Development Team: Zoology Immunology Structure and Function of Major Histocompatibility Complex
Development Team: Zoology Immunology Structure and Function of Major Histocompatibility Complex
: 10 : Immunology
Module : 07 : Structure and function of Major Histocompatibility Complex
Development Team
Principal Investigator: Prof. Neeta Sehgal
Head, Department of Zoology, University of Delhi
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
Description of Module
Contents
1. Learning Objectives
2. Introduction
3. MHC Haplotypes
3.1 MHC Class-I
3.1.1 Structure of MHC Class-I
3.2 MHC Class-II
3.2.1 Structure of MHC Class-I
3.3 MHC Class-III
4. Structure of Peptide Binding Cleft
4.1 Peptide –MHC interaction
5. MHC Class-I and MHC Class-II Antigen Presentation
5.1 Class-I MHC Antigen Presentation
5.2 Class-II MHC Antigen Presentation
6. Role of MHC in Self/Nonself Recognition
7. The Genetic Architecture of MHC
8. Natural Selection and MHC Genes Polymorphisms
9. Epigenetics and MHC
10. MHC in Transplantation
10.1 Allogenic Immune Responses
10.2 HLA- Testing and Clinics
11. Summary
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
1. Learning Objectives
After studying this module, you shall be able to
2. Introduction
For the body to rein an attack on any pathogen; it must be recognized as self or non-self and this is
accomplished by the set of cell surface receptors which not only distinguishes self from non-self but
also plays a pivotal role in tissue transplantation and rejection, autoimmunity and susceptibility to
infections. These cell surface receptors are encoded by the most diverse gene loci in vertebrates
known as the major histocompatibility complex (MHC Class I and II loci). In humans, MHC spans a
considerable part of the DNA i.e. about 0.1% of total genome containing 200 coding loci. These are
highly polymorphic genes that can be attributed to the natural selection over long periods of
evolutionary time.
The history behind MHC genes dates back to 1916 when Little and Taylor showed that tissue could be
transplanted only in the same strain mice; followed by George D. Snell’s observation in 1935 that
several closely linked histocompatible genes are responsible for tissue rejection. He discovered that in
tumor transplants on mice from their congenic cousins were immediately rejected but were successful
in syngeneics trains. The mystery was solved in 1975 by Zinkernagel and Doherty, who found that T-
cell responses were restricted to antigens and the MHC’s presented on the cell surface. The 1980’s
and 90’s finally revealed the structure of MHC molecules and their encoded proteins. The Nobel Prize
in Physiology or Medicine in 1980 was awarded jointly to Baruj Benacerraf, Jean Dausset and George
D. Snell for their contribution in understanding that similar antigens are controlled by dominant
autosomal genes termed immune response (Ir) genes located on the MHC loci of all the vertebrates.
Other than any region of the genome, the MHC genes are the most associated with autoimmune and
infectious diseases. These disease-associated variations in the MHC regions are due to very subtle and
minute changes in the gene loci. There is still more to learn about MHC like (i) what mechanism
drives MHC polymorphism, (ii) which are naturally and positively selected variations having
functional importance, (iii) which signaling pathways does the MHC genes follow at the molecular
level.
The MHC class-I heavy chains are encoded by three genes (HLA-A, HLA-B, HLA-C) and presents
small peptides to CTLs (cytotoxic T-lymphocytes) enabling each cell of the body to the immune
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ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
system with possible chances of infection, therefore every nucleated cells of the body present MHC-I
molecules. Mostly these peptides are self-peptides, but in case of virus-infected cell or cancer cell or
parasite infected cell, the foreign peptides are presented resulting in their elimination by CTLs. The
CTLs are very unique cells with highly specific TCR (T-cell receptor) that scan for MHC-antigen
complexes and CD8+ molecules to stabilize and help bind the cells to MHC – antigens. The cells with
foreign antigens help activate the CTLs, resulting in the clonal selection of these specific CTLs, thus
eliminating other infected cells. But it is still very difficult to envisage that how MHC molecules
achieve the specificity in immune recognition with such small pool of TCR. In fact, MHC molecules
can bind to a varied range of peptides rather than specific peptides.
It is composed of α and β polypeptide chains held together with non-covalent bonds with a short
cytoplasmic tail (Figure 1). The α-chain is the MHC-encoded integral membrane glycoprotein of
~45000 Da; while β2-microglobulin is non-MHC encoded extracellular ~12kDa peptide. The α-chain
is of 350 aminoacids with three globular domains (α1, α2, and α3). α2 and α3, both have intra-chain
disulphide linkages. It also has a 26 hydrophobic amino acids transmembrane segment spanning the
plasma membrane. The α1 and α2 forms the platform as the peptide-binding unit mainly comprising
of β-sheets. The groove formed between them is blocked at both the ends and is enough to hold 8-
10amino acids, so it is imperative for the foreign residues to be processed and presented before they
bind to MHC-I. The β2-m chain is non-polymorphic ~90a.a. peptide is encoded by chromosome 15
and interacts non-covalently with α3. This part of the MHC molecule binds to CTL. Class-I MHC
molecule is a glycoprotein with one or two N-linked oligosaccharide.
Class II peptides are expressed on specialized cells called antigen presenting cells (APCs); mainly the
macrophages, dendritic cells and B-lymphocytes. MHC –II molecules are encoded by three
polymorphic genes (HLA-DR, HLA-DQ and HLA-DP) that bind to different peptides. The
immunodominant epitopes are largely confined to the pathways dependent on lysosomal degradation
and peptide binding of newly synthesized MHC-II molecules in the acidic endosomal compartment.
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
The MHC-II molecules that are involved in the CD 4+ T-cell response against intracellular pathogens
may trigger immunity towards infection, thymic selection, autoimmunity and tumor immunity
Like MHC-I, the class-II MHC molecules have an extracellular domain, a transmembrane domain and
an intracellular carboxy terminal tail. The extracellular domain is composed of two non-covalently
associated domains (α1, α2 or β1, β2) of ~90 a.a anchored to the cell membrane. The heavy chain (α 1
and α 2) has a molecular weight of ~30-34 KDa and the light chain (β 1 and β 2) has a molecular
weight of ~26-29 KDa. α2, β1 and β2 domain contains disulphide linkages, while α1, α2 and β1
domains are glycosylated but β2 domain is not. The transmembrane domain is comprised of ~25
hydrophobic amino acids and the cytoplasmic tail of ~10-15 hydrophilic amino acids. Peptide antigen
binds in a groove formed from a pair of α-helices (on a floor of anti-parallel β-strands) involving α1
or β1 segments, respectively. The antigenic peptide – binding domain can hold 13-18 a.a. amino acid
residues as it is open at both the ends of the peptide binding groove (Figure 2).
Peptide-binding groove
Transmembrane stretch
Cytoplasmic tail
These molecules are mainly comprised of serum proteases and complement enzymes and do not have
any role in antigen presentation. The complement component includes C2 (serine protease), C4A and
C4B (pro-proteins of the multichain forms) and factor B (component of alternate complement
pathway). They also include the TNF-α and TNF-β, as well two heat-shock proteins.
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
different chains to construct the peptide‐ binding groove. Peptides bind to MHC molecules through
primary and secondary anchor residues protruding into the pockets in the peptide‐ binding grooves.
Peptide preferences are dependent on the amino acids polymorphisms comprising the anchor pockets,
which are related to the various alleles of MHC. The conformations of peptides presented by MHC I
molecules are length dependent. The modified groups of post-translationally modified peptides have
important roles in the peptide–MHC interaction (Figure 3).
Figure 3. Peptide binding domain and cleft of MHC Class-I and Class-II. Source : Liu et al., (2011). Major
Histocompatibility Complex: Interaction with Peptides. DOI: 10.1002/9780470015902.a0000922.pub2
Peptide binding to MHC class-I and Class-II molecules requires classic anchor peptides at the N and
C-terminus of the peptide, these residues being termed as ANCHOR RESIDUES. These anchor
peptides bind to the pocket of the binding cleft of the MHC molecules. Class-I MHC anchor residues
are mainly comprised of hydrophobic amino acids. At C-terminus, the anchor residues are at the
9tha.a. position mainly comprising leucine and isoleucine, while the anchor at the N-terminus is
mostly present at the 2nd or 3rda.a. position e.g. N-terminus anchor residue in H-2Kd is tyrosine, H-2Dd
is glycine. The mid-portion arches away from the floor of the MHC complex to make a contact with
the TCR.
Peptides that bind to Class-II MHC have 7 to 8 a.a. that provides maximum contact. It has hydrophobic
residues at N-terminus, C-terminus as well as 3 a.a. in the middle, thereby providing a constant elevation
from the floor of the binding cleft (Figure 4).
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
Figure 4. Peptide-binding grooves of MHC class I and II proteins. (A) Peptides binding in an MHC class I
groove are usually 8–10 amino acids long. The residues at both the N-terminus and C-terminus end of the
peptide are anchored in the groove. (B) Peptides binding in an MHC class II groove are usually 13–18 amino
acids long. The residues bind at both the ends as well as in the middle. Source: Major histocompatibility
Complex in Primer to the Immune Response (Second Edition), 2014.
Figure 5. Antigen processing and presentation pathways in dendritic cells. MHC-I pathway: Intrinsic pathogens
(like viruses) or endogenous proteins are either degraded in cytosol or nucleus by immunoproteasomes and are
presented as peptides along with MHC-I to CD8+ T-CTL cells. MHC-II pathway: The exogenous peptides are
endocytosed and via endomembrane system binds to MHC-II to be presented to CD4+Th cells. Cross-
presentation pathway: Indicates that the exogenous peptide which has been sequestered via endocytic
mechanism may compete for the peptide either to MHC class II or MHC class I pathway. Some DCs have a
unique ability to deliver exogenous antigens to the MHC class I pathway. Source: Villadangos J. A. &
SchnorreP. (2007). Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo.
Nature Reviews Immunology 7, 543-555 (July 2007) |
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ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
5.1 Class-I MHC Antigen Presentation
The endogenous antigens are first degraded by cytosolic and nuclear proteasomes. These are
immunoproteasomes which are mainly involved in extending the peptide pool rather than be selective
for MHC-I peptides, this thus explain the wide diversity of peptides presented. The peptides thus
formed are transported via Transporter associated with antigen presentation (TAP) and assemble in
ER with the MHC-I chaperone molecule (Tapasin). The binding of the peptide to MHC-I molecule is
aided by two more chaperones proteins; calreticulin and ERp57. The complex thus formed after
loading of the peptide along with TAP, tapasin, MHC-I, ERp57 and calreticulin is called peptide
binding complex (PLC). The MHC class-I complex is finally released of the chaperones and is
transported to plasma membrane (Figure 6).
Figure 6. In the MHC class I molecule pathway, endogenous proteins are broken down by proteasomes into
smaller peptides. In the endoplasmic reticulum (ER), an antigenic peptide binds to the peptide binding site in an
MHC class I molecule. The peptide-MHC complex then migrates through the Golgi apparatus to the cell
surface. Source: Neefjes et al., (2011). Towards a systems understanding of MHC class I and MHC class II
antigen presentation .Nature Reviews Immunology 11, 823-836.doi:10.1038/nri3084
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
5.2 Class-II MHC Antigen Presentation
MHC-II molecules are synthesized de novo and assembled in the ER. They are transported to golgi
where they associate with the chaperone the invariant chain (Ii). MHC-II-Ii complex move to the
endomembrane system, there together with other chaperones, DM and DO regulate the loading of
peptide within the MHC-II peptide binding cleft. MHC class-II associated Ii peptide (CLIP) remains
associated with the peptide binding groove of MHC heterodimer until DM catalyzes the dissociation
of CLIP and binds the processed antigen at a low pH. HLA-DM then transfers the CLIP with the
specific peptide from the exogenous pathogen brought in through the endosomal pathway to be
presented to CD4+ TH cells. Thus, cytosol-to-endosome transport may lead to the processing of
proteins translocated into the host cytosol by intracellular pathogens (Figure 7).
Figure 7. In the MHC class II molecule pathway, α and βsubunits of the MHC class II molecule bind to the
invariant chain (Ii) in the ER. Ii is partially degraded in an endosomal compartment. The portion of Ii that
occupies the antigenic peptide binding site on the MHC class II molecule (called CLIP) is removed with the
help of HLA-DM, freeing the molecule for binding processed antigen. Once antigenic peptide has bound to an
MHC class II molecule, the complex migrates to the cell surface. Source: cellular and molecular Immunology,
8th Edition (2015) by Abbas et al.
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
olfaction. The MHC in mice is termed as H-2 (Histocompatible-2) complex and in humans as HLA
(human leukocyte antigens) complex. The immune response specific regions of MHC gene cluster
(also called classical) are closely linked loci called class I and class II loci and are the most divergent
among vertebrates. Among the most studied, humans have six class I loci (A, B and C) and eight class
II loci (DP, DQ and DR), whereas mice have three class I loci (K, D and L) and 2 class II loci (A and
E) (Figure 8). The polymorphism can be observed with over 170-100 alleles per locus with each locus
having multiple coding genes. Close linkage among these loci means each individual inherits a
particular combination of MHC alleles as a single haplotype; thereby there are little or no chances of
recombination separating these loci resulting in highly polymorphic status. This condition suggests
that the MHC are inherited from a common ancestor and is been maintained by natural selection
(Figure 9). Till date, it is extremely difficult to unravel the functional aspect of each loci owing to
high gene density, extreme polymorphism, strong linkage disequilibrium and clustering of genes with
related functions. Hence, our understanding of MHC-related diseases are very poor because (i) natural
selection should have eliminated the neutral and deleterious alleles and must have kept only the
disease-protective allele (ii) which specific variation among the array of MHC gene variation is
functionally relevant (iii) the MHC-gene related diseases may not be in the genes but the discrete
changes in the amino-acids or the changes in the groove residues.
Figure 8. The genetic architecture of MHC in humans and mice. Source: Bradford M. Elmer, A. Kimberley
McAllister, (2012). Trends in Neuroscience; 35(11), p660–670.
Figure 9. The immunoglobulin multigene superfamily gave rise to various immunoglobulins, T-cell receptors,
antibodies as well as MHC genes; suggesting that these molecules evolved from a common ancestor. Source:
Dustin J. Penn, 2001. DOI: 10.1038/npg.els.0000919.
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8. Natural Selection and MHC Genes Polymorphisms
There are many evidences that confirm that MHC polymorphism is maintained due to natural selection
(Figure 10). First, high number of alleles in these gene segments or gene densities may arise when neutral
alleles are not selected and are randomly fixed through genetic drift. Second, there is lot more gene density
and uniformity in the proportions of allelic frequencies. Third, the selection pressure resulted in fewer
MHC homozygotes which are not expected of a randomly mating population. Fourth, high linkage among
allelic loci in individuals and their strong linkage disequilibrium are more often than chance expectations.
Fifth, the high dS/dN (synonymous substitution to non-synonymous substitution) ratio in the antigen-
binding site of MHC-genes to both neutrality and purifying selection, maintains diversity. Sixth, the
divergence of MHC alleles is pretty ancient in evolutionary time scale, thus the persistence of Class-I and
II MHC diversity is clarified by Darwinian Natural selection process.
Figure 10. Chromosomal location of the MHC locus in man depicting allelic polymorphism. Source:
International Immunogenetics information systems (IMGT) (http:/www.imgt.org/)
MHC polymorphism though is important for disease resistance; it is also involved in promoting
outbreeding. MHC polymorphism is aimed to provide “Herd Immunity” i.e. if large numbers of
variables are present in a population then it is more likely that the infection may not get an escape
route and the spread is restricted. Thus, it can be related to the potential of certain populations to their
relationship with the pathogen-associated selection.
MHC polymorphism among humans can also be explained on the basis of admixture with archaic
humans. Whenever a new species is territorially isolated, it acquired a new set of MHC alleles thus,
evolving novel alleles. The best example may be given in human evolution where HLA-B*73 allele of
HLA-B found in West Asian population has been traced to Denisovnas, who are related to
Neanderthals. Hence, certain haplotypes of MHC were introgressed into Eurasian and Oceanian
populations much before they were introduced into the African population. The polymorphism among
MHC has been useful in tracing ancient population migration; for example, this can help deduce the
divergence of early humans from their African origins.
ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
the offspring’s of mothers carrying the affected allele of MHC-II, HLA-DRB1*15 for multiple sclerosis
(an autoimmune disorder) are more likely to get infected rather than fathers carrying the affected gene. The
master regulator of MHCII genes has been the MHCII transactivator (CIITA), which play the pivotal role
in normal immune function. Significant epigenetic modifications such as methylation, acetylation and
histone modification have been demonstrated to alter CIITA expression. Therefore, many diseases
associated with aging and autoimmunity is linked to polymorphism of CIITA.
When a tissue transplant is performed, the HLA of the donor is recognized by the recipient’s immune
system, triggering an allogenic immune response. The divergent alloantigens caused due to large
numbers of genetic differences between donors and recipients are the easy targets for T-cell
recognition, thereby increasing the risk of both graft rejection and graft-versus-host disease (GVHD).
Allogenic immune responses may happen by three recognizing mechanisms: (1) Indirect recognition-
the donor’s HLA is processed by APC and presented as peptides with MHC molecules to the
recipient’s T-cells. This mechanism is mainly dominant in chronic rejection. (2) Direct recognition-
the donor’s HLA molecules is directly recognized on the donor’s presenting cells; i.e. the receptor
recognizes the foreign HLA as own molecule with foreign peptide. This causes acute rejection. (3)
Semi-direct recognition- the Immunoglobulin-like receptors (KIRs) on NK cells recognize the
polymorphic sequences of HLA-C, B or A in the target cells, causing acute rejection. It has been
shown recently that the naïve and memory CD4+ and CD8+ T-cells are cross-reactive against
allogenic HLA molecules presenting self-peptides (Figure 11).
Figure 11. Direct recognition: recognition by both CD8+ and CD 4+ T-cells of an intact MHC molecule
displayed by donor APC in the transplanted graft. Indirect recognition: donor’s MHC with the foreign peptide is
processed and presented by the recipient’s APC to be presented to only the C4+ Tcells.
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Structure and function of Major Histocompatibility Complex
10.2 HLA-Testing and Clinics
Traditionally, HLA typing was performed by serologic testing by using antiserum in complement-
dependent cytotoxic assays. Lately, more precise DNA-based HLA typing methods using molecular
techniques have been developed. Techniques like sequence-specific oligonucleotide probe
hybridization; sequence-specific primer amplification, sequencing-based typing, and reference strand-
based conformation analysis are frequently used. The high polymorphism among HLA with more
than 800 alleles have restricted to standard HLA typing at the HLA-A, HLA-B, HLA-C, HLA-DRB1,
and HLA-DQB1 genetic loci. An HSCT donor is referred to as a "10/10 allele match" or "perfect
match" when both HLA alleles are identical at each of the HLA-A, HLA-B, HLA-C, HLA-DRB1, and
HLA-DQB1 loci. While searching for an unrelated donor, high-resolution (4-digit) genetic typing of
both the patient and the donor is necessary.
11. Summary
a) Major histocompatibility complex (MHC Class I and II loci) are the cell surface receptors
encoded by the most diverse gene loci in vertebrates and spans about 0.1% of total genome
containing 200 coding loci.
b) The MHC genes are located in autosomes, Y-chromosomes and even in mitochondrial genome. It
is a large chromosomal region of chromosome # 6; that has over 200 coding regions. The MHC in
mice is termed as H-2 (Histocompatible-2) complex and in humans as HLA (human leukocyte
antigens) complex.
c) MHC not only distinguishes self from non-self but also plays a pivotal role in tissue
transplantation and rejection, autoimmunity and susceptibility to infections. Apart from specific
immune functions; these genes also impact growth, development, reproduction, odor and
olfaction.
d) The MHC class-I heavy chains are encoded by three genes (HLA-A, HLA-B, HLA-C) and
presents small peptides to CTLs (cytotoxic T-lymphocytes) enabling each cell of the body to the
immune system with possible chances of infection, therefore every nucleated cells of the body
present MHC-I molecules.
e) MHC Class II peptides are expressed on specialized cells called antigen presenting cells (APCs);
mainly the macrophages, dendritic cells and B-lymphocytes. These molecules are encoded by
three polymorphic genes (HLA-DR, HLA-DQ and HLA-DP) that bind to different peptides.
f) Both MHC-I and MHC-II present peptides to the CD8+ and CD4+ T-cells, the sources of these
peptides may differ. The MHC-I are presented with intracellular peptides and the exogenous
peptides are presented by MHC-II.
g) Both MHC classes have similar three-dimensional structure; they originate from the common
parent gene by gene duplication and a similar function in antigen presentation to the T-
lymphocytes.
h) MHC genes influence the development of T-cell repertoire through thymic selection as only those
T-cells that recognize peptide-MHC complex are able to mature and enter the circulation.
i) There are many evidences that confirm that MHC polymorphism is maintained due to natural
selection like high number of alleles, uniform allelic frequencies, deficiencies of homozygotes,
linkage disequilibrium among loci, high dN/dS substitution, ancient allelic linkages and
disassortative mating preferences.
j) Expression of MHC genes was shown to have an epigenetic effect as many diseases associated
with aging and autoimmunity is linked to polymorphism of MHCII transactivator (CIITA).
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ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex
k) Allogenic immune responses happen due to divergent MHC alloantigens between donors and
recipients which are the easy targets for T-cell recognition, thereby increasing the risk of both
graft rejection and graft-versus-host disease (GVHD).
l) Precise DNA-based HLA typing methods using molecular techniques like sequence-specific
oligonucleotide probe hybridization; sequence-specific primer amplification, sequencing-based
typing, and reference strand-based conformation analysis are frequently used for HLA typing in
clinics.
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ZOOLOGY Immunology
Structure and function of Major Histocompatibility Complex