION per trasferimento
intercellulare attraverso gap junctions
Peptidi citosolici di cellule non APC professioniste
trasferiti attraverso gap-junctions attraverso i citoplasmi delle
2 cellule.
Un emicanale formato da 6 molecole di connessina si
assembla con un emicanale analogo sulla cellula adiacente.
Gli oncogeni ras, src, neu e le proteine virali di HSV-2 e HPV16
chiudono queste connessioni.
immunoevasione tumorale e virale
Peptide transfer mediato da
gap-junctions
••MHC-II
MHC-IIprevalentemente
prevalentementeda
daAPC
APCprofessioniste
professioniste
(interazione
con
T CD4+)
(interazione
con
T CD4+)
••MHC-I
MHC-Ièèespresso
espressoda
datutte
tuttelelecellule
cellulenucleate
nucleate(interazione
con
T CD8+) con T CD8+)
(interazione
••Anche
Anchecellule
cellulenon
nonAPC
APCprofessioniste
professionistepossono
possonopresentare
peptidi
tramitepeptidi
MHC-I,
peptidiMHC-I,
endogeni
sintetizzati
nel loro
presentare
tramite
peptidi
endogeni
cytosol
sintetizzati nel loro cytosol
Qualsiasi cellula
cellula MHC-I+
MHC-I+ infettata
infettatada
da parassiti
parassiti
Qualsiasi
endocellulari
può
presentare
peptidi
derivati
dalle
proteine
endocellulari
può
presentare
peptidi
derivati
dalle
deiproteine
parassiti
essere lisata
dai Tlisata
CD8+dai
citotossici,
deiedparassiti
ed essere
T CD8+ con
eliminazione
del parassita
citotossici, con
eliminazione del parassita
Presentation of extracellular and cytosolic antigens to different subsets of T cells. A. Extracellular antigens
are presented by macrophages or B lymphocytes to CD4+ helper T lymphocytes, which activate the macrophages
or B cells and eliminate the extracellular antigens.B. Cytosolic antigens are presented by nucleated cells to CD8+
CTLs, which kill (lyse) the antigen-expressing cells.
Immunodominance of peptides. Protein antigens are processed to generate multiple peptides; immunodominant
peptides are the ones that bind best to the available class I and class II MHC molecules. The illustration shows an
extracellular antigen generating a class II-binding peptide, but this also applies to peptides of cytosolic antigens that
are presented by class I MHC molecules. APC, antigen-presenting cell.
The end