21 MHC Proteins and transplantation

Dr. M. N. Gupta

1. Objectives

• To understand the role of MHC proteins in graft rejection

• To learn about the various mechanism by which polymorphism of MHC molecules arise

• To understand MHC restriction of T-cells

• To learn the various mechanisms of graft rejection and various kinds of grafts.

2. Concept Map

3. Description

A major histocompatibility complex (MHC) is present on all nucleated cells of bodies of mammals. This gene complex expresses antigens (cell surface proteins) which are called MHC antigens. The discovery and the understanding of the role(s) of MHC has a long and instructive history. Its evolution is deeply rooted in the concepts of population genetics and diversity of a specie. MHC play roles in grafting of tissues and organs. But that role is incidental, its role is central to the functioning of the immune response.

Transplants

Jacques Louis Reverdin was a surgeon in Paris who around 1860s introduced skin grafting. The early plastic surgeons mistakenly believed that skins of even frogs and rabbits could be transplanted on humans.

In 1911, Erich Lexer tried to convince others that skin grafts between even different humans are not possible. Ultimately, with time people grudgingly accepted this truth

This is inspite of the fact that the skin transplanted from another person becomes the site of inflammation and drops away after a week or so!

The transplant of skin from one part to another of the same individual is accepted. Skin can also be exchanged between identical twins. In that era (decades before an enzyme from a thermophile called Taq polymerase made PCR technology possible!) skin grafts became a tool for deciding paternity. With the exception of hamster, every vertebrate species have this characteristic behaviour Given earlier understanding of blood transfusion reactions (thanks to the work of Karl Landsteiner), the rejection of the graft was naturally believed to be an immune reaction.

It was surprising to find that a high concentration of antibodies in the blood against the graft tissue antigens delayed the graft rejection.

G.H.Algire, J.M. Weaver and R.T. Prehn at the National Research Institute in Bethesda with mouse as the animal model provided the first experimental evidence that graft rejection involved cell mediated immune response.

Cornea has no blood vessels and hence cells cannot reach the cornea. Cornea grafts are exempted from these mechanisms of graft rejection. Brain has no lymphatic drainage system, so antigens released from any grafted tissue do not travel out of the brain and evoke immune response. Thus, cornea and brain are two organs in which graft rejection immunologically does not take place.

The second transplant between the same pair of individuals is rejected faster. That also indicates operation of immunological memory.

In 1975, F.M.Burnet remarked that while successful kidney transplantation is a “spectacular clinical achievement of applied immunology” yet it was possible by empirical use of immunosuppressive drugs rather than based upon the knowledge of graft rejection and tolerance. If a graft is accepted, it shows immunological tolerance.

If the spleen cells from a donor were injected in a neonatal mouse (or its foetus), the mouse became tolerant to the subsequent grafts from the donor strains but not from the mouse from other strains.

We will return to tissue and organ transplantation immunology later to take this further. Let us familiarize ourselves with the basic concepts and knowledge of MHC genes. We will see that large number of gene loci are involved in graft rejection but MHC are the most important ones.

Human Leukocyte Antigen (HLA)

The MHC complex locus in humans is called Human Leukocyte Antigen (HLA). The corresponding loci in mice and rats are H-2 complex and RT-1 respectively. In mice, these are called H-2 genes.

The name HLA is derived from the fact that earlier it was believed that MHC antigens are present on the leukocyte only. Their genetic diversity was carried out by serological typing. In recent years, variations in MHC are investigated by genotyping. In current (unfortunate) usage MHC refers to both the genes or the cell surface proteins (antigens) and one has to rely upon the context to understand which one is being referred to.

MHC genes are most polymorphic genes known so far, that is, within a population, large variants of each gene exist. Secondly, large number of MHC Class I and MHC Class II genes exist.

Major Histocompatibility Complex (MHC)

Apart from the receptors present on the B-cells (immunoglobulins) and TCR, MHC proteins are the most diverse class of proteins. It was found out that during the graft rejection, the recipient‟s immune system responds to the MHC proteins of the donor. Unrelated individuals express different set of MHC proteins.

While the B-cell receptor and TCR differ from cell to cell (unless both cells are from the same clone!); MHC proteins differ between individuals. Hence, MHC proteins are sometimes called markers of identity.

Decades after the role of MHC proteins in graft rejection was discovered, people wondered what is their role in vivo as transfer of tissues between individuals is extremely rare in nature.

Their role in directing the T-cells to the infected cells came to be discovered while many groups were trying to characterize TCR. We will revert to that when we discuss the discovery of the nature of the TCR.

It turned out that the MHC molecules bind to the antigen derived peptides from the pathogen on the cell surface. As we have mentioned earlier, this role of MHC proteins is critical t and hence pathogens in order to survive try to down regulate the expression of the MHC protein.

Let us look at the complete picture now. The organization of the principal MHC genes is shown for both humans (where the MHC is called HLA and is on chromo-some 6) and mice (in which the MHC is called H-2 and is on chromosome 17). The organization of the MHC genes is similar in both species. There are separate clusters of MHC class I genes (shown in red) and MHC class II genes (shown in yellow). In humans, it may span about 4 x 106 base pairs.

All nucleated cells express MHC Class I molecules. The level of their constitutive expression varies between cell types. Cells of the immune system have high expression levels: liver cells (hepatocytes), brain cells and kidney cells have low expression levels.

RBC in mammals have practically no MHC I proteins. Thus Tc cells cannot destroy any infection in RBC. However, RBC cannot support viral replication, so that does not hurt. Malarial parasite Plasmodium, thereby, however finds RBC an immunologically previledged site.

MHC class II proteins are expressed on a subset hematopoietic cells and thymic stromal cells. Thus, these are formed on B-lymphocytes, dendritic cells and macrophages.

The expression of both class I and class II proteins is regulated by cytokines (especialy interferons), IFN-γ increases the expression of both classes. It also induces the expression of MHC class II on some cell types (which do not normally express these proteins) during inflammation.

Unlike humans in which activated T-cells express class II proteins, in mice T-cells do not express these proteins. In brain, except in microglia, cells do not express class II proteins.

MHC Class I Proteins

These molecules are integral membrane proteins. Each has one polypeptide chain that spans the plasma membrane. The extracellular portion has 3 domains (α1, α2, α3). The α-chain is a 44-kDa in size and is a glycoprotein. Each of the 3 extracellular or external domains contains about 90 amino acids.

The α3 domain is linked to a domain of about 40 amino acid long transmembrane region which is followed by the cytoplasmic region of 30 amino acid α1 domain is noncovalently linked to a single polypeptide β2-microglobulin of about 11.5 kDa. β2-microglobulin gene is on a separate chromosome number 15 on humans and 2 in mice. β2-microglobulin is critical for transport of the MHC Class I proteins to the cell surface and helps it to retain its native conformation. It is very similar to α3 domain in its amino acid composition.

In fact α3 domain and β2-microglobulin have amino acid composition (and hence fold structures) similar to the C-domain in Ig.

MHC Class I Proteins: Peptide Binding Cleft

α1 and α2 domains fold to form two segmented α-helices resting on eight stranded antiparallel beta sheet. α1 and α2 domains form a cleft which is the site which MHC Class I uses to bind the antigenic peptide fragment. Hence, this is the site wherein polymorphism is seen!

Interferons are produced in the early phase of the viral infections as part of the innate immune response. Once again, we can see the linkage between innate immune response and adaptive immune response. Interferons IFN-α, β or γ can increase the transcription of MHC Class I α-chain and β-2 microglobulin.

The human genome project has made the sequence of the HLA complex and reading frames available.

MHC Class II Proteins

MHC Class II molecules are expressed on B-cells, macrophages, monocytes, APCs and some T-cells. These resemble Class I molecules in many respects. They consist of two transmembrane glycoprotein chains α and β of 34 kDa and 29 kDa respectively. Each chain consists of two domains: α1, α2 and β1, β2. The two chians associate noncovalently to form 4 domain appearance which is very similar to MHC Class I + β-2 microglobulin complex.

Here the bottom of the peptide binding cleft consists of eight anti-parallel beta strands and its walls have anti-parallel alpha helices. In MHC Class II, the domains part of the cleft is formed by different chains The clefts in both MHC Class I and Class II are thus very similar. That is understandable as both do the same function: bind antigenic peptide fragments.

Sequences of the cloned MHC Class I and Class II genes show that each domain is encoded by a separate axon In both mice and humans, Class I and II genes have 5‟ leader sequence which allows these to enter the endoplasmic reticulum and reach the cell surfaces.

In addition to the highly polymorphic Class I and Class II MHC genes, there are MHC Class IB genes. These encode β-2 microglobulin associated cell surface molecules. These cell surface molecules vary in amount from cell to cell and are distributed differently on each tissue.

One such molecule of the MHC Class IB kind is H2-M3 and it can present peptides which are formylated at the N-end. This is of significance as bacterial protein synthesis starts with N-formylmethionine. So, these are able to present antigenic fragments (from bacterial pathogens) which arise from the N-terminals.

HFe gene, also part of the same gene complex, is expressed on intestinal tract cells. Humans defective in Hfe gene have an iron storage disease called hemochromatosis. In this disease liver and other organs retain high amount of iron. Mice lacking β-2 microglobulin have similar problems.

Other genes which are part of the MHC gene complex includes those which code for some complement components, some cytokines like TNF-α and TNF-β. These are sometimes called MHC Class III genes. These also include those coding for HSP7 and adrenal steroid 21-hydroxylase.

MIC gene family belongs to Class IB genes. Their prtoducts MIC A and MIC B are recognized by NK cell rceptors. This allows killing of MIC expressing cells as a part of the innate immunity even when interferons are not yet produced. These class IB genes are expressed in fibroblasts and epithelial cells especially intestinal epithelial cells.

The need for highly polymorphic nature of the MHC Class I and Class II genes is obvious. At the level of the protein products, several mechanisms exist which increases their diversity. Both polygeny and polymorphism contribute to the diversity within an individual and also in the population.

There are >200 alleles (the variant genes occupying a gene locus within a specie) of some class I and class II genes. Each allele has a high frequency in the population. Hence, there is a strong probability that both homologous chromosomes on an individual will have different allele.

Human MHC genes are highly polymorphic

Expression of MHC alleles is codominant

This heterozygous nature at MHC loci is the source of diversity (of protein products) as the expression of MHC alleles is codominant.

Both gene products are available to present antigen fragments. This makes the number of different MHC molecules on an individual 2 times. This is inaddition to the polygeny.

Polymorphism and Polygeny

Polygeny is the availability of different related genes with similar functions. In humans, three MHC Class I genes and four sets of MHC Class II genes are available on chromosome 6.

For MHC Class II genes, the diversity is increased further by combination of α and β chains encoded by different chromosomes.

The diversity is more in the segments which are responsible for binding the antigenic peptide fragments

MHC Polymorphism

Several genetic mechanisms contribute to the generation of new alleles. Apart from point mutations, gene conversion in which one sequence s partly replaced by another generates MHC polymorphism.

That the selective evolutionary pressure favours MHC polymorphism is seen by the fact that point mutations are more frequently replacement mutations rather than silent mutations (change in codon but leaving the amino acid unchanged)  Gene conversions result in change in the several amino acid simultaneously. As there is a close linkage between MHC genes, gene conversion is not rare in the MHC allele evolution.

It appears that genetic recombination between different alleles at a locus may play even more important role than gene conversions in generating MHC diversity.

In genetic recombination, DNA segments are exchanged between alleles on the two homologous chromosomees. Analysis of MHC sequences indicated that this has occurred frequently during the evolution.

Closely related mice strains show one or two segments having been exchanged.

Unrelated strains indicated accumulated effects of recombinations.

MHC Class I like molecules

Some MHC Class  like molecules are coded by genes outside the MHC complex.

CD1 is expressed on dendritic cells, monocytes and tymocytes. In antigen processing, it behaves more like MHC Class II proteins.

Apart from protein antigens, CD1 is also capable of presentation of glycolipids which are components of the myobacterial membranes. These include mycolic acid, glucose monomycolate, phosphoinositol mannosides and lipoarabinomannans. CD1 gene may have evolved for presentation of microbial lipids.

MHC Class I proteins bind to peptides from cytosolic degradation whereas MHC Class II prefer peptides generated in endocytic vescicles.

Wide variety of infectious agents is one reason behind the evolutionary selective pressure for maintaining MHC diversity.

The role of MHC in Thymic education

T-cells recognize antigenic peptide fragments only in combination with MHC molecules. The diversity of MHC molecules thus widens the range of peptides that can be presented to T-cells.

T-cell responses are said to be MHC-restricted. The „non-self‟ peptide must be present along with a „self‟ marker: either MHC Class I or Class II antigen.

Not only that, in an individual, these have to be its own MHC proteins. These are the ones, T-cells during their development, encountered during thymic education to be positively selected. Non-self MHC cannot do this function. Hence, T-cell responses are actually self-MHC restricted.

MHC and Transplantation

Tissue graft rejection is T-cell mediated immune rejection. MHC matching between donor and recipient increases the chance of graft acceptance. The broad guidelines for tissue grafting emerge from studies with skin grafting.

Grafting from another organ/site of the same individual is called autograft.

Graft from a genetically identical individual is called syngeneic graft.

Graft from an unrelated individual is called allogeneic or allograft.

Tissue typing is carried out by a specific antiserum to cells. Complement is added. If the cell had antigen corresponding to the antibodies in the antisera, cells are lysed.

In mixed lymphocyte culture/reaction (MLC/MLR), lymphocytes from prospective donor and recipient are mixed. If the cells do not divide, they have a shared MHC specificity.

Tissues commonly transplanted in clinical medicine

Currently it is possible to graft many organs. During grafting (except in the case of corneal graft and some bone marrow grafts), immunosupppressive drugs have to be used. Kidney and heart transplants are the most common, liver and lungs are less common but not infrequent.

Alloantigens in grafted organs are recognized in two different ways

Both antibody and cell mediated mechanisms are known to operate but T-cells are involved in all cases except blood transfusion.

Direct recognition of a grafted organ (red in upper panel) is mediated by T cells whose receptors have specificity for the allogeneic. MHC class I or class II molecule in combination with peptide. These alloreactive T cells are stimulated by donor antigen-presenting cells (APC), which express both the allogeneic MHC molecule and co-stimulatory activity (bottom left panel). Indirect recognition of the graft (bottom right panel)

is mediated by T cells whose receptors are specific for allogeneic peptides that are derived from the grafted organ. Proteins from the graft are processed by the recipient’s antigen-presenting cells and are therefore presented by self (recipient) MHC class I or class II molecules.

Types of Allograft Rejection

Minor H antigens are peptides derived from polymorphic proteins of the donor‟s cell which are different from the corresponding ones on the recipient‟s cell.

Work with kidney transplants has led to classifying allograft rejection into three types:

Hyperacute rejection: This occurs during time period which ranges from few minutes to few hours. Pre existing circulatory antibodies are involved and cause type II hypersensitivity. Prior blood transfusion/organ transplant definitely favours this.

Acute rejection: This takes weeks to months and is T-cell mediated. Delayed type hypersensitivity and cytokines from monocytes/macrophages are involved.

Chronic rejection: This takes place after months/years. It is also cell mediated and may occur due to differences in minor transplantation antigens

Mechanism of Allograft Rejection

The % of involvement of direct and indirect recognition mechanisms is not always known. Acute rejection is largely due to direct recognition. In indirect recognition, T-cells may activate macrophages leading to tissue damage and fibrosis. This leads to antibody mediated graft rejection. The direct recognition is best understood in terms of skin grafts. Here langerhans cells act as APC. These as APCs migrate to lymph nodes where they interact witrh recirculating naïve T-cells specific for graft antigens and activate them. The effector T-cells reach blood via thoracic duct and from blood to the grafted tissue. As only graft is destroyed, it is mediated by adaptive immune response and not by just innate immunity processes like inflammation.

Xenotransplants

Animals have been considered as organ/tissue donors. Pigs have many organs of sizes similar to humans and have been studied widely. Such transplants from different specie are called xenotransplants.

Unfortunately, human shave natural haemagglutinins which bind to the cell surface carbohydrates of pigs. This leads to hyperacute rejection.

MHC molecules on pig cells would be another problem. Over and above these problems, ethical and social issues are worrisome factors. So is the possibility of unknown viruses being transferred and become infected in the germ line.

The discoveries of the MHC molecules during organ/tissue transplantation and later their involvement in T-cell responses shows how sometime two different lines of investigations can merge and help understanding biological phenomenon. Finally, MHC restriction of T-cell responses completes the circle.

Foetus is a “graft” which is tolerated and not rejected under normal circumstances.

That puzzling phenomenon involves both “grafting” and immunological tolerance.

Summary

• MHC proteins were recognized in skin transplant studies

• MHC molecules have different classes

• Many mechanisms contribute to the polymorphism of the MHC molecules

• All T-cell responses are MHC restricted

• Graft rejection always involves T-cell mediation