MHC protein products
The protein products of MHC molecules, expressed on the surface of all nucleated cells, are responsible for the immune response to allogeneic tissues. Of all the genes included in this region, two highly variable groups are central in allorecognition. These are the class I and class II molecules. Class I molecules are known as human leukocyte antigens (HLA)-A, -B and -C in humans and H2-K, -D, -L in mice and are constitutively expressed on most nucleated cells. Class II molecules are known as HLA-DR, -DP and -DQ in humans and H2-A and -E in mice and are constitutively expressed only by bone marrow-derived APCs, such as macrophages, DC, B lymphocytes and by thymic epithelial cells. The convention is to identify the genes in Roman letters (e.g. HLA-DRB or H2-D) and the encoded proteins in corresponding Greek symbols (e.g. HLA-DR βor H2-Aβ) (Figure 2).
The MHC contains the most variable functional genes described in vertebrates. At three of the more variable human MHC loci, HLA-A, HLA-B and HLA-DRB1, 243, 499 and 321 alleles have been resolved worldwide, respectively, and nucleotide diversity in the human MHC is up to two orders of magnitude higher than the genomic average (30). This polymorphism underlies the extreme difficulty in finding perfectly matched organs or unrelated bone marrow donors that will not induce a strong anti-MHC alloresponse. MHC genes are inherited from the parents as a whole set or haplotype, and because each individual has two sets of chromosomes, one haplotype will come from the mother and the other from the father.
These molecules play a critical role in the normal immune system, namely the presentation of peptides in a form that can be recognized by T cells. In particular, CD8+ T cells recognize peptides presented by class I molecules and CD4+ T cells recognize those presented by class II molecules and this is valid both for the self-MHC molecules as well as the allo-MHC molecules. From the crystal structures of the extracellular portions of human class I and class II molecules, it is now clear that the MHC molecules form a ‘groove’ where the peptide to be presented is bound (31). The peptides presented are the result of the natural processing of cellular and serum proteins. The peptide-binding groove of the MHC molecules on each cell is thus occupied by a very diverse (several hundreds) set of different peptides. Class I molecules are mainly occupied by peptides originating from intracellular proteins, whereas those presented by class II molecules have mainly an extracellular origin (32); although cross-presentation of peptides of extracellular origin has been widely demonstrated in class I molecules (33). It has been confirmed that the TCR recognizes a complex of two MHC helixes and a bound peptide. In the allorecognition setting in a direct pathway response, these MHC–peptide complexes recognized are from the allogeneic tissue. Recently, it has been shown that complementarity-determining region (CDR) 3α could undergo rearrangements to adapt to structurally different peptide residues. This CDR3 loop flexibility helps to explain TCR binding cross-reactivity and thus supports the conundrum of T cells responding to MHC molecules that they have not been selected to recognize (34).
The MHC class I-related chain (MIC) system
In 1994, two new polymorphic families of MHC class I-related genes, termed MHC class I-related chain A (MICA) and B (MICB), were described (35). These genes are located near the HLA-B locus on chromosome 6 and encode cell surface glycoproteins that do not associate with β-2 microglobulin. These molecules function as restriction elements for intestinal γ/δT cells and they behave as cell stress molecules. MICA is expressed in endothelial cells, keratinocytes and monocytes, but not in CD4+, CD8+ or CD19+ lymphocytes (36). It is, therefore, likely that the polymorphic MICA molecule may be a target for specific antibodies and T cells in solid organ grafts or in graft vs host disease (GvHD) (37). The anti-MICA antibodies induce a prothrombotic state, characterized by a loss of surface heparan sulphate and thrombomodulin from cultivated endothelial cells (38). In fact, in kidney transplants, two prospective trials, after 1 and 4 years, have provided strong evidence that HLA and MICA antibodies are associated with graft failure (39).
Minor histocompatibility antigens
A different set of polymorphic non-MHC proteins have been identified that are important in provoking transplant rejection, they were defined by Snell and colleagues as mHAg, as the rejection reactions they induced in mice were slower (40). In principle, any protein that has polymorphisms within a species can become mHAg. Peptides from these proteins are presented to T cells in an MHC class I or class II restricted manner (41). The number of possible mHAgs in transplants performed between genetically unrelated, MHC-matched individuals, is very large. However, the reactions seem to be restricted to a few epitopes, thus dubbed immunodominant (41). The molecular basis for this phenomenon is incompletely understood, although it has recently been shown that both the duration of individual mHAg presentation and the avidity of T-cell antigen recognition influence the magnitude of the cytotoxic response that ensues (42).
The frequency of T cells responding to these antigens in non-transplanted individuals is very small and can only be measured in vitro after in vivo immunization or repeated stimulations, as opposed to direct pathway responses. When alloresponses of mHAgs have been measured, the cells that respond to these antigens are generally CD8+ T cells, implying that most mHAgs are peptides bound to self-MHC class I molecules. However, peptides bound to self-MHC class II molecules can also participate in the response to MHC-identical grafts (43). The in vivo correlate of an immune response to an mHAg is transplant rejection, or in MHC-matched individuals, GvHD (44). GvHD is a series of manifestations and symptoms that appear after bone marrow transplantation (BMT) and results from an immune response of the immunocompetent cells of the donor against the tissues of the recipient. The effector immune responses are specifically described later on. Notably, even though mHAgs are named minor, and the frequency of responders to these antigens is very low, after transplantation, a single immunodominant mHAg can induce GvHD. Apart from gene polymorphisms, homozygous gene deletions can also serve as mHAgs as it has recently been described for an autosomal gene in the UDP-glycosyltransferase 2 family (45).
Minor HLA antigens important in transplantation have been described from different cellular origins.
(a) Encoded by sex chromosomes: The most thoroughly studied are a set of proteins encoded on the male-specific Y chromosome that are known collectively as H-Y antigens. The absence of Y-chromosome-specific gene products in females induces responses to male antigens. In fact, these responses are very frequent (37–50%) in women with previous male pregnancies (46), whereas male anti-female responses are not seen (because both males and females express X-chromosome-derived genes). To date, the number of H-Y epitopes described in humans that are important in transplantation is 10 (47). These are restricted by either class I or class II molecules and originate in six different loci of the Y chromosome (DFFRY, SMCY, TMSB4Y, UTY, DBY and RPS4Y1).
(b) Encoded by autosomes: Non-Y-linked mHAgs have also been shown by T cells from patients with GvHD after BMT between HLA identical individuals. The first example identified in humans was named ‘HA’(48) after the patient. Recognition of this peptide was restricted by class I molecules. In the interim, other antigens have been identified for humans (HA-1, -2, -3, -8, HB-1, ACC-1, etc.); their cellular origin is varied: Mysoin 1G, LBC oncogen, BCL2A1, and some not yet identified genes (47) are examples.
(c) Encoded by mitochondrial DNA (mtDNA): Tracking of an mHAg to the small mitochondrial genome from the studies of a maternally transmitted transplantation antigen informed that such peptides could become histocompatibility antigens (49). Cytotoxic T lymphocytes (CTL) were used to test candidate peptides derived from polymorphic regions of the enzyme mt-ND1. A simple amino acid difference in the peptide was found to account for immunogenicity. Subsequently, additional mitochondrial genes in mouse and rat have been found to encode mH peptides, and several are presented to T cells by non-classical, MHC class I molecules (50). In the humans, however, no effect was observed on cumulative disease-free survival or incidence rate of GvHD when the clinical effect of mtDNA mismatches was studied in a Japanese cohort (51).