Chapter 8

The Major Histocompatibility

Complex and Antigen Presentation
 Major histocompatibility complex (MHC),
products play roles in intercellular recognition and in
discrimination between self and nonself.

T lymphocytes function against Intracellular microbes and towards

activation of B cells and macrophages
The task of displaying cell associated antigens for recognition by T
cells is performed by specialized proteins encoded by genes in the
MHC
There are two main types of MHC gene products, called class
I MHC and Class II MHC
General Organization and Inheritance of the MHC
MHC genes are present on chromosome 6 in humans and chromosome 17 in
mice
Allelic Forms of MHC Genes Are Inherited in Linked Groups
Called Haplotypes
MHC loci is highly polymorphic
The genes of the MHC loci lie close together; recombination
frequency within the H-2 complex is only 0.5%.
Thus, most individuals inherit the alleles encoded by these
closely linked loci as two sets, one from each parent.
Each set of alleles is referred to as a haplotype.
In outbred populations, the offspring are generally
heterozygous at many loci and will express both maternal
and paternal MHC alleles.
The alleles are codominantly expressed.
If mice are inbred (that is, have identical alleles at all loci), each H-2 locus
will be homozygous because the maternal and paternal haplotypes are
identical, and all offspring therefore express identical haplotypes
Certain inbred mouse strains have been designated as prototype
strains, and the MHC haplotype expressed by these strains is
designated by an arbitrary italic superscript
If two mice from inbred strains having different MHC haplotypes are
bred to one another, the F1 generation inherits haplotypes from both
parental strains and therefore expresses both parental alleles at
each MHC locus
Because such an F1 expresses the MHC proteins of both parental strains
on its cells, it is histocompatible with both strains and able to accept grafts
from either parental strain.
Can parent accept from F1?
When the father and mother have different haplotypes:
there is a one-in-four chance that siblings will inherit the same paternal and maternal
haplotypes and therefore be histocompatible with each other
none of the offspring will be histocompatible with the parents
Although the rate of recombination by crossover is low within the HLA, it still
contributes significantly to the diversity of the loci in human populations.
The high number of intervening generations since the appearance of humans as a
species has allowed extensive recombination, so that it is rare for any two unrelated
individuals to have identical sets of HLA genes.
Inbred mice have aided the study of MHC

MHC Congenic Mouse Strains Are

Identical at All Loci Except the
MHC
 α chain, 44-47kD
MHC Molecules and Genes
β2 microglobulin12kD non MHC encoded
α1 and α2,90 residues long, interact to form
a platform of an eight stranded, antiparallel β
pleated sheet supporting two parallel strands
of α helix
Forms peptide binding cleft
Ends closed, larger peptides cannot be
accommodated
Polymorphic residues confined to α1 and
α2
α3 domain has amino acids conserved among
all class I molecules, binding site for CD8
Is followed by 25 hydrophobic amino acids
30 residues located in cytoplasm, contains
basic amino acids which interact with head
group of phospholipids
Class II Molecules Have Two Nonidentical Glycoprotein Chains
Class II MHC contain two different
polypeptide chains, a 33-kDa chain and a 28-
kDa chain, which associate by non-covalent
interactions.
Each chain in a class II molecule contains two
external domains:
The membrane-proximal domains, bear
sequence similarity to the Ig-fold structure.
The membrane-distal portion forms the antigen
binding cleft.
The peptide binding cleft is composed of a
floor of eight antiparallel strands and sides of
antiparallel helices.
However, it lacks the conserved residues that
bind to the terminal residues of short peptides
and forms instead an open pocket; an open-
ended groove
The membrane-distal, peptide-binding cleft of a human class II MHC molecule, HLA-
DR1 (blue), superimposed over the corresponding regions of a human class I MHC
molecule, HLA-A2 (red)
The Exon/Intron Arrangement of Class I and II Genes Reflects
Their Domain Structure
Class I and II Molecules Exhibit Polymorphism in the Region That
Binds to Peptides
Single MHC molecule can bind multiple peptides can be expected as every
individual contains few MHC molecules (Class I: 6 and class II: 10-20) which must
bind enormous number of peptides
The peptides that bind to MHC molecules share structural features that promote
this interaction
Size, class I: 8-11 residues; class II: 10-30 residues.
Contain amino acids complementary to MHC.
Residues that bind MHC distinct from those that bind T cells.
The association of antigenic peptides and MHC molecules is a saturable
interaction with a very low off rate
Complexes stable with long half life.
Persist for long on surface of antigen presenting cells.
Enables few T cells specific to the antigen to locate the antigen.
The MHC molecules of an individual do not discriminate between foreign peptides
and individual peptides
MHC displays both peptides
T cells survey for foreign antigens
Structural basis of peptide binding to MHC molecules
The binding of peptides to MHC is a noncovalent interaction mediated by
residues both in the peptides and clefts of MHC
Peptides bind in extended conformation
Associated water molecules fill clefts making contact with  strands and 
helix
 Strands on the floor of cleft contain pockets which house R groups which
bind through hydrophobic interactions (anchor residues)
Anchor residues of peptides may be located in the middle or at ends
Each MHC contains 1 or 2 anchor residues, and this presumably allows
more variability to the peptide at other residues (residues recognized by T
cells)
Anchor residues not always present (especially class II)
Interaction with  helix (hydrogen bonds or charge interaction) also
contributes
Class II peptides contain hydrophobic or basic amino acids at C terminal
which also contributes
 CLASS I MHC–PEPTIDE INTERACTION
The ability of an individual class I MHC molecule to bind to a diverse spectrum of
peptides is due to the presence of the same or similar amino acid residues at several
defined positions along the peptides

These amino acid residues

anchor the peptide into the
groove of the MHC molecule,
are called anchor residues.

Because a single nucleated

cell expresses about 105
copies of each class I
molecule, many different
peptides will be expressed
simultaneously on the
surface of a nucleated cell by
class I MHC molecules
Conformation of peptides bound to class I MHC molecules

Schematic diagram of conformational difference in bound peptides of different

lengths. Longer peptides bulge in the middle, whereas shorter peptides are more
extended. Contact with the MHC molecule is by hydrogen bonds to anchor
residues 1/2 and 8/9.
Representation of 1 and 2 domains of HLA-B27 and a bound antigenic peptide
based on x-ray crystallographic analysis of the cocrystallized peptide–HLA
molecule. The peptide (purple) arches up away from the strands forming the floor
of the binding cleft and interacts with twelve water molecules (spheres)
CLASS II MHC–PEPTIDE INTERACTION
Present peptides to CD4+ T cells
Can bind a variety of peptides
Most of the peptides associated with class II MHC molecules derived from
membrane bound proteins or proteins associated with the vesicles of the endocytic
processing pathway.
Peptides contain 13–18 amino acid residues
The peptide-binding cleft in class II molecules is open at both ends
Peptides maintain constant elevation on the floor of the binding cleft
The binding characteristics are determined by the central 13 residues.
They lack conserved anchor residues
Hydrogen bonds between the peptide and class II molecule distributed throughout
the binding site
Internal sequence comprising 7–10 amino acids provide the major contact points
This sequence has an aromatic or hydrophobic residue at the amino terminus and
three additional hydrophobic residues in the middle portion and carboxyl-terminal
end of the peptide.
Class I and Class II Molecules Exhibit Diversity Within a Species and
Multiple Forms Occur in an Individual
An enormous diversity is exhibited by the MHC molecules within a species and within
individuals
• but different from BCR and TCR diversity
• The MHC molecules expressed by an individual are fixed in the genes and do not
change over time

• stems from polymorphism; presence of multiple alleles

 For MHC I:2013 A alleles, 2605 B alleles, and 1551 C alleles
 1.7 billion different class I haplotypes
 For MHC II: 1015 different class II combinations
 Total 1.7 x 1024

The high level of MHC polymorphism may provide a survival advantage by

supplying a broad range of MHC molecules and thus a broad range of
presentable antigens
MHC Polymorphism Has Functional Relevance
Although the sequence divergence among alleles of the MHC within a species is
very high, this variation is not randomly distributed along the entire polypeptide
chain. Instead, polymorphism in the MHC is clustered in short stretches, largely
within the membrane-distal α1 and α2 domains of class I molecules

Location of polymorphic amino acid

residues (red) in the α 1/ α 2 domain
of a human class I MHC molecule.

Similar patterns of diversity are observed in the α1 and β1 domains of class II.
Genomic Map of MHC Genes
The human class I region spans 2000kb at the telomeric end of HLA
complex
Contains 20 genes
Included are genes encoding classical MHC I: HLA-A, HLA-B, HLA-C
Non classical include HLA-E, HLA-F, HLA-G, HFE, HLA-J, HLA-X, MICA to MICE
no polymorphism
• HLA-G plays a role in antigen recognition by NK cells
• play a role in self/nonself discrimination
Some non classical are pseudogenes, others such as HLA-G and HFE encode class I like
products with highly specialized function
MIC family has only 15% to 30% identity to classical class I; MICA are highly polymorphic
MIC expressed at low levels in epithelial cells and induced by heat or other stimuli that
influence heat shock protein
The class II MHC genes are located at the centomeric end of HLA
Genes encoding  and  chains of class II designated HLA-DR, DP and DQ
Multiple  and  chain genes
All the  chain and  chain gene product expressed together, thus increasing number of
different antigen presenting molecules on the cell
Within class II, genes that encode proteins and play role in antigen processing are present:
Transporter associated with antigen processing (TAP), a heterodimer that transports
peptides from cytosol into ER, where peptides can associate with newly synthesized
class I molecule.
Cytosolic protease complex, proteasome, degrades cytosolic proteins into peptides
to be presented by class I MHC
HLA-DMA & HLA-DMB, encode non polymorphic heterodimeric Class II like molecule,
HLA-DM involved in peptide loading to class II molecules.
Human MHC class III genes are between class I and class II
Encode several components of complement, two cytokines (TNF-, TNF-), two
HSPs and two steroid 21 hydroxylases.
Play important role in diseases (eg. Mutation in genes coding 21 hydroxylase,
congenital adrenal hyperplasia)
Conserved in all species with MHC
Cellular expression of MHC molecules
Class I expressed on all nucleated cells but level of expression differs among different cell
types.
Highest level expressed by lymphocytes (1% of total plasma proteins; 5 x 105 molecules per
cell)
Fibroblasts, muscle cells, liver hepatocytes and neural cells express very low levels.
Low levels on liver cells may contribute to considerable success of liver transplants by
reducing chances of graft rejection by Tc cells of the recipient.
A few cells like, neurons and sperm cells at certain stage of development lack MHC
altogether
Heterozygous individuals express on their cells gene products encoded by both the alleles
Cheetah female with two nearly full grown cubs.
Polymorphism in MHC genes of the cheetah is very limited, presumably because of a
bottleneck in breeding that occurred in the not too distant past. It is assumed that all cheetahs
alive today are descendants of a very small breeding pool.
MHC and Immune responsiveness
Self-MHC Restriction of T Cells
Strain-2 antigen-pulsed macrophages activated
strain-2 and F1 T cells but not strain-13 T cells.
Strain-13 antigen-pulsed macrophages activated
strain-13 and F1 T cells but not strain-2 T cells.
Subsequently, using congenic mice it was
confirmed that the CD4 TH cell is activated and
proliferates only in the presence of antigen-
pulsed macrophages that share class II MHC
alleles. Thus, antigen recognition by the CD4
TH cell is class II MHC restricted
In 1974 R. Zinkernagel and P. Doherty demonstrated the self-MHC
restriction of CD8+ T cells

In 1996, Doherty and Zinkernagel were

awarded the Nobel prize for their major
contribution to the understanding of cell-
mediated immunity

LCM: lymphocytic choriomeningitis

Role of Antigen-Presenting Cells
Processing of Antigen Is Required for Recognition by T Cells

Experimental demonstration that

antigen processing is necessary
for TH-cell activation.
(a) When antigen-presenting cells
(APCs) are fixed before exposure
to antigen, they are unable to
activate TH cells.
(b) APCs fixed at least 1 h after
antigen exposure can activate TH
cells.
(c) When APCs are fixed before
antigen exposure and incubated
with peptide digests of the
antigen (rather than native
antigen), they also can activate
TH cells.
TH-cell activation is determined
by measuring a specific TH-cell
response
Most Cells Can Present Antigen with Class I MHC; Presentation with
Class II MHC Is Restricted to APCs
Evidence for Two Processing and Presentation Pathways

Class I presentation requires internal synthesis of virus protein, as

shown by the requirement that the target cell be infected by live virus,
and by the inhibition of class I presentation observed when protein
synthesis was blocked by the inhibitor.

• Class II presentation can occur with either live or replication-

incompetent virus; protein synthesis inhibitors had no effect, indicating
that new protein synthesis is not a necessary condition of class II
presentation.

• Class II, but not class I, presentation is inhibited by treatment of the

cells with an agent that blocks endocytic processing within the cell (e.g.,
chloroquine).
Overview of endogenous and exogenous pathways for
processing antigen
Endogenous Antigens: The Cytosolic Pathway
Intracellular proteins are degraded into short peptides by a cytosolic proteolytic
system present in all cells, called the proteasome

The large (20S) proteasome is composed of 14 subunits arrayed in a barrel-like structure of

symmetrical rings.
Many proteins are targeted for proteolysis when ubiquitin is attached to them.
These ubiquitin-protein conjugates enter the proteasome complex, through a narrow channel at the 19S
end.
A distinct high efficiency proteasome, the immunoproteasome, is found in APCs & infected cells.
This proteasome, has some unique components that are induced by exposure to interferon - or TNF-
and are coded by LMP2 and LMP7 genes (class I region)
 Peptides Are Transported from the Cytosol to the Rough
Endoplasmic Reticulum
Insight into the role that peptide transport, the delivery of peptides to the MHC molecule,
plays in the cytosolic processing pathway came from studies of cell lines with defects in
peptide presentation by class I MHC molecules

The transporter protein, designated

TAP (for transporter associated
with antigen processing) is a
membrane-spanning heterodimer
consisting of two proteins: TAP1
and TAP2
TRANSPORT OF PEPTIDES FROM THE CYTOSOL TO THE
ER
Peptides generated in the cytosol are translocated by a specialized transporter
into the ER, where newly synthesized class I MHC molecules are available to
bind the peptides.

Delivery of cytosolic peptides into the

ER is brought about by the transporter
associated with antigen processing
(TAP).
 Its genes located within the MHC and
are homologous to the ABC
transporter family of genes, which
encode proteins that mediate (ATP)-
dependent transport.
TAP1 and TAP2 genes are next to the
genes encoding LMP-2 and LMP-7 in
the MHC, and the synthesis of the TAP
protein is also stimulated by IFN-γ
Assembly and stabilization of class I MHC molecules

Within the RER membrane, newly synthesized class I chain associates with calnexin, a
chaperone, and ERp57 until β2-microglobulin binds.
Binding of β2microglobulin releases calnexin and allows binding to calreticulin and to
tapasin, which is associated with TAP.
This association promotes binding of an antigenic peptide.
Antigens in the ER processed via exopeptidases such as ERAP1, producing fragments
ideally suited for binding to class I. Peptide association stabilizes the class I-peptide
complex, allowing it to be transported from the RER to plasma membrane.
Exogenous Antigens: The Endocytic Pathway

Antigen-presenting cells can internalize antigen by phagocytosis, endocytosis,

or both.

Macrophages internalize antigen by both processes, whereas most other APCs

are not phagocytic or are poorly phagocytic and therefore internalize
exogenous antigen only by endocytosis
Peptides Are Generated from Internalized Molecules in Endocytic
Vesicles
The Invariant Chain Guides Transport of Class II MHC Molecules
to Endocytic Vesicles
This trimeric protein interacts with the peptide-binding cleft of the class II
molecules, preventing any endogenously derived peptides from binding to the
cleft while the class II molecule is within the RER

A short fragment of the invariant chain termed CLIP (for class II–associated
invariant chain peptide) remains bound to the class II molecule after the invariant
chain has been cleaved within the endosomal compartment
 Processing of Endocytosed Antigens for Class II MHC-Associated Presentation

1. Surface receptors on APCs-Fc & C3b,; B cells- sIg internalize proteins.

2. Antigen processing requires time & cellular metabolism; mimicked by in vitro proteolysis.
3. MHC II are transported from ER to endosomes with an associated protein called the
invariant chain.
4. In MIIC, the Ii dissociates from class II MHC molecules by the action of proteolytic enzymes
and HLA-DM, and antigenic peptides bind peptide-binding clefts of the class II molecules
5. Class II MHC molecules are stabilized by the bound peptides, delivered to the surface of the
APC, and displayed for recognition by CD4+ T cells.
Class II MHC molecules are synthesized in the ER and transported to endosomes
with an associated protein called the invariant chain (Ii), which occupies the
peptide-binding clefts of the newly synthesized class II molecules.

One additional nonclassical member of the class II MHC family is HLA-DO.

HLA-DO is a negative regulator of antigen binding, modulating the function of
HLA-DM and changing the repertoire of peptides that preferentially bind to
classical class II molecules. In cells that express both DO and DM, these two
molecules strongly associate in the ER and maintain this interaction all the way to
the endosomal compartments.
Comparison of three dimensional structures showing the binding groove of HLA
class II molecules (1 and 1) containing different antigenic peptides or CLIP.
The red lines show HLA-DR4 complexed with collagen II peptide, yellow lines are
HLA-DR1 with influenza hemagglutinin peptide, and blue lines are HLA-DR3
associated with CLIP.

No major differences in the structures of the class II molecules or in the conformation of

the bound peptides are seen. This comparison shows that CLIP binds the class II molecule
in a manner identical to that of antigenic peptides
Cross-Presentation of Exogenous Antigens
Presentation of Nonpeptide Antigens

Recent reports indicate that various types of T cells (expressing 

as well as  T–cell receptors) can react against lipid antigens, such
as mycolic acid, derived from well-known pathogens, such as
Mycobacterium tuberculosis.

These antigens are presented by members of the CD1 family of

nonclassical class I molecules.
• three extracellular domains,which associate
noncovalently with 2-microglobulin.
• trafficking and expression profile resembles MHC class
II proteins
• expressed by many immune cell types, including
thymocytes, B cells, and DCs
 The lipid moiety fits into deep pockets within the CD1
binding groove and the hydrophilic head group remains
exposed, allowing recognition by T cells

It is now hypothesized that short-

chain self-lipids with relatively low
affinity are loaded onto CD1
molecules in the ER, shortly after
translation, and allow proper CD1
protein folding.

Cartoon of the deep binding pockets (A, C, and F)

and the deep binding tunnel (T) that accommodate
lipid antigens in a foot-in-shoe fashion in the
members of the CD1 family of nonclassical class I
molecules