CHAPTER 8 - MHC molecules were initially discovered during studies

aimed at understanding the molecules responsible

for rejection of transplanted tissues.

Major Histocompatibility Complex (MHC) - Hence the name “Major Histocompatibility Complex”
(MHC).

What is MHC?
– HLA - The term “Major Histocompatibility Complex” actually
refers to a region of the genome that encodes a number
– H-2 of genes (hence Complex) that play an important (hence
Major) role in tissue transplantation (hence
– Minor histocompatibility antigens
Histocompatibility).
– Peter Gorer & George Sneell (1940)
- The term “MHC molecule” or “MHC antigen” refers to a
molecule encoded by a gene within this region.

Chromosome 17

Significance of the MHC

L

role in immune response

role in organ transplantation
Chromosome 6

role in predisposition to disease

In humans:
In the Mouse:
Class I = A, B and C (also called HLA-A,
HLA-B and HLA-C) Class I = K, D and L molecules (also called
- Ag (peptide) presentation to CD8+ cells H-2D, H-2K and H-2L)

Class II = DP, DQ and DR (also called Class II = A and E (also called I-A and I-E)
HLA-DP, HLA-DQ and HLA-DR)
- Ag (peptide) presentation to CD4+ cells Class III = Complement proteins, Tumor
necrosis factor (TNFs)-α, β
Class III = Complement proteins, Tumor
necrosis factor (TNFs)-α, β

1
 MHC- Polymorphism Polymorphism of MHC antigens
(based on phenotype)
• MHC loci are highly polymorphic – presence
of many alternative forms of the gene or 250 221
alleles in the population 188
200
• Inherited from mother and father 150
76 85
• New haplotypes are generated by 100
32 42
50 10 18
recombination
0
DPA DPB DQA DQB DRB B C A

MHC-II MHC-I

MHC polymorphism
Polymorphism of MHC genes The loci that encode class I and class II MHC molecules are the most
(based on DNA sequence/ PCR) polymorphic known in higher vertebrates.

460 Within any species, there are many different alleles for each class I
500 and class II MHC molecule.
400 360
Humans:
300 220
HLA Class-I genes: A (240), B (470), C (110) alleles (1.2 x 107)
200 96 110 HLA Class-II genes:
100 20 22 48
1 DP= DPB1 (96) alleles
0 DQ= DQA1 (22), DQB1 (49) alleles
DR= DRB1 (304), DRB1 (1), DRB1 (35), DRB1 (11), DRB1 (15) alleles
1.8 x 1011 different Class II combinations, and
A
PA

B
B
PB

A
B

C
Q

R
Q
D

D
D

(1.2 x 107) x (1.8 x 1011) = 2.25 x 1018 different combinations of

Class I and Class II possible combinations
MHC-II MHC-I

MHC- Polymorphism
• MHC loci are highly polymorphic –
presence of many alternative forms of the
gene or allele in the population
• Inherited from mother and father
• New haplotypes are generated by
recombination
**

Rafa Ashley Nicole Morgan Amber

2
 MHC- Polymorphism
• MHC loci are highly polymorphic –
presence of many alternative forms of the
gene or allele in the population
• Inherited from mother and father
• New haplotypes are generated by
recombination

Cross-Over

Terminology: Mouse Strains

• Haplotype: set of alleles present in each
parental chromosome (two sets). • Thus, the strain C57BL/6 was designated H-2b
• Inbred mouse strains: same set of alleles haplotype and said to possess the ‘b’ allele at each
(homozygous) at each locus (K, IA, IE, L, D). MHC locus.
• Inbred strains are SYNGENIC = identical at
all genetic loci Thus, it is: H-2b = Kb, Db, Lb, I-Ab, I-Eb
• Inbred strains have been bred by brother-sister • Another strain, CBA/2 was found to possess
mating for > 20 generations
different alleles than C57BL/10 and was
• Outbred mouse strains: different set of alleles arbitrarily designated as having the k haplotype
at each locus ~ like humans. (I.e. H-2k).
• Congenic strains = genetically identical
except at a single loci Thus, it is: H-2k = Kk, Dk, Lk, I-Ak, I-Ek

INHERITANCE OF MHC HAPLOTYPES

MOUSE HAPLOTYPES – INBRED STRAINS

Syngenic
Syngenic

3
 MHC-I Molecule - Heterodimers
Recipient
DONOR Recipient - Two noncovalently bound
chains

- Alpha chain
- encoded in the MHC
- transmembrane
- 3 domains (1, 2, 3)

- β2-microglobulin
- not encoded in MHC
- not transmembrane
- 1 domain
DONOR
- Class I MHC cannot be
expressed without
β 2-microglobulin

Kuby Figure 8-3

Kuby Figure 7-10a

− α1 and α2 domains
MHC-I Molecule Form the peptide-
binding cleft

− α3 domain performs a
structural role but
has no direct role in
peptide binding

- CD8 binds to the α3

domain
*
- Both, MHC-1 and β2-
microglobulin belong
to the Ig superfamily
− β 2- microglobulin shown in blue; antigen shown in red
-*Papain cleavage (*)
- Closed peptide-binding cleft - binds peptides 8-11 amino acids in
Kuby Figure 8-3
length

MHC-II Molecule MHC-II Molecule

- Heterodimers (different - α1 and β1 domains form
molecules) the peptide-binding
cleft
- Two noncovalently bound
chains - α2 and β2 domains
perform a structural
- alpha chain and beta role but have no direct
chains role in peptide binding
- both encoded in the MHC
- both transmembrane - the β2 domain is bound
by CD4
- both chains are necessary
for expression of class II
MHC

Kuby Figure 8-5 Kuby Figure 8-5

4
 Kuby Figure 7-10b

− β -chain shown in blue; antigen shown in red

8-11 amino acids 13-18 amino acids
- Open peptide-binding cleft - binds peptides 13-18 amino acids in
length

Class II genes
Class I genes - classical and non-classical
* *

* *

Figure 4.13 Figure 4.14

Class III genes Peptide-MHC Interaction

1. Complement components (C2, C4)
• Peptide binding by MHC molecules is not as specific
2. Tumor necrosis factor (alpha, beta) as antigen binding by antibodies or T cell receptors.
3. Heat shock proteins (HSP)
• Any particular MHC molecule will bind a large
range of peptides - but only ONE at a time.

• A given MHC molecule will bind peptides that have

certain amino acids at key positions in the peptide
(anchor residues).

• Each MHC molecule binds a unique set of peptides.

Keep in mind that each allelic variant also binds a
unique set of peptides!!

Figure 4.15

5
 Aromatic Hydrophobic
MHC-Peptide Interaction
MHC-I:
- Each unique molecule (A, B or C) binds a
unique set of peptides
- Single nucleated cell express 105 of each class
I molecule (~ 300,000 MHC-I molecules!)
- As few as 100 peptide-MHC complexes are
enough to target a cell for killing by CD8+
- Requirements:
*
1) 8-11aa length,
2) key amino acids at positions 2 and 9 *

*
Kuby Figure 7-11

MHC-Peptide Interaction MHC-II-Peptide Interaction

MHC-II: P1 P4 P6 P9

- IA, IE bind a unique set of peptides

- Although there are a few MHC-II molecules
on the surface of APC, MHC-II molecules
are UP-REGULATED after activation
(cytokines!)

- Requirements: F = phenyl alanine K = Lysine

1) Larger peptides (8-11aa length, D = Aspartic Acid Y = Tyrosine
N = Asparagine P = Proline
2) Key amino acids at positions 1, 4, 6, 9 E = Glutamic acid A = Alanine
R = Arginine

Peptide-binding grooves for class I

and class II MHC are structurally
Aspects of MHC
similar 1. Recognition by T cells requires cell-cell
contact.
• Both have a peptide-binding groove
2. Peptides from cytosol associate with class
• Close-ended groove for class I MHC requires I MHC and are recognized by Tc cells.
an 8-11 amino acid-length peptide to bind
3. Peptides from endocytic vesicles
• Open-ended groove for Class II MHC lets it associate with class II MHC and are
bind a peptide 13-18 amino acids long, not all recognized by Th cells.
of which lie in the groove
• Anchor site rules apply to both classes in
particular Class I MHC (P2 and P9)

6
 Aspects of MHC (continued) Aspects of MHC (continued)
3. Although there is a high degree of 4. Mature T cells must have a T cell
polymorphism for a species, an receptor that recognizes the peptide
individual has maximum of six different associated with MHC. This is the
class I MHC products and eight class II second level of control!!!!.
MHC products. 5. Each MHC molecule has only one
4. A peptide must associate with a given binding site. The different peptides a
MHC of that individual, otherwise no given MHC molecule can bind ….
immune response can occur. That is bind to the same site, but only one at
one level of control!!!!. a time.

Aspects of MHC (continued)

Aspects of MHC (continued)
6. MHC polymorphism is determined only
in the germline. There are no 9. Alleles for MHC genes are co-dominant.
recombination mechanisms for Each MHC gene product is expressed on
generating diversity. the cell surface of an individual nucleated
7. Because each MHC molecule can bind cell.
many different peptides, binding is
termed degenerate. [Link] the high degree of polymorphism?
8. Cytokines (especially interferon-γ)
increase level of expression of MHC. Survival of species!

Where is INHERITANCE OF MHC HAPLOTYPES

polymorphism
located in the
molecule?

7
 Crossing Inbred Strains - 6 MHC-I molecules:
Kk Kb, Dk Db, Lk Lb
H-2b = Kb, Db, Lb, I-Ab, I-Eb
X
H-2k = Kk, Dk, Lk, I-Ak, I-Ek - 8 MHC-II molecules:
IAαkβk, IAαbβb, IAαkβb, IAαbβk,
What would be the MHC complex of a liver IEαkβk, IEαbβb, IEαkβb, IEαbβk,
cell in the F1?

In a macrophage?

Regulation of MHC Expression

• 1) Cytokines:
• IFN-α, β, and γ - ↑ Class-I expression.
• IFN- γ - ↑ Class-II expression in MO and DC
• IL-4 ↑ expression of MHC-II in resting B cells
• IFN- γ ↓ expression of MHC-II in B cells
• 2) Corticosteroids and Prostaglandins
• ↓ expression of MHC-II
• 3) Viruses (↓ expression of MHC-I)
• Human cytomegalovirus (CMV)
• Hepatitis B virus (HBV)
• Adenovirus 12 (Ad12)

Kuby Figure 7-16

MHC and immune responsiveness: The term “restricted” is used in various other
ways:
- In many cases, the ability of an inbred mouse strain to respond to
a given antigen will depend on which alleles the strain carries at its
- T cells are MHC-restricted i.e. they must recognize antigen
MHC loci…..low vs high responders!!
presented on self MHC.
- The reason is that if an antigen cannot bind to an MHC molecule,
- CD4+ T cells are class II MHC-restricted i.e. they must recognize
it cannot be presented to T cells and therefore an immune antigen presented on self class II MHC.
response cannot be made to it.
- CD8+ T cells are class I MHC-restricted i.e. they must recognize
- To respond to an antigen, the first criterion that must antigen presented on self class I MHC.
be met is that the individual must have an MHC molecule
that can bind and present the antigen. - A particular T cell clone may be I-Ek-restricted i.e. it recognizes
its antigen ONLY when presented on self I-Ek.
- The second criterion that must be met is that the
individual must have T cells capable of responding to the ("restricted" = "recognizes antigen on…")
antigen.

8
Associations between MHC and disease
The risk of developing immunological diseases is often influenced Associations between MHC and disease
by the presence or absence of specific MHC alleles.
Disease Relative Risk Allele
• Ankylosing Spondylitis 90 B27
• Hereditary hemochromatosis 90 A3/B14
• Narcolepsy 130 DR2

Classic
Self MHC Restriction Experiment to
show self -MHC H-2K

• Both MHC-I and MHC-II molecules can restriction

CD8 T cells
only recognize antigens when presented by
SELF-MHC molecules.
• No value for individual to have T cells
that recognize foreign antigen
associated with foreign MHC H-2K H-2K H-2b

• Self MHC restriction occurs in thymus

(-) (+) (-)

Role of Antigen-Presenting Cells (APC) Antigen presenting cells

- Helper T cells: recognize antigen after processing and • Remember: 1) MHC-II, 2) deliver co-stimulatory
presentation by MHC-II on APC (dendritic cells, macrophages, B signals
cells).
• Professional APC: DC> MΦ Φ > B cells, why?
- Cytotoxic T cells: recognize antigen when it is presented on • DC: Always express high levels of MHC-II
MHC-I. molecules and co-stimulatory activity (B7
molecule)
- Since most nucleated cells in the body express class I MHC,
most cells in the body can present antigen to cytotoxic T cells.
• MΦ Φ : requires activation to up-regulate MHC-II
Although they are presenting antigen, these cells are usually molecules and co-stimulatory molecules (B7
not referred to as “antigen-presenting cells”. If they are molecules)
presenting antigen that will cause them to be killed by cytotoxic • B cells: always express MHC-II molecules but
T cells, they are referred to as “target cells”. needs to be activated to express co-stimulatory
activity (B7 molecule)

9
 Role for APC?

Points Concerning Antigen

Ag processing is required Processing and Presentation
• Classical experiment showing that B and T 1. Location of pathogen
cells have different requirement for antigen • viruses in cytosol, MHC class I pathway, Tc
recognition. response (Cytosolic pathway)
• extracellular bacteria, MHC class II
• Processing is required for Th activation pathway, Th2 response  Ab formation
• Processing is a metabolic active process (Endocytic pathway)
• intracellular bacteria, MHC class II
pathway, Th1 response  cellular response
(Endocytic)

Points Concerning Antigen

Processing and Presentation
2. Peptides derived from both self and
non-self proteins can associate with
MHC class I and class II molecules.
3. Chemical nature of MHC groove
determines which peptides it will
bind.

10
 Endogenous Pathway
• Peptides are generated by proteasome
degradation
• Peptides are transported from cytosol to the
RER
• Peptides loading onto MHC-I is aided by
chaperones

- Size
- Hydrophobicity

- The cytosolic antigen processing pathway - 2. The role of the TAP The cytosolic antigen processing pathway - 3. Assembly of the class I-peptide
(Transporter associated with Antigenic Processing). complex

1. The class I alpha

- Peptides from proteasome degradation of cytoplasmic proteins are transported
chain is stabilized
across the membrane of the rough endoplasmic reticulum by a heterodimeric
by calnexin.
protein designated as TAP.
2. When the alpha
- TAP is composed of two subunits - TAP1 and TAP-2
chain binds beta-2-
- TAP-mediated transport is ATP-dependent
microglobulin:
The genes for TAP1 and TAP2 are encoded within the MHC. - calnexin is lost
- calreticulin, ERp57,
and tapasin bind

3. Tapasin, ERp57 &

calreticulin bring the
class I molecule into
the vicinity of the
ERAAP TAP.
- Facilitate peptide
loading
Kuby Figure 8-6b

The cytosolic antigen processing pathway - 3. Assembly of the class I-peptide

complex

4. A cytoplasmic
peptide transported
through the
TAP is loaded onto
the class I molecule.

*. ERAAP – trims
peptides to right size
or complete
degradation.

5. Class I MHC
dissociates from
calreticulin, ERp57,
and tapasin.
ERAAP
Class I MHC-peptide
complex is
Kuby Figure 8-6b transported to Golgi
and to the cell surface.

11
 Receptor-Mediated Endocytosis
Class II Processing: (Exogenous)

- The endocytic antigen processing pathway –

processing of externally-derived peptides

- Antigen can be taken into cells by various means:

phagocytosis, endocytosis, pinocytosis, receptor-
mediated endocytosis

- Antigen taken up in these ways passes through a

series of intracellular compartments of increasing
acidity - early endosome (pH 6.5-6.0), late endosome
(pH 6.0-5.0), phagolysosome (pH <5.0)

The endocytic antigen processing

pathway - processing of
externally-derived peptides The endocytic antigen processing
pathway - processing of
Three major events occur in the externally-derived peptides
endosomal pathway:
The endosomal compartment is
- Degradation of material that was taken completely separate from the
in – endosome goes through endoplasmic reticulum ---> So,
acidification and fusion with externally derived peptides are
lysosome which contain a wide usually not loaded on to MHC-I.
array of degradative enzymes

- Loading of peptides from this material

on to class II MHC molecules

- Transport of class II MHC - peptide

complexes back to the cell surface

Figure 5-18 Figure 5-18

The endocytic antigen processing Invariant Chain- guides transport to

pathway - processing of endocytic Vesicles
externally-derived peptides

Class II MHC is synthesized in the ER, but

is not loaded with peptides there
because it's peptide binding site is
blocked by the invariant chain (Ii).

Once class II MHC enters the endosomal

compartments, the invariant chain is
degraded, leaving a small fragment -
CLIP - in the peptide binding site.

CLIP is removed by HLA-DM, which loads

a peptide on to class II MHC

HLA-DO inhibits HLA-DM until the cell is CLIP= Class II-associated HLA-DM – mediates exchange
activated invariant chain peptide HLA-DO – inhibits HLA-DM
Figure 5-18

12
 Presentation of Non-Peptide
Antigens
• CD1 molecules (CD1a-e)
• Structurally related to MHC-I
• Encoded outside the MHC region
• Present in APC (DC>MØ>B cells)
• Presents peptides of 12-22 aa in size
• Presents to CD4, CD8 and NK cells
• Present LIPIDS and glycolipids
• Third Ag-processing pathway?

Class I MHC Pathway

Peptide is presented
by MHC-I to CD8
cytotoxic T cell 1 Viral protein is made
Plasma membrane on cytoplasmic
6
The End!! ribosomes

Globular viral
5
protein - intact
Peptide passes 2
with MHC from Golgi Proteasome
body to surface rER degrades
Peptide associates protein to
with MHC-I complex peptides
Peptide transporter
protein moves
4 peptide into ER
3
MHC class I alpha
Golgi body Peptide with MHC
and beta proteins
goes to Golgi body
are made on the rER

Peptide MHC-II
Class II MHC Pathway
complex is presented CD4 helper T cell Globula
to CD4 helper T cell r protein
Globular protein

Endosome fuses with Endosome

Endocytosis
plasma membrane Fusion of endosome
1
3 and exocytic vesicle
Immunodominant Lysosome
peptide binds
to class II MHC 2
Exocytic vesicle fuses
with endosome Protein is processed to
Golgi releasing Ii from αβ dimer peptides in endosome
body or lysosome

Class II MHC
α Synthesis
β Ii 3 chains: α,β and Ii
Endoplasmic reticulum

13