Advanced molecular immunology-2011

Adaptive Immunity

Youmin KangYoumin Kang

Tel: 62731278Tel: 62731278Email: kymin1012@163.com

Lab: CLS Room 3076Lab: CLS Room 3076

03/23/2011

C t f i itC t f i itComponents of immunityComponents of immunityi t d tiinnate adaptive

ll l t hil T l h tcellular neutrophils T lymphocytesmonocytes-macrophagesmacrophages, DC, NK cells

humoral complement antibodiesacute phase (B lymphocytes)proteins (CRP,MBL..)

Innate vs adaptive immunityInnate vs. adaptive immunity• Innate immunity

– First line of defense– Immediate (0 – 4 hours)

N ifi– Non-specific– Does not generate lasting protective immunity

• Adaptive (acquired) immune response (late: > 96 hours)Is initiated if innate immune response is not adequate (>– Is initiated if innate immune response is not adequate (> 4 days)

– Antigen-specific immunityg p y– Generates lasting protective immunity

Humoral and cell-mediated

Adaptive Immunity Features

Highly specific for the invading organism

Adaptive Immunity Features

g y p g g

Discrimination between “self and “non self” molecules

The response only occurs to “non self” molecules

Diversity- It can respond to millions of different antigens- Lymphoctes population consists of many

diff t l ( ll d it )different clones (one cell and its progeny)- Each clone express an antigen receptor and

responds only to one antigenic epitoperesponds only to one antigenic epitope

Unmethylated CpG motif Toll like receptors

Specificity of Ag

Ab1 Ab2 Ab3

Antibody-Antigen Interactionsy g

Compare Epitope StructuresCompare Epitope Structures

Classification of antigenic determinantClassification of antigenic determinant

(1)According to the structure of Ag determinants( ) g g

• Conformational determinants• Conformational determinants

• Sequential (or linear) determinantsq ( )

(2)According to types of cells recognizing antigenic determinantsantigenic determinants

T ll d t i t (T ll it )• T cell determinants (T cell epitopes)

• B cell determinants (B cell epitopes)

MOLECULES OF LYMPHOCYTE RECOGNITION

Although B and T cell receptor genes are rearranged similarly to generate a high degree of specificity for

ti th t “ ” ti i ti l diff t

T-CELL

antigen, the receptors “see” antigen in entirely different ways.

CD3B-CELL

Ig Ig

TCRSurface Ig

P d

11

Processed peptide(linear

epitope)

Complete molecule

(conformational

MHC2 2

(conformational epitope)

ANTIGEN PRESENTING CELL

CD4 T-CELLCD3

CD8 T-CELL

CD3

TCR CD4

CD8

TCR

CD4

9 aa peptide15 aa

peptide 2 1peptide

MHCCLASS II

11

2 2 CLASS IMHC 2m

2 1

3

ANTIGEN PRESENTING CELL

2

ANTIGEN PRESENTING CELL

Adaptive ImmunityImmunity

Humoral ImmunityHumoral Immunity

胸腺依赖性抗原胸腺非依赖性抗原

（thymus dependent antigen, TD抗原）（thymus independent antigen, TI抗原）

TD抗原和TI抗原的比较

TD抗原 TI抗原

本质 多数为蛋白质重复排列的长链

聚合物

刺激B细胞产生抗体是否需要T细胞

参与+ -

参与

产生抗体的类型 主要IgG IgM

引起细胞免疫 + -

免疫记忆性 + -

Production of antibodiesProduction of antibodiesPathogen (virus or(virus or bacteria)

Pathogen is internalized

and degradedSignal 1

B cell

B cell binds pathogenPlasmaPlasma

cellsSignal 2 IFN-g,IL-4,IL-5 Tfh

TH

MHC II

B cell

B cell proliferation

B cells differentiate into antibody-secreting

plasma cells

Produce antibodies

Peptides from the pathogen are presented (MHC II) to the T cell resulting in the

i i f h ll Produce antibodies against pathogen

activation of the B cell

C ll di t d I itCell mediated Immunity

I l T ll h i f i ll i• Involves T cells that act against foreign cells or tissue• T cells mature in the thymus gland• Also regulate the activation and proliferation of other

immune system cells• Cell mediated immune response is directed against

bacteria and viruses inside phagocytic cells or i f d h ll f i d h l i hinfected host cells, fungi, protozoa, and helminths

• This also causes rejection in implanted tissue

BB B BB Y

B

Y

BB Y

BBB Y

BBY

YY Y Y YYYY Y

YYY

YY

YY Y YY

Proliferation and antibody production

Cross-linking of surface membrane Ig antibody production

T T N lif ti

YT

YT No proliferationNo cytokine releaseY Y

T ll d t i ti tiT cells do not recognise native antigens

Antigens must be processed in orderto be recognised by T cellsto be recognised by T cells

YT Y

Cell surface peptides of Ag presented by cells that

C ll f

Solublenative Ag

Soluble peptides

express MHC antigens

Cell surfacepeptides

of Ag

Cell surfacenative Ag

of Ag

ANTIGEN

T cellNo T cellNo T cellNo T cell No T cell

ANTIGENPROCESSING

T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

Early evidence that antigens are catabolised

MM

Macrophages and radiolabelledListeria monocytogenes

Rapid binding to cell surfaceListeria monocytogenes

M Degradationof bacteriaM of bacteria

and release ofRadiolabelled

t i i t

Internalisation

protein into supernatant and cells

How is antigen catabolism linked to T cell proliferation?

The interaction of T cells with macrophagesrequires antigen catabolism

Listeria-specific

requires antigen catabolism

NO T CELLSBIND

T

T cells BINDListeriacoatedplasticT

Li t i

plastic

Listeria NO T CELLSBIND

T CELLSBIND

NO T CELLSBIND

NO T CELLSBIND

MM MM

0mins 60minsT cell do not bind stably to antigen presenting cells unless the

antigen is catabolised

Only metabolically active cells can process antigen

TListeria-specific TT cells

MMM

Fix withparaformaldehyde

or poison with

Pulse withListeria for 60min

Add Listeriaspecific T cells

catabolic activity is dependent upon the viability of macrophages

or poison withsodium azide & wash cells NO T CELLS BIND

catabolic activity is dependent upon the viability of macrophages

Antigen presenting cells must be viable to PROCESS antigen

APCs?

Ph t d l ti f DC b tPhenotype and location of DC subsets

A general modelfor interactionbetween DC

Subsets and T cellsduring infection.g

Stages of endogenous and exogenousantigen processing

UPTAKEUPTAKEAccess of native antigens and pathogens to intracellular

pathways of degradationpathways of degradation

DEGRADATIONLimited proteolysis of antigens to peptidesLimited proteolysis of antigens to peptides

ANTIGEN-MHC COMPLEX FORMATIONL di f tid t MHC l lLoading of peptides onto MHC molecules

ANTIGEN PRESENTATIONTransport and expression of peptide-MHC complexes on the

surface of cells for recognition by T cells

Uptake of exogenous(外源的）antigens

Membrane Igreceptor mediated

p g (外源的 g

Y Phagocytosis

receptor mediateduptake

YComplement receptor

Pi i

Complement receptormediated phagocytosis

Pinocytosis

Y Fc receptor mediated phagocytosis

Uptake mechanisms direct antigen into intracellular vesiclesfor exogenous antigen processing

Exogenous pathway

Cell surface

Protein antigens

Endosomes

Uptake Protein antigensIn endosome

Endosomes

Increasein acidity

To lysosomes

Cathepsin B, D and L proteases are activated by the decrease in pHProteases produce ~24 amino acid long peptides from antigensDrugs that raise the pH of endosomes inhibit antigen processing

Surface expression of MHC class II-peptide complexes

Exported to the cell surface

MIIC compartment sorts peptide MHC complexes for surface expression or

Sent to lysosomes for degradation

MIIC compartment sorts peptide-MHC complexes for surface expression orlysosomal degradation

Non-lysosomal antigen processingInactive virus raises a weak CTL response

The processing of antigens from inactive viruses is sensitive to

y g p g

The processing of antigens from inactive viruses is sensitive to lysosomotrophic drugs

ANTIGENS FROM INACTIVE VIRUSES ARE PROCESSED VIA THE EXOGENOUS PATHWAY

Infectious virus raises a strong CTL responseg p

The processing of antigens from infectious viruses is NOT sensitive to lyosomotrophic drugs

Most CTL recognise antigens generated via a non-lysosomal pathway

Protein synthesis is required for non-lysosomal antigen processing

ANTIGENS FROM INFECTIOUS VIRUSES ARE PROCESSED VIA THE ENDOGENOUS PATHWAY

Do the two pathways generate the same type of T cell receptor ligand?

Degradation in the proteasome

Cytoplasmic cellular proteins, including non-self proteinsare degraded continuously by a multicatalytic protease of 28 subunits

The components of the proteasome include MECL-1, LMP2, LMP7These components are induced by IFN- and replace constitutive

components to confer proteolytic properties.

LMP2 & 7 encoded in the MHC

Proteasome cleaves proteins after hydrophobic and basic amino acids and releases peptides into the cytoplasm

Peptide antigens produced in the cytoplasm are physically separated from newly formed MHC class Iphysically separated from newly formed MHC class I

ENDOPLASMIC RETICULUM

Newly synthesisedMHC class I molecules

CYTOSOLPeptides need

access to the ER inorder to be loaded onto MHC class I molecules

Transporters associated withantigen processing (TAP1 & 2)antigen processing (TAP1 & 2)

Hydrophobic

Lumen of ERPeptidePeptidePeptidePeptidePeptide

Lumen of ER

y ptransmembranedomain

ER membrane

PeptidePeptidePeptidePeptidePeptide

ER membrane

Cytosol PeptidePeptidePeptidePeptidePeptideCytosol Peptide

ATP-binding cassette(ABC) d i

Peptide antigens

Transporter has preference for >8 amino acid peptides

(ABC) domainfrom proteasome

Transporter has preference for >8 amino acid peptideswith hydrophobic C termini.

Maturation and loading of MHC class I

PeptidePeptidePeptidePeptidePeptidePeptide

E d l i ti l

PeptidePeptidePeptidePeptidePeptide

Endoplasmic reticulum

C l i bi d B2 M Tapasin calretic lin TAP C t l i tidCalnexin bindsto nascent

class I chainuntil 2 M binds

B2-M binds and stabilises

floppy

Tapasin, calreticulin, TAP 1 & 2 form a complex with

the floppy MHC

Cytoplasmic peptides are loaded onto the

MHC molecule and the structure becomesuntil 2-M binds floppy

MHCstructure becomes

compact

Fate of MHC class IFate of MHC class I

E t d t th ll fExported to the cell surface

S t t l f d d tiSent to lysosomes for degradation

The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway useduptake determines the antigen processing pathway used

Y Vesicular Compartment

EXTRACELLULAR ORENDOSOMAL REPLICATION

Y Vesicular CompartmentContiguous with extracellular fluid

Exogenous processing(St t l M b t i l ti )(Streptococcal, Mycobacterial antigens)

INTRACELLULAR REPLICATIONCytosolic compartment

Endogenous processing(Vi l i )(Viral antigens)

Distinct mechanisms of antigen generation are used to raiseDistinct mechanisms of antigen generation are used to raiseT cells suited to the elimination of endogenous or exogenous pathogens

Three pathways of antigen presentationp y g p

How DCs activate T cells?

Intravital two-photon imaging of T-cell–DC interactionsp g g

a

b

Intravital two-photon imaging of T-cell–DC interactionsIntravital two photon imaging of T cell DC interactions

c

c

Activation of CD8+ T cells by DCs in the lymph node

highhigh velocities rapid

shapechanges

7.3 new T cells—1 DC/ minute

T cell migration promotes very efficient scanning of theT cell migration promotes very efficient scanning of theT cell repertoire by an individual DC.

NATURE IMMUNOLOGY 2003,4:579

Priming of CD8+ T cells by DCs occurs as long lasting interactions

engaged42 T cell-DC interactions

Average duration-11 minutes

Priming of CD8+ T cells by DCs occurs as long-lasting interactions

free engaged with DCs

Average duration 11 minutes

P14 TCR T cells

Sl li f th T ll th DC f

Green-P14 TCR T cellsred -DCs pulsed with 1 μM of gp33 peptide

Q: DC density?

Slow crawling of the T cells on the DC surface;passive movement of the T cell owing to shape changes of the DCs.

NATURE IMMUNOLOGY 2003,4:579

Impact of DC density on T cell-DC contacts

One DC

DCs was virtually saturated with P14 T cells

NATURE IMMUNOLOGY 2003,4:579

Low-avidity interactions result in inefficient recruitmentof peptide-specific T cells by antigen bearing DCs

75 individual P14 TCR T cells

DCs-high densities of peptide:marked by green arrows;

DCs-low densities of peptide: k d b d

NATURE IMMUNOLOGY 2003,4:579

marked by red arrows.

T cell cluster formation

T cell-DC contacts

antigen absence : DCs -- 500 different T cells/hour;

ti b i DC 10 T ll i lt lantigen-bearing: DCs -- 10 T cell simultaneously;

interaction: hours, not minutes, low DCs

density, high peptide density .

Rapid Formation of Stable CD8+ T Cell-DC Contacts in the Lymph Nodes

Immunity 2010, 33:412

The Two-Signal Model of T-cell Activation

DC T cell

CD4 or CD8

1TCRMHC

1

2

COSTIMULATION

An important costimulator - CD28p

Costimulation signalingCostimulation signaling

T cell

3

IFNIFN--

ILIL--44

ILIL--22

B cellsB cellsYYYYYYYYYYYYYYYYYYYYYY

Effector Th cell differentiation

The discovery of TFH cell

Spleenp

Human TFH in a lymph node from a patient with angio-immunoblastic T cellLymphoma (血管原始免疫细胞性T细胞淋巴瘤 ). Note the interactions between

haematologica | 2010; 95(3):356

PD-1+ (in green) and CD4+ (in blue) Tfh cells and CD20 (in red) B cells(confocal microscopy x500).

Q: Tfh-cytokines?

TFH cells express distinct genes from TH1, TH2 and TH17 cells

KLH(昆虫血蓝蛋白)/ CFA; CD4+CD44hiCXCR5+BTLA+ (TFH) cells were (昆虫 蓝蛋白) ; ( )sorted and restimulated with anti-CD3 for 4 hours for gene profiling analysis

Immunity. 2008, 29(1): 138

TFH cells express distinct genes from TH1, TH2 and TH17 cells

real-time PCR analysis

Immunity. 2008, 29(1): 138OT-II cells-anti-CD3

Th2ELISA

Th1Th1

Intracellular staining

Th17

Immunity. 2008, 29(1): 138

Generation of TFH cells is independent of TH1 and TH2 lineagesp g

OVA/CFACD4+ T cells from CD45.2/OT-II mice were transferred into C57BL/6 (CD45.1) mice OVA/CFA

Immunity. 2008, 29(1): 138KLH/CFA

IL-21 is necessary for TFH cell developmentIL 21 is necessary for TFH cell development

Immunity. 2008, 29(1): 138Q: Tfh-costimulator-B7H?

B7H expressed on B cells is required for generation of TFH cells

ELISA

Immunity. 2008, 29(1): 138B7h BKO:B7h B-cell conditional knockoutQ: Tfh-cytokines?

Generation of TFH cells requires IL-6 and STAT3

Immunity. 2008, 29(1): 138Q: Tfh-Bcl-6?

Bcl-6 Is Required for Tfh Cell Formation In Vivo

SRBC (sheep red blood cells) immunizationgroup A: Bcl6+/+ CD45.1:Bcl6+/+ CD45.2 g pgroup B: Bcl6+/+ CD45.1:Bcl6-/- CD45.2 fetal liver chimeric mice

Immunity 2009, 31:457

Bcl-6 Is Required for Tfh Cell Formation In Vivo

C

mixed bone marrowhi i

SRBC (sheep red blood cells) immunizationgroup A: Bcl6+/+ CD45.1:Bcl6+/+ CD45.2 group B: Bcl6+/+ CD45 1:Bcl6-/- CD45 2 fetal liver chimeric mice

chimera mice

Immunity 2009, 31:457

group B: Bcl6 CD45.1:Bcl6 CD45.2 fetal liver chimeric mice

I t t l l f Tfh ll f tiImportant molecules for Tfh cell function.

Annu. Rev. Immunol. 2008. 26:741

Extrafollicular and Follicular pathways to humoral immunity

Pre-Tfh can either enter the follicle to become GC Tfh or interact with plasmablasts outside the follicle.

signaling lymphocyte activation molecule (SLAM)

lymphoid tissue inducer (LTi) cells:CD4+CD3-Accessory cells localised at the T/B border and within the folliclesborder and within the follicles

Cellular interactions required for Tfhformation and maintenancemaintenance

The signaling pathways in TFH cells

J Immunol 2010, 184: 6563

ReferencesReferences1. Philippe Bousso. T-cell activation by dendritic cells in the lymph node:

lessons from the movies. Nature Reviews Immunology 2008,8:6752. Philippe Bousso, Ellen Robey. Dynamics of CD8+ T cell priming by

dendritic cells in intact lymph nodes. Nature Immunology 2003,4:579 3. He´le`ne Beuneu, et al. Visualizing the Functional Diversification

of CD8+ T Cell Responses in Lymph Nodes.Immunity 2010, 33: 4124. Camille Laurent , et al. A novel subset of T-helper cells: follicular T-

helper cells and their markers.Haematologica 2010; 95:3565. Julia Rolf, et al. Signaling Pathways in T Follicular Helper Cells. J

Immunol 2010, 184: 65636. Cecile King, et al. T Follicular Helper (TFH) cells in Normal and

Dysregulated Immune Responses. Annu. Rev. Immunol. 2008. 26:7417. Di Yu, et al. The Transcriptional Repressor Bcl-6 Directs

T Follicular Helper Cell Lineage Commitment. Immunity,2009,31:4578. Roza I. Nurieva, et al. Generation of follicular helper T cells is mediated by

IL-21 but independent of TH1, TH2 or TH17 lineages. Immunity. 2008, 29: 138