CORSO DI IMMUNOLOGIA per il corso di Laurea in biotecnologie a. a 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 vladia.monsurro@univr.it 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 (in preparazione del talk) 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 linfocitaria, trasduzione del segnale. 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’ cellulo-mediata, 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 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 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 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 DC al microscopio microscopio ottico elettronico a scansione DC nei LN DC della cute, Cellule di Langerhans (LC)

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 ATTIVA- ZIONE SOPRAMOLE- COLARE (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 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

E PRESENTAZIONE Ag VIA MHC-II PROCESSAZIONE E PRESENTAZIONE Ag VIA MHC-II

Antigen processing requires time and cellular metabolism and can be mimicked by in vitro proteolysis. If an antigen-presenting 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.)

E PRESENTAZIONE Ag VIA MHC-I PROCESSAZIONE E PRESENTAZIONE Ag VIA MHC-I

Pathways of antigen processing and presentation 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 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 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 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 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.

GILT = IFN--Inducible Lysosomal Thiol reductase, 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 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 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 CROSS- PRESENTAZIONE 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 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 There is a marked increase of MHC-I synthesis during DC maturation Several proteasome subunits of the immunoproteasome are induced during DC maturation DC appear very efficient in presenting exogenous internalized antigens in the context of MHC-I molecules (cross-presentation)

CROSS-PRESENTATION Quindi, la 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 HPV-16 chiudono queste connessioni. immunoevasione tumorale e virale

Peptide transfer mediato da gap-junctions

MHC-II prevalentemente da APC professioniste (interazione con T CD4+) MHC-I è espresso da tutte le cellule nucleate (interazione con T CD8+) Anche cellule non APC professioniste possono presentare peptidi tramite MHC-I, peptidi endogeni sintetizzati nel loro cytosol Qualsiasi cellula MHC-I+ infettata da parassiti endocellulari può presentare peptidi derivati dalle proteine dei parassiti ed essere lisata dai T CD8+ citotossici, con eliminazione del parassita MHC-II prevalentemente da APC professioniste (interazione con T CD4+) MHC-I è espresso da tutte le cellule nucleate (interazione con T CD8+) Anche cellule non APC professioniste possono presentare peptidi tramite MHC-I, peptidi endogeni sintetizzati nel loro cytosol Qualsiasi cellula MHC-I+ infettata da parassiti endocellulari può presentare peptidi derivati dalle proteine dei parassiti ed essere lisata dai T CD8+ 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 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