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1.

HLA- disease associations and transplantation

1. Review of the function and polymorphisms of HLA molecules

Figure The distribution of the main genes that encode MHC class I and MHC class II molecules on the short arm of
chromosome 6; the figure also shows the large number of allelic forms of most of these genes.
Between the class I and class II genes lie a third class of proteins which include 21a and 21b-hydroxylase, genes for the
complement proteins C4A, C4B, C2 and factor B, and also genes for TNF and Heat shock protein 70. Diseases associated
with these genes have strong HLA associations because of their close linkage genetically. It is important to realise that it is
wrong to assume that all HLA disease linked autoimmune disease is due HLA. For example SLE is HLA linked, but this is
probably due to associations with null C4 alleles.
Within the class II cluster of genes between DP and DQ lie genes for DM (class II antigen processing) LMP and TAP (class I
processing transports).
b2microglobulin which is not encoded on chromosome 6 is not polymorphic i.e. has only one allele.
2. Antigen-processing and presentation.

Antigen-processing and presentation in association with MHC molecules:

Antigen-presentation of peptides derived from proteins taken up by a cell is mainly in association with class II MHC molecules
(right picture) and involves pinocytosis of antigen by antigen-presenting cells. The pinocytotic vesicle fuses with a specialised
antigen-processing endosomal compartment; this has class II MHC molecules inserted in its wall. Enzymes within the antigenprocessing compartment are activated and ingested proteins are broken down to peptides. These peptides are loaded into the

antigen-presenting grooves of the class II MHC molecules and the complexes are transported to the cell surface. Antigenpresentation of peptides from proteins produced within cells is mainly the property of class I MHC molecules (left picture). A
proportion of proteins produced on ribosomes of almost all cells is broken down to peptides within a cytoplasmic organelle
known as a proteasome. The resulting peptides are actively transported by TAP proteins through the wall of an endosomal
compartment specialised in loading MHC class I molecules. Loaded molecules are then transported to the cell surface.
3. The polymorphism of MHC class I and class II molecules
We have 6 gene clusters that encode for MHC class II molecules 3 loci on each chromosome 6; the molecules encoded on
these loci are respectively termed MHC class II DR, DP and DQ. There are also 3 MHC class I loci on each chromosome 6
and the molecules encoded at these loci are termed MHC class I A, B and C. Cross-over during meiosis within the MHC
region of chromosome 6 is relatively rare; consequently the alleles on one of our chromosome 6 are inherited en bloc from one
of our maternal chromosomes and those on our other chromosome 6 from one of our paternal chromosomes. The alleles
present on a chromosome 6 are termed the MHC haplotype of that chromosome. There is almost a 1 in 4 chance of two
siblings having the same MHC haplotype on both chromosomes; in this situation the siblings are said to be HLA identical.
Tissue transplantation between HLA identical siblings is associated with far less tendency to graft rejection than transplantation
between unrelated donors. It will be appreciated that the chance of finding two unrelated individuals who share all the same
HLA alleles is remote.
Self-T cells recognise the allelic structure of MHC molecules
Foreign MHC molecules are targets for graft rejection because they are recognised as foreign by the graft recipient. Each of
our ab T cells recognises a peptide structure held in association with one of our MHC class I or class II molecules. If the T
cell expresses CD4 it will recognise a peptide associated with a class II allele; if it expresses CD8 it will recognise a peptide
held in the peptide binding-groove of a class I allele. A T cell that recognises a particular peptide held in HLA-B allele is
unlikely to recognise the same peptide held in another HLA-B allele. There is, however, some level of cross-recognition of
some MHC alleles in combination with some peptides.
Some MHC molecules bind some peptides better than others
For many years it has been recognised that individuals vary in their responsiveness to certain protein antigens. Highly inbred
strains of mice have the same MHC haplotype on both chromosomes and accept grafts from each other. Analysis of immune
responses of such mice indicates that some strains of mice are poor responders to certain antigens while other strains produce
high responses to the same antigen. Analysis of the response to different antigens does not necessarily show the same pattern of
high and low responders. Cross-breeding experiments show that strains of mice responding well to a certain protein have an
MHC allotype that is particularly good at presenting a peptide from the antigen to T cells. It is possible to rank the capacity of
particular alleles to present particular peptides; but this only relates to a particular T cell receptor. Different T cell receptors
might recognise the same peptide better presented on another MHC molecule.
Particular MHC alleles are associated with better protection against certain infections, but certain alleles are
associated with a greater chance of developing autoimmunity.
This heading summarises the positive and negative features that might lead to a selective advantage during evolution for
particular MHC alleles. It seems logical that most alleles have been retained because they have certain advantages without
having too many disadvantages. At the same time the advantages of an allele might relate to a particular infection that is
prevalent in one part of the world, but almost unknown in another; for example a prevalence of a potentially lethal form of
malaria in West Africa is associated with a high frequency of HLA-B53, which is associated with relative protection from the
lethal effects of infection. However, TNF also maps in the HLA region and is known to play a role in cerebral malaria, so this
could be an example where the HLA linkage is by association with other genes. Where risks are multiple, populations are more
likely to survive if they are heterogeneous in their repertoire of MHC alleles.
Some diseases are distinctly more common in individuals with a particular MHC allele or MHC haplotype. There are numerous
examples of this. No situation has been identified where all individuals with a particular allele develop a disease, but some
diseases have been identified where most individuals developing a disease have a particular MHC allele. Diseases with a strong
association with certain MHC alleles include: insulin-dependent diabetes; Graves' disease; multiple sclerosis and ankylosing
spondylitis.
4. HLA-associated disease
The essential feature of autoimmune diseases is the stimulation of clonal expansion of T cells which have not been clonally

deleted in the thymus, by any of the ways listed above. The HLA molecules inherited by an individual govern which peptide
sequences within a microbial antigen that individual will present most efficiently, and will have the same, major influence upon
which (conserved) self-sequences that individual will be able to present. This will in turn determine how easily an autoimmune
disease may develop.
Most autoimmune disorders are HLA-associated, that is they occur much more frequently in individuals who have certain HLA
alleles. The strongest HLA-associations are found in Insulin Dependent Diabetes Mellitus (IDDM) and Rheumatoid Arthritis.
In type 1 Insulin-Dependent Diabetes Mellitus (IDDM), b -cells in the pancreatic islets of Langerhans are the targets of
autoimmune attack. A number of autoantigens have been identified as targets for both antibody and T-cell responses in both
humans and a commonly used mouse model (NOD, or non-obese diabetic mice who spontaneously develop IDDM). Three
antigens (GAD [glutamic acid decarboxylase], insulin, and Hsp [heat shock protein] 60) are leading candidates. Autoantibodies
to pancreatic islet cells are present in the serum of >90% of patients with IDDM at diagnosis and disappear over the following
year or so, but the immunopathology is thought to be due mainly to cell-mediated immune responses.
In man, IDDM is most strongly associated with expression of HLA-DQb MHC class II alleles. This b-chain carries a neutral
amino acid (serine, alanine or valine) at position 57. By contrast, class II alleles with an aspartic acid at residue 57 are strongly
associated with resistance to IDDM. Aspartate-57 forms a salt bridge which may stabilise bound peptide. Here you can see a
computer-generated model of the IDDM-associated DQ molecule. See if you can indentify position 57. However,
polymorphisms elsewhere in the DQb chain can sometimes produce critical modifications. This suggests that subtle changes in
the presentation of the peptide/MHC complex to CD4+ T cells can have a dramatic effect on T cell autoreactivity. Various
studies indicate that in individuals with the susceptible MHC class II allele, the MHC/peptide complex stimulates a
predominantly Th1 inflammatory T-cell response, which promotes b -islet cell destruction. On the other hand IDDM-resistant
class II alleles lead to the development of aTh2 response to islet cell autoantigens. However, it should be noted that multiple
other genes appear to participate in predisposition to diabetes. In NOD mice, at least 14 non-MHC loci (scattered over many
chromosomes) have been implicated in the susceptibility to diabetes.
Most associations are more strongly linked to MHC class II (i.e. HLA-D loci) than MHC class I (HLA-A,B,C), the latter
arising from linkage disequilibrium between class II + I genes. Ankylosing Spondylitis is a disorder which is strongly associated
with HLA-B27, but the lifetime-risk of an HLA-B27 positive individual developing it is only about 2%. This illustrates that in
diseases with HLA associations, it is not the disease itself, but the susceptibility to it that is inherited. This may be due to the
existence of other genes in linkage disequilibrium with those now known to be associated with disease, or alternatively chance
interaction with an environmental agent, or both. The existence of HLA associations provides evidence, but not proof, that
immune mechanisms are involved in the pathogenesis. The Table below gives some examples of HLA-associated diseases, the
relative risk being the chance of an individual with that particular allele developing the disease compared with an individual
lacking that allele.
Disease

HLA allele

Relative risk

Rheumatoid arthritis

DR4

IDDM

DR3

DR4

6-7

DR3/DR4

20

DR3, DQw8/DQw2

30

DR2

0.25

Pemphigus

DR4

24

Chronic Active Hepatitis

DR3

14

Coeliac Disease

DR3

12

In some cases it is likely that the main factor determining which sequence will be presented may not be which the MHC class I
molecule binds best, but which peptides are made available by the Antigen-processing machinery in order to be made available
for such binding. It is thus possible that polymorphism in the LMP and TAP genes may result in different sets of peptides

derived from the same Ag being presented to T cells in different individuals. In addition to MHC polymorphism, this
polymorphism may thus contribute to some of the associations between MHC genotype and disease.
5. Transplantation
Xenogeneic - tissue transplanted from a different species
Allogeneic - tissue transplanted from a member of the same species
Autologous - tissue transplanted from the same individual
Syngeneic - tissue transplanted from a genotypically identical twin
6. Role of HLA in transplantation
The major loci important in organ transplantation are HLA A, B and DR. When both alleles for these three antigens are
matched, it is termed a 6-antigen match.
With a few exceptions (such as the gonads) all nucleated cells express all HLA antigens, although the quantity on the cell
surface at any moment is highly controlled. In transplantation this appears to have 2 major consequences. Firstly, allogeneic
HLA molecules are recognised by T cells resulting in a powerful cytotoxic (Th1) inflammatory response (acute rejection of a
graft by host T-cells, or acute graft-versus-host disease in the case of engrafted T cells attacking an immunosuppressed host).
Secondly, in the case of bone marrow transplantation, antigen presenting cells and T cells are of dissimilar MHC type, so that
cellular co-operation is very inefficient resulting in prolonged immunosuppression and downregulation of normal immune
responses.
7. HLA typing
In the past HLA typing was done using microcytotoxicity testing first sera from multiparous women with anti-HLA antibodies,
and then monoclonal antibodies. Now tissue typing is done using the polymerase chain reaction (PCR). Sometimes functional
reactivity is tested by setting up a mixed lymphocyte reaction where donor and recipient cells are setup in stimulation cultures.
Absence of reaction of recipient lymphocytes against donor cells was useful in predicting graft acceptance.
T cells which recognise donor HLA are termed alloreactive. Direct recognition of donor graft antigen-presenting cells by
alloreactive T cells is the most important mechanism of donor graft rejection.
8. Organs that are transplanted
Organ

Matching and immunosuppression

Bone marrow

requires complete or near complete HLA identify at major loci. Immunosuppression

generally required for only (6 12 months

Kidney, Heart, Lung, Pancreas

Better results with HLA matched organs (the closer the better) but 6 antigen
mismatches may perform perfectly well as long as the
Lung recipient was not 'sensitised' beforehand to the incoming HLA type

Small bowel

Very immunogenic

Liver

Generally only cross matched for blood group antigens. Less immunogenic than
other grafts but requires lifelong immunosuppression.

9. Mechanisms of rejection
1. Acute Rejection
Several factors determine whether a graft will be rejected - e.g. in HLA-mismatched kidney transplants prior

sensitisation to the kidney's HLA type (perhaps from previous pregnancy or blood transfusion), the state of the innate
immunity of the recipient (proinflammatory or anti-inflammatory, which can be influenced by infection) and any infection
of the organ or host (particularly with the herpes virus CMV). CMV can alter both the quantity and even the quality
(structure) of HLA molecules. Hyperacute rejection which occurs in minutes or hours is usually do to antibodies. Donor
graft endothelium express ABO and HLA. Recipient antibodies can lead complement fixing antibodies precipitiating
acute vasculitis and coagulation of blood vessels.
2. Acute Graft Versus Host Disease
After bone marrow transplantation, the incoming immunocompetent immune system may attack sites of high HLA DR
expression such as skin, gut and liver. In the skin a measles-like hypersensitivity reaction occurs (grades I-IV depending on severity) and similarly in the gut (manifested by severe bloody diarrhoea) and the liver (jaundice). It
classically occurs as the bone marrow engrafts (14-21 days) but can occur at any time within the first few months. It is
caused by the proliferation and activation of those mature T cells in the bone marrow graft which are specifically reactive
towards the recipients HLA type.
3. Chronic Rejection/Chronic Graft Versus Host Disease
This may follow acute rejection or occur de-novo. The de-novo process is due to the presence of autoreactive T cells (that
would normally have died earlier by apoptosis). In solid organ transplantation this is probably an effect of the
immunosuppressive agent cyclosporin preventing surveillance mechanisms killing autoreactive cells. In bone marrow
transplantation where donor cells are conditioned by the host, there is less stringent T-cell surveillance by the 'foreign' thymus
(young children) or skin and other sites used in the adult for processing T-cells. Immunosuppressive drugs have dramatically
improved the survival of grafts after 1 year, but have had little impact on 5 year survival which is attributable to chronic
rejection. The immune mechanisms involved in chronic rejection are poorly understood.
10. Blood Transfusion
A blood transfusion is also (usually) an allograft. Normally it is quickly rejected without viable discomfort on the part of the
recipient. Certain groups of immunosuppressed patients are at risk of getting lymphoid engraftment from transfused blood and
this can cause fatal graft versus host disease. These are: During foetal and early neonatal life, very heavily immunosuppressed
patients on some chemotherapies, for 6 months after a bone marrow transplant; patients with certain malignancies (e.g.
Hodgkin's disease), patients with certain primary immunodeficiency diseases.
The antigens in the foreign blood can also induce an alloreactive response in the host, preventing further transfusions. This can
be of a narrow specificity (e.g. a specific minor blood group antigen or platelet specific antigen) or a broad type (pan-HLA)
which can prevent transfusion of blood products (especially platelets).
11. Immunosuppressive Treatments
Drug

Application

Steroids

widely employed for solid organ transplants

Azathioprine

widely employed for solid organ transplants

Cyclosporin

an inhibitor of calmodulin that prevents expression of the T cell growth factor receptor
IL2R

Tacrolimus

This acts similarly to cyclosporin, formerly being known as FK506

Thalidomide

Antagonises the effects of TNF, useful in graft versus host disease

Monoclonal antibodies

Anti-T cell antibodies (e.g. OKT3, CAMPATH-1)

12. Immunology of Pregnancy

The foetus is effectively a mismatched allograft which is not usually rejected. There is evidence of tolerance induction in the
maternal peripheral T cell pool (there is downregulation of the T cell receptor on alloreactive T cells) although the mechanism
by which this occurs is unknown. Pregnant woman make anti-class I and class II antibodies so foetal antigens are
immunogenic. There is a suggestion that the pattern of T cell responses is shifted towards a Th2 phenotype, and the
susceptibility of pregnant woman to intracellular infections like Listeriosis may be a consequence of this. Many autoimmune
disorders like SLE also improve in pregnancy and relapse after birth, suggesting that the T cell immunosuppression is not
selective for the foetus.

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