Antigen Processing and Presentation

Chapter 8

2T - cytolytic cell (Tc) with

T cell receptor and CD8

marker

Virus-infected cells with

viral antigen along with

MHC I molecules on the

surface

T - helper cell (Th) with

T cell receptor and CD4

marker

Antigen-presenting cell with

processed antigen along with

MHC II molecules on the surface

Cell-Mediated Immune Response

Properties of Antigens recognized by T Lymphocytes

Most T lymphocytes (alpha beta) recognize peptides

T cells are specific for AA sequences

Peptide + MHC = recognition

T cell activation is self-MHC restricted

4T cell recognition of Peptide MHC Complex

MAJOR HISTOCOMPATIBILITY

COMPLEX (MHC)

1) Role in organ transplantation

2) Role in immune response

Vaccine immunity

Infectious diseases

3) Role in predisposition to diseaseInfectious diseases

Cancer

Autoimmune diseases

Cell Membrane

PeptideMHC class I MHC class II

MHC MOLECULES

Foreign PeptidesVirusBacteriaParasiteFungiAllergenTransplant

Self PeptidesCryptic self peptides

(autoimmune Ag)Altered self (cancer)

What are the MHC molecules? And what do they do ?

MHC presents foreign or self peptide to TCR on T cells.

Peptide

Binding

Cleft or

Groove

Peptide

Binding

Cleft or

Groove

MHC Properties Are the most polymorphic genes

(alternative forms of a gene/ allele) in genome (population)

Also polygenic (several genes with similar, but not identical structures and functions), on individual level

The set of MHC alleles on each chromosome is a haplotype (HLA-A2,HLA-B5, HLA-DR7,etc

Each person gets 2 haplotypes- one from mom and the other from dad

9

GENERAL ORGANIZATION OF MOUSE & HUMAN MHC

(Human leukocyte antigen complex)

(Histocompatibility-2 complex)

Chromosome 17

Chr. 6

Glycoprotein (Gp)or Protein (P)

Gpor P

MHC Properties

Much of our initial knowledge of MHC came from mice (KO mice-MHCIKO mice)

20 inter-matings make identical (syngenic) mice

Syngeneic inbred mice have a single haplotype

Allogeneic, (distinct strains of mice- eg. Balb/c versus C57BL/6) may have differing haplotypes)

Congeneic strains identical except at 1 locus

Are people syngeneic or allogeneic?11

MHC Properties

MHC molecules have been found in every mammalian species examined

MHC alleles are Co-dominantly expressed

In humans, each HLA allele is given a number designation

In mice, each H-2 allele is given a letter designation

12

MHC Properties

MHC molecules consist of extracellular peptide binding cleft (groove), Ig-like domain, a transmembrane domain, and cytoplasmic region

Polymorphic AA of MHC located in and adjacent to peptide binding groove

Consist of paired a helices and a floor of 8 stranded b pleated sheets

Non-polymorphic Ig domains of MHC interact with CD4 and CD8 molecules

13

MHC

Peptides that bind to a given MHC share structural features that promote interaction

Association of antigenic peptide / MHC has slow off rate

MHC molecules dont discriminate between foreign and self peptides

Hydrophobic interactions, hydrogen bonding and charge interactions play large role in association between peptide and MHC

14

MHC Class I

Consist of an a chain and a Non-MHC encoded subunit (b2-Microglobulin)

Present endogenously derived peptide to Tc

Binds peptides 8-11 amino acids in length

This means that native proteins have to be processed prior to binding

15

MHC Class II

Consist of MHC encoded a chain and b chain subunits

Present exogenously derived peptide to Th

Binds peptides 13-20 amino acids in length

Again, the native proteins have to be processed prior to binding

16

GENOMIC MAP OF MOUSE & HUMAN MHC GENES

Class III

Within Class II

DP2, DP2 - pseudogenesDQ2, DQ2, DQ3 Not expressed

18

Question:

Female Mouse of MHC Haplotype H-2b

(C57BR) is mated with male Mouse of MHC Haplotype H-2d (Balb/c).

What would be the MHC haplotype of the progeny?

Would transplanted tissue from the mom be accepted or rejected by the progeny?

Would transplanted tissue from the progeny be accepted or rejected by the mom?

Would transplanted tissue from the dad be accepted by the mom? 19

20

21

22

MHC of an individual

2 MHC Haplotypes: one from mom and dad

Each haplotype has one HLA-A, B, C

Each haplotype has at least one HLA-DR, DP, DQ, however there are multiple alpha and beta chains

Therefore it is estimated that each individual has 6 MHC I and up to 12 MHC II

23

MHC

Analysis of human MHC alleles in 2006 have revealed 370 A alleles, 660 B alleles, 190 C alleles.

Numbers likely to be significantly larger as data determined from European descent. In fact, many non-European groups cannot be typed using available MHC serological typing reagents

24

b a b a aB C A

DP DQ DR

b1

Polygeny

1) The combination of alleles on a chromosome is an MHC HAPLOTYPE.

Variant alleles

polymorphism

Additional set

of variant alleles

on second

chromosome

2) MHC molecules are CODOMINANTLY expressed.

3) Two each of 3 MHC-I molecules (-A, -B, -C) are expressed (6 MHC-I molecules).

4) Two each of the and chains from the 3 MHC-II molecules (-DR, -DQ, -DP)are also expressed (12 MHC-II molecules).

DIVERSITY OF MHC GENES IN AN INDIVIDUAL

b a b a aB C ADP DQ DR

b1

b a b a aB C ADP DQ DR

b1

HAPLOTYPE 1

HAPLOTYPE 2

DP DQ DR C4B Bf 21OH TNF- B C AC4A C2 TNF - HSP

CHROMOSOME 6

Centromere

Class III

HLA HAPLOTYPE

HUMAN LEUKOCYTE ANTIGEN ( HLA ) GENES TO PROTEINS

a

b

DP11DQ33

DR55B7 C9 A11

APC

B3DQ66 DR448

DP81

DQ36DQ63 DR45

DR54

Class II Class I

MHC molecules are

CODOMINANTLY

expressed

(2 x A) + (2 x B) + (2 x C) = 6 MHC-I

(4 x DR) + (4 x DP) + (4 x DQ) = 12 MHC-II

INHERITANCE OF

HLA

HAPLOTYPES

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

X

ParentsDP-1,2

DQ-3,4

DR-5,6

B-7,8

C-9,10

A-11,12

DP-9,8

DQ-7,6

DR-5,4

B-3,2

C-1,8

A-9,10

Children

DP-2,9

DQ-4,7

DR-6,5

B-8,3

C-10,10

A-12,9

DP-1,8

DQ-3,6

DR-5,4

B-7,2

C-9,8

A-11,10

DP-2,8

DQ-4,6

DR-6,4

B-8,2

C-10,8

A-12,10

DP-1,9

DQ-3,7

DR-5,5

B-7,3

C-9,1

A-11,9

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

B C ADP DQ DR

What is a haplotype ?

A set of linked MHC alleles present onone parental chromosome.

Thus, an individual has two haplotypes.

COOH COOHCOOH

Description MHC-I Molecules MHC-II Molecules

Peptide-binding domain 1 / 2 1 / 1

Peptide-binding cleft Closed at both ends Open at both ends

General bound peptide size 8-10 aa (8-11 aa) 13-18 aa (10-30 aa)

Peptide binding motifs Anchor 2 & 9 residues Anchor residues along peptide length

Bound peptide structure Ends anchored w/ middle arched up Flat along floor of cleft

SCHEMATIC DIAGRAMS OF MHC-I AND MHC-II MOLECULES

Papain

a1

a3

a2

MHC-encoded a-chain of 45kDa

MHC-I MOLECULE:SCHEMATIC DIAGRAM & DESCRIPTION

a3 domain & b2-m have structural & amino acid

sequence homology with Ig constant domains members of Ig GENE SUPERFAMILY

b2-m

b2-microglobulin, 12kDa, non-MHC encoded,

non-transmembrane, non covalently bound to a-

chain

Peptide antigen in a groove formed

from a pair of 1/2-helixes on a floor of anti-parallel b strands

a-chain anchored to the cell membrane

3-DEMENSIONAL STRUCTURE OF MHC CLASS I

b2

b1

and a b-chain of 28kDaMHC-encoded, a-chain of 33kDa

a2

a1

a and b chains anchored to the cell membrane

a2 & b2 domains have structural & amino acid

sequence

homology with Ig constant domains - members of

Ig GENE SUPERFAMILY

No b2-microglobulin

Peptide antigen in a groove formed from a pair of

a1/1-helixes on a floor of anti-parallel b strands

MHC-II MOLECULE:SCHEMATIC DIAGRAM & DESCRIPTION

Peptide

b-chain

a-chain

PeptidePeptide

b-chainb-chain

a-chaina-chain

PEPTIDE - MHC BINDING MOTIF

b2-m

a-chain

Peptide

a-chain

b-chainPeptide

MHC class I accommodate

peptides of 8-10 amino acids

MHC class II accommodate

peptides of 13-18 amino acids

33

MHC class-I molecule - chain with 2-microglobulin

MHC class-II molecule - and chains

Features of MHC-binding peptides

How does the limited number of MHC moleculespresent >1015 peptides?

MHC CLASS-I AND II STRUCTURES

AND MHC-PEPTIDE BINDING

MHC-BINDING PEPTIDES

Each human usually expresses:

6 types of MHC class I (A, B, C) and

at least 12 types of MHC class II (DR, DP, DQ)

Total of 18 MHC molecules per person

The number of different T-cell antigen receptors is estimated to be

1,000,000,000,000,000 (1x1015).Each of which may potentially recognizes a different peptide antigen.

How can 18 fairly invariant MHC molecules have the capacity to

bind to 1,000,000,000,000,000 (1015) different peptides?

Flexible binding site allows binding of many different peptides.

The binding site is flexible at an early, intracellular stage of maturation and

forms by folding the MHC molecules around the peptide.

Floppy Compact

Allows a single type of MHC molecule to:

bind many different peptides bind peptides with high affinity form stable complexes at the cell surface Export only molecules that have captured a peptide to the cell surface

Why few MHC molecules can react to so many different peptides ?

Complementary anchor residues (often hydrophobic aa) & pockets provide

the broad specificity of a particular type of MHC molecule for peptides.

2) MHC molecules bind peptides of different length.

Peptide sequence between anchors can vary.

Number of amino acids between anchors can vary.

Arched

peptide

1) MHC polymorphism allows for more peptide antigen bindings.

MHC-I moleculeMHC-I molecule

Why few MHC molecules can react to so many different peptides ?

P S

ASI

K

S

YI

P2

P9

I

P SA IK S

Y

P3P9

S

P1

P4 P5P8

P2 P3 P

6

NH3+

P7 P9

COO-

W L S L L V P F VHLA-A*0201 L L F G V P V Y V

I L K E P V H G Y

R L R P G G K K KHLA-A3 I L R G S V A H K

R L R A E A G V K

K T G G P I Y K RHLA-A*6801 E V A P P E Y H R

A V A A V A A R R

G P G P Q P G P LHLA-B7 I P Q C R L T P L

P P P I F I R R L

R R V K E V V K KHLA-B27 G R I D K P I L K

R R I K E I V K K

A B C D E F

P1 P2 P3 P4 P5 P6 P7 P8 P9

Peptide

PEPTIDES

A

B

Adapted from Klein J, Sato A. 2000. New England J Med 343:702

HUMAN MHC:

Number of HLA Class-I Alleles:

660 HLA-B > 370 HLA-A > 190 HLA-C

4.6x107 MHC-I combinations

HLA Class-II for Antigen Presentation:

3 HLA-DRA, 480 HLA-DRB,

(15) HLA-DPA1, 118 HLA-DPB1,

28 HLA-DQA1, 62 HLA-DQB1

8x1011 MHC-II combinations

4x1019 MHC-I & -II combinations

SUMMARY ON HLA AND

BINDING INTERACTIONS

Pockets

of an HLA

molecule

Pocket A

Pocket B

Pocket C

Pocket D

Pocket E

Pocket F

BINDING GROOVE

EXTRACELLULAR REGION

Transmembrane Domain

Cytoplasmic DomainSignal Peptide

HLA-A/B ( 362 / 365 aa )

h

-

-

h

h

h

h

- -

h = hydrophobic aa

T cells can only be activated by interaction between the T-cell receptor(TCR) and peptide antigen in an MHC molecule.

Without T cells there can be no effective immune response.

There is strong selective pressure on pathogens to evade the immune response.

The MHC has evolved two strategies to prevent evasion by pathogens:

More than one type of MHC molecule in each individual

Extensive differences in MHC molecules between individuals

MHC MOLECULES AS THE TARGETS

FOR IMMUNE EVASION BY PATHOGENS

Example: If MHC X was the only type of MHC molecule

Population threatened with

extinction

Survival of

individual

threatened

Pathogen that

evades MHC X

MHC

XX

Example: If each individual could make two MHC molecules, MHC X and Y

Impact on the

individual depends

upon genotype

Pathogen that

evades

MHC X

MHC

XX

MHC

XY

Population survives

MHC

YYbut has

sequences

that bind to

MHC Y

Example: If each individual could make two MHC molecules, MHC X and Yand the pathogen mutates

Population threatened with

extinction

Survival of individual

threatened

Pathogen that

evades

MHC X but has

sequences that

bind to MHC Y

MHC

XX

MHC

XY

MHC

YY

The number of types of MHC molecule can not be increased ad infinitum.

.until it mutates to

evade MHC Y

Populations need to express allelic variants

of each type of MHC molecule

The rate of replication by pathogenic organisms is faster than humanreproduction.

In a given time, a pathogen can mutate genes more frequently than humans

and can easily evade changes in MHC molecules.

The number of types of MHC molecules are limited.

To counteract the flexibility of pathogens:

The MHC has developed many variants of each type of MHC molecule.

These MHC variants may not necessarily protect all individuals from every pathogen, but will protect the population from extinction.

How diverse is the MHC distribution in a population?

~4 x 1019 unique combinations

IF each individual had 6 types (HLA-A, -B, -C, -DR, -DP, -DQ) of MHC

In reality MHC alleles are NOT randomly distributed in the population.

Alleles segregate with lineage and race.

15.18

28.65

13.38

4.46

0.02

5.72

18.88

8.44

9.92

1.88

4.48

24.63

2.64

1.76

0.01

CAU AFR ASI

Frequency (%)

HLA-A1

HLA- A2

HLA- A3

HLA- A28

HLA- A36

Group of alleles

the alleles of each MHC type were randomly distributed in the population

any of the 1,900 alleles could be present with any other allele

Antigen processing by antigen presenting cells (APC).

MHC-II restricted antigen presentation by APC.

MHC-I restricted antigen presentation by target cells.

MHC-RESTRICTION IN

ANTIGEN PROCESSING AND PRESENTATION

ANTIGEN (Ag) PROCESSING AND PRESENTATION

Fixation blocksmetabolic activity.

No Ag Processing orPresentation

Ag Processing Ag Presentation

Ag PeptideBinding

Ag Presentation

Ag Processing & Presentation by APC to TH cell MHC Restriction of TC Activity

MHC restriction: Effector (Tc) cells and target cellsneed to be MHC identical.

Uninfected

APC process Ag and present Ag peptide in context

of MHC-II to TH cells.

Chromium Release Assay

DIFFERENTIAL DISTRIBUTION OF MHC MOLECULESTissue MHC class I MHC class II

T cells +++ - / + (activation)

B cells +++ ++

Macrophages +++ ++ (activation)

Dendritic cells +++ +++

Thymic epithelial cells + +++ (activation)

Glial cells + ++ (activation)

Vascular endothelial cells + human + ++ (activation)

Neutrophils +++ -

Hepatocytes + -

Kidney + -

Brain + -

Erythrocytes - -

1) Cell activation affects the level of MHC expression.

2) The pattern of expression reflects the function of MHC molecules:

a. Class I is involved in anti-microbe immune responses.

b. Class II involved in activation of other cells of the immune system.

HLA - AInteract with TCR of CTLs (Tc)

HLA - B MHC-I to Induce CD8+ CTL Activity: (all nucleated cells) Foreign graft antigens

HLA - C Virus-infected cellsBacteria-infected cellsParasite-infected cells

HLA - DRInteract with TCR of TH Cells

HLA - DQ MHC-II to Induce CD4+ TH Activity: (APC) Foreign graft antigens

HLA - DP Virus-infected cellsBacteria-infected cellsParasite-infected cells

GENE PROTEIN FUNCTION

MAJOR FUNCTIONS OF MHC MOLECULES

Classical antigen presentation:

Endogenous antigen presentation by MHC-I (cytosolic pathway) to T cells.

Exogenous antigen presentation by MHC-II (endocytic pathway) to T cells.

Non-classical antigen presentation:

Cross-presentation of exogenous antigens by MHC-I to T cells.

Non-peptide (lipid, sugar) antigen presentation by CD1 to T cells

MHC AND CD1

IN ANTIGEN PRESENTATION

Intracellular (cytoplasmic)

Intracellular (cytoplasmic)

CYTOSOLIC PATHWAY (Endogenous Ag presented by MHC-I)

ENDOCYTIC PATHWAY (Exogenous Ag presented by MHC-II)

CYTOSOLIC AND ENDOCYTIC PATHWAYS

SCHEMATIC DIAGRAM OF

In nucleated cells

In APCs

PROTEASOME

PEPTIDES

TAP Transporter in Antigen Processing

GOLGI

ER

MHC I - RESTRICTED PRESENTATION OF ENDOGENOUS AG

Cytosolic Processing of Endogenous Proteins

Synthesis of MHC-I Chain & -Microglobulin

Transport of Peptide-MHC IThrough Golgi

Peptide DrivenMHC-I Assembly

Presentation of Peptide-MHC I to CD8+ CTL

PLASMA MEMBRANE

Step 1

2-m

Synthesis of MHC-I chain and 2-microglobulin

Step 2Step 3

VIRALINFECTION

(viral Agproduction)

Protein

A

Ubiquitin = small protein

B

CYTOSOLIC PROTEOSOMIC SYSTEM FOR DEGRADATION

OF PROTEIN INTO PEPTIDES ( Step 1 )

C IFN and TNF enhance immunoproteosome, which is found in virus infected cells and has a more rapid degradation activity than

regular proteosome.

TAP2TAP1Step 2

In Rough ER (RER)

Step 3

STEP 2: TAP MEDIATED TRANSFER

OF PEPTIDES INTO RER

STEP 3: MHC-I -CHAININTERACTION WITH 2-mAND LOADING OF PEPTIDE

TAP = Transporter Associated

with Antigen Processing

Processing of Cytosolic Antigens for CI Associated presentation

1) Peptides are derived from cytosolic proteins

2) Proteolytic degradation of cytosolic proteins-mediated by proteosome- large multi-protein enzyme w/ broad range of proteolytic activity-present in most cells

3) Transport of peptides from cytosol to ER-2 genes located within MHC that mediate ATP dependent transport of Low molecular weight compounds across cellular membranes- TAP-1, TAP-2 located in ER transports from cytosol to ER lumen 6-30 aa long basic or hydrophobic peptides

55

4) Assembly of peptide-Complex in ER- Proper alpha chain folding is dependent upon ER chaperone proteins calnexin and Calreticulin. Empty CI dimers remain attached to TAP complex by tapasin- peptide enters, is trimmed by ERAP, peptide binds, the peptide/CI complex is released, and exits the ER

56

Processing of endocytosed Antigen for CI associated Presentation

5) Surface expression of peptide/ CI complex associate w/ CD8+ T cells

57

Processing of endocytosed Antigen for CI associated Presentation

Proteosome A 700 kD form appears as a cylinder w/ stacked array

of 2 outer/ 2 inner rings composed of 7 subunits

3 of 7 subunits are catalytic sites for proteolysis

2 subunits LMP-2 and LMP-7 are encoded by genes of the MHC

Performs basic housekeeping in cell- proteins targeted for degradation are coated w/ ubiquitin

IFN gamma increases synthesis of LMP-2 and LMP-7

58

GOLGI

ERAlpha Beta

MHC II - RESTRICTED PRESENTATION OF EXOGENOUS AG

Fusion of Endosomewith Vesicle Containing MHC; Ii released.

MHC-I I Synthesis

Presentation of Peptide-MHC II toCD4+ T-Helper Cell

Plasmamembrane

Ii

Endosome

Lysosome

Exocytic vesicleBinding of Peptide to MHC-II

Endocytosis of Exogeneous Aginto Endosomes

Processing of Exogeneous Ag in Endosome or Lysosome

Step 1

Step 2

(invariant chain)

Step 3

STEP 1: THREE MODES OF

EXOGENOUS Ag UPTAKE

1) Receptor mediated uptake /phagocytosis

2) Phagocytosis

3) Pinocytosis

STEP 2: ENDOSOMAL

DEGRADATION OF

FOREIGN PROTEIN

1) Proteinases activated by acidity

2) Degraded to ~24 aa peptides

3) Peptide size suitable for MHC-II

Step 1

Step 2

STEPS 1-3: 1) Receptor mediated exogenous

Ag uptake

2) Endosomal degradation of Ag

3) Loading peptide on MHC-II

STEP 3: 1) MHC-II interact with invariant

chain

2) CLIP production

3) HLA-DM assist peptide loading

on MHC-II

4) HLA-DO block

HLA-DM activity

Steps 1-3

Step 3

CLIP = class II-associated invariant chain peptide

62

SUMMARY OF

MHC-I ENDOGENOUS AND MHC-II EXOGENOUS PATHWAYS

64

Chloroquine

endocytosis

Emetine

Protein synthesis

Classical Presentation:

Endogenous antigen presentation by MHC-I to CD8+ T cells.

Exogenous antigen presentation by MHC-II to CD4+ T cells.

Non-classical Presentation:

Cross-presentation of exogenous antigens by MHC-I to CD8+ T cells.

Non-peptide antigen presentation by CD1 to T cells and NK T cells.

MHC-I AND CD1

IN NON-CLASSICAL ANTIGEN PRESENTATION

1) Exogenous Ag uptake &

endosomal degradation

2) Peptide loading on MHC-I in RER?

3) Exogenous Ag peptide

presented in context of MHC-I

to CD8+ T cells.

CROSS-PRESENTATION OF EXOGENOUS Ag BY MHC-I

NON-PEPTIDE Ag PRESENTATION BY CD1 1) Presentation of lipid and glycolipid antigens to T cells (CD1) and NK T cells (CD1d)

2) Humans express CD1a, CD1b, CD1c, CD1d, and CD1e. Mice express only CD1d (CD1d1 &CD1d2).

3) CD1a, CD1b, and CD1c on immature thymocytes and professional APCs (DC); CD1c on B cells;

CD1d1 on nonprofessional APCs and on certain B-cell subsets (mouse T, B, DC, hepatocyte, some epithelial cells).

4) CD1 pathway is still unclear, but different from MHC-I and -II pathways:

a) CD1a found in early endosomes, recycling endocytic compartments, & on cell surface.

b) CD1b and CD1d found in late endosome and lysosomal compartment.

c) CD1c found throughout the endocytic system.

Thought to work directly in endocytic compartments.

5) Structure similar to MHC-I with 2-m.

6) CD1 expression enhanced by

GM-CSF and IL-3.

Group 1Group 2

Group 2

Line 5

Line 2

CD1 H-2Kb

68

69

70