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Antigen Processing and

Presentation
Chapter 8

Cell-Mediated Immune Response


T - 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
2

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

T 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 disease


Infectious diseases
Cancer
Autoimmune diseases

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

MHC MOLECULES
Peptide
Binding
Cleft or
Groove

MHC class I

Peptide
Foreign Peptides
Virus
Bacteria
Parasite
Fungi
Allergen
Transplant

MHC class II

Peptide
Binding
Cleft or
Groove

Self Peptides
Cryptic self peptides
(autoimmune Ag)
Altered self (cancer)

Cell Membrane

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

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,HLAB5, HLA-DR7,etc
Each person gets 2 haplotypes- one from
mom and the other from dad

GENERAL ORGANIZATION OF MOUSE & HUMAN MHC


(Histocompatibility-2 complex)

Chromosome 17
Glycoprotein (Gp)
or Protein (P)
(Human leukocyte antigen complex)

Chr. 6

Gp
or 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 - pseudogenes
DQ2, 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?

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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

DIVERSITY OF MHC GENES IN AN INDIVIDUAL


DP

DQ

DR

b a

b a

b1 a

A
Polygeny

DP
b a

DQ
b a

DR
b1 a

DP
b a

DQ
b a

DR
b1 a

A
Variant alleles
polymorphism

HAPLOTYPE 1

HAPLOTYPE 2

Additional set
of variant alleles
on second
chromosome

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


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).

HUMAN LEUKOCYTE ANTIGEN ( HLA ) GENES TO PROTEINS

SOME 6

mere

HLA HAPLOTYPE
DP DQ DR

MHC molecules are


CODOMINANTLY
Class II
expressed

C4B Bf 21OH TNF- b


C4A C2 TNF - a HSP

Class III

B C

Class I

DR55
DQ63
DR
45
DQ
33
B7 C9
DQ36
DR54
DP
11
DQ66
DR44
81
(2 x A) + (2 x B) + (2 x C) = 6 MHC-I
8
APC
(4 x DR) + (4 x DP) + (4 x DQ) = 12 MHC-II

A11

INHERITANCE OF
HLA
HAPLOTYPES
DP-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

Parents
DP

DP

DQ

DQ

DR

DR

B C

B C

Children

DP

DQ

DR

B C

DP

DQ

DR

B C

What is a haplotype ?
A set of linked MHC alleles present on
one parental chromosome.
Thus, an individual has two haplotypes.

DP-1,9
DQ-3,7
DR-5,5
B-7,3
C-9,1
A-11,9
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-2,9
DQ-4,7
DR-6,5
B-8,3
C-10,10
A-12,9

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

DP

DQ

DR

B C

SCHEMATIC DIAGRAMS OF MHC-I AND MHC-II MOLECULES

Papain

COOH

Description
Peptide-binding domain
Peptide-binding cleft
General bound peptide size
Peptide binding motifs

Bound peptide structure

COOH

COOH

MHC-I Molecules

MHC-II Molecules

1 / 2

1 / 1

Closed at both ends


8-10 aa (8-11 aa)
Anchor 2 & 9 residues

Ends anchored w/ middle arched up

Open at both ends


13-18 aa (10-30 aa)
Anchor residues along peptide length

Flat along floor of cleft

MHC-I MOLECULE:
SCHEMATIC DIAGRAM & DESCRIPTION
MHC-encoded a-chain of 45kDa
a-chain anchored to the cell membrane

a2

a1

a3

b2-m

Peptide antigen in a groove formed


from a pair of 1/2-helixes on a
floor of anti-parallel b strands
b2-microglobulin, 12kDa, non-MHC encoded,
non-transmembrane, non covalently bound to achain
a3 domain & b2-m have structural & amino acid
sequence homology with Ig constant domains
members of Ig GENE SUPERFAMILY

3-DEMENSIONAL STRUCTURE OF MHC CLASS I

MHC-II MOLECULE:
SCHEMATIC DIAGRAM & DESCRIPTION
MHC-encoded, a-chain of 33kDa
and a b-chain of 28kDa
a and b chains anchored to the cell membrane

b1
b2

a1
a2

No b2-microglobulin
Peptide antigen in a groove formed from a pair of
a1/1-helixes on a floor of anti-parallel b strands
a2 & b2 domains have structural & amino acid
sequence
homology with Ig constant domains - members of
Ig GENE SUPERFAMILY
Peptide
a-chain

b-chain

PEPTIDE - MHC BINDING MOTIF

a-chain

a-chain

Peptide
b2-m

MHC class I accommodate


peptides of 8-10 amino acids

Peptide

b-chain

MHC class II accommodate


peptides of 13-18 amino acids

33

MHC CLASS-I AND II STRUCTURES


AND MHC-PEPTIDE BINDING
MHC class-I molecule - chain with 2microglobulin
MHC class-II molecule - and chains
Features of MHC-binding peptides
How does the limited number of MHC molecules
present >1015 peptides?

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?

Why few MHC molecules can react to so many 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 ?

1) MHC polymorphism allows for more peptide antigen bindings.


2) MHC molecules bind peptides of different length.
Arched
peptide
P2

P S S
P9
I
A K

I
MHC-I molecule

P3

S A

P9

K S I P S
Y

I
MHC-I molecule

Complementary anchor residues (often hydrophobic aa) & pockets provide


the broad specificity of a particular type of MHC molecule for peptides.
Peptide sequence between anchors can vary.
Number of amino acids between anchors can vary.

SUMMARY ON HLA AND


BINDING INTERACTIONS

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
Signal Peptide

EXTRACELLULAR REGION

A
HLA-A*0201

W
L
I

L
L
L

S
F
hK

L
G
E

L
V
P

V
P
V

P
V
H

F
hY
G

V
V
Y

HLA-A3

R
I
R

L
L
L

R
R
R

P
G
A

G
S
E

G
V
A

K
A
G

K
H
- V

K
K
K

HLA-A*6801

K
E
A

T
V
V

G
hA
A

G
P
A

P
P
V

I
E
A

Y
Y
A

K
H
- R

R
R
R

HLA-B7

G
I
P

P
P
P

G
Q
hP

P
C
I

Q
R
F

P
L
I

G
T
R

P
P
hR

L
L
L

HLA-B27

R
R
G
R
R - R

V
I
I

K
D
K

E
K
E

V
P
I

V
I
V

K
L
- K

K
K
K

Cytoplasmic Domain

BINDING GROOVE
Pocket
Pocket
Pocket
Pocket
Pocket
Pocket

A
B
C
D
E
F

PEPTIDES
P1 P2 P3 P4 P5 P6 P7 P8 P9

Transmembrane Domain

NH3+

P4 P5

P8 P

P1

HLA-A/B ( 362 / 365 aa )

P2 P3

Pockets
of an HLA A B
molecule Adapted from
h = hydrophobic aa

P
6
D

P7

P9

Klein J, Sato A. 2000. New England J Med 343:7

MHC MOLECULES AS THE TARGETS


FOR IMMUNE EVASION BY PATHOGENS
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

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

MHC
XX

Pathogen that
evades MHC X

Survival of
individual
threatened

Population threatened with


extinction

Example: If each individual could make two MHC molecules,


MHC X and Y

Pathogen that
evades
MHC X
but has
sequences
that bind to
MHC Y

MHC
XX

MHC
YY
MHC
XY

Impact on the
individual depends
upon genotype

Population survives

Example: If each individual could make two MHC molecules,


MHC X and Yand the pathogen mutates
MHC
XX
Pathogen that
evades
MHC X but has
sequences that
bind to MHC Y
.until it
mutates to
evade MHC Y

MHC
YY
MHC
XY

Survival of individual
threatened

Population threatened with


extinction

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

Populations need to express allelic variants


of each type of MHC molecule
The rate of replication by pathogenic organisms is faster than human
reproduction.

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?


IF

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

~4 x 1019 unique combinations


In reality MHC alleles are NOT randomly distributed in the population.
Alleles segregate with lineage and race.
Frequency (%)
Group of alleles

CAU

AFR

ASI

HLA-A1

15.18

5.72

4.48

HLA- A2

28.65

18.88

24.63

HLA- A3

13.38

8.44

2.64

HLA- A28

4.46

9.92

1.76

HLA- A36

0.02

1.88

0.01

MHC-RESTRICTION IN
ANTIGEN PROCESSING AND PRESENTATION

Antigen processing by antigen presenting cells


(APC).
MHC-II restricted antigen presentation by APC.
MHC-I restricted antigen presentation by target
cells.

ANTIGEN (Ag) PROCESSING AND PRESENTATION


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

Fixation blocks
metabolic
activity.

No Ag
Processing or
Presentation

Ag Presentation

Ag Processing

Ag Peptide
Binding

Uninfected

Ag Presentation

APC process Ag and present Ag peptide in context


of MHC-II to TH cells.

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

Chromium Release Assay

DIFFERENTIAL DISTRIBUTION OF MHC MOLECULES


Tissue

MHC class I

T cells

+++

- / + (activation)

B cells
Macrophages
Dendritic cells

+++
+++
+++

++
++ (activation)
+++

Thymic epithelial cells


Glial cells
Vascular endothelial cells
(activation)

Neutrophils
Hepatocytes
Kidney
Brain
Erythrocytes

+
+
+

+++
+
+
+
-

MHC class II

+++ (activation)
++ (activation)
human + ++

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.

MAJOR FUNCTIONS OF MHC MOLECULES


GENE

PROTEIN

FUNCTION

HLA - A
HLA - B

MHC-I
(all nucleated cells)

HLA - C

Interact with TCR of CTLs (Tc)


to Induce CD8+ CTL Activity:
Foreign graft antigens
Virus-infected cells
Bacteria-infected cells
Parasite-infected cells

HLA - DR
HLA - DQ

MHC-II
(APC)

HLA - DP

Interact with TCR of TH Cells


to Induce CD4+ TH Activity:
Foreign graft antigens
Virus-infected cells
Bacteria-infected cells
Parasite-infected cells

MHC AND CD1


IN ANTIGEN PRESENTATION
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

SCHEMATIC DIAGRAM OF

CYTOSOLIC AND ENDOCYTIC PATHWAYS


CYTOSOLIC PATHWAY (Endogenous Ag presented by MHC-I)

Intracellular (cytoplasmic)

In nucleated cells

ENDOCYTIC PATHWAY (Exogenous Ag presented by MHC-II)

Intracellular (cytoplasmic)

In APCs

MHC I - RESTRICTED PRESENTATION


OF ENDOGENOUS AG

Presentation of
Peptide-MHC I
to CD8+ CTL

PLASMA MEMBRANE
VIRAL
INFECTION
(viral Ag
production)
Transport of
Peptide-MHC I
Through Golgi

GOLGI

Step 1

Cytosolic Processing of
Endogenous Proteins

PROTEASOME
Peptide Driven
MHC-I Assembly

PEPTIDES

Step 3

TAP
ER

2-m

Step 2

Transporter in
Antigen Processing

Synthesis
MHC-I
Chainand
& -Microglobulin
Synthesis
of of
MHC-I

chain
2-microglobulin

CYTOSOLIC PROTEOSOMIC SYSTEM FOR DEGRADATION


OF PROTEIN INTO PEPTIDES ( Step 1 )
A

Protein

Ubiquitin = small protein

C IFN and TNF enhance immunoproteosome, which is found in


virus infected cells and has a more rapid degradation activity than
regular proteosome.

STEP 2: TAP MEDIATED TRANSFER


OF PEPTIDES INTO RER
TAP = Transporter Associated
with Antigen Processing
Step 2

STEP 3: MHC-I -CHAIN


INTERACTION WITH 2-m
AND LOADING OF PEPTIDE

Step 3

In Rough ER (RER)

TAP1

TAP2

Processing of Cytosolic Antigens for CI


Associated presentation
1) Peptides are derived from cytosolic proteins
2) Proteolytic degradation of cytosolic proteinsmediated 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

Processing of endocytosed Antigen for CI


associated Presentation
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

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

Presentation of
Peptide-MHC II to
CD4+ T-Helper Cell

MHC II - RESTRICTED PRESENTATION


OF EXOGENOUS AG

Step 1 Endocytosis of

Plasma
membrane

Exogeneous Ag
into Endosom es

Exocytic vesicle

Binding of Peptide
to MHC-II

Endosome

Fusion of Endosome
with Vesicle Containing
MHC; Ii released.

Step 3

Processing of
Exogeneous Ag
in Endosom e
or Lysosom e

Lysosome

GOLGI

Step 2

MHC-I I Synthesis

Alpha

Beta

ER

Ii (invariant chain)

STEP 1: THREE MODES OF


EXOGENOUS Ag UPTAKE

Step 1

1) Receptor mediated uptake / phagocytosis

2) Phagocytosis
3) Pinocytosis

Step 2

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

Steps 1-3

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
Step 3
4) HLA-DO block
HLA-DM activity

CLIP = class II-associated invariant chain peptide

62

SUMMARY OF

MHC-I ENDOGENOUS AND MHC-II EXOGENOUS PATHWAYS

Chloroquine
endocytosis

Emetine
Protein synthesis

64

MHC-I AND CD1


IN NON-CLASSICAL ANTIGEN PRESENTATION

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.

CROSS-PRESENTATION OF
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.

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.

CD1

H-2Kb
Line 2

Group 2

Line 5
Group 2

Group 1

68

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70