C C

a2 ß2

Fig. 3.8 Structure of class I and class II major histocompatibility complex (MHC) molecules. Schematic diagrams

(at left) and models of the crystal structures (at right) of class

I MHC and class II MHC molecules illustrate the domains of

the molecules and the fundamental similarities between them.

Both types of MHC molecules contain peptide-binding clefts

and invariant portions that bind CD8 (the a3 domain of class I)

or CD4 (the a2 and ß2 domains of class II). Ig, Immunoglobulin;

ß2m, ß2-microglobulin. (Crystal structures courtesy Dr. P. Bjorkman, California Institute of Technology, Pasadena, CA.)

60 CHAPTER 3 Antigen Capture and Presentation to Lymphocytes

cells and endothelial cells and can be induced on

other cell types by the cytokine interferon-?.

Inheritance Patterns and Nomenclature of HLA

Genes

Three polymorphic class I genes, called HLA-A, HLA-B,

and HLA-C, exist in humans, and each person inherits one of these genes from each parent, so any cell can

express six different class I molecules. In the class II

locus, every individual inherits from each parent two

separate genes encoding the a chain and the ß chain of

HLA-DP, two encoding DQa and DQß, one or two for

DRß (HLA-DRB1 always and sometimes HLA-DRB3,

HLA-DR4, or HLA-DR5), and one for DRa. The polymorphism resides mainly in the ß chains for class II

genes and exclusively in the a chain for class I genes.

Epithelial cells

Parental

chromosomes

T cells

MHC

molecules

Co-dominant

expression:

 Both parental

 alleles of each

 MHC gene are

 expressed

MHC-expressing

cell types:

 Class I:

 All nucleated

 cells

 Class II:

 Dendritic cells,

 macrophages,

 B cells

Increases number of

different MHC molecules

that can present peptides

to T cells

CD4+ helper

T lymphocytes interact

with dendritic cells,

macrophages,

B lymphocytes

CD8+ CTLs can kill any

type of virus-infected cell

Leukocytes

Mesenchymal

cells

Dendritic cell Macrophage B cell

Polymorphic

genes:

 Many different

 alleles are

 present in the

 population

Different individuals are

able to present and

respond to different

microbial peptides

Feature Significance

Fig. 3.9 Properties of major histocompatibility complex (MHC) molecules and genes. Some of the

important features of MHC molecules and their significance for immune responses. CTLs, Cytotoxic T lymphocytes.

CHAPTER 3 Antigen Capture and Presentation to Lymphocytes 61

Because of several reasons, including the extra DRß

genes in some individuals (not everyone has the extra

HLADRB3/4/5 locus), and the fact that some a chains

encoded on one chromosome can associate with ß

chains encoded from the other chromosome, the total

number of expressed class II molecules may be considerably more than six.

The set of MHC genes present on each chromosome

is called an MHC haplotype. The genes in an MHC

haplotype are tightly linked and inherited together in a

Mendelian fashion. Therefore, the chance that two siblings will inherit identical sets of HLA alleles is 25%.

This is why siblings are often tested before unrelated

individuals for their suitability as donors for transplantation—the chance of finding an HLA match with the

recipient is much greater for siblings. In humans, each

HLA allele is given a numeric designation. For example,

an HLA haplotype of an individual could be HLA-A2,

B5, DR3, and so on. In the modern terminology, based

on molecular typing, individual alleles may be called

HLA-A*0201, referring to the 01 subtype of HLA-A2,

or HLA-DRB1*0401, referring to the 01 subtype of the

DR4B1 gene, and so on.

Peptide Binding to MHC Molecules

The peptide-binding clefts of MHC molecules bind

peptides derived from protein antigens and display

these peptides for recognition by T cells (Fig. 3.10).

There are pockets in the floors of the peptide-binding

clefts of most MHC molecules. Some of the amino

acids in the peptide antigens fit into these MHC pockets and anchor the peptides in the cleft of the MHC

molecule; these amino acids are called anchor residues. Other residues of the bound peptide project

upward and are recognized by the antigen receptors

of T cells.

Several features of the interaction of peptide antigens

with MHC molecules are important for understanding the peptide display function of MHC molecules

(Fig. 3.11):

• Each MHC molecule can present only one peptide

at a time, because there is only one binding cleft, but

each MHC molecule is capable of presenting many

different peptides. As long as the pockets of the MHC

molecule can accommodate the anchor residues

of the peptide, that peptide can be displayed by the

MHC molecule. Therefore, only one or two residues

in a peptide determine if that peptide will bind to

the cleft of a particular MHC molecule. Thus, MHC

molecules are said to have a broad specificity for peptide binding; each MHC molecule can bind many

peptides as long as they have the optimal length and

amino acid sequence. This broad specificity is essential for the antigen display function of MHC molecules, because each individual has only a few different

MHC molecules that must be able to present peptides

derived from a vast number and variety of protein

antigens.

Peptide

Peptide

Pockets in floor of peptide

binding groove of class II

MHC molecule

Peptide

A

B

Anchor

residue

of peptide

Class II

MHC

molecule

Class I

MHC

molecule

Fig. 3.10 Binding of peptides to major histocompatibility

complex (MHC) molecules. A, The top views of the crystal

structures of MHC molecules show how peptides (in yellow)

lie on the floors of the peptide-binding clefts and are available

for recognition by T cells. B, The side view of a cutout of a

peptide bound to a class II MHC molecule shows how anchor

residues of the peptide hold it in the pockets in the cleft of the

MHC molecule. (A, Courtesy Dr. P. Bjorkman, California Institute of Technology, Pasadena, CA. B, From Scott CA, Peterson

PA, Teyton L, Wilson IA: Crystal structures of two I-Ad-peptide

complexes reveal that high affinity can be achieved without

large anchor residues, Immunity 8:319–329, 1998. Copyright

Cell Press; with permission.)

62 CHAPTER 3 Antigen Capture and Presentation to Lymphocytes

+ +

Cytosolic

protein Proteasome Class I MHC

Class II MHC

Peptides from proteins

in cytosol

Feature Significance

Each T cell responds to

a single peptide bound

to an MHC molecule

Class I and class II

MHC molecules provide

immune surveillance

for microbes in

different locations

Many different peptides

can bind to the same

MHC molecule

MHC molecule displays

bound peptide for long

enough to be located

by T cell

Only peptide-loaded

MHC molecules

are expressed on the

cell surface for

recognition by T cells

MHC-restricted T cells

respond mainly to

protein antigens*

ß2-

microglobulin

a Days

MHC

molecule with

bound peptide

"Empty"

MHC

molecule

Lipids

Carbohydrate

sugars

Proteins

Peptides

Peptide

Nucleic

acids

Endosome/

lysosome

Peptides from

internalized proteins

in endocytic vesicles

Endocytosis of

extracellular protein

Each MHC

molecule

displays one

peptide at a time

Broad specificity

Very slow

off-rate

Stable surface

expression of

MHC molecule

requires bound

peptide

MHC molecules

bind only

peptides

Class I and

class II MHC

molecules

display peptides

from different

cellular

compartments

Fig. 3.11 Features of peptide binding to MHC molecules. Some of the important features of peptide binding

to MHC molecules, with their significance for immune responses. ER, Endoplasmic reticulum; Ii

, invariant chain.

*Some small chemicals and heavy metal ions may directly alter MHC molecules and are recognized by T cells.

CHAPTER 3 Antigen Capture and Presentation to Lymphocytes 63

• MHC molecules bind mainly peptides and not other

types of antigens. Among various classes of molecules, only peptides have the structural and charge

characteristics that permit binding to the clefts of

MHC molecules. This is why MHC-restricted CD4+

T cells and CD8+ T cells can recognize and respond to

protein antigens, the natural source of peptides. The

MHC is also involved in the reactions of T cells to

some nonpeptide antigens, such as small molecules

and metal ions. The recognition of such antigens is

discussed briefly later in the chapter.

• MHC molecules acquire their peptide cargo during

their biosynthesis, assembly, and transport inside cells.

Therefore, MHC molecules display peptides derived

from protein antigens that are inside host cells (produced inside cells or ingested from the extracellular

environment). This explains why MHC-restricted T

cells recognize cell-associated microbes and not free

antigens in the circulation, tissue fluids, or mucosal

lumens. Class I MHC molecules acquire peptides

from cytosolic proteins and class II molecules from

proteins that are taken up into intracellular vesicles.

The mechanisms and significance of these pathways

of peptide-MHC association are discussed later.

• Only peptide-loaded MHC molecules are stably

expressed on cell surfaces. The reason for this is that

MHC molecules must assemble both their chains

and bound peptides to achieve a stable structure,

and empty molecules are degraded inside cells. This

requirement for peptide binding ensures that only

useful MHC molecules—that is, those displaying

peptides—are expressed on cell surfaces for recognition by T cells. Once peptides bind to MHC molecules, they stay bound for a long time, up to days for

some peptides. The slow off-rate ensures that after an

MHC molecule has acquired a peptide, it will display

the peptide long enough to allow a particular T cell

that can recognize the peptide-MHC complex to find

the bound peptide and initiate a response.

• In each individual, the MHC molecules can display

peptides derived from the individual’s own proteins,

as well as peptides from foreign (i.e., microbial) proteins. This inability of MHC molecules to discriminate between self antigens and foreign antigens raises

two questions. First, at any time, the quantity of self

proteins in an APC is likely to be much greater than

that of any microbial proteins. Why, then, are the

available MHC molecules not constantly occupied by

self peptides and unable to present foreign antigens?

The likely answer is that new MHC molecules are constantly being synthesized, ready to accept peptides,

and they are adept at capturing any peptides that are

present in cells. Also, a single T cell may need to see a

peptide displayed by only as few as 0.1% to 1% of the

approximately 105

 MHC molecules on the surface of

an APC, so that even rare MHC molecules displaying

a peptide are enough to initiate an immune response.

In addition, during viral infections, host protein synthesis is suppressed and viral proteins dominate and

therefore are preferentially presented by MHC molecules. The second problem is that if MHC molecules

are constantly displaying self peptides, why do we not

develop immune responses to self antigens, so-called

autoimmune responses? The answer is that T cells

specific for self antigens are either killed or inactivated (see Chapter 9). Thus, T cells are constantly

patrolling the body, looking at MHC-associated peptides, and if there is an infection, only those T cells

that recognize microbial peptides will respond, while

self peptide–specific T cells will either be absent or

will have been previously inactivated.

MHC molecules are capable of displaying peptides

but not intact protein antigens, which are too large to fit

into the MHC cleft. Therefore, mechanisms must exist

for converting naturally occurring proteins into peptides able to bind to MHC molecules. This conversion,

called antigen processing, is described next.

PROCESSING AND PRESENTATION OF

PROTEIN ANTIGENS

Proteins in the cytosol of any nucleated cell are

processed in proteolytic complexes called proteasomes and displayed by class I MHC molecules,

whereas extracellular proteins that are internalized by specialized APCs (dendritic cells, macrophages, B cells) are processed in late endosomes

and lysosomes and displayed by class II MHC molecules (Fig. 3.12). These two pathways of antigen

processing involve different cellular proteins (Fig.

3.13). They are designed to sample all the proteins

present in the extracellular and intracellular environments. The segregation of antigen-processing

pathways also ensures that different classes of T

lymphocytes recognize antigens from different

compartments. Next we discuss the mechanisms of

antigen processing, beginning with the class I MHC

pathway.

64 CHAPTER 3 Antigen Capture and Presentation to Lymphocytes

Processing of Cytosolic Antigens for Display

by Class I MHC Molecules

The main steps in antigen presentation by class I MHC

molecules include the tagging of antigens in the cytosol or nucleus for proteolysis, proteolytic generation of

peptide fragments of the antigen by a specialized cytosolic enzyme complex, transport of peptides into the ER,

binding of peptides to newly synthesized class I molecules, and transport of peptide-MHC complexes to the

cell surface (Fig. 3.14).

Proteolysis of Cytosolic Proteins

The peptides that bind to class I MHC molecules

are derived from cytosolic proteins following digestion by the ubiquitin-proteasome pathway. Antigenic proteins may be produced in the cytoplasm

from viruses that are living inside infected cells, from

some phagocytosed microbes that may leak from or

be transported out of phagosomes into the cytosol,

and from mutated or altered host genes that encode

cytosolic or nuclear proteins, as in tumors. All of these

proteins, as well as the cell’s own misfolded cytosolic and nuclear proteins, are targeted for proteolytic

digestion by the ubiquitin-proteasome pathway. These

proteins are unfolded, covalently tagged with multiple copies of a peptide called ubiquitin, and threaded

through a protein complex called the proteasome that

is composed of stacked rings of proteolytic enzymes.

The proteasomes degrade the unfolded proteins into

peptides. In cells that have been exposed to inflammatory cytokines (as in an infection), the enzymatic

composition of the proteasomes changes. As a result,

these cells become very efficient at cleaving cytosolic

and nuclear proteins into peptides with the size and

sequence properties that enable the peptides to bind

well to class I MHC molecules.

Antigen

processing

MHC

biosynthesis

Peptide-MHC

association

Antigen

uptake

Class II

MHC pathway ER

Endocytosis of

extracellular

protein

CD4+

T cell

Invariant

chain (Ii)

Endosome/

lysosome

Class II MHC

ER

Cytosolic

protein Proteasome

TAP CD8+

CTL

Peptides in

cytosol

Class I

MHC

Class I

MHC pathway

Fig. 3.12 Pathways of intracellular processing of protein antigens. The class I MHC pathway converts

proteins in the cytosol into peptides that bind to class I MHC molecules for recognition by CD8+ T cells. The

class II MHC pathway converts protein antigens that are endocytosed into vesicles of antigen-presenting cells

into peptides that bind to class II MHC molecules for recognition by CD4+ T cells. CTL, Cytotoxic T lymphocyte; ER, endoplasmic reticulum; TAP, transporter associated with antigen processing.

CHAPTER 3 Antigen Capture and Presentation to Lymphocytes 65

Binding of Peptides to Class I MHC Molecules

In order to form peptide-MHC complexes, the peptides must be transported into the endoplasmic reticulum (ER). The peptides produced by proteasomal

digestion are in the cytosol, while the MHC molecules

are being synthesized in the ER, and the two need to

come together. This transport function is provided by a

molecule, called the transporter associated with antigen processing (TAP), located in the ER membrane.

TAP binds proteasome-generated peptides on the cytosolic side of the ER membrane, then actively pumps

them into the interior of the ER. Newly synthesized

class I MHC molecules, which do not contain bound

peptides, associate with a bridging protein called tapasin

that links them to TAP molecules in the ER membrane.

Thus, as peptides enter the ER, they can easily be captured by the empty class I molecules. (As we discuss

later, in the ER, the newly synthesized class II MHC

molecules are not able to bind peptides because of the

associated invariant chain.)

Transport of Peptide-MHC Complexes to the Cell

Surface

Peptide loading stabilizes class I MHC molecules,

which are exported to the cell surface. Once the class

I MHC molecule binds tightly to one of the peptides

generated from proteasomal digestion and delivered into the ER by TAP, this peptide-MHC complex

Feature Class II MHC Pathway

Composition of

stable peptide-MHC