why we need a immune system? - unicz.it · mancanza di espressione delle molecole mhc di classe i...

WHY WE NEED A IMMUNE SYSTEM?

• TO FIGHT PATHOGENS

•TO KEEP OUR ORGANISM DISTINCT TO THE OTHERS (SELF)

•TO KEEP THE ORGANISM OMEOSTASIS (ELIMINATION OF PATHOLOGICAL SELF CELLS)

PATHOGENS

Antigen

• A substance (antigen) that is capable of reacting with the products of a specific immune response, e.g., antibody or specific sensitized T-lymphocytes.

• A “self” component may be considered an antigen even though one does not generally make immune responses against those components.

Immunogenicità

• Estraneità dell’ antigene• Peso molecolare (100.000 Da, buona

immunogenicità, 1000Da scarsa.• Composizione chimica la presenza di aa

come tyr e phe aumentano l immunogenicità• Capacità di essere processati e presentati

al S.I.• Aduvanti. completi ed incompleti

Formula leucocitaria

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The Immune System Evolution

1798 Edward JennerVaccinazione

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THE IMMUNE SYSTEMAND PATHOLOGY

ACQUIRED IMMUNODEFICIENCY

SYNDROME (AIDS)

BARRIERE ANATOMICHE

THE CELLS OF THE IMMUNE SYSTEM

ONTOGENESI

ORGANI LINFATICITessuti Linfoidi Primari:•Midollo osseo •TimoTessuti Linfoidi Secondari:•Linfonodi•Milza•Fegato.

Lymphatic System



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CIRCOLAZIONE LINFOCITARIA

5x106 lymphocytes travel from blood to the lymph every minute.

I LINFONODI

LA MILZA

Mucosa Associated Lymphoid Tissue (MALT)

THE IMMUNE SYSTEM ORGANIZATION

Innate immunity(0-4 hr)

Pathogen

Risposta adattativa precoce

4-96 hr Risposta adattativa tardiva >96 hr

Espansione clonale cellule della memoria

Figure 8-28

Which line is which:

1. Lacks T and B cells2. Lacks innate immunity3. Normal response

Property Innate immune system Adaptive immune system

Receptors Fixed in genome Encoded in gene segments Rearrangement is not necessary Rearrangement necessary

Distribution Non-clonal Clonal All cells of a class identical All cells of a class distinct

Recognition Conserved molecular patterns Details of molecular structure (LPS, LTA, mannans, glycans) (proteins, peptides, carbohydrates)

Self-Nonself Perfect: selected over Imperfect: selected in individualdiscrimination evolutionary time somatic cells

Action time Immediate activation of effectors Delayed activation of effectors

Response Co-stimulatory molecules Clonal expansion or anergy Cytokines (IL-1, IL-6) IL-2 Chemokines (IL-8) Effector cytokines: (IL-4, IFN)

Innate and adaptive immunity

Janeway CA Jr, Medzhitov R.Innate immune recognition.Annu Rev Immunol. 2002;20:197-216.

Innate Immune Recognition• All multi-cellular organisms are able to

recognize and eliminate pathogens• Despite their extreme heterogeneity, pathogens

share highly conserved molecules, called “pathogen-associated molecular patterns” (PAMPs)

• Host cells do not share PAMPs with pathogens• PAMPs are recognized by innate immune

recognition receptors called pattern-recognition molecules/receptors (PRMs/PRRs)

I RECETTORI DELL IMMUNITA’ INNATA

Endogenous Signals Induced by PAMPs

• Mediate inflammatory cytokines• Antigen-presenting cells recognize

PAMPs– Same APC processes pathogens into specific

pathogen-derived antigens and presents them within MHC encoded receptors to T-cells

– T-cell responds only when presented with both signals

– Different Effector Cytokines in Response to Different Pathogens (Th1 vs. Th2)

TLR STRUCTURE

TLR AND THEIR PAMPs

HOW LPS ACTIVATE INNATE IMMUNITY

TLR SIGNALING

Response to Initial Infection

FAGOCITOSI

Maturazione cellula dendritica APC

• CD14 low

• CD80 high

• CD86 high

• CD40 high

Biological effect of microsphere on DC

Phagocytes

Phagocytes can bind foreign particles via:1) Antibody mediated mechanisms2) “innate” mechanisms, ie receptors present on the

phagocyte’s cell surface-Mannose receptor -Glucan receptor-CD14 (LPS receptor)-TLR2: bacterial lipoproteins-TLR4: LPS-TLR9: CpG DNA-Mannose binding protein receptor-probably have receptors for SP-A and SP-D (collectins)-complement receptors

MIGRAZIONE PER DIAPEDESI

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FAGOCITOSI ED ATTIVAZIONE PMN

NK physiology

• Who are the NK cells?• Which is their biological

task?• How the NK cell think

and take decision?• What happen when a NK

cells meet a virus infected or a tumor cells?

NK effector function I: natural cytotoxicity

NK effector functions II:ADCC

NK effector functions III:Differences between NK and

LAK killing

• IFNγ −> Th1, MHC-I induction and anti viral effect

• TNFα−> Maturation of DC and direct citotoxic effect

• GM-CSF-> DC maturation

• IL-4-> DC maturation , Th2 induction

• MIP1-α−> Macrophage recruitment at the inflammation site

NK effector function IV: Cytokines and Chemokines

NK cells functions are activated by:

• Virus infected cells : CMV, HSV, HIV

• Parassites: Toxoplasma

• Transformed cells

• Tumor cells:Hematopoietic origen, melanoma.

MIP1-αChemokines

M

IL-12

Th2

CMV

NK-DC positive interaction

M

CD40CD40L

limphonodes

cytokines release, T cytotoxicity,adaptative immune response start

NK-DC negative interaction

IL-10 release

kill

NK

DC

DC-CD40+

end of the immune response

IFN-γTNF

Liver

NK

NK

imm.DCM

1 2

3 4

T

IFN-α

M

M

M

MIG Chemokines

HOW NK ACTIVATING RECEPTORS WORK

Two receptors model

Figure 8-32

MICA recognition - “Stress”



GRANULOMATOSI WEGNER LIKE, MANCANZA DI ESPRESSIONE DELLE

MOLECOLE MHC DI CLASSE I

The Lancet, 2000; 354:1598:1603

A

B

LetteraturaAlternative activation of macrophages

Siamon Gordon

Nature Reviews Immunology 3, 23-35 (01 Jan 2003) Review

Close encounters of different kinds: Dendritic cells and NK cell take centre stage

Mariapia A. Degli-Esposti, Mark J. Smyth

Nature Reviews Immunology 5, 112-124 (01 Feb 2005) Review

Vivier E, Anfossi N.

Inhibitory NK-cell receptors on T cells: witness of the past, actors of the future.

Nat Rev Immunol. 2004 Mar;4(3):190-8. Review

Antigen Presentation and Processing

25/11/ 2008

Odontoiatria Corso Patologia Generale

The Processing for endo (MHC-I) and extra (MHC-II) cellular

antigens.



CD1 present lipid antigens



Peptide binding sites and binding sites for CD4 or CD8 on MHC class I and MHC class II

The binding sites for CD4 and CD8 on MHC class II molecules or MHC class I lie in the immunoglobulin domain, nearest to the membrane

Base of β2 domain(green)

α chain(purple)

β chain (white)

β2-Microglobuline(purple)

α−Chain (white)

Base of α3 domain(green)

A COMPLEX CHAPERON INTERPLAY IS NECESSARY

FOR MHC-I PEPTIDE LOADING





tempi di assemblaggio

A COMPLEX CHAPERON INTERPLAY IS NECESSARY

FOR MHC-II PEPTIDE LOADING





Structure of MHC class I

Computer graphic representationand ribbon diagramms of of the human MHC class I moleculeHLA-A2.

Heterodimer: α chain (43 kDa): polymorphicβ2-microglobin (12 kDa): non-polymorphic, non-covalently bound

α1 and α2: peptide binding, cleft formed by single structureα3: transmembrane

T CELL MATURATION

POSITIVE AND NEGATIVE SELECTION

PRE-TCR

FACS ANALYSIS OF THYMOCYTES

POSITIVE AND

NEGATIVE SELECTION

MHC-RESTRICTIONZinkernagel e Doherty

Nobel Per la Medicina 1997



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NO

NO

YES

THE SELECTIVE KISS OF DEATH



MHC dependent T cell maturation

K.O. mice and T cell maturation

Kuby Immunology, Ed. Freeman 2000

TAP and Peptides in positive selection

TCR and T cell maturation

TC

R a

ffin

ity

for

self

-M

HC

/sel

f p

epti

de

No to extremely low affinity ⇒ Lack of positive selection ⇒ Failure to rescue from programmed cell death

Low to intermediate affinity ⇒ Positive selection ⇒ Rescue from programmed cell death

High affinity ⇒ Negative selection ⇒ immediate death signal

Thymocyte Fate

Death in thymic medulla

Immunocompetent CD4+ and CD8+ T cells exit to periphery

Death in thymic cortex

Huang CY, Sleckman BP, Kanagawa O.

Revision of T cell receptor {alpha} chain genes is required

for normal T lymphocyte development.

Proc Natl Acad Sci U S A. 2005 Oct 4;102(40):14356-61.

• These are distinguished by cell surface markers

• CD8 bearing cells are the Cytotoxic T cells & recognise MHC Class I.They kill cells infected with viruses

• CD4 bearing cells are the Helper cells & recognise MHC Class II & secrete cytokines that activate other cells of the IS.

EFFECTOR T CELLS

HOW AND WHERE T CELLS GET ACTIVATED?



Selectine L,P, ELegano CD34, GlyCAM-I,MAdCAM-I

LFA-1, ICAM-I-3

HOW LONG T CELL SURVIVE?

Naive T cells SEVERAL MONTHSTCR low affinity binding to MHC-II (CD4) or MHC-I (CD8) partial phosphorilation CD3 zeta chain +IL7Memory T cells SEVERAL YEARSMHC independent IL-15 dependent survival

T CELL ACTIVATION TWO SIGNAL MODEL

Costimulatory moleculesCD40, CD80, CD86, CD27, OX40, ICOS

Receptor

CD40L, CD28, CTLA-4, OX40L, CD27, LICOS

TCR

CD28

MHC-I

CD40

CD40L CD80

CD28

TCR MHC

CD40L-CD40

CD80

DIALOGO COSTIMOLATORIO T/APC

I

II

SUPERMOLECULAR SYNAPSE ORGANIZATION

IL2 production and its receptor

Th pcell

Th 1cell

Th 2cell

Regulation of CD4+T cell Responses

IL-2IFNγTNFαLT

IL-4IL-5IL-13IL-10(TGFβ)

INTERLEUKINS and CHEMOKINS

• INTERLEUKINS 1-21 (monokines, lymphokines) molecular mass 30kDa they derive from three gene family: hematopietin, interferon, tumor necrosis factor.

• CHEMOKINE: IL-8, MIP1-a , MIG

They act nearby seldom in endocrin fashion.

EFFECTOR FUNCTIONS:CYTOKINES

TH1 vs TH2 Priming by Innate Immunity

Cytokines Receptors

• Immunoglobulin superfamily receptors (IL1, M-CSF, C-kit)

• Class I cytokines receptors family (hematopoietin receptor family) (IL2-12)

• Class II (Interferon receptor family)(IFNs and IL10)• TNF receptor family (TNFa/b, CD40, NGF, FAS)• Chemokines receptor family(IL8, RANTES, MIP)

HOW THEY WORK?

Phosphorylation

Binding of Stat 1α

JAK1/2

TH2 immune response function

PlasmaCell

B Cell

_

Th cell MemoryB Cell

1. B cell help: Th2 > Th1

TH2 FUNCTION MECHANISMTh2 clones can efficiently help resting B cells

Requires at least 3 signals:-TCR-MHC-II interaction- Cell contact; later identified as CD40-CD40L

(cannot be achieved by Th2 sup. alone)- IL-4 and IL-5 both required for optimal proliferation and differentiation (blocking Abs)

NaïveB Cell

_

Th2

cell

IL-4 + IL-5

CD40L-CD40

Th1 support cell mediated immunity:

Eradication of intracellular pathogens (viruses, ingested bacteria). IFNγ and LT activate phagocytes.

Recruits NK cells and cytotoxic (CD8) cells; perforin killing.

Often accompanied by production of complement fixing antibodies (eg IgG2a)

Uncontrolled implicated in autoimmune diseases (eg. diabetes, Multiple sclerosis).

Th2 support humoral immunity:

Activation of mast cells and eosinoiphils (IL-4, IL-5, IL-13), helps eradicate parasites and extracellular organisms.

Efficient B cell help leading to proliferation and differentiation; antibody production.

Uncontrolled implicated in atopic conditions (Asthma, allergy)

++++Macrophage Activation

+/-++Selectin Ligands (E,P)

++

++

+/-

+/-

+/-

++

Chemokine Receptor

CCR3 (eotaxin)

CCR4

CCR5, CXCR3

++

-

-

++

Cytokine ReceptorIFNγββ

γ

Th pcell

Th 1cell

Th 2cell

Cytokine Regulation of T cell Responses

IL-2IFNγα

β

Presence early (during initiation of response) = Strongest signals.

IL-12 (macrophage, NK cells ) drives naïve T cells towards Th1 differentiation.

IL-4 drives naïve T cells towards Th2 differentiation (early source: ?).

IL-12 suppresses Th2 response, while IL-4 suppresses Th1 response (counter-regulation).

Factors differentiation that influence - Cytokines

O’Garra, Immunity (8):275-283, 1998

Strength of interaction via TCR and MHC/Ag complex (signal 1) or dose of Ag can directly affect differentiation (Constant, Ann Rev Immunol (15):297, 1997)

• Mechanism unclear, but appears related to altering balance between IL-4 and IL-12/IFNγ production by T cells and/or APCs (O’Garra, Immunity (8):275-283, 1998

Role of costimulatory molecules: B7.1 may favor Th1 responses and B7.2 may favor Th2 responses (Kuchroo, Cell (80):707, 1995)

Factors that influence differentiation - Signal strength and duration

(Lanzavecchia et al 2000 Science, 290:92-97)

AICD

Neglect

Non-polarized

Polarized

Homing

FunctionRegulation,

Effector prescursor

LNTissues

InflammationCytotoxicity

Duration of TCR and Cytokine stimulation

Mechanisms of Th subset development: GATA-3 is necessary and sufficient for Th2 differentiation.

Differential gene display: GATA-3 levels high in naïve T cells and in Th2 cells, but become minimal as naïve cells commit to Th1 subset.

Antisense GATA-3 inhibits expression of all Th2 cytokine genes (IL-4, IL-5, IL-13) in Th2 clone.

GATA-3 directly activates an IL-4 promoter-luciferase reporter gene.

Inducing expression of GATA-3 in developing Th1 cells in Tg mice, resulted in Th2 cytokine expression in these Th1 cells.

(Zheng, Cell (89):587, 1997)

Mechanisms of Th subset development: T-bet directs Th1 lineage commitment

The transcription factor T-bet controls the expression of the hallmark Th1 cytokine IFNγ.

T-bet expression correlates with IFNγ production in Th1 and NK cells.

Ectopic expression of T-bet transactivates the IFNγ gene and induces IFNγ production.

Retroviral gene transduction of T-bet into polarized Th2 (and Tc2) cells redirects them into Th1 (and Tc1) cells

Szabo, 2000 Cell (100):655

• Mechanisms that are used by the immune system to eliminate pathogens (or other substances) from the body

• Cellular effector mechanisms– Activated T cells– Natural killer cells

• Humoral effector mechanisms (antibody)– Neutralization– Opsonization– Complement activation– Antibody-dependent cell-mediated cytotoxicity (ADCC)

Effector Mechanism

Effector Activity Against Pathogens

CTL-Cytotoxic T Lymphocytes NK--Natural Killer Cells

Lymphocytelineage

Bone marrow stem cell, maturein thymus, CD8+

Bone marrow stem cell, thymusindependent, non T

Occurence Absent in normal lymphoidorgans; differentiate from non-cytolytic precursors in responseto antigen

Present in normal lymphoidorgans (e.g., 5-10% oflymphocytes in human blood)

Targetrecognition

T cell receptor binds to peptide-class I MHC complex, + CD8binds to class I MHC

Multiple triggering receptors;specialized receptors for selfclass I MHC give negativesignal; CD16 IgG FcR (ADCC)

Lytic pathways Granule exocytosis + FasL/Fas Granule exocytosis

In vivo role Protection against non-cytopathic viruses; tumorsurveillance and immunotherapy

Early responses against viralinfections; ?tumor surveillanceand prevention of metastasis

CTL and NK Cells: Two lineages of cytotoxic lymphocytes

NecrosisNecrosisProgrammed cell death or apoptosisProgrammed cell death or apoptosis

T CELL MEDIATED CYTOTOXICITY

ACTIVECASPASES

Nuclear DamageDNA fragmentation

Chromatin condensation

PARP cleavage

Lamin cleavage

Apoptotic death Membrane PS flip Mitochondrial Ψ drop Membrane blebbing LYSIS

Fas DNA damage

CorticosteroidTcR EtoposideIrradiation

p53

Spontaneous

Receptor

Transcription

CTL/NK

Granzymes

FasL Anti-Fas

FADD

FLICE

Degranulation

Perforin TargetPermeabilization

97076.ppt Henkart

Cytoplasmic ApoptoticDeath Pathway

Control of Caspase Activation

Membrane Recycling

Target cell death pathways induced by cytotoxic lymphocytes



Coniugato citotossico



Referenze1) Zheng, Cell (89):587, 19972) Kuchroo, Cell (80):707, 19953) Lanzavecchia et al 2000 Science, 290:92-974) O’Garra, Immunity (8):275-283, 19985) Constant, Ann Rev Immunol (15):297, 19976) Szabo, Cell (100):655 . 20007) Braud, Nature 391:795. 19988) Lanier, Annu. Rev. of Immunol. 16:359.19989) Kagi, Science.265:528.199410)Long, Annu.Rev.of Immunol. 17:875.19991) Radaev and Sun. Ann.Rev.Bioph.Biom.Str.2003



REGOLAZIONE MHC CLASS I DELLA CITOTOSSICITA NK

IG LIKE NK INHIBITORY RECEPTORS





STRUTTURA DEI RECETTORI INIBITORI IG

LIKE.1

STRUTTURA RECETTORI INIBITORI IG LIKE.2



RECETTORI INIBITORI ED ATTIVATORI LECTIN LIKE



LIGANDI DI NKG2D



Annual Review of Biophysics and Biomolecular StructureSergei Radaev and Peter D. Sun. 2003



NK ACTIVATING RECEPTOR SIGNALING

INTEGRATIVE REGULATION OF THE EFFECTOR MECHANISMS

Science, Vol 306, Issue 5701, 1517-1519 , 26 November 2004

RICONOSCIMENTO DELL’Ag DA PARTE DELL’ IMMUNITA

ADATTATIVA

T B

TCR BCR

IMMUNOGLOBULINE



Struttura delle Immunoglobuline 1



Struttura delle Immunoglobuline 2



CHI PRODUCE LE IMMUNOGLOBULINE



Dissezione enzimatica delle Ig



1. PM 150 kDa2. Light chain 25kDa3. Heavy chain 50kDa

5 Classi di Ig





Light Chain Structural Organization

REGIONI IPERVARIABILI



SITO DI LEGAME DELL’ ANTIGENE



EPITOPOCONFORMAZIONALE

EPITOPOLINEARE

AGRETOPO

mAbs Clinic use

1. Against human Chorionic gonadotropin (HCG) Pregnancy test (ELISA)

2. AIDS (ELISA)3. Radioterapy, Radioimmunodetection4. Bacteria Strain typing5. OKT3 as immunosuppressive durg in transplanted patients

GENETICA DELLE IMMUNOGLOBULINE



RICOMBINAZIONI SOMATICHE



COME AUMENTARE LA VARIABILITA’1



Sequenza segnale di ricombinazione (RSS)



RSS

RIARRANGIAMENTO 12/23



COME AUMENTARE LA VARIABILITA’2

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AggiuntaNucleotidi P ed NNei siti giunzionali

COMPLESSI ENZIMATICI

V(D)J RECOMBINASI

RAG1

RAG2DNA ligasi IVTdT Desossinucleotidil transferasi terminaleDNA PK

SWITCH ISOTIPICO



Complement Binding Site

Phagocyte Receptor Binding Site

Antibody Structure

TCR like this portion of Ab

OMOLOGIE TRA Ig e TCR

T cell Membrane

βα

T Cell Receptor

Antigen Recognition

30.000 TCR per Lymphocyte

All T cells are “Antigen specific”

• Mediated by “T cell receptor” − TCR

– Surface molecule homologous to part of Ab

• Diversity is generated by rearrangement of TCR gene locus

Variable-region (V) Genes

Diversity (D) Gene

Joining (J) Genes

β Constant Domain

Germline DNA

Somatic Recombination

Transcription

Translation

Message

DNA in T cell

Generation of TCR Diversity

Splicing

TCR β Chain

Generation of TCR Diversity

Variable-region (V) Genes

Joining (J) Genes

α Constant Domain

Germline DNA

Somatic Recombination

Transcription

Translation

Message

DNA in T cell

Splicing

TCR α Chain

why we need a immune system? - unicz.it · mancanza di espressione delle molecole mhc di classe i...

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