HLA system- HLA typing and

its role in tissue transplantation

Presenter: Dr. Animesh Debbarma

Moderator: Dr. Sharmila
Objectives
 Major histocompatibility complex

 MHC polymorphism

 Regulation of MHC expression

 HLA nomenclature, typing, methods, application

 Organ transplantation
 Major histocompatibility complex
 Group of genes coding for a set of host surface molecules that bind to a
peptide fragments derived from pathogens and foreign antigens, and
display them on host cell surface for recognition by the appropriate T cells.

 Also called human leukocyte antigens (HLA).

 Serves as a unique identification marker for every individual.

 Following transplantation of a graft the recipient mount an immune

response against the graft’s MHC molecules and vice versa.

 Also called histocompatibility antigens.

 HLA complex (MHC genes) and their
products
 In humans, HLA complex coding for MHC proteins are located on short
arm of chromosome 6.
 Around 4000 kbp in length covering >100 genes. Genes are clustered in
three regions namely MHC region-I, II and III.
MHC region I

 About 2000 kbp in length. Comprises of three class I genes called

HLA-A, HLA-B and HLA-C genes encoding HLA-A, HLA-B and
HLA-C proteins respectively.

 Each protein is capable of forming the α-chain of MHC class I

molecules.

 MHC class I molecules are present on the surface of all nucleated

cells (except sperm cells) and platelets.

 Present the peptide antigen to CD8+ T cells.

MHC region II

 About 1000 kbp length. Comprises of three genes namely DP, DQ and DR genes
encoding DP, DQ and DR proteins respectively.

 Each protein is capable of forming α and β-chain of MHC class II molecules.

 In addition MHC region also contain non classical genes such as DM, DO, LMP
and TAP that help in antigen processing and presentation.

 MHC-II proteins are located on the surface of APC.

 Present the peptide to CD4+ T cells.

MHC region III

 About 1000 kbp in length. Not involved in Ag presentation.

 Comprises of genes that code for complement factors, heat shock

proteins (HSP), tumor necrosis factor (TNF α and β), steroid 21
hydroxylases etc.
STRUCTURE OF THE MHC MOLECULES
MHC class I molecules

 They are heterodimer composed of polymorphic α-chain (glycoprotein, 45 KDa, coded

by HLA class I genes) linked non-covalently to smaller non polymorphic β2
microglobulin (non glycosylated 12 KDa protein, encoded by non MHC gene from
chromosome 15).
 α-chain is organized into
 3 extracellular globular domain (N terminal) α1, α2 and α3 (each containing 90 AA);
 A hydrophobic transmembrane domain of about 25 AA followed by a short stretch of
charged (hydrophilic) amino acids;
 Cytoplasmic anchor segment of 30 amino acids.
 Antigen peptide groove is formed by the cleft between α1 and α2 domains. Polymorphic
amino acid residues lines the side and base of the peptide binding groove.
 The α3 domain bind to CD8 molecules of cytotoxic T cell during Ag presentation.
MHC class II molecules

 They are heterodimer consisting of noncovalently associated α chain

(33 KDa) and β chain (28 KDa), both of them are polymorphic.
 Like class I molecule they have external domain, a transmembrane
segment and a cytoplasmic anchoring segment.
 Each chain contain 2 extracellular domain: α1, α2 and β1, β2
respectively.
 The Ag peptide groove is formed by the α1 and β1 domains.
 β2 domain interacts with CD4 molecules of Helper T cell during Ag
presentation
 Difference between MHC I and MHC II
molecules
MHC class I MHC class II

Present on All nucleated cells (except sperm Ag presenting cells

cells) and platelets
Peptide Ag is presented to CD8+ T cells CD4+ T cells

Nature of peptide Ag Endogenous or intracellular Exogenous

(viral/tumor Ag)
General size of bound antigens 8-10 amino acids 13-18 amino acids

Peptide binding site α1 and α2 groove α1 and β1 groove

CD4 or CD8 binding site α3 binds to CD8 molecules on Tc β2 binds to CD4 molecules on TH
cells cells.

Ag presentation pathways Cytosolic pathway Endocytic pathway

 MHC polymorphism
Three mechanism:

1. Multiple gene loci: Ex-

MHC I molecules (α chain) are encoded by any of the three loci of
MHC I region, i.e. HLA-A, HLA-B or HLA-C loci.

MHC II molecules (α and β chain) are encoded by any of the three

loci of MHC II region, i.e. DP, DQ or DR loci.
2. Multiple allele for each locus: Ex-
 For Class I MHC region in humans, there are 240 alleles for HLA-A, 470
alleles for HLA-B and 110 alleles for HLA-C. So MHC Class I region of
any of the individual would have one of the allele from each HLA-A,
HLA-B and HLA-C allele bank.

 So there are total 240X470X110 theoretical combinations possible for

MHC Class I region. These allele encode for products that differ from
each other by 5-10 % of their DNA sequence.

 Similar polymorphism also exists for alleles of Class II DP, DQ and DR

loci.

3. Codominant expression: MHC genes are expressed in codominant

fashion, i.e. the alleles inherited from parents (one from father and one
from mother) are simultaneously and equally expressed.
 Regulation of MHC expression
 Transcription factors: MHC genes have promoter sequence at their 5’
end which are regulated by certain transcription factors such as CIITA and
RFX (both bind to MHC II molecules and increase their transcription).
Defect in CIITA and RFX- Bare lymphocyte syndrome.

 Cytokines:
 IFN-γ activates both MHC-I and II promoter genes.
 IL-4 increases the expression of MHC II molecules in resting B cells.
 Corticosteroids and prostaglandins decrease the expression of
MHC II molecules.

 Viral infection: Some viral antigens inhibit various components of

MHC I (e.g. adenovirus protein inhibit TAP, cytomegalovirus protein
inhibit β2 microglobulin). As a result, MHC-I expression is
suppressed.
 HLA Nomenclature

 There are a number of ways of writing an HLA antigen. For example, it

may be expressed as HLA-DR3, HLA-DRI7, HLA-DRB* 03 or HLA-
DRBI*0301. These could all refer to the same antigen.

 Firstly, “HLA” is the name for the gene cluster which tends to be inherited
en-bloc.

 Second part -e.g. DR- is the name of the specific locus. There are 6 loci
normally referred to. These are A, B, C, DR, DQ and DP.
 Third part, the number, e.g. 3, 17, 03, 0301, refers to the actual
antigen at the locus. For example, the DNA in the HLA-DR locus
tends to be different from person to person. This difference will
result in a different type of HLA-DR molecule. These different types
of HLA-DR molecules are given names, such as DR17.

 When we look at the antigens above: -HLADR3 is the broadest

description of the antigen. It is the name for a specific group of
antigens. The DR3 group can be divided into HLA-DR17 and HLA-
DR18 by using antibodies (serology).
 When we look at this antigen at the DNA level we call the DR locus
DRB 1 and the antigen 03 and 01 for the specific variant of the 03.
So, HLA-DR17 is now called HLA-DRB1*0301. This is similar for
other antigens in the system, at either HLA Class II or Class I e.g.
HLA-B60 (HLA-B*4001 molecularly).

Fig: HLA nomenclature

 HLA TYPING
 In this test, donor’s antigens expressed on the surface of leukocytes or
their genes are matched with that of the recipient.

 The closer the HLA antigens on the transplanted organ match the recipient,
the more likely that the recipient’s body will not reject the transplant.

 Value of HLA matching between donor and recipient varies in different

solid organ transplantation. In kidney transplants, there is substantial
benefit if all the polymorphic HLA alleles are matched.
 Every person inherits each of the following antigens from each parent:
 HLA-A antigen
 HLA-B antigen
 HLA-C antigen
 HLA-DR antigen
 HLA-DQ antigen and
 HLA-DP antigen

Fig: Major histocompatibility complex at chromosome 6

 When performing an HLA typing test for a kidney transplant,
the following HLA antigens are looked at:
 HLA-A
 HLA-B
 HLA-DR

 Six HLA antigens are looked at for each person.

 Each person has two of each of the antigens (one inherited from
the mother and one inherited from the father).
 By analyzing which six of these HLA-antigens both the donor and
recipient have, scientists are able to determine the closeness of
tissue matching.

 A six-antigen match is the best compatibility between a donor and

recipient.

 This match occurs 25% of the time between siblings who have the
same mother and father.
 METHODS OF HLA TYPING
A. Phenotypic method:
 Serology: Microcytotoxicity
 Tissue typing: Mixed lymphocyte reaction

B. Genotypic methods:
 PCR detecting HLA genes
 PCR-RFLP (restriction fragment length polymorphism)
 Variable number tandem repeat (VNTR) typing
 Short tandem repeat (STR) typing
 DNA sequence based typing
 Karyosome analysis
 Serology: Microlymphocytotoxic test:

 Viable WBC’s of the individual to be typed are incubated with HLA

(class I and II) specific antibodies. If the specific Ag is present on
the cell, the antibody is bound.

 Complement is added and incubated.

 If the antibody is bound, it will activate the complement which

damages the cell membrane making it permeable to vital stains.
Pros Cons
Easily performed, does not require Require large volume of blood
expensive equipments

3 hrs Require viable WBC’s

With good antisera results are reliable Difficult to find good antisera for rarer
antigens.
 Cellular: Mixed lymphocyte culture (MLC)

 Used to quantify the degree of class II MHC compatibility between

potential donors and recipients.

 It is based on the principle that if immunocompetent T cells from one

individual is incubated with APC’s of a genetically different
individual, the T-cell of the first individual will proliferate.

 Proliferation of the recipient T-cells is measured by the uptake of

thymidine into the cell DNA.
Molecular methods: Commonly used molecular techniques of the HLA
typing utilizes DNA extraction and direct DNA typing

 The phenotypic methods were used widely in the past.

 But with the advent of molecular methods, they are not preferred now.
 VNTR and DNA sequence based typing are the most reliable method of
HLA typing.
 Application of HLA typing
1. Organ and tissue transplantation
 In organ and tissue transplantation, HLA antigens of the donor
identified as invaders by the recipient causing rejection. Careful
selection of the matched donor and recipient critically affect the
outcome of transplantation.

2. Diagnosing some disease :

 In autoimmunity: Many HLA combination are potentially indicative
of autoimmune disorders, e.g.
HLA allele Associated disease

HLA B27 Ankylosing spondylitis, Reactive

arthritis, Reiter’s syndrome

DR2 Multiple sclerosis, Good pasture’s

syndrome
DR3 Myasthenia gravis, SLE

DR3/DR4 Insulin dependent DM

DR4 Rheumatoid arthritis

A3/B14 Hereditary hemochromatosis

  Susceptibility to viral infections: There is a link between certain
HLA antigens and susceptibility to some viral infections such as
AIDS (HIV virus), Hepatitis B (Hep B), Hepatitis C (Hep C),
Infectious mononucleosis (EVB), Rubella (Rubella virus) etc.

3. Paternal testing
 HLA typing can be used alongside other test for paternity testing.
4. Infertility (recurrent pregnancy loss):
 Infertility due to recurrent pregnancy loss can be attributed to
immune factors (40%) one of which is presence of certain common
HLA antigens between the parents.

5. Phylogenetic studies:
 Some HLA haplotypes have distinctive geographical distribution
and are found only in some population. These haplotypes can be
used to trace human migration.
 ORGAN TRANSPLANTATION
Based on the genetic relationship between the donor and the recipient:

 Autograft: Self tissue transferred from one part of the body site to
another in the same individual (e.g. transfering healthy skin to a
burned area in burned patients).

 Isograft of syngeneic graft: Tissue transferred between genetically

identical individuals (e.g. monozygotic twins).
 Allograft: Tissue transferred between genetically non-identical
members of the same species (e.g. kidney or heart transplant).

 Xenograft: Tissue transferred between different species (e.g. the graft

of a baboon heart into a man).
 In humans allograft are the most commonly used graft in transplant
centers.

 Histocompatibility between the graft and the recipient would decide

whether the graft is going to be accepted or rejected.

 Transplantation antigens are the antigens of allograft against which

the recipient would mount an immune response. MHC molecules are
the most important transplantation antigens. Apart from that, ABO
and Rh group system also play an important role in determining the
histocompatibility.
Mechanism of recognition and rejection of
allograft
 The major antigenic differences between a donor and the recipient
that result in rejection of transplants are differences in HLA alleles.

 Following transplantation, the recipient’s T cells recognize donor

antigens from the graft (the allogeneic antigens, or alloantigens) by
two pathways, called direct and indirect.
 Direct pathway of allorecognition.

 In the direct pathway, T cells of the transplant recipient recognize

allogeneic (donor) MHC molecules on the surface of APCs in the graft.

 CD8+ T cells recognize class I MHC molecules and differentiate into

active CTLs.

 CD4+ helper T cells recognize allogeneic class II molecules and

proliferate and differentiate into TH1 (and possibly TH17) effector cells
Indirect pathway of allorecognition:

 In the indirect pathway, recipient T lymphocytes recognize MHC

antigens of the graft donor after they are presented by the recipient’s
own APCs.

 This process involves the uptake and processing of MHC molecules

from the grafted organ by host APCs. The peptides derived from the
donor tissue are presented by the host’s own MHC molecules, like
any other foreign peptide.
 T cell mediated rejection:

 Acute cellular rejection:

 Most commonly seen within the initial months after transplantation and is
heralded by clinical and biochemical signs of organ failure.
 Old concept was direct killing of graft cells by CD8+ CTLs is a major
component of the reaction.
 Recent concept- an important component of this process is an inflammatory
reaction in the graft triggered by cytokines secreted by activated CD4+ T
cells. The inflammation results in increased vascularpermeability and local
accumulation of mononuclear cells and graft injury is caused by the
activated macrophages.
 Chronic rejection:
 Lymphocytes reacting against alloantigens in the vessel wall secrete
cytokines that induce local inflammation and may stimulate the
proliferation of vascular endothelial and smooth muscle cells.
 Antibody mediated reaction:
 Hyperacute rejection
 Occurs when preformed antidonor antibodies are present in the circulation
of the recipient.

 Such antibodies may be present in a recipient who has previously rejected a

transplant. Multiparous women who develop antibodies against paternal
HLA antigens shed from the fetus may have preformed antibodies that will
react with grafts taken from their husbands or children, or even from
unrelated individuals who share HLA alleles with the husbands.

 Prior blood transfusions can also lead to presensitization, because platelets

and white blood cells are rich in HLA antigens and donors and recipients are
usually not HLA-identical.
 Acute antibody-mediated rejection:
 caused by antidonor antibodies produced after transplantation.

 In recipients not previously sensitized to transplantation antigens, exposure to

the class I and class II HLA antigens of the donor graft, as well as other
antigens that differ between donor and recipient, may evoke antibodies.

 The antibodies formed by the recipient may cause injury by several

mechanisms:-
a. complementdependent cytotoxicity,
b. inflammation, and
c. antibodydependent cell-mediated cytotoxicity.
 Chronic antibody-mediated rejection:

 usually develops insidiously, without preceding acute rejection, and

primarily affects vascular components.
References:

1. Robbins and Clotran Pathologic Basis of Disease

2. Essential of medical microbiology by Dr. Apurba Sankar
3. Owen Kuby’s Immunology
4. Internet source
5. Slide of Dr. Anupama’s
THANK YOU
For patience hearing