Hla typi ng pg seminar final 0604

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.


• 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.



• 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.



• 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.

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.

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.

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

HLA Molecules

MHC- I & MHC-IIMHC- I & MHC-II

• 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: 1, 2, & 3

1 and 2 interact to present processed Ag

• Process Ag is optimally a monomer

Microglobulin (12 kDa) associates non-covalently with 3Microglobulin and 3 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. 1-1 interact to present

processed Ag• Processed Ag is optimally 13-

18 aa

2 & 2 are part of immunoglobulin super family

Isotypes of MHC molecules

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

What are the genetic mechanisms?

• Polygenecity

• Polymorphism

• Co-dominance

• Linkage disequilibrium

What does the “syntenic” organization of a haplotype look like?

Remember:

polygenecitypolymorphismco-dominance

linkage disequilibrium

There are no rearrangements!

What is polygenecity?• Humans have DP, DQ, and DR “regions”

specifying and chains of MHC-II.

• Why are these called “regions”?

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 allelesHLA B locus ~110 allelesHLA 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.

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.

• 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.

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.

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.

• 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

• 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

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.

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.


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….• 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.


• 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.


• 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.

• 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.

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

[ 3H] thymidine

Incorporation of radioactivity into

Cell Nuclear DNA

Mixed lymphocyte reaction to determine identity of class II HLA antigens between a potential donor and recipientLymphocytes from the donor are irradiated or treated with mitomycin C to prevent cell division and then added to cellsfrom 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 intothe newly synthesized nuclear DNA. The amount of radioactive nucleotide uptake is roughly proportionate to theMHC class II differences between the donor and recipient lymphocytes.

Mixed Lymphocyte Reaction

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

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.

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.

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.

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.


• 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.


• PCR SSOP ( Sequence Specific Oligonucleotide Probes).

• Initially 32P- labelled allele- specific oligonucleotides were hybridised to an amplified conserved region of exon 2 of HLA-DQ gene, but soon after biotin was used as a label.

• PCR – SSOP also used for other loci DP, DQ & DR 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.

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.


• 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.


• 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.


• 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 ELISAMonoclonal antibodies to different MHC alleles have been generated.Using panels of these antibodies, HLA typing before transplantation is possible.

• RFLP: Restriction Fragment Length PolymorphismDigestion of genomic DNA with certain restriction enzymes followed by hybridization with radio-labeled MHC gene probes gives MHC isotype-specific digestion patterns.

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.

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

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)

Diseases associated with specific HLA antigensIf 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

That’s all for Now!Stay tuned for more exciting

topics on immunology!

Education

Hla typi ng pg seminar final 0604