MHC class I and II

The MHC is divided into three regions, namely the class I, II and III regions, and these are described in Chapter 4. A large array of genes has been localised to the MHC by sequencing, but two major classes of transplantation antigens were those first mapped by classical genetic techniques. The structure of these molecules is shown in Fig. 3.2.

Figure 3.2 Structure of MHC class I and II molecules. Schematic diagram of MHC class I and II proteins showing their basic structure, membrane association and peptide-binding groove. α₁, α₂, α₃ of MHC class I are membrane integral and form a non-covalent bond with β₂-microglobulin, which is not membrane bound. MHC class II is formed from α and β chains, each with two domains and each membrane integral. β_2-M, β₂-microglobulin.

MHC class I proteins are cell-surface glycoproteins comprising a heavy chain (≈︀45 kDa), which is highly polymorphic, and a less variable light chain, β₂-microglobulin (≈︀12 kDa), which is encoded outside of the MHC. β₂-Microglobulin is not anchored in the membrane and binds non-covalently to the heavy chain. MHC class I proteins are expressed on most nucleated cells and generally engage T lymphocytes bearing the cell-surface protein CD8. CD8 is required for effective TCR engagement of MHC class I, resulting in intracellular signalling.

MHC class II proteins consist of two membrane anchored glycoproteins (α-chain, ≈︀35 kDa; β-chain, ≈︀28 kDa), which are both encoded within the MHC. The tissue distribution of MHC class II proteins is more restricted than that of MHC class I. MHC class II is expressed constitutively by B lymphocytes and dendritic cells, which have specialised functions in the generation of the immune response. It is also expressed on some endothelial cells in humans and can be induced by inflammation on many cell types. MHC class II generally engages T lymphocytes bearing the CD4 surface protein. CD4 is required for effective TCR engagement of MHC class II and resultant intracellular signalling.

In humans these transplantation antigens were defined serologically on leucocytes and so are referred to as human leucocyte antigens (HLAs).

Structure of MHC class I and II proteins

MHC class I and II proteins have a similar three-dimensional structure forming a ‘peptide binding groove’ between two alpha helices (Fig. 3.2). The amino acids that constitute this groove are those which vary most between allotypes. During synthesis and transport of MHC class I and class II proteins to the cell surface the groove binds a wide range of short peptides. The groove in MHC class I has closed ends and the peptides bound are therefore 8–10 amino acids long. The groove in MHC class II is open and can accommodate peptides of considerably greater length. The sequences flanking those bound within the groove whilst not contributing directly to antigen specificity may contribute to the physicochemical properties of the complex. The peptides bound to MHC class I are primarily derived from intracellular proteins (but see ‘Indirect/cross-presentation’ below) and those from MHC class II primarily from extracellular proteins (Fig. 3.3). This difference arises from their distinct intracellular trafficking. In the presence of infection, foreign protein is processed to generate peptides bound to MHC forming a compound antigenic determinant, which engages the TCR and stimulates T-lymphocyte activation. This compound determinant is the structural basis for self-restricted antigen recognition by a T-cell repertoire that is skewed, by thymic selection, toward the engagement of peptide presented in the context of self-MHC. In the absence of foreign protein, all peptides are derived from self-proteins to which the individual is, to varying degrees, tolerant. A significant proportion of self-peptides are derived from MHC proteins themselves and this has consequences for allorecognition.

Figure 3.3 Antigen processing and presentation in the MHC class I and II pathways. (a) Processing of endogenous antigens occurs primarily via the class I pathway. Peptides are produced and loaded into MHC class I proteins as shown in steps 1–4. During the synthesis of MHC class I proteins (steps 1–3) the α chain is stabilised by calnexin before β₂-microglobulin binds. Folding of the MHC class I/β₂-microglobulin remains incomplete but the complex is released by calnexin to bind with the chaparone proteins, tapaisin and calreticulin. Only when the TAP transporter delivers peptide to the MHC class I/β₂-microglobulin can folding of this complex be completed and transport to the cell membrane occur (steps 5 and 6). (b) Processing of exogenous antigens occurs primarily via the class II pathway. Antigens are taken up into intracellular vesicles where acidification aids their degradation into peptide fragments (steps 1 and 2). Vesicles containing peptides fuse with trans-Golgi, containing CLIP–MHC class II complexes (step 3). HLA-DM aids removal of CLIP and loading of peptide before the class II peptide complex is displayed on the cell surface (steps 4 and 5). MHC class II proteins are synthesised in the endoplasmic reticulum, where peptide binding is prevented by invariant chain. Invariant chain is cleaved leaving the CLIP peptide still in place (steps A and B) before fusing with acidified vesicles containing peptide. In B lymphocytes and epithelial cells of the thymus an atypical class II protein, HLA-DO, is expressed; this is a dimer of HLA-DNα and HLA-DOβ. It, like HLA-DM, is not expressed at the cell surface and inhibits the action of HLA-DM. Its precise role is unknown. ATP, adenosine triphosphate; CLIP, class II-associated invariant chain peptide; ER, endoplasmic reticulum; MHC, major histocompatibility complex; TAP, transporters associated with antigen processing.

CD8⁺ T lymphocytes engage antigenic peptide bound to MHC class I and are typically cytotoxic. The normal function of these lymphocytes is to lyse cells infected intracellularly with, for example, virus. CD8⁺ cells also produce a range of cytokines and some have regulatory properties.

CD4⁺ T lymphocytes engage antigenic peptide bound to MHC class II. Antigen generally derives from extracellular proteins. These cells have a variety of functions, summarised in their designation as ‘helper cells’. These functions include help for cytotoxic T-cell generation, B-cell maturation and promoting delayed-type hypersensitivity (DTH) inflammation. CD4⁺ T-cell responses are evidently central to many forms of allogeneic rejection and their activation by dendritic cells is an important component of this response. In some rodent models absence of CD4⁺ T-cell stimulation can abolish rejection despite the presence of CD8⁺ T cells and fully allogeneic MHC class I mismatches. In such cases tolerance to alloantigen may emerge rather than productive immunity, illustrating the crucial importance of CD4⁺ T-cell responses in rejection.

The specific structure of the binding groove determines the peptide that can be bound by any given MHC and there are a limited number of peptides from a given protein that form a stable complex. The ontogenetic drive for extreme polymorphism in the MHC is therefore likely to reflect the need to counter a wide variety of infectious organisms that rapidly mutate and could evolve protein sequences that do not bind an individual’s MHC repertoire. This is unlikely to occur across a whole species if there is a full range of polymorphism as observed in humans. Certain species with limited polymorphism at either MHC class I or II can be devastated by infections that, in closely related species with polymorphic MHC, do not threaten the population.

It is now possible to predict the sequences of peptides likely to bind to a given MHC from its structure and to then confirm this from peptide elution and peptide-binding studies. The peptide can be orientated in the groove and those amino acids in contact with MHC and those in contact with the TCR can be predicted. This is likely to be a powerful tool in future vaccine development.

Non-classical MHC

The MHC locus is large and it encodes a wide range of proteins other than the classical histocompatibility antigens described above. Some of these have a structure similar to classical histocompatibility antigens but are not polymorphic. They may present specialised antigens such as lipids (e.g. mycolic acid and lipoarabinomannan from mycobacterium) or peptides of various sequences but with common characteristics (e.g. with N-formylated amino termini). The relevance of some of these molecules to transplantation is unclear but they do not behave as classical transplantation antigens.

Molecules such as HLA-DM, TAP and LMP that have a role in antigen processing are encoded within the MHC locus as are other molecules more broadly involved in immunity, for example tumour necrosis factors α and β (TNF-α and TNF-β) and complement components C2 and C4. Polymorphisms linked to these genes have been implicated in disease expression and transplantation responses.⁶

HLA-E and HLA-G are expressed in the placenta and are thought to play a role in modulating alloimmune responses to the fetus. There is emerging evidence that the expression of these molecules may also be associated with improved outcome in allogeneic transplantation.^7,8 HLA-E modulates alloimmunity through its interaction with CD94-NKG2A, an inhibitory receptor expressed on NK and cytotoxic T cells. HLA-G interacts with inhibitory receptors ILT-2 and -4 expressed by APCs, NK and T cells. In a large series of renal transplant patients, the formation of soluble HLA-G was strongly correlated with diminished acute rejection rates.⁸

MHC class I-related chain

MHC class I-related chain A (MICA) genes are located within the class I region of the chromosome and are highly polymorphic. Although constitutively expressed at low levels by various cell types, including monocytes, epithelial cells, fibroblasts and endothelial cells, they may be upregulated by stress signals of oxidative stress and sepsis. Expression of MICA in response to stress may activate NK and T cells through its interaction with cytolytic activating receptor NKG2D. MICA is polymorphic and its alloantigenicity has been demonstrated in animal models. There is now good evidence that formation of antibody to MICA is associated with poor graft outcomes in renal transplantation⁹ and increased rates of acute rejection in cardiac transplantation.¹⁰

Minor histocompatibility antigens

The study in rodents of genetic loci determining organ rejection identifies a range of sites encoding transplant antigens that are distinct from the MHC. These minor histocompatibility (miH) antigens are less polymorphic and typically induce a weaker response than disparities in the MHC. It is almost impossible to raise antibodies to these antigens, although they induce a well-defined cellular response.

The crystallographic structure of the MHC with bound peptide provides a structural explanation for these properties of miH antigens. They are peptides derived from proteins of limited polymorphism that, when bound to syngeneic MHC, constitute an antigenic determinant in the same way as any conventional antigen. This explains why it is difficult to raise antibodies to miH antigens, since antibodies typically engage conformational determinants on proteins and the B-lymphocyte receptor is less discriminating of subtle differences in the structure of peptide–MHC composites than is the TCR.

The most easily defined miH antigen is the male-specific H-Y antigen, which is in fact a series of antigenic peptides derived from proteins encoded on the Y chromosome, accounting for its consistent detection across a wide range of background MHC (although in certain strain combinations there is a single dominant antigen¹¹). In rodent studies the role of miH proteins as transplantation antigens can be readily demonstrated, particularly when there has been prior sensitisation to the relevant antigen.

In rare cases of rejection in renal transplants between HLA-identical siblings, miH are thought to be the inciting antigens but their importance in other forms of solid-organ transplantation is generally limited. This is not true, however, in bone marrow transplantation, in which rejection is easily stimulated by such differences causing graft vs. host or graft vs. tumour response.¹²

Direct allorecognition

Allogeneic MHC on DCs derived from the graft will be occupied by any number of different endogenous peptides (Fig. 3.4). The frequency of alloreactive T lymphocytes stimulated through this pathway is high, of the order of 1/10². Although this appears to contradict the principles of ‘self-MHC’ restriction resulting from thymic education, it emerges as a consequence of the vast array of endogenous peptides that occupy the MHC generating an equivalent number of compound epitopes. It also arises from the relatively limited differences in the structure of MHC alloantigens outside the peptide-binding groove and the corresponding fact that T lymphocytes can distinguish subtle differences in the kinetics of TCR–peptide–MHC interactions.

Figure 3.4 Direct, indirect and semi-direct pathways of antigen presentation. Sensitisation of the recipient can occur by antigen presentation delivered through passenger leucocytes or dendritic cells of donor origin (a) or recipient origin (b,c). APC, antigen-presenting cell; MHC, major histocompatibility complex; TCR, T-cell receptor.

These facts apply equally to the response to recall antigens; that is, other than in naive animals any direct alloresponse will include cross-reactions with self-restricted secondary responses to conventional antigens.^13–16 As most studies on experimental transplantation, particularly those on the induction of tolerance, use naive rodents these are unlikely to accurately reflect the alloresponse observed clinically. Indeed, strategies to induce transplantation tolerance which succeed in the naive animal are unsuccessful in previously infected animals,^15,16 providing one possible explanation for the difficulties in translating such strategies into preclinical models.

Indirect allorecognition

Proteins from extracellular infectious organisms are generally processed by APCs and presented in the context of MHC class II. As discussed above, only a small proportion of potential peptide sequences from a protein will be processed, bind MHC and generate a response.

In the setting of allogeneic transplantation any donor protein can be processed and presented in the context of MHC by recipient APCs. As most proteins have little or no polymorphism within a species, they will not always initiate an alloimmune response, although occasionally transplant rejection is associated with the development of auto-immune responses. However, polymorphic proteins such as those of the MHC can stimulate an immune response via this conventional or ‘indirect pathway’ of antigen presentation.

The potential importance of this route of allorecognition is demonstrated by experiments in which immunisation with MHC class I peptides prime for subsequent rejection of a skin allograft bearing the intact class I antigens from which the peptides were derived.¹⁷ Studies in other models have demonstrated that priming with MHC class I- and II-derived peptides lead to chronic allograft rejection, with accelerated vasculopathy.^18,19 These peptides are most likely to be presented in the context of recipient class II since they are extracellular for the self-APCs. However, there is now evidence of crossover or ‘cross-presentation’ between the two pathways, so there may also be presentation of such peptides in the context of MHC class I.^20,21

The importance of indirect presentation of alloantigen is also illustrated by transplantation of skin allografts from MHC class II deficient onto normal mice. Donor APCs are unable to stimulate recipient CD4⁺ T cells and yet rejection remains dependent upon CD4⁺ T cells. It is likely that this reflects recipient CD4⁺ T-cell stimulation through indirect presentation of donor alloantigens by self-MHC.^20–22 In this latter case, of course, peptides are not artificially introduced into the recipients but produced by normal processing of MHC from apoptosed donor-derived cells.

Semi-direct allorecognition

If host T cells are stimulated by recipient-derived DCs via indirect antigen presentation, the MHC restriction of the effector cell population will be to host rather than donor. A problem could arise if a cytotoxic T cell, once stimulated with self-MHC and allogeneic peptide, comes to lyse its target cell – in the case of graft rejection, the foreign transplanted tissue, which does not express self-MHC molecules. This problem is overcome if the foreign MHC on the target cell cross-reac with self insofar as the T cell is concerned, if the effector arm of the immune response does not require MHC restriction (e.g. macrophages, DTH) or if the effector population is primed by the donor-derived MHC. How could this latter situation arise if the T cells are primed by recipient-derived DCs? It has been known for several years now that intact proteins can be exchanged between cells in cell culture systems and indeed that MHC proteins transferred in this fashion can stimulate alloreactive responses.^23–25 The importance of this in stimulation of alloreactive responses in the whole animal has been highlighted in recent work,²⁶ although its importance in inducing graft rejection has yet to be established.

Activation of dendritic cells

Although the immunostimulatory properties of DCs are most apparent in the experimental data described above, it is now evident that marked phenotypic and functional differences exist between dendritic cells, depending upon the context in which they are studied. The factors that contribute to acquisition of such properties include:

The precise mechanisms of dendritic cell differentiation and maturation are currently under intense investigation. For example, DCs are activated by TLR engagement and by other facets of the innate immune system, including complement, NK cells and γδ T cells.

These set DC phenotype, which may then determine the phenotype of the subsequent adaptive immune response. Generally ‘immature’ DCs play a role in maintaining peripheral tolerance by both deletional and regulatory mechanisms, and these properties may themselves be regulated by T lymphocytes. DCs therefore bidirectionally connect innate and acquired immunity, and by doing so link the nature of antigen-specific memory to the context in which antigen is first encountered.^42,43

CD28–B7

The role of CD28 has been the most intensively investigated in the field of co-stimulation.^44–46 CD28 is a homodimeric glycoprotein present on the surface of T cells, which interacts with two counter- receptors, CD80 and CD86, expressed on the surface of APCs. CD86 is expressed constitutively at low level by APCs and is upregulated rapidly following interaction with the T lymphocyte. It has a rather low affinity for CD28 whereas CD80, which is not constitutively expressed, is upregulated with slower kinetics but has an approximately 10-fold greater binding affinity for CD28 than does CD80. The result of ligation of CD28 by either CD86 or CD80 appears to be increased cytokine synthesis and proliferation, and they do not appear to be qualitatively distinct. The different kinetics of expression and affinity for other ligands such as CTLA4 (CD152) may result in different effects on cellular phenotype.

CD28 ligation by CD80/86 promotes initial T-cell activation but also plays a role in CTLA4 (CD152) regulatory T-cell homeostasis. Blocking the CD28 pathway in rodents can have dramatic effects on the generation of primary immune responses and may result in prolonged graft survival or even tolerance of grafts in some experimental models,^47,48 but mice with a disrupted CD28 gene can make productive immune responses, albeit with sometimes altered kinetics. This may be due to redundancy of co-stimulatory pathways, including other members of the B7 and CD28 families.

CTLA4 ligation by CD80/86 inhibits T-cell activation. The severe phenotype of CTLA4 −/− mice, which die from uncontrolled lymphoid proliferation shortly after birth,⁴⁹ suggests that there is less redundancy in this aspect of immune regulation.

Other CD28 family members, e.g. ICOS, PD-1 and BTLA (B- and T-lymphocyte attenuator), are inducible on T cells and seem to have roles regulating previously activated T cells.

There are five other B7 family members, ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3 and B7-H4 (B7x/B7-S1), that are expressed on various cell types and are likely to provide further control of T-cell activation and tolerance in the periphery. B7 molecules seem to be able to bidirectionally deliver signals into B7-expressing cells as well as those expressing their ligands.

The most widely used method to block CD28–B7 interactions has been through the use of CTLA4-Ig but this will also block CTLA4–B7 interaction, and inhibition of negative CTLA4 signalling may account for diverse observations on immune responses in different experimental systems. A high-affinity analogue of CTLA4-Ig has been developed as an immunosuppressive for use in clinical practice,⁵⁰ and the development of reagents that preferentially inhibit the CD28–B7 interaction is also ongoing.