Transcript Class II MHC

MAJOR
HISTOCOMPATIBILITY
COMPLEX (MHC)
THE MHC GENE COMPLEX
The human MHC is located on chromosome 6.The MHC
complex contains a number of genes that control several
antigens, most of which influence allograft rejection.
These antigens (and their genes) can be divided into
three major classes :class I ,class II and class III .
The class I and class II antigens are expressed on cells
and tissues whereas as class III antigens are
represented on proteins in serum and other body fluids
(e.g.C4, C2, factor B, TNF). Antigens of class III gene
products have no role in graft rejection
A. History
1.In transplantation studies, MHC gene products were identified
as responsible for graft rejection
2.In studies on responses to antigens, MHC gene products
were found to control immune responses, called the immune
response (Ir) genes
3.It was determined that antigen-specific T cells recognize
protein antigens that are bound non-covalently to MHC gene
products
a. Helper T cells recognize peptide bound to class II MHC
gene products,Cytolytic T cells peptide bound to class I MHC
4.The complete three-dimensional structure for both class I and
class II determined by x-ray crystallography.
Class I MHC
The class I gene complex contains three major loci ,
B ,C and A and other undefined minor loci. Each
major locus codes for a polypeptide; the alphachain that contains antigenic determinants, is
polymorphic (has many alleles).
‫ يك فرد مشابه اما افراد مختلف داراي اللهاي مختلفي‬MHC ‫مولكولهاي‬
.‫از هر لوكوس ژنتيكي هستند‬
It associates with beta-2 microglobulin (beta-chain),
encoded by a gene outside the MHC complex, and
expressed on the cell surface. Without the beta-2
microglobulin, the class I antigen will not be
expressed on the cell surface. Individuals with a
defective beta-2 microglobulin gene do not express
any class I antigen and hence have a deficiency of
cytotoxic T cells.
Structure of Class I MHC Molecules
The MHC class 1 molecule has three globular
domains alpha 1 (yellow), alpha 2 (green) and
alpha 3 (blue).
The alpha 3 domain is closely associated with
the non-MHC -encoded beta 2 microglobulin
(pink). The latter is stabilized by a disulfide
bridge (red) and is similar to an
immunoglobulin domain in three-dimensional
structure.
The allo antigenic sites which carry determinants
specific to each individual are found in the alpha
1 and 2 domains.
The latter also has a carbohydrate chain (blue,
CHO).
There is a phosphate in the cytoplasmic domain.
Papain cleaves near the outer surface of the
plasma membrane.
Class I MHC molecules contain two
separate polypeptide chains
1) MHC-encoded alpha (or heavy) chain;
43 kDa
2) non-MHC-encoded beta chain (
microglobulin) 12 kDa
b. There are four separate regions
1.peptide-binding region is a groove formed from
the alpha1 and alpha 2 regions which interact to
form a "floor" of an 8-stranded, beta-pleated
sheet with two opposite "walls" consisting of
parallel strands of an alpha-helix. (alpha 1 and
alpha 2 each contribute 4 strands of beta-pleated
sheet and one alpha-helix).
A peptide 8-10 amino acids long sits in the groove.
The greatest variability in amino acids occurs in
the alpha1 and alpha 2 sequences that form the
groove that interacts with amino acids of the
peptide fragment.
Thus, the polymorphism among class I MHC gene
products creates variation in the chemical
surface of the peptide-binding groove.
For any given MHC molecule, binding of a peptide
usually requires the peptide to have one or more
specific amino acids at a fixed position, frequently
the terminal or penultimate amino acid of the
peptide.
Binding of the specific amino acid in the groove of
the MHC molecule occurs in what is termed
anchor site(s).
The other amino acids can be variable so
that each MHC molecule can bind many
different peptides.
Other polymorphic residues of the MHC
molecule are those in contact with the T
cell receptor (TCR), which interacts with
both peptide and the MHC molecule itself.
2.immunoglobulin-like region is composed of an
alpha3 segment that is highly conserved and is
homologous to Ig constant domains and noncovalently bound to beta2 microglobulin, an
invariant molecule, also homologous to Ig
constant domains.
These two interact with alpha1 and alpha2 to
maintain their proper conformation.
The importance of the highly conserved region of
alpha3 is that CD8, a molecule expressed on
cytotoxic T cells that recognize class I MHC
molecules, binds to this region.
(3) Transmembrane region is a stretch of ~25
hydrophobic amino acids.
(4) Cytoplasmic region is the carboxy terminal 30
amino acids. Contains phosphorylation sites and
provides binding sites for cytoskeletal elements.
Most variability in amino acids at different positions along the alpha chain of class I
MHC molecules occurs in the alpha 1 and alpha 2 regions. The greatest
polymorphism is found for amino acids that line the wall and floor of the
groove that binds the peptides.
The regions of variability of MHC I molecules and the interaction of the alpha
chain with other subunits of the MHC I complex and the bound peptide
2.Class II MHC
MHC class II molecules comprise two non-identical
proteins (alpha and beta) which are noncovalently associated and traverse the plasma
membrane with the N terminus to the outside of
the cell.
The domains closest to the membrane in each
chain are structurally related to immunoglobulins.
With the exception of the alpha 1 domain, all
domains are stabilized by disulfide bridges (red).
Both the alpha and beta chains are
glycosylated.
The beta chain is shorter than the alpha
chain (beta mol. wt = 28,000) and contains
the alloantigenic sites.
There is some polymorphism in the alpha
chain of some MHC II molecules.
Class II MHC
The class II gene complex also contains at least
three loci ,DP ,DQ and DR ;each of these loci
codes for one alpha- and one beta-chain
polypeptide which associate together to form the
class II antigens.
Like the class I antigens, the class II antigens are
also polymorphic. The DR locus may contain
more than one, possibly four, functional betachain genes.
MHC class II
a. contains two non-covalently associated
polypeptide chains, both MHC-encoded
and polymorphic, especially the beta chain
in human class II MHC
1.alpha chain; 34 kDa
beta chain; 28 kDa
b. There are four separate regions
1.peptide-binding region is formed by
interaction of the alpha1 and beta1
segments.
There is a groove having a "floor" of 8
beta-pleated strands and two "walls"
with alpha-helices; alpha1 and beta1
make equal contributions to this
structure.
As in the case for class I MHC, the greatest
polymorphic variability is in the amino
acids facing the groove.
Thus, as for class I MHC, the genetic
polymorphism determines the chemical
structure of the groove and influences
the specificity and affinity of peptide binding
and T cell recognition.
Peptides associated with class II MHC are
13-25 amino acids long;
the longer peptides =project from the ends
of the groove.
As with class I MHC, anchor sites for one or
more amino acids also exist in the groove
of the class II MHC molecule, but these
occur at more variable locations.
‫توزيع مولكولهاي كالس دو نسبت به كال س يك محدود تر است به‬
‫طوري كه به طور طبيعي و مداوم يعني بدون تحريك خارجي در‬
‫انفوسيتهاي ‪ ، B‬سلولهاي دندريتيك‪ ،‬و سلولهاي اپي تليال تيموس‬
‫ظاهر مي گردند اگر چه بعضي سلولهاي ديگر مثل سلولهاي‬
‫اندوتليال و ماكروفاژها نيز ممكن است در اثر تحريك عوامل فعال‬
‫كننده اي مثل اينتر فرون گاما وادار شوند مولكولهاي كالس دو را‬
‫ظاهر سازند‪.‬‬
‫مهمترين تفاوت ‪ MHC‬كالس يك و دو در داشتن فرم دايمر كالس دو‬
‫است(يك جفت آلفا‪-‬بتا) كه شايد به توانايي آن مولكول در اتصال به‬
‫دو مولكول گيرنده سطح لنفوسيت ‪ T‬بر مي گردد كه منجر به فعال‬
‫شدن لنفوسيت ‪ T‬مي گردد‪.‬‬
The greatest polymorphism for the beta chain of class II MHC molecules is found
for those amino acids in the beta I region that line the wall and floor of the groove
that binds the peptide.
2. Immunoglobulin-like region formed by alpha 2
and beta 2 is folded into Ig-like domains. These
are largely non-polymorphic.
The correlation of CD4 expression on helper T cells
with a specific TCR for class II MHC molecules
is due to binding of the CD4 molecules to the Iglike non-polymorphic beta 2 domain of the class
II MHC molecules.
3,4.Transmembrane and cytoplasmic regions have
proposed functions similar to their counterparts
in class I MHC.
Similarities between class I and class II MHC molecules
Despite differences in the two-chain composition of class I
and class II MHC molecules, they are quite similar
structurally.
Important aspects of MHC
1. Because MHC molecules are membrane-associated and
not soluble, T cells must make cell to cell contact with
cells expressing MHC molecules.
2 . In general, peptide fragments of proteins in the cytosol
associate with class I MHC; those of vesicular
proteins associate with class II MHC. Each is
recognized by functionally distinct T cell populations. Tc
recognize class I MHC-peptide; Th recognize class II
MHC-peptide.
3.There are many different MHC gene products for class I
and class II in the human population (polymorphism),
only some of which are found in an individual. The
maximum number of class I MHC gene products
expressed in an individual is six; that for class II
MHC products can exceed six but is also limited.
Whether or not a peptide fragment can associate with a
given MHC product determines whether there will be an
immune response. This is one level of control of immune
responses.
4. Mature T cells respond to foreign antigens, but not self
protein. The repertoire of antigen recognition is based on
selection processes involving MHC molecules that occur
mainly in the thymus.
This is another level of control of immune responses.
5. Only a single binding site exists on a class I or class II
MHC molecule; all peptides it is capable of binding must
bind to the same site.
6. The MHC polymorphism is determined only in the
germline.
There is no somatic DNA recombination that occurs for
antibodies and for the TCR, so the MHC genes lack
recombinational mechanisms for generating diversity. As
a result, the affinity and selectivity of MHC molecules for
foreign proteins are considerably lower than those of
antibodies and T cell antigens.
7.Because each MHC molecule can bind many different
peptides, the binding is said to be degenerate. Compare
with the restricted binding of a hormone to a receptor, for
example, where a difference in even one amino acid may
impair binding.
8.cytokines, especially interferon gamma (IFN-gamma),
increase the level of expression of class I and class II
MHC molecules.
9. Alleles for MHC genes are co-dominant, i.e. each gene
product is expressed on the cell surface. (Contrast this
with allelic exclusion.)
10. Why the high polymorphism of MHC molecules?
Although an individual may not possess MHC molecules
capable of binding certain antigenic peptides, say for
example against a virulent organism, the likelihood is
great that other MHC in the species can do so.
Overall this affords an evolutionary protection for the
continuation of that species.
MHC ANTIGENS
HLA specificities are identified by a letter for locus and a
number (A1, B5 ,etc ).and the haplotypes are identified
by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10,
DR8).
Specificities which are defined by genomic analysis (PCR),
are names with a letter for the locus and a four digit
number (e.g. A0101, B0701, C0401 etc) .
Specificities of mouse MHC (H-2) are identified by a
number. Since laboratory mice are inbred, each strain is
homozygous and has a unique haplotype. The MHC
haplotype in these strains is designated by a 'small' letter
(a, b, d, k, q, s ,etc ;).for example, the MHC haplotype of
Balb/c mice is H2d.
Inheritance
MHC genes are inherited as a group haplotype ,)one from
each parent (.
Thus, a heterozygous human inherits one paternal and one
maternal haplotype, each containing three class-I (B, C
and A) and three class II (DP, DQ and DR) loci.
A heterozygous individual will inherit a maximum of 6 class
I specificities.
Similarly, the individual will also inherit DP and DQ genes
and express both parental antigens.
Since the class II MHC molecule consists of two
chains (alpha and beta), with some antigenic
determinants (specificities) on each chain, and
DR alpha- and beta-chains can associate
in either cis( both from the same parent) or
trans( one from each parent) combinations, an
individual can have additional DR specificities.
Also, there are more than one functional DR betachain genes. Hence, many DR specificities can
be found in any one individual.
MHC antigen expression on cells
MHC antigens are expressed on the cell surface in
a co-dominant manner: products of both parental
genes are found on the same cells.
However, not all cells express both class I and
class II antigens.
While class I antigens are expressed on all
nucleated cells and platelets (and red blood cells
in the mouse), the expression of class II antigens
is more selective. They are expressed on B Cells,
a proportion of MQs and monocytes, skin
associated (Langerhans) cells, dendritic cells and
occasionally on other cells.
MHC detection by serological test
The MHC class I antigens are detected by
serological assays (Ab and C).
Tissue typing sera for the HLA were obtained, in the
women who were multiparous past, from
exposed to the child's paternal antigens
during parturition and subsequently developed
antibodies to these antigens.
More recently, they are produced by monoclonal
.antibody technology
MHC detection by mixed leukocyte reaction (MLR)
It has been observed that lymphocytes from one donor,
when cultured with lymphocytes from an unrelated donor
are stimulated to proliferate.
It has been established that this proliferation is due to a
disparity (differences, inequality) in the class II MHC
(DR) antigens and T cells of one individual interact with
allogeneic class II-MHC antigen-bearing cells. (B cells,
dendritic cells, Langerhans cells etc.)
This reactivity was termed mixed leukocyte reaction
(MLR) and has been used for typing some class II MHC
antigens .
The test lymphocytes were mixed with irradiated or
mitomycin-C treated homozygous leukocytes, containing
B-lymphocytes and monocytes (stimulator cells).
In culture (over 4 - 6 days), T-cells (responder cells)
recognize the foreign class II antigen and undergo
transformation (DNA synthesis and enlargement:
blastogenesis) and proliferation (mitogenesis).
These changes can be recorded by the addition of
radioactive (tritiated3 ,H) thymidine into the culture and
monitoring its incorporation into DNA.
Most modern laboratories, however, are switching to PCR
technology for tissue typing using specific probes for MHC
specificities
‫تجزيه پروتئين به پپتيدها در دو مرحله صورت مي گيرد‪:‬‬
‫الف)درون وزيكولهاي اسيدي(به ‪ MHC‬كالس دومتصل مي‬
‫شوند)‪.‬‬
‫ب)درون سيتوپالسم و رتيكولوم آندوپالسميك(به ‪ MHC‬كالس‬
‫يك متصل مي شوند)‪.‬‬