HLA typing

2. HLA = Human Leucocyte Antigen system
History
Early work of Gorer (1930) on antigens responsible
for allograft rejection in mice led to the discovery of
the ‘MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)’.
Development of congenic & recombinant strains of
mice by Snell enabled the detailed analysis of various
loci of this complex.

3. HLA = Human Leucocyte Antigen system
• History contd.-----
• Dausset pioneered studies on HUMAN LEUKOCYTE
ANTIGENS MAJOR HISTOCOMPATIBILITY
ANTIGENS in human being.
• Benacerraf & colleagues established genetic basis of
immune response.
• For their work on MHC & genetic control of immune
response, Snell, Dausset & Benacerraf were awarded
Nobel Prize for medicine in 1980.

4. HLA = Human Leucocyte Antigen system
• History contd.-----
• Early studies on MHC were carried out on mice.
• All species of animals including HUMAN BEING were
found to possess a similar complex of genes on a
segment of one chromosome pair.

5. HLA = Human Leucocyte Antigen system
• History contd.-----
• These genes coding for………….
1.Class I proteins ---- determining histocompatibility
& acceptance or rejection of allograft.
2. Class II proteins ---- regulating the immune
response.
3. Class III proteins ---- including some
components of the complement system.

6. Histocompatibility complex
• Name histocompatibility complex because
of its discovery based on transplantation
experiments.
• Human MHC antigens are found on surface of
leucocytes hence synonymous with
Human Leucocyte Antigens (HLA) & MHC complex of
genes with the HLA complex.

7. HLA complex
• HLA complex of genes located on short arm of chromosome 6.
• It is comprised of three separate clusters of genes:
1. HLA class I A,B & C loci.
2. Class II or D region DR DQ & DP loci.
3. Class III or the complement region genes for
complement components C2 & C4 of the classical
pathway, properdin factor B of alternative pathway, heat
shock proteins, tumor necrosis factors C.

8. HLA complex
• HLA loci -- multiallelic
• 24 alleles at HLA locus A & 50 alleles at HLA locus B

9. HLA Molecules

10. MHC- I & MHC-IIMHC- I & MHC-II

11. • MHC-I
• Heavy chain (alpha) and
• “microglobulin” (beta
two)
• Heavy is 45 kilodaltons, has
three domains + a
transmembrane component
(40 aa) + a cytoplasmic tail
(30 aa)
• The three alpha domains are
called: α1, α2, & α3
∀ α1 and α2 interact to
present processed Ag
• Process Ag is optimally a
monomer
Microglobulin (12 kDa) associates non-covalently with α3
Microglobulin and α3 are part of immunoglobulin
superfamily
Microglobulin is the only member of the superfamily that
does not have a component linking it to a membrane
• MHC-II
• An alpha and beta chain,
33 kDA and 28 kDa,
respecitvely.
• Chains are non-
covalently associated.
• Each chain has two
domains.
∀ α1-β1 interact to present
processed Ag
• Processed Ag is
optimally 13-18 aa
∀ α2 & β2 are part of
immunoglobulin super
family

12. Isotypes of MHC molecules

13. What are the genetic mechanisms?
Nota bene: whatever are the genetic
mechanisms, they must account for the
huge diversity of “haplotypes”
“Haplotype”: “the set of alleles of linked
genes present on one parental
chromosome…” cf. synteny
“Synteny”: the association of genes in a
distinct region of a chromosome

14. What are the genetic mechanisms?
• Polygenecity
• Polymorphism
• Co-dominance
• Linkage disequilibrium

15. What does the “syntenic” organization
of a haplotype look like?
Remember:
polygenecity
polymorphism
co-dominance
linkage
disequilibrium
There are no
rearrangements!

16. What is polygenecity?
• Humans have DP, DQ, and DR “regions”
specifying α and β chains of MHC-II.
• Why are these called “regions”?

17. There are no rearrangements!
Thus, MHC proteins (from the “haplotype”)
constitute a life-long cell surface character
for any vertebrate.
This circumstance is very different from Ig’s which are constantly
being generated in response to new foreign proteins and
carbohydrates in the environment.
The loci which specify MHC’s are polymorphic.
Many alleles may exist at a locus:
HLA A locus has ~60 alleles
HLA B locus ~110 alleles
HLA C locus ~40 alleles
The high level of allelism creates diversity within a species
(thus restricting allografting) but does not produce diversity
within an individual.

18. MHC Antigen NomenclatureMHC Antigen Nomenclature
• Nomenclature
• 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.

19. • 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 ether 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 beta-
chain genes. Hence, many DR specificities can be found in
any one individual.

20. Crossover
• Haplotypes, normally, are inherited intact and hence
antigens encoded by different loci are inherited together
(e.g., A2; B27; Cw2; DPw6; DQw9; DRw2). However, on
occasions, there is crossing over between two parental
chromosomes, thereby resulting in new recombinant
haplotypes. Thus, any one specificity encoded by one
locus may combine with specificities from other loci.
This results in vast heterogeneity in the MHC make-up
in a given population.
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
lymphocytes, a proportion of macrophages and
monocytes, skin associated (Langerhans) cells,
dendritic cells and occasionally on other cells.

21. HLA - Typing
• SEROLOGY used to be the ‘gold’ standard. Now
being superseded by molecular techniques as they
become more robust and time efficient.
• CELLULAR rarely used now. Originally used for
Class II typing.
• MOLECULAR fast becoming the method of choice.
Many laboratories test of choice.

22. • Earlier antisera were obtained from multiparous
women ,as tending to have antibodies to HLA
antigens of their husbands owing to sensitization
during pregnancy.
• Now monoclonal antibodies to HLA antigens have
been developed.
SEROLOGY

23. • HLA Typing is done serologically by
MICROCYTOTOXICITY ( microlymphocytotoxicity)
which tests for complement mediated lysis of peripheral
blood lymphocytes with a standard set of typing sera.
• Viable peripheral blood lymphocytes are obtained by
discontinuous density gradient centrifugation using
Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density
of 1.077 at 19º - 22ºC.
• Microlymphocytotoxic test: 3 stages
SEROLOGY

24. Microlymphocytotoxic 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.

25. Microlymphocytotoxic test Contd….
• 3.Results are visualised by adding dye usually a
fluorochrome eg Ethidium Bromide although both
Trypan Blue and Eosin Y 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. A cocktail
of Ethidium Bromide and Acridine Orange, quenched
using Bovine Haemoglobin to allow simultaneous
visualisation of both living and dead cells.

26. Microlymphocytotoxic test Contd….

27.  White blood cells from potential donors and the
recipient are added to separate wells of a microtiter plate. The
example depicts the reaction of donor and recipient cells with a
single antibody directed against an HLA-A antigen. The reaction
sequence shows that if the antigen is present on the
lymphocytes, addition of complement will cause them to become
porous and unable to exclude the added dye.
Microlymphocytotoxic test Contd….

28. Microlymphocytotoxic test Contd….
• Because cells express
numerous HLA
antigens, they are
tested separately with
a battery of antibodies
specific for various
HLA-A antigens.
• Here, donor 1 shares
HLA-A antigens
recognized by antisera
in wells 1 and 7 with
the recipient, whereas
donor 2 has none of
HLA-A antigens in
common with the
recipient.

29. Microlymphocytotoxic test Contd….
• Test is left for 10 minutes and then read using an
inverted fluorescent 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.

30. Microlymphocytotoxic test Contd….
• In the past neuraminidase
treated sheep red blood cell
rosetting and nylon wool have
been used.
• Immunomagnetic bead
separation is the current method
of choice.
• It utilises polystyrene
microspheres with a
magnetisable core coated in
monoclonal antibody for a HLA
Class II b chain monomorphic
epitope. Positive selection.

32. • 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 primarily due to a disparity 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 studying the
degree of histocompatibility. In this test, the test lymphocytes
(responder cells)are mixed with irradiated or mitomycin C treated
leukocytes from the recipient, containing B-lymphocytes and
monocytes (stimulator cells). The cells are cultured for 4 6 days.
The responder T cells will recognize the foreign class II antigens
found on the donor and undergo transformation (DNA synthesis
and enlargement: blastogenesis) and proliferation (mitogenesis).
The T cells that respond to foreign class II antigens are typically
CD4+ TH-1 type cells. These changes are recorded by the
addition of radioactive (tritiated, 3H) thymidine into the culture
and monitoring its incorporation into DNA.

33. Mixed Lymphocyte Reaction
Allele A
Recipient cell sharing
Class II MHC of donors
No reaction
Irradiation
Allele A
Donor cells
Allele B
Recipient cells lacking
Class II MHC of donor
Activation & proliferation
of recipient cells
[ 3
H] thymidine
Incorporation of radioactivity into
Cell Nuclear DNA

34. Mixed lymphocyte reaction to determine identity of class II
HLA antigens between a potential donor and recipient
Lymphocytes from the donor are irradiated or treated with
mitomycin C to prevent cell division and then added to cells
from the recipient. If the class II antigens on the two cell
populations are different, the recipient cells will divide rapidly
and take up large quantities of radioactive nucleotides into
the newly synthesized nuclear DNA. The amount of
radioactive nucleotide uptake is roughly proportionate to the
MHC class II differences between the donor and recipient
lymphocytes.
Mixed Lymphocyte Reaction

35. Pros and cons
Pros:
• Easily performed does
not require expensive
equipment.
• Takes around three
hours to perform.
• Low level resolution,
with good antisera
reliable results.
Cons:
• Requires large volumes
of blood
• Requires viable
lymphocytes
• Difficult to find good
antisera for rarer
antigens in different
populations

36. Cellular typing
• Not / Rarely used by laboratories these days.
• Requires panels of homozygous typing cells.
• Cell culture method therefore takes a long time.
• Labour intensive involves use of radioisotopes.

37. Molecular typing
• All methods rely on DNA extraction from the nucleated
cells following cell lysis and protein digestion.
• The application of molecular techniques to HLA typing
began around 1987 when the Southern Blot technique
was used to identify restriction fragment length
polymorphisms (RFLP’s) associated with known
serological DR/DQ and cellular Dw defined
specificities.
• Around 1992 polymerase chain reaction (PCR)
methods were developed.
• Most methods currently used have a PCR element
within the technique.

38. Molecular typing Contd…….
• PCR
• Three steps per cycle– denaturation, annealing and
extension. Amplification is exponential yielding 2
power n where n = number of cycles.
• The introduction of the programmable Thermal
Cycler revolutionised the use of PCR within the
routine laboratory.

39. Molecular typing Contd…….
• PCT SSP (Sequence Specific Priming)
• The principle is that a completely matched primer will be more
efficiently used in PCR reaction than a primer with one or several
mismatches. Specificity determined by use of sequence specific
primers in which a 3’ single base mismatch inhibits the priming of non
specific reactions. Because Taq polymerase lacks 3’ to 5’ exonuclease
activity, even if primer pairs do anneal non specifically , they will not
amplify efficiently. Thus only desired allele or alleles will be amplified
& amplified product can then be detected by agarose gel
electrophoresis.
• Can be used for HLA Class I and II typing using a panel of primer pairs
either for low to medium resolution whereby primers amplify groups
of alleles of high resolution while primer pairs amplify specific alleles.
Each PCR reaction takes place in a separate tube therefore the
number of tubes depends on the level of resolution. Each tube also
contains a pair of primers for part of the human growth hormone gene
as an internal control. These are at a much lower concentration thus
do not compete with specific primers.

40. Molecular typing Contd…….
• 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 photography.

41. Molecular typing Contd…….
• PCR SSOP ( Sequence Specific Oligonucleotide Probes).
• Initially 32
P- labelled allele- specific oligonucleotides were
hybridised to an amplified conserved region of exon 2 of HLA-
DQα gene, but soon after biotin was used as a label.
• PCR – SSOP also used for other loci DPβ, DQ β & DR β by using
P, biotin or horseradish peroxidase labelled probes.
• ‘Dot blot’ in house method usually whereby one labels ones own
probes with Digoxigenin.
• ‘Reverse dot blot’ normally commercial where specific
oligonucleotide probes are attached to a nylon membrane.

42. Molecular typing Contd…….
• Amplification: DNA of interest is amplified by a
single pair of biotinylated primers which flank the
whole of exon eg exon 2 of the HLA DRB1 gene. PCR
amplifies all the alleles in the exon.
• Hybridisation: PCR product is denatured and then
added to a ‘well’ containing the nylon membrane
with the bound probes and incubated with
hybridisation buffer . PCR product hybridises to
probes with complementary sequences.
• Excess product is washed away during a series of
wash steps.
• Temperature is VERY important during these stages.

43. Molecular typing Contd…….
• Visualisation of results is achieved by incubating
with a conjugate and enzyme often streptavidin and
horse radish peroxidase which binds to the biotin of
the PCR product and then adding a substrate. Band
with PCR product turn blue.
• Strips will have internal control bands to show the
test has worked.
• Interpretation is usually achieved by entering the
band pattern into a computer programme.
• This is an excellent method for low resolution batch
testing.
• Can be semi automated.

44. Molecular typing Contd…….
• Sequence Based Typing (SBT)
• DNA sequencing is the determination of the sequence of
a gene and thus is the highest resolution possible.
Sequence based typing involves PCR amplification of
the gene of interest eg HLA DRB1 followed by
determination of the base sequence. The sequence is
then compared with a database of DRB1 gene
sequences to find comparable sequences and assign
alleles. This method also allows for detection on new
alleles.

45. Molecular typing Contd…….
• Other molecular methods:
• Reference Strand Conformational Analysis (RSCA) Offers
sequence level typing without the need to sequence. Assigns
HLA type on the basis of accurate measurement of
conformation i.e. shape dependent on DNA mobility in
Polyacrylamide gel electrophoresis (PAGE). Complex and
difficult technique not taken up by labs for routine use.
• Luminex technology – SSOP based. Just beginning to be
introduced into laboratories for routine use on non urgent
samples.
• Flow cytometry or ELISA
Monoclonal antibodies to different MHC alleles have been
generated.
Using panels of these antibodies, HLA typing before
transplantation is possible.
• RFLP: Restriction Fragment Length Polymorphism
Digestion of genomic DNA with certain restriction enzymes
followed by hybridization with radio-labeled MHC gene probes
gives MHC isotype-specific digestion patterns.

46. Pros and cons
• Pros:
• Does not require viable
cells.
• Samples do not have to
arrive in the lab the day
they are taken.
• PCR SSOP good for
batch testing.
• Can be semi automated.
• Cons:
• Requires good quality
DNA.
• Require a degree of
redundancy within the
primers used.
• Sequence of alleles must
be known.

47. MHC-Linked Diseases
• Defects in MHC gene expression lead to
immunodeficiencies (MHC molecules are required
for both T cell development and activation)
• Some MHC alleles are associated with susceptibility
or resistance to autoimmune diseases

48. MHC-Linked Immunodeficiencies
Bare Lymphocyte Syndromes lead to loss of MHC
molecule expression:
• Defects in TAP genes prevent MHC Class I protein
surface expression (even though MHC proteins are
normal), so no CD8+
T cells - surprisingly mild
immunodeficiency (respiratory and skin infections)
• Defects in TF’s controlling Class II gene expression
(CIITA, RFXANK, RFX5, RFXAP) block CD4+ T cell
development - result in SCID (severe combined
immunodeficiency)

49. Diseases associated with specific HLA antigens
If you have the MHC allotypes (left), you have a higher chance of
getting the following diseases (right):
HLA-DR3/DR2 Systemic Lupus Erythematosus
(b) HLA-DR4  Rheumatoid Arthritis
(c) HLA-B7 and DR2  Multiple Sclerosis
(d) HLA-B8, DR3/DR4  Type 1 diabetes
(e) HLA-B27  Ankylosing Spondylitis

52. That’s all for Now!
Stay tuned for more exciting
topics on immunology!