HLA TYPING AND TRANSPLANT

IMMUNOLOGY

Rajeswari S
Moderator : Dr BIMAN SaIKIA
What is an MHC ?

 The Major Histocompatibility Complex (MHC) is a large

multigene family whose products are associated with the
intercellular recognition and with self or non-self discrimination.
 The MHC is critical for immunological specificity,
histocompatibility and susceptibilty for autoimmune diseases.
 In humans, the ~4 Mb (4,000,000bp) MHC region on
Chromosome 6 contains about 140 genes, more than 20% of
which have functions in immunity.
 Shows extensive conservation with the MHC of other other
mammals (in mice MHC is termed H-2 complex), which helps
in understanding MHC
Discovery of MHC
• The MHC was discovered as the genetic locus whose
products were responsible for rapid rejection of tissue
graft exchanged between inbred strains of mice.
• The particular locus that was identified in mice by Snell's
group was linked to a gene on chromosome 17 encoding
a polymorphic blood group antigen called antigen II, and
therefore this region was named histocompatibility-2 or,
simply, H-2.
• Genes that determine tissue compatibility between
individuals- histocompatibility genes
• George Snell was awarded the Nobel Prize in 1980
 20% genes are The most gene
Genome of HLA: functional dense region

20 HLA
genes;112 non
HLA genes
Properties of MHC Molecules :
q
Each MHC molecule consists of an extracellular peptide-
binding cleft a pair of immunoglobulin (Ig)-like domains
anchored to the cell by transmembrane and cytoplasmic
domains
 Properties of MHC Molecules:
 The polymorphic amino acid residues of MHC molecules are located
in and adjacent to the peptide-binding cleft
 The nonpolymorphic Ig-like domains of MHC molecules contain binding
sites for the T cell molecules CD4 and CD8
Feature Class I MHC Class II MHC
Distribution On nucleated cells; maximum on B macrophages, dendritic
cells; very low levels in fibroblasts, cells and B-cells, thymus
liver cells, muscle cells, neurons; epithelial cells,
sperm cells at certain stages and Langerhans cells.
cells of placenta lack

Polypeptide α (44-47 kD) α (32-34 kD)

chains β2-Microglobulin (12 kD) β (29-32 kD)

polymorphic α1 and α2 domains α1 and β1 domains

residues &
antigen binding
site
Binding site for T α3 region binds CD8 β2 region binds CD4
cell coreceptor

Antigen Endogenous protein Both endogenous and

processed exogenous
Properties of MHC and peptide interactions:

 MHC molecules have a broad specificity

for peptide binding (the fine specificity
rests with the TCR).
 Each Class I or Class II molecule has a
single peptide-binding cleft that can
accommodate many different
peptides
 The association of peptide and MHC
groove is a saturable, low- affinity
interaction, with a slow on rate and a
very slow off rate.
 The peptide molecules (anchor residues
 Peptide binding by class I and class
II MHC molecules
Feature Class I Class II
Peptide-binding α 1 and α 2 α 1 and β 1
domain
Nature of peptide- Closed at both ends Open at both ends
binding cleft
General size of 8–10 amino acids 13–18 amino acids
bound peptides

Peptide motifs Anchor residues at both ends Anchor residues distributed

involved in of the peptide ;generally
hydrophobic
carboxyl-terminal anchor
Nature of bound Extended structure in which
peptide both ends interact with MHC
cleft but middle
arches up away from MHC
molecule
along
binding to MHC molecule
peptide
Extended structure that is
held at a constant elevation
above the floor of MHC cleft.
Class I Class II
Genetic characteristics of
HLA
HLA INHERITANCE:
 They follow Mendelian inheritance
 Expression of MHC alleles is codominant,
meaning that the protein products of both the
alleles at a locus are expressed equally in the cell,
and both gene products can present antigens to T
cells.
 The particular combination of MHC alleles found
on a single chromosome is known as an
MHC haplotype.
HLA inheritance:
 Assuming no recombination, 4 different haplotypes
are possible in the offspring. The inheritance pattern
iimportant for compatible donors for transplantation.
 The chance that two siblings will be genotypically
HLA identical is 25%. The chance that any one
patient with “n” siblings will have at least one HLA-
identical sibling is 1 – (3/4) n .
HLA inheritance
 Having two siblings provides, a priori, a 44%
chance, and having three siblings provides a
58% chance that one sibling will be HLA identical.
 The probability will never be 100% for
finding an HLA-identical sibling.
 Each time a new sibling is tested, that new
sibling has (only) a 25% chance of being a match,
no matter how many siblings have previously
been tested.
HLA GENE DIVERSITY:
 The MHC is polygenic: it contains several different MHC
class I and MHC class II genes, so that every individual
possesses a set of MHC molecules with different ranges
of peptide-binding specificities.
 The MHC is highly polymorphic; that is, there are
multiple variants, or alleles, of each gene within the
population as a whole. The MHC genes are, in fact, the
most polymorphic genes known.
 MHC class I and class II are polygenic
(several loci encoding products with essentially the same function)

a chain

a chain a chain

b2 microglobulin is not encoded in

MHC
 Allelic variation in MHC occurs at the peptide
binding site and on the floor /sides of the groove

MHC polymorphism in
the peptide binding
groove explain the
preference of different
MHC proteins for
different sequence
motifs.
Polymorphisms in
MHC show how TCRs
recognize MHC-
allele-specific epitopes
for restricted
recognition of antigens
For MHC class I, a2 and a1 domains are polymorphic;
b2microglobin and the a3 domain are not polymorphic
For MHC class II, b1 domains are polymorphic;
a2 and b2 domains are not polymorphic
FOR 2010
Mechanisms:
 Point mutation
(replacement
substitutions/
silent mutations)
 Gene conversion
 Tandem
duplication
Why Polymorphism and
Polygenism in that
 Polygeny, ensures MHC?
each individual produces
a number of different MHC molecules
 The high polymorphism of the classical MHC
genes ensures a diversity in MHC gene
expression in the population as a whole.
 However, no matter how polymorphic a gene, no
individual can express more than two alleles at
a single gene locus.
T-cells are specific for amino acid sequence and
MHC molecule
MLR (Mixed Lymphocyte
reaction):
 Studies have shown that roughly 1-10% of all T
cells in an individual will respond to stimulation by
cells from another, unrelated, member of the same
species.
 This type of T-cell response is called an
alloreaction or alloreactivity, because it represents
the recognition of allelic polymorphism in
allogeneic MHC molecules as explained by Mixed
lymphocyte reaction.
 Linkage Disequilibrium:
 Expected frequencies for HLA haplotypes are derived by
multiplication of the frequencies of each allele
 In reality the haplotypes are overrepresented than expected,
which can be explained by Linkage Disequilibrium
 Assuming random distribution of haplotypes in European
population, calculated as follows
Crossing Over:
 HLA genes occasionally demonstrate
chromosome crossover in which segments
containing linked genetic material are
exchanged between the two chromosomes
during meiosis or gametogenesis haplotypes
in offspring.
 It is related to the physical distance
between the genes and their resistance
or susceptibility to recombination.
HLA Nomenclature, Loci and Alleles
 HLA nomenclature established by the WHO nomenclature
committee in 1967 adheres to the following format:
1.HLA antigen nomencltaure ( by serology)
2. HLA allele nomenclature (molecular technique)

25
SEROLOGICAL SPECIFICITIES
 Nomenclature : Name of HLA Molecule
 Order in which the serological specificity defined
( eg HLA – A9 )
 Over the time old serological specificities were split
( eg HLA – A9 : A 23 , A24 )
 The most narrow definition of the specificity :
Subtypic or PRIVATE SPECIFICITY
 The broader shared specificities : Supertypic
specificities
SO A CELL THAT IS A9 + MUST ALSO BE POSITIVE
FOR A23 & A24.
HLA ANTIGEN
NOMENCLATURE:
 Splits”: Refinement of serologic
methods permitted antigens that were
previously believed to represent a
single specificity to be “split” into
specificities that were serologically
(and, later, genetically) distinct.
 Cross-Reactive Groups (CREGs): HLA
antigens and antigen groups may
have other epitopes in common.
Antibodies that react with the shared
determinants often cause cross-
Serology Based Typing:
 A panel of known anti HLA antibodies
are incubated with viable lymphocytes of
unknown HLA type
 The HLA type of the sample is
determined from the pattern of cell killing
(cytotoxicity) that results from the
antigen antibody reactions
 Advantages:
1.Rapid
2. Assesses HLA cell surface expression
SCORING OF
LYMPHOCYTOTOXICITY
TEST:
 HLA ALLELIC
NOMENCLATURE:
HLA refers to the genetic complex
The second part is the name of the specific locus 6 loci- A, B, C, DR, DQ,
DP .An allele is defined as a unique nucleotide sequence for a gene as
defined by the use of all of the digits in a current allele name.
MOLECULAR TYPING
:PRINCIPLE
 Molecular HLA typing methods can detect any
polymorphic nucleotide sequence
 Advantages:
 Allele level matching
 Minimum no of cells required
 no viable cells required
 Improved accuracy and specificity : Specific
oligonucleotide probes and primers
 Flexibility: New reagents can be designed as new
alleles are discovered
 HLA resolution techniques-
Venn diagram
High-resolution:
HLA typing
defines the
specific DNA
sequence of the
antigen binding
site.
Allelic
resolution
defines a single
allele as defined
by a unique DNA
sequence for the
HLA gene
 METHODS :
Methods Clinical application Resolution

SSP (PCR) Solid organ, related and unrelated Serologic to allele level, higher
HPC transplantation resolution with large number of
primers

DNA Unrelated HPC transplantation, Allele level

sequencing resolution of typing problems with
other methods, characterization of
new alleles

SSOP Solid organ and HPC transplanta- Serologic to allele level

hybridization tion
ROLE OF HLA:
 Solid organ transplantation
 Allogenic Hematopoietic stem cell
transplantation
 Alloimmunization in multiple transfused
patients
 Platelet refractoriness
 Neonatal alloimmune thrombocytopenia
 TRALI
 FNHTR
 Immunologic Basis of Graft
 Rejection:
The degree of immune response to a graft varies with the
type of graft.
 The following terms are used to denote different types of
transplants:
 Autologous (self)
e.g., BM, peripheral blood stem cells, skin, bone
 Syngeneic (identical twin)
 Allogeneic (another human except
identical twin)
 Xenogeneic (one species to another)
HLA and Hematopoietic cell
transplantation:
GRAFT REJECTION
 Rejection is a complex process in which both cell-
mediated immunity (cellular)and circulating antibodies
(humoral) play a role.
 Cellular rejection: Acute and chronic
 Allorecognition occurs by 2 ways: Direct and indirect
allorecognition
MECHANISMS OF
ALLORECOGNITION:
 DIRECT:

 T cells of the transplant recipient recognize allogeneic (donor)

MHC molecules on the surface of APCs in the graft through the
DONOR dendritic cells.
 CD8+ T cells recognize class I MHC molecules active CTLs
 kill the graft cells.
 CD4+ helper T cells recognize allogeneic class II molecules
and proliferate and differentiate into TH1 (and possibly TH17)
effector cells.
 Cytokines secreted by the activated CD4+ T cells trigger a
delayed hypersensitivity reaction in the graft-----increased
vascular permeability and local accumulation of mononuclear
cells (lymphocytes and macrophages), and graft injury
MECHANISM OF
ALLORECOGNITION:
 INDIRECT:

 The recipient T lymphocytes recognize MHC antigens of the

donor cells after they are presented by the recipient's own
APCs.
 The principal mechanism T-cell cytokine production and
delayed hypersensitivity.
 CD8+ CTLs that may be generated by the indirect pathway
cannot directly recognize or kill graft cells, because these
CTLs recognize graft antigens presented by the host's
APCs
 Direct pathway is the major pathway in acute cellular rejection
and indirect pathway is important in chronic rejection. This
separation is by no means absolute
 HUMORAL REJECTION:
 IN SENSITIZED PATIENTS (HYPERACUTE):
Antibodies produced against alloantigens in the graft cause
humoral rejection  occurs within minutes after
transplantation. kidney rapidly becomes cyanotic
 Immunoglobulin /complement are deposited in the vessel wall
endothelial injury and fibrin-platelet thrombi.
 PREVIOUSLY NOT SENSITIZED (ACUTE): Antibodies cause
complement-dependent cytotoxicity and ADCC causing
rejection vasculitis and renal atrophy
Graft versus host disease -
Transplantation of
 Two problems that are unique to bone marrow
hematopoietic stem cells
transplantation are graft-versus-host (GVH) disease and
immunodeficiency.
 GVHD is a complex disease resulting from donor T-
cell recognition of a genetically disparate recipient that is
unable to reject donor cells after allogeneic HSCT.
 Billingham criteria:
 Immuno-incompetent host
 Infusion of competent donor T-cells
 HLA disparity between host and donor
Risk Factors of GVHD
 HLA disparity and unrelated mismatched donors
 Allo stem cell source (More with PBSC)
 Donor Age
 Immunosuppression
 Increased dose of TBI
 Intense conditioning regimen
 Multi transfused donor
 Multiparous female donor /Male recipient
Incidence :
 The incidence of GVHD with HLA-nonidentical marrow
donors who are related or in HLA-matched unrelated
donors  rates of 70-90%.
 Chronic GVHD is observed in 33% of HLA-identical
sibling transplantations, in 49% of HLA-identical related
transplantations, in 64% of matched unrelated donor
transplantations.
 The rate could be as high as 80% in 1-antigen HLA-
nonidentical unrelated transplantations
(Atkinson K. Chronic graft-versus-host disease. Bone
Marrow Transplant. Feb 1990;5(2):69-82)
Steps of GVHD
Stages of GVHD
 Hyperacute Day 0 – 7
 Acute Day 7 – 100
 Chronic Day 100 ≥
Acute GVHD
• Acute GVH disease occurs within days to weeks after
allogeneic bone marrow transplantation.
• Although any organ may be affected, the major clinical
manifestations result from involvement of the immune
system and epithelia of the skin, liver, and intestines.
• Although tissue injury may be severe, the affected
tissues are usually not heavily infiltrated by
lymphocytes. It is believed that in addition to direct
cytotoxicity by CD8+ T cells, considerable damage is
inflicted by cytokines released by the sensitized donor T
cells.
Acute GVHD
• Dermal: Maculopapular
Palms / Soles
Pruritic ±
Cheeks/ Ears/ Neck / Trunk
Necrosis / Bullae
• Hepatic : Hyperbilirubinemia
• Gastrointestinal : Diarrhea
Abdominal pain
Nausea ,Vomiting
Grading of acute GVHD
Chronic GVHD
 Multi system disorder
 Clinical & pathological findings resemble
autoimmune diseases
Pathological Classification
 Limited chronic  Extensive Chronic
GvHD
GvHD
 Generalised skin
 Localised skin involvement
 Hepatic dysfunction
 Hepatic
involvement  Aggressive hepatitis
 Ocular involvement
 May not always
 Involvement of the
require treatment salivary gland
& has a favorable  Fatal if not treated
outcome
Effects of Chronic GvHD
 Skin (95%) cases  Sinuses –
–increase in (increase risk of
collagen deposits gram +ve
-atrophy of the bacterial
dermis infections
-development of  Lungs-
sclerosis bronchiolitis
-hyperpigmentation obliterans
-patchy permanent  Small intestine
hair loss ( diarrhoea, Copyright Marvelle Brown
GVL EFFECT
 Minor Histocompatibility
 antigens:
Unless donor T cells are depleted from the stem-cell graft,
GVHD also frequently occurs after HLA-matched stem-cell
transplantation because of recognition of minor
histocompatibility antigens
 Polymorphic peptides that are displayed by HLA molecules of
recipient cells.
 Endogenous proteins in recipient cells that differ from those
of the donor, because of genetic polymorphisms, can
provide distinct HLA-binding peptides and serve as minor
histocompatibility antigens for donor T cells.
 The identification of haematopoietic-restricted minor
histocompatibility antigens such as HA-1, HA-2,HB-1 and
BCL2A1 is crucial for combining targeted immunotherapy with
PLS( Passenger lymphocyte
syndrome):
 Three different groups of ABO incompatibility minor,
major, and bidirectional ABO incompatibility.
 Major ABO-incompatible (e.g., A into O)  presence of
preformed antidonor A/B Ab directed against donor ABO
Ag expressed on transplanted cells.
 Recipients of minor ABO-incompatible transplantation
(e.g., O into A) express ABO Ag that are not expressed in
the donor and are at risk for graft-versus-host (GvH)
reactions such as delayed hemolysis of recipient red
blood cell (RBC) due to PLS.
 PLS:
 Bidirectional ABO incompatibility (e.g., A into B) represents
a combination of major and minor ABO incompatibility and
puts the recipient at risk for both host-versus-graft and
GvH .
 Immunocompetent donor memory B lymphocytes produce
antibodies in a secondary immune response against the
recipient’s red cells.
 The massive red cells destruction is thought to be
complement-mediated.
 Occurs in solid organ transplantation
 It usually occurs in 1–3 weeks posttransplant and resolves
within 3 months posttransplant, and is a self-limited
process.
Methods to Reduce the
Immunogenicity of Allografts
 ABO blood typing;
 Determination of HLA alleles expressed on donor and
recipient cells, called tissue typing.
 The detection of preformed antibodies in the recipient that
bind to antigens of an identified donor's leukocytes, called
crossmatching.
 Use of A2 organs with low titre antibodies for group B and O
recipients
 Rituximab, splenectomy, plasmapheresis with
intravenous immune globulin.
 Why is typing necessary ?
• MHC matching - profound influence on graft survival
before modern immunosuppressive drugs.
• Clinical experience has shown that of all the class I
and class II loci, matching only HLA-A, HLA-B, and
HLA-DR is important for predicting outcome.
• HLA-C is not as polymorphic as HLA-A or HLA-B, and
HLA-DR and HLA-DQ are in strong linkage
disequilibrium, so matching at the DR locus often also
matches at the DQ locus. DP typing is not in common
use, and its importance is unknown.
 GRAFT
SURVIVA
L WITH
HLA
MISMATC
H
Hematopoietic Progenitor Cell
 Candidate donors and recipients are typed for their
Transplants:
HLA-A, -B, -C, -DR, and -DQ alleles and, in some cases,
for their HLA-DP allees.
 Match at allele level
 Some transplant programs additionally attempt to
match for HLA-DQ or -DP alleles, or both.
 Molecular HLA typing is performed on samples from
both the donor and recipient for optimal assessment
of Class I and Class II region compatibility.
CONT…
• Because two codominantly expressed alleles are
inherited for each of these MHC genes, it is
possible to have zero to six MHC antigen
mismatches between the donor and recipient.
• Zero-antigen mismatches predict the best living
donor graft survival, and one-antigen-matched
grafts do slightly worse.
• The survival of grafts with two to six MHC
mismatches all are significantly worse than zero-or
one-antigen mismatches
 Crossmatching

 A pre-transplant test
 Ensures absence of donor reactive
antibodies
 Prevents hyperacute rejection
 Prevents accelerated antibody mediated
rejection.
Techniques:
1. Serology ( CDC test )
2. Solid phase assays ( Flow cytometry or
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