HLA Molecules, Biosynthesis and Expression
M.Tevfik Dorak, M.D., Ph.D.
For a recent review, see Chaplin DD: Overview of the immune response. J Allergy Clin Immunol 2010 (open access)
MHC class I molecules bind to and present endogenous antigens, e.g. viral peptides or tumour antigens synthesized within the cytoplasm of the cell, to CD8+ cytotoxic T cells. Their function is the activation of cytotoxic T-lymphocytes to kill cells compromised through environmental effects which may be infection, irradiation, chemical modification or some other causes of malignancy. MHC class II molecules, however, present exogenously derived proteins, e.g. bacterial proteins or viral capsid proteins, to CD4+ helper T cells (see Poster: T Cell Subsets). There are exceptions that class I may handle exogenous antigen and class II may present endogenous peptides that have not come from endosomes 1-4. The biosynthesis and expression of each class of MHC molecules are tailored to meet their different roles.
The HLA class I and class II proteins have similar structures with subtle functional differences. Class I molecules are made up of one heavy chain (45 kD) encoded within the MHC and a light chain called b 2-microglobulin (b2m; 12 kD) whose gene is on chromosome 15. Class II molecules consist of one a (34 kD) and one b chain (30 kD) both of which are encoded within the MHC. The class I heavy chain has three domains of which the membrane-distal first (a 1) and the second (a 2) are the polymorphic ones. Within these domains, polymorphisms concentrate on three regions: positions 62 to 83; 92 to 121; 135 to 157. These areas are called hypervariable regions (HVR) 5. The two polymorphic domains are encoded by the exons 2 and 3 of the class I gene 5;6. Diversity in these domains are very important in that these two domains form the antigen binding cleft (ABC) or peptide binding region (PBR) of MHC class I molecule. The sides of the antigen binding cleft is formed by a 1 and a 2, while the floor of the cleft is comprised of eight anti-parallel b sheets 7;8. The antigenic peptides of eight to ten amino acids (typically nonamers) bind to the cleft with low specificity but high stability 9. The a 3 domain contains a conserved seven amino acid loop (positions 223 to 229) which serves as a binding site for CD8 10. This domain also contains the TAP interaction site between amino acid positions 219 and 233 11. Another site in this segment is also of importance. The amino acid residue at position 227 (in the a 3 domain) is critical for the interaction of MHC class I molecule with the chaperon calreticulin 12. On the other hand, class I heavy chain residues of 77 to 83 (of the a 1 helix) are important in natural killer (NK) cell recognition 13;14 (see NK Cell Receptors). On the HLA-B molecule, this segment is contained within the Bw4 / Bw6 supertypic epitope.
In the class II molecule, generally both a and b chains are polymorphic. In these chains a 1 and b 1 domains form the ABC, therefore, diversity is located mainly in these domains (except in HLA-DRa which is not polymorphic). These domains are encoded by the exon 2 of their class II A or B genes. Hypervariable regions tend to be found in the walls of the cleft. Antigenic peptides of 12 to 24 amino acids long bind to the cleft and extend on either side 9. In a region analogous to the CD8-binding site on class I molecules, a major CD4-binding site is contained within residues 241 to 255 in b 2 domain 15. In the same domain, the polymorphic residues between positions 180 and 189 determine the quality of CD4 interaction 16. Among the class II molecules, HLA-DR53 encoded by HLA-DRB4 is known to interact poorly with CD4 16.
MHC class I molecule is synthesized in the rough endoplasmic reticulum (RER). It is the signal peptide encoded by the first exon of the class I molecule which directs the insertion of the molecule into the RER during translation. The intracellular proteins are targeted for degradation to the RER by binding of ubiquitin. The peptide / ubiquitin complex is transported to the proteasome complex where it is degraded by low molecular mass polypeptide (LMP2 and LMP7) - proteasome 17. Both LMPs required for this function are encoded within the class II region. The resulting antigenic peptides are then transported into the RER by the heterodimer of transporters-associated-with-antigen-processing (TAP1 and TAP2) whose genes are also in the MHC class II region 18.
Newly synthesized class I heavy chain - b2m dimer first associates with the ER chaperone calnexin and calreticulin acting sequentially 12. In the presence of tapasin (encoded just outside the class II region 19), empty MHC class I molecules complexed with calnexin / calreticulin and the reductase ERp57 can then associate with TAP transporters. Peptide binding releases the class I - b2m dimer from all auxiliary molecules for transport to the cell surface via the Golgi body, while lack of binding results in proteasome-mediated degradation 20;21.
The role of class I molecules as indicators of the intracellular protein composition is reflected in this scheme which does not allow them to leave the ER unless they have bound to a peptide with sufficient affinity. Cytotoxic T cells regularly patrol to see if any of the presented peptides are non-self. In healthy cells, the peptides are derived from normal cellular proteins, and the immune system is rendered tolerant to these peptides during development. Therefore, the complexes of self peptides and MHC molecules are necessary to establish the repertoire of T-cell receptors (TCR). It is believed that this fact has been the limiting factor in the number of HLA class I loci in evolution 22-24. Having the maximum number of antigen presenting MHC alleles would result in maximum number of T cells (interacting with self MHC) positively selected, but, at the same time it would reduce the number due to negative selection (elimination of T-cells reacting with self-peptides) to avoid autoimmunity. Depending on the heterozygous allele combination, the net effect could be a reduced number of T cells compared to a homozygous situation. In no species, more than three classical class I loci have been found so far. This suggests that further expansion is deleterious possibly due to the erosion of the TCR repertoire. Because of the negative selection of self-reactive T cells during ontogeny, the more MHC molecules present, the fewer the T cells that are available 25. Therefore, in heterozygotes, there is a trade-off between an advantage as an increased ability to present foreign peptides and disadvantage as an erosion of the T cell repertoire. Considering heterozygosity as an ability to mount twice as potent an immune response as the homozygote may be too simplistic.
Cytotoxic T cells can recognise the nonself peptides only in conjunction with the self MHC molecule 26;27. The only exception to that is a nonself MHC molecule (as in the case of mismatched transplantation) which does not require presentation by the host MHC molecules 28;29. The obligate recognition of foreign peptides in the context of MHC molecules is called 'MHC restriction' of T-cell recognition 5;26;27;30.
It is estimated that there are up to 250,000 of each HLA class I molecules on the surface of a somatic cell 31. MHC class I molecules are unstable in the absence of a bound peptide. Once formed, the complex of antigenic peptide and MHC are generally very stable with a half life of about 24 hours. Typically the population of molecules of a single allele will have approximately 1000 different peptides bound on any cell. The expression patterns of each class of MHC molecules are different. Nearly all somatic cells express class I molecules but it would be wrong to say that class I molecules are ubiquitously expressed since there are certain cell types that lack expression 6. It is believed that having no DNA, red cells cannot support virus replication, thus, they do not need class I molecules. HLA-C expression is low and about 10% of the average level of HLA-A and -B 6. This is because HLA-C heavy chains inefficiently assemble with b2m 32 and also the cis-regulatory element of transcriptional control called enhancer A (or region I) is mutated in the promoter of HLA-C which affects the expression 33.
MHC class II molecule expression is more restricted. MHC class II genes are regulated in a tissue-specific manner, normally in a co-ordinate fashion 34. Only antigen presenting cells (APC) express class II molecules. These are B cells, macrophages, Langerhans and related dendritic cells, and activated T cells. Unscheduled expression of class II genes has been observed in autoimmunity 34. Normally, the level of expression decreases in the order of DR > DP > DQ 34;35.
Both chains of the class II molecules are synthesized in the ribosomes which are associated with the RER. They enter the RER and are brought together with the assistance of a chaperone molecule. When the a and b chains join together, a segment of the invariant chain (Ii or CD74) blocks the peptide binding site temporarily to prevent the acquisition of immunogenic peptides. The nested set of peptides that are derived from amino acids 80 to 104 of Ii are called CLIP (class II-associated invariant chain peptides) 36. The class II - CLIP complex is then transported to the specialised endosomal compartment MIIC (MHC class II-containing compartment), a subpopulation of lysosomes 37. It is in this lysosomal compartment that the complex meets with the antigenic peptides entered the cell in membrane vesicles. The acidic conditions of the compartment causes the CLIP to be released, and the peptide with the appropriate sequence motif binds to the class II molecule. The non-classical class II molecule HLA-DM acts as a dedicated chaperone in the lysosomal compartment to prevent the functional inactivation and aggregation of empty HLA-DR ab dimers 38;39. These empty class II molecules that are chaperoned by HLA-DM enable the antigen-processing system to respond promptly to the challenge by newly entering antigens. The DR-peptide complex is transferred to the cell surface by means of membrane bound vesicles. Class II molecules present the peptide to CD4+ T-helper cells. Class II molecules often occur in the cell membrane as a dimer of dimers. In this case, the two molecules must be identical to be recognised by TCR.
T cell activation occurs following recognition of peptide / MHC complexes on an APC. T cell activation can be viewed as a series of intertwined steps, ultimately result in the ability to secrete cytokines, replicate, and perform various effector functions. During antigen presentation, CD4 and CD8 are intimately associated with the TCR and bind to the MHC molecule. Besides this interaction between T cells and APCs, ligation between counter-receptors on the T cell and accessory molecules on the APC is also required as additional signals for T cell activation. The major accessory molecules and their receptors are: B7-1/2 (CD80/86) and CD28/CTLA-4; ICAM-1/2/3 (CD54/102/50) and LFA-1 (CD11a/CD18); LFA-3 (CD58) and CD2 40;41 (see eBiosciences Poster: Costimulation). Among these CD28 engagement of its ligands, the B7 molecules, represent the main co-stimulatory interactions 42;43. Co-stimulatory activity largely involves the coupling of many intracellular signalling pathways that form an integrated network, eventually leading to the production of interleukin-2 (IL-2) and proliferation. The importance of co-stimulation is that TCR occupancy in the absence of adequate co-stimulation fails to generate T cell responses, and may result in the induction of anergy in T cell clones 43;44. Another implication is that the mere expression of class II molecules does not necessarily make a cell a functional APC.
The expression of MHC molecules on the cell surface is also important for NK cell activity. Non-specific immunity provided by NK cells is governed by the expression levels of MHC molecules. Normally, the presence of class I molecules provide inhibitory signals for NK cells. When the expression is down-regulated as happens in viral infections or malignant transformations, NK cells are activated by the lack of inhibitory MHC antigens (missing self) and they eliminate such cells 45;46. NK cells have different membrane receptors that bind to MHC class I ligands which inhibit the lysis of class I-bearing target cells. Each family of these receptors interacts with different class I molecules. Human NK cells express receptors encoded by the Killer Inhibitory Receptor (KIR) gene family present on chromosome 19q13.4 47. Different KIR molecules are displayed on overlapping subsets within the total NK cell population and the repertoire of expressed receptors is heterogeneous in different individuals. The specificity of KIRs maps to the a1 domain of HLA-C and HLA-B molecules 14;48. Another group of human NK cell inhibitory receptor is CD94/NKG2 which is specific for the non-classical class I molecule HLA-E 49. This recent understanding of the regulation of NK cell function implies an important role for MHC class I molecules in tumour immunity. The loss of MHC class I molecules may help a transformed cell escape from cytotoxic T cell attack but makes it susceptible to NK cell-mediated lysis.
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Last edited on 13 August 2013