HUMAN LEUKOCYTE

ANTIGENS

Dr. B.Vijayasree
1st year Post-graduate
Department of Microbiology
 CONTENTS
History
Introduction
Classification of HLA
Polymorphism
Regulation
HLA Typing
Applications
MHC restriction
HISTORY
The primary function of the Immune system is the recognition and
elimination of foreign cells or antigens that enter the body.
Tissues and organs grafted from one individual to another member of the
same species (ALLOGRAFTS) are recognised as foreign material and
rejected.
It was the early work of Gorer in the 1930s on the antigens responsible for
allograft rejection in inbred mice that led to the discovery of the MAJOR
HISTOCOMPATIBILITY COMPLEX(MHC).

Gorer identified 2 blood group antigen systems in mice: antigen 1 was

common to all the strains, antigen 2 was found only in some strains and
appeared to be responsible for allograft rejection.
This was called the H-2 antigen (H for Histocompatibility).
The Histocompatibility antigens are cell surface antigens that induce an
immune response leading to rejection of allografts.
The H-2 Antigen system was found to be the Major Histocompatibility
Antigen for mice and to be coded for by a closely linked multiallelic
cluster of genes called the Major Histocompatibility Complex.
Dausset pioneered studies on Human Leukocyte antigens, which were
later found to be the Major Histocompatibility antigens in human beings.
The major antigens determining histocompatibility in human beings are
alloantigens, characteristically found on the surface of leukocytes.
Human MHC antigens are therefore synonymous with Human
Leukocyte Antigens (HLA) and the MHC complex of genes with the HLA
complex.
For their work on MHC and the genetic control of immune response,
Snell, Dausset and Benacerraf were awarded the Nobel Prize for Medicine
in 1980.
In humans, HLA complex coding for HLA proteins are located on the short
arm of chromosome 6.
The HLA complex extends over 4000kbp length covering >100 genes.
The genes are clustered in 3 regions namely MHC/HLA region-I,II,III.
HLA antigens are 2 chain glycoprotein molecules anchored on the surface
membrane of cells.

HLA REGION-I :
It is about 2000kbp in length, comprises of 3 class I genes called HLA-A,HLA-
B,HLA-C genes which code for HLA-A, HLA-B, HLA-C proteins respectively,
each one is capable of forming the α-chain of HLA class I molecules.
HLA-I proteins are located on the surface of all nucleated cells (except sperm
cells) and platelets.
They are absent in RBCs.
HLA CLASS-I MOLECULE :
It is composed of α chain (glycoprotein,45kDa) coded by HLA class I
genes and β2 microglobulin (Beta chain-non-glycosylated 12kDa protein,
encoded by a non MHC gene from chromosome 15).
The α chain is folded further and organized into 3 extracellular globular
domains- α1, α2 and α3 (each containing 90 amino acids) and a small
length of transmembrane C terminus reaching into the cytoplasm
(cytoplasmic tail).
The association of β2 microglobulin with α chain(non-covalent bond) is
necessary for the expression of MHC I molecules onto the cell surface.
In DAUDI cells (a type of human B cell tumour cell which are not able
to produce β2 microglobulin), it is observed that they synthesize MHC-I
but don’t express them on cell surface.
Role of HLA Class I Molecules :
The antigen peptide binding groove of class I HLA molecule i.e. the site
where the antigen peptide binds ) is formed by the cleft between α1 and
α2 domains.
The α3 domain binds to CD8 molecule present on cytotoxic T cells
during antigen presentation so they present the peptide antigen to CD8 T-
cells.
They are the principal antigens involved in graft rejection and cell-
mediated cytolysis.
Class I molecules may function as components of hormone receptors.
HLA REGION-II :
It spans over 1000 kbp length; comprises of 3 regions-DP,DQ,DR genes
encoding DP,DQ,DR proteins respectively, each one is capable of
forming α and β chain of HLA class II molecules.
In addition, HLA II region also contains certain other non-classical
genes, such as DO, DM, LMP and TAP (transporter associated with
antigen processing) that help in antigen processing and presentation.
HLA Class II Molecule :
It comprises of one α chain (33 kDa) and one β chain (28kDa).
The α and β chains in turn consist of 2 domains each –α1 and α2 and β1
and β2, respectively and cytoplasmic tails.
The antigen peptide binding groove is formed by the cleft between α1
and β1 domains.
The β2 domain interacts with CD4 molecule of helper T cells during
antigen production.
MHC-II proteins are located on the surface Antigen Presenting Cells
(Macrophages, dendritic cells, activated T cells and particularly on B
cells).
ROLE OF HLA CLASS-II MOLECULE :
They present the peptide antigen to CD4 T-cells.
They are primarily responsible for the Graft versus Host response and
Mixed Leukocyte reaction.
Both class I and II molecules are members of the immunoglobulin gene
superfamily.
The immune response (Ir) genes which control immunological
responses to specific antigens are believed to be situated in the HLA class
II region, probably associated with the DR locus.
Ir genes have been studied extensively in mice and located in the region
I of mouse MHC. And they code for Ia(I associated) antigens consisting
of 1A and 1E proteins.
However the relevance of the Ir genes in humans is not clear.
HLA REGION III :
It is also 1000kbp in length.
HLA class III proteins are heterogenous.
It carries genes that code for complement factors (C2,C4,C3
Convertase, Factor B and Properdin), Heat Shock Protein(HSP) and
Tumour Necrosis Factor (TNF-A AND B) and steroid 21-hydroxylases.
It is not involved in antigen presentation.
They also show polymorphism.
MHC POLYMORPHISM
Each MHC molecule specifically presents a certain antigenic peptide to the T
cells.
Since there is a wide spectrum of different antigenic peptides (with specific
sequences) derived from various antigens; there is a likewise need of different
MHC molecules capable of recognizing these peptides.
This is made possible by the polymorphic feature of MHC genome; by which
it is capable of producing a wide array of MHC molecules with vast antigenic
specificities.
There are 3 mechanisms by which MHC molecules show such a high level of
polymorphism. They are
1.Multiple gene loci
2.Multiple alleles for each locus
3.Codominant expression
1.MULTIPLE GENE LOCI :
Both MHC class I and II molecules are coded by multiple genes.
Gene of each locus codes for a similar but not identical chain.
Eg : MHC class I molecules (α chain) are coded by any of 3 loci of
class I region i.e. HLA-A,HLA-B,HLA-C loci.
MHC class II molecules ( α and β chains) are coded by any of the 3 loci
of class II region i.e. DP or DQ or DR loci.
2.MULTIPLE ALLELES FOR EACH LOCUS :
In a given species, extraordinarily large no. of different alleles are known to exist
for each locus.
Eg : Class I MHC-240 alleles for HLA-A
470 alleles for HLA-B
110 alleles for HLA-C
The MHC class I region of any individual would have one of the allele from each
HLA-A,B and C allele bank.
So there are 240 × 470 × 110 no. of combinations possible for class I MHC region.
These alleles encode for products that vary from one another by 5-10% of their
DNA sequence.
Similar polymorphism also exists for alleles of class II DP,DQ and DR loci.
3.CO-DOMINANT EXPRESSION :
MHC genes are expressed in co-dominant manner i.e.the alleles
inherited from parents (1 from father and 1 from mother) are
simultaneously and equally expressed.
REGULATION OF MHC EXPRESSION :
There are several regulatory mechanisms that control the expression of
MHC genes in different cell types. They are
1.TRANSCRIPTION FACTORS :
MHC genes have promoter sequences at their 5’ end which are
regulated by certain transcription factors such as CIITA (class II
transactivator) and RFX (both bind to MHC II promoter genes and
increases their transcription).
Defects in CIITA and RFX cause one of the form of BARE
LYMPHOCYTE SYNDROME.
2.CYTOKINES : They also influence MHC expression.
IFN-γ activates both MHC –I and II promoter genes.
IL-4 increases expression of class II MHC molecules on resting B cells.
3.CORTICOSTEROID and PROSTAGLANDINS decrease the
expression of MHC II molecules.

4.In many viral infections, the viral antigens inhibit various components
of MHC-I
eg : 1.Adenovirus E19 proteins inhibit TAP (by binding class I
molecules and retains them in endoplasmic reticulum).
2.CytoMegaloVirus proteins inhibit β2 microglobulin.
As a result , MHC-I expression is suppressed.
HLA TYPING
Antisera for HLA typing were obtained principally from multiparous women as they
tend to have antibodies to HLA antigens of their husbands due to sensitisation during
pregnancy.
Monoclonal antibodies to HLA antigens have been developed.
Typing is done serologically by Microcytotoxicity, which tests for complement
mediated lysis of peripheral blood lymphocytes with a standard set of typing sera.
However serological typing is not possible for HLA-DR antigens which are
detected by the Mixed Leukocyte Reaction (MLR) and Primed Lymphocyte
Typing(PLT) respectively.
Genetic methods are being used increasingly for HLA typing.
These employ Restriction Fragment Length Polymorphism(RFLP) and gene
sequence specific oligonucleotide probe typing.
MICROLYMPHO-
CYTOTOXICITY TEST
In this test, purified lymphocyte suspensions from donor and recipient are added to
microwells containing a standard panel of typing antisera (containing monoclonal
antibodies), complement and tryphan blue stain.
Cells carrying antigens corresponding to the HLA antiserum are killed by
complement mediated membrane damage.
These cells can be detected by using vital stains such as Eosin or Tryphan blue.
Cell death indicates that the test cells (recipient and donor cells) possess the
antigens being tested for namely HLA-A, HLA-B and HLA-DR antigens.
Although this method of HLA typing is preliminary it doesn’t provide direct
information about sequence variation in alleles.
TISSUE MATCHING/TISSUE TYPING :
It is a procedure in which the tissues of a prospective donor and recipient are tested
for compatibility prior to transplantation.
This is done by the functional assay called Mixed Lymphocyte Reaction or
Culture (MLR OR MLC).
This assay measures the proliferative response of lymphocytes on exposure to HLA
incompatible antigens from one individual(recipient) to lymphocytes from another
individual (donor).
The intensity of the reaction (proliferative response) is a measure of the antigenic
disparity between the donor and recipient lymphocytes.
This is a one-way test in which donor lymphocytes are killed and only recipient
lymphocytes are permitted to be transformed in response to the incompatible antigens
on the donor cells.
The MLR can be executed on lymphocytes from the alike individual/monozygotic
twin (AUTO-MLR), from different individuals of the same species (ALLO-MLR) or
individuals from different species (XENO-MLR).
APPLICATIONS
Due to the extreme pleomorphism of the HLA system, delineation of
the HLA type provides a method of typing of individuals that is far more
discriminating than blood grouping.
1.Transplantation : HLA typing is used primarily for testing
compatibility between recipients and potential donors before tissue
transplantation.
2.Paternity : it also has applications in disputed Paternity.
3.Anthropological studies : As the prevalence of HLA types varies
widely between different human races and ethnic groups, HLA typing is
used in anthropological studies. Population studies of HLA polymorphism
suggest the origin of the human species in Africa and emigration as
different subtypes to other continents.
4.Genetic predisposition to Disease :
An association between HLA types and certain diseases has been
observed.
Such diseases are generally of uncertain origin, associated with
immunological abnormalities and exhibit a hereditary tendency.
The relative risk of occurrence of the disease in presence of the
identified allele varies.
HLA B27 is strongly associated with Ankylosing Spondylitis (90 times
higher risk than those not expressing HLA 27).
HLA allele Associated disease
HLA B27 Ankylosing spondylitis
Reactive arthritis (Yersinia, Salmonella,
Gonococcus)
Reiter’s syndrome

DR-2 Multiple Sclerosis

Goodpasture’s syndrome

DR-3 Myasthenia gravis

Systemic Lupus Erythematosis

DR-3/DR-4 Insulin-dependent Diabetes Mellitus

DR-4 Rheumatoid Arthritis
A3/B14 Hereditary Hemochromatosis
MHC RESTRICTION
The importance of MHC antigens in immune reaction is indicated by the finding
that T cells respond to processed antigens on Macrophages and other accessory cells
only when they are presented along with the self-MHC antigen. This is known as
MHC restriction.
Both class I and II Antigens are involved in this phenomenon.
Cytotoxic T-lymphocytes from immunised mice are able to kill and lyse virus
infected target cells only when the T cells and target cells are of the same MHC type,
so the T cells can recognise class I MHC antigens on the target cells.
Helper T cells can accept antigens presented by Macrophages/Dendritic cells only
when they bear the same class II MHC molecules on the surface.
For T cells participating in Delayed type hypersensitivity, the antigen has to be
presented along with class II MHC determinants.
REFERENCES
Kuby’s Immunology
Textbook of Microbiology and Immunology by Parija
Textbook of Microbiology -Ananthanarayana
THANK YOU