1. Major histocompatibility Complex and
antigen processing
Dr.Kayeen Vadakkan

3. Major histocompatibility Complex
• MHC molecules are membrane proteins on APCs that
display peptide antigens for recognition by T lymphocytes.
• In all vertebrates, the MHC contains two sets of highly
polymorphic genes, called the class I and class II MHC genes.
• The human MHC, called the human leukocyte antigen (HLA)
complex, and the mouse MHC, called the H-2 complex.
• Therefore, MHC can be classified into 3 based on structure
and site of action
1. Class I MHC
encode glycoproteins expressed on the surface of nearly all
nucleated cells; the major function of the class I gene products
is presentation of peptide antigens to TC cells
2. Class II MHC
encode glycoproteins expressed primarily on antigen-
presenting cells (macrophages, dendritic cells, and B cells),
where they present processed antigenic peptides to TH cells
3. Class III MHC
encode, in addition to other products, various secreted
proteins that have immune functions, including components of
the complement system and molecules involved in
inflammation

4. Structure of MHC Molecules
Class I MHC Molecules
• Class I MHC molecules contain a 45-kilodalton (kDa) α chain associated noncovalently with a
12-kDa β2-microglobulin molecule. The α chain is a transmembrane glycoprotein encoded by
polymorphic genes within the A, B, and C regions of the human HLA complex. β 2-
Microglobulin is a protein encoded by a highly conserved gene located on a different
chromosome.
• The α chain is anchored in the plasma membrane by its hydrophobic transmembrane
segment and hydrophilic cytoplasmic tail.
• Structural analyses have revealed that the αchain of class I MHC molecules is organized into
three external domains (1, 2, and 3), each containing approximately 90 amino acids; a
transmembrane domain of about 25 hydrophobic amino acids followed by a short stretch of
charged (hydrophilic) amino acids; and a cytoplasmic anchor segment of 30 amino acids.
• The β2-microglobulin is similar in size and organization to the α3 domain; it does not contain
a transmembrane region and is noncovalently bound to the class I glycoprotein.
• The amino-terminal α1 and α2 domains of the α chain form two walls and a peptide-binding
cleft, or groove, that can accommodate peptides typically 8 to 9 amino acids long.
• The floor of the peptide-binding cleft contains amino acid residues that bind peptides for
display to T lymphocytes, and the tops of the cleft walls make contact with the T cell
receptor.
Class II MHC Molecules
• Class II MHC molecules contain two different polypeptide chains, a 33-kDa α chain and a 28-
kDa β chain, which associate by noncovalent interactions
• Each class II MHC molecule consists of two transmembrane chains, called α and β. Each chain
has two extracellular domains, followed by the transmembrane and cytoplasmic regions.
• The amino-terminal regions of both chains, called the α1 and β1 domains, contain
polymorphic residues and form a cleft that is large enough to accommodate peptides of 10 to
30 residues.

5. Cellular Distribution of the MHC Antigens
• Essentially, all nucleated cells carry classical class I molecules.
• These are abundantly expressed on lymphoid cells, less so on liver, lung and kidney, and only sparsely on brain and
skeletal muscle.
• Class II molecules are restricted in their expression, being present only on antigen presenting cells (APCs) such as B-
cells, dendritic cells and macrophages and on thymic epithelium.
• When activated by agents such as interferon g, capillary endothelia and many epithelial cells in tissues other than the
thymus, they can develop surface class II and increased expression of class I.
Properties of MHC Genes and Proteins
• MHC genes are highly polymorphic, meaning that many different alleles (variants) are present among the different
individuals in the population.
• MHC genes are co-dominantly expressed, meaning that the alleles inherited from both parents are expressed equally
• Class I molecules are expressed on all nucleated cells, but class II molecules are expressed mainly on dendritic cells,
macrophages, and B lymphocytes.

6. Inheritance Patterns of HLA Genes
• An individual inherits one haplotype from the mother and
one haplotype from the father. In outbred populations, the
offspring are generally heterozygous at many loci and will
express both maternal and paternal MHC alleles.
• The alleles are co-dominantly expressed; that is, both
maternal and paternal gene products are expressed in the
same cells.
• In an outbred population, each individual is generally
heterozygous at each locus.
• The human HLA complex is highly polymorphic and multiple
alleles of each class I and class II gene exist.
• When the father and mother have different haplotypes,
there is a one-in-four chance that siblings will inherit the
same paternal and maternal 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.
• Genetic recombination generates new allelic combinations ,
and 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

7. Antigen Processing
and Presentation
• Recognition of a foreign antigen by a T cell requires that
peptides derived from the antigen be displayed within the
cleft of an MHC molecule on the membrane of a cell.
• The formation of these peptide-MHC complexes requires that
a protein antigen be degraded into peptides by a sequence
of events called antigen processing.
• The degraded peptides then associate with MHC molecules
within the cell interior, and the peptide-MHC complexes are
transported to the membrane, where they are displayed
(antigen presentation).
• Class I and class II MHC molecules associate with peptides
that have been processed in different intracellular
compartments.
• Class I MHC molecules bind peptides derived from
endogenous antigens that have been processed within the
cytoplasm of the cell (e.g., normal cellular proteins, tumor
proteins, or viral and bacterial proteins produced within
infected cells).
• Class II MHC molecules bind peptides derived from
exogenous antigens that are internalized by phagocytosis
or endocytosis and processed within the endocytic pathway.
• Based on the type of cell where the processing occurs and
the type of MHC the antigen processing can be classified into
1. Endogenous pathway / Cytosolic Pathway
2. Exogenous pathway / Endocytic Pathway

8. Endogenous
pathway /
Cytosolic
Pathway
This is the pathway by which endogenous antigens are
degraded for presentation with class I MHC molecules.
This occurs in four stages
1. Proteolysis of Cytosolic Proteins
2. Intake of peptides into Endoplasmic reticulum
3. Synthesis of Class I MHC and binding of peptide on it
4. Transport of Peptide-MHC Complexes to the Cell Surface

9. 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.
• These proteins are first bind to a ubiquitin molecule which
unfolded the protein and take the molecule to proteosome.
• This reaction, requires ATP, links several ubiquitin molecules to
a lysine-amino group near the amino terminus of the protein.
• Proteasome is composed of stacked rings of proteolytic
enzymes that degrade the unfolded proteins into peptides.

10. 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
molecule

11. Synthesis of Class I
MHC and peptide
binding
• The α chain and β 2-macroglobulin components of the class I MHC molecule are
synthesized on polysomes along the rough endoplasmic reticulum.
• The assembly process involves several steps and includes the participation of molecular
chaperones, which facilitate the folding of polypeptides.
• The first molecular chaperone involved in class I MHC assembly is calnexin, a membrane
protein of the endoplasmic reticulum.
• Calnexin associates with the free class I α chain and promotes its folding.
• When β 2-microglobulin binds to the α chain, calnexin is released and the class I molecule
associates with another chaperone calreticulin and with tapasin.
• Tapasin (TAP-associated protein) brings the complex near to the TAP transporter and
allows it to acquire an antigenic peptide

12. 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 becomes stable and is delivered to the cell surface.
• If the MHC molecule does not find a peptide it can bind, the empty molecule is unstable and is
eventually degraded in the ER.

13. Exogenous pathway
/ The Endocytic
Pathway
• This pathway is for antigens which are
generated outside of cell and processed by
class II MHC.
This pathway can be studied in three stages
1. Internalization and Proteolysis of
Antigens
2. Peptide Assembly with Class II MHC
Molecules
3. Transport of Peptide-MHC Complexes to
the Cell Surface

14. Internalization and
Proteolysis of Antigens
• Antigen-presenting cells can internalize antigen by phagocytosis,
endocytosis, or both.
• Once an antigen is internalized, it is degraded into peptides
within compartments of the endocytic processing pathway.
• The endocytic pathway appears to involve three increasingly
acidic compartments: early endosomes (pH 6.0–6.5); late
endosomes, or endolysosomes (pH 5.0–6.0); and lysosomes (pH
4.5–5.0).
• Internalized antigen moves from early to late endosomes and
finally to lysosomes, encountering hydrolytic enzymes and a
lower pH in each compartment.
• Within the compartments of the endocytic pathway, antigen is
degraded into oligopeptides of about 13–18 residues, which bind
to class II MHC molecules.

15. Peptide Assembly with
Class II MHC Molecules
• When class II MHC molecule are synthesized within the RER, it remains
associate with a preassembled trimer of a protein called invariant chain
(Ii, CD74).
• 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.
• Class II MHC–invariant chain complexes are transported from the RER to
the endocytic pathway, moving
• from early endosomes to late endosomes, and finally to lysosomes.
• As the proteolytic activity increases in each successive compartment, the
invariant chain is gradually degraded. However, a short fragment of the
invariant chain termed CLIP (for class II–associated invariant chain
peptide) remains bound to the class II molecule.
• CLIP physically occupies the peptide-binding groove of the class II MHC
molecule, preventing any premature binding of antigenic peptide.
• The CLIP molecule will be replaced by antigen peptide catalyzed by a
molecule called HLA-DM and this reaction is regulated and controlled by
HLA-DO molecule.

16. Transport of
Peptide-MHC
Complexes to the
Cell Surface
• Peptide loading stabilizes class II MHC
molecules, which are exported to the cell
surface.
• If a class II molecule binds a peptide with
the right fit, the complex is stabilized and
transported to the cell surface, where it can
be recognized by a CD4+ T cell.
• Class II molecules that do not find peptides
they can bind are eventually degraded by
lysosomal proteases