Transcript Major Histocompatibility complex OR

Major Histocompatibility
complex
OR
MHC
MHC

The principal function of T cells are :
Defense against intracellular microbes
Activation of other cells(B cells ,Macrophages )

peptide recognition by T and B cells is
different.
B cells recognize soluble as well as cell associated Ags
In contrast ,T cells recognize peptide which is displaying only
by APC in associated with MHC proteins.
MHC





MHC genes are the most polymorphic genes
MHC genes are codominantly expressed in each
individual
There are two main type of MHC:class I & II
The physiologic function of MHC molecules is
presentation of peptides to T cells ,control of
immune responsiveness to all proteins and graft
rejection
MHC-I present cytosolic peptides to T cytotoxic
cells and MHC-II present endocytosed peptides
to Th cells
MHC genes



HLA-G ( a role in Ag recog.by NK cells)
HLA-H (involved in iron metabolism
HLA-DM (peptide binding to class II)
Class III
 C4,B,C2
 TNF,LT
joined to class II
Proteasome genes,TAP,DM
MHC –I structure
MHC-II structure
Conformational structure
MHC-II
MHC-I
Peptide location
Characteristics of peptide-MHC interaction



Each class –I or II has a single peptide
binding cleft that are accommodate many
different peptides
Slow on-rate and very slow off-rate
The MHC molecules of an individual don’t
discriminate between self and non self
T cell receptor and MHC
interaction
Polymorphism of class II





HLA-DPA
HLA-DPB
HLA-DQA
HLA-DQB
HLA-DR
12
88
17
42
>400
Polymorphism of class I



HLA-A
HLA-B
HLA-C
>280
>500
>130
MHC genes and graft rejection
HLA expression
Peptide presentation by MHC-II
Peptide presentation by MHC-I
MHC –I and peptide presentation
Features of peptide binding to MHC
HLA and diseases



Testing of DNA sequences permits detection of
many more subtypes or "splits" of HLA antigens
or alleles.
In serological typing, some antigens are difficult
to identify and may even mask the presence of
others. DNA typing can routinely define antigens
at the allele level, assuring no ambiguity in
interpretations.
DNA typing does not require live blood cells from
the patient, permitting more flexible sample
requirements. Thus, LabCorp can perform DNAbased HLA typing on:
More interesting facts



Erythrocytes will adsorb some Class I
antigens viz. Bg blood group system
(B7,A28, B57….)
HLA B most polymorphic system and
studies have shown is most significant
followed by A and then C
45Kd glycoprotein comprising three heavy
chain domains, non-covalently associated
TYPING METHODS



SEROLOGY used to be the ‘gold’ standard.
Now being superceded by molecular
techniques as they become more robust
and time efficient
CELLULAR rarely used now. Orginally used
for Class II typing
MOLECULAR fast becoming the method of
choice. Many laboratories test of choice.
SEROLOGY



Complement Dependent Cytotoxicity
(CDC)
Viable peripheral blood lymphocytes are
obtained by discontinous density gradient
centrifugation using Ficoll / Tryosil or Ficoll
/ Sodium Metrizoate at a density of 1.077
at 19º - 22ºC.
Microlymphocytotoxic test: 3 stages
Microlymphocyototoxic test


1.Viable lymphocytes are incubated with
HLA specific antibodies. If the specific
antigen is present on the cell the antibody
is bound.
2.Rabbit serum as a source of complement
is added, incubate. If antibody is bound to
the HLA antigen on the cell surface it
activates the complement which damages
the cell membrane making it permeable to
vital stains.
Microlymphocyototoxic test 2



3.Results are visualised by adding dye usually a
fluorochrome eg Ethidium Bromide although
both Trypan Blue and Eosin have been used in
the past.
If the reaction has taken place the EB enters the
cell and binds to the DNA.
For ease double staining is normally used. We
use a cocktail of Ethidium Bromide and Acridine
Orange, quenched using Bovine Haemoglobin to
allow simultaneous visualisation of both living
and dead cells.
Microlymphocytotoxicity test 3




Test is left for 10 minutes and then read
using an inverted fluorescient microscope.
A mixture of T and B lymphocytes can be
used for HLA Class I typing.
B lymphocytes are required for HLA Class
II typing by serology. (Normal population
85-90% T and 10-15% B cells)
This can be achieved using a number of
methods.







Easily performed does not require expensive
equipment.
Takes around three hours to perform
Low level resolution, with good antisera reliable
results
Requires large volumes of blood
Requires viable lymphocytes
Difficult to find good antisera for rarer antigens
in different populations
molecular


DNA extraction from the nucleated cells
following cell lysis and protein digestion.
polymerase chain reaction (PCR)
Molecular Methods 4



Electrophoresis is used following amplification.
PCR product is run out on an agarose gel
containing ethidium bromide. Each product
moves according to its size and is compared to a
molecular weight marker.
Interpretation: every tube should produce an
identical sized product as internal control and
either a specific band or not dependent on
whether the allele(s) is/are present or not.
Results are visualised using 312nm UV
transillumination and recorded either by video
imaging or polaroid photograghy.