Inroduction

The Major Histocompatibility complex (MHC) system known as the human leukocyte antigen (HLA) in humans is located on the short arm of chromosome 6 (6p21.3) and contains the most polymorphic gene cluster of the entire human genome. Furthermore, the HLA consists of three regions which have been designated as class I, class II, and class III based on the structure and function of gene products. The main function of HLA class I gene products (HLA-A, -B, and -C) is to present endogenous peptides to responding CD8⁺ T Cells while the class II coded molecules HLA-DR, -DP, and –DQ have restricted expression and process exogenous peptides for presentation to CD4⁺ helper T Cells. The class III region, in turn, contains genes which encode for immune regulatory molecules, e.g., tumor necrosis factor (TNF), factors C3, C4, and C5 of complement and heat shock proteins. This chapter will discuss the genetic, structural and functional characteristics of HLA.

History

The major Histocompatibility Complex (MHC) was initially discovered as a genetic locus associated with the acceptance or rejection of transplanted organs in mice. In 1954, the same genetic system was described in humans by Jean Dausset and Jan van Rood and was called human leukocyte antigens (HLA). This discovery was made as the result of the presence of antibodies against antigens expressed on leukocytes from patients who had received multiple blood transfusions. Also, these antibodies were present on leukocytes from multiparous women and patients who had gone through kidney transplants. Subsequently, two distinct classes of HLA molecules were defined: HLA class I antigens and HLA class II antigens. HLA class I antigens are expressed on all nucleated cells and platelets (except those of the central nervous system) while the HLA class II antigens are expressed on antigen presenting cells (APC) such as B lymphocytes, dendritic cells, macrophages, monocytes, Langerhans cells, endothelial cells, and thymic epithelial cells. All this knowledge led to the practice of serological typification from different HLA variants in patients and donors before the performance of organ transplants. The HLA system revolutionized the transplant practice, and subsequent studies allowed the elucidation of its importance in the development of various diseases (e.g., autoimmune diseases), its application in genetic population studies and in genomic medicine (1,2).

Genetic organization of HLA

The HLA system is located on the short arm of chromosome 6 on band 6p21.3. This gene system is the largest cluster in the human genome, and it is divided into three main sub-regions: the genes of class I, class II, and class III, which are all involved in immune response and suppression (3) (Figure 1).

Figure 1

Map of the human HLA. The complex is conventionally divided into three regions: I, II, and III. Each region contains numerous loci (genes), only some of which are shown. Abbreviations: tapasin (TAPBP); large multifunctional proteases 1 and 2 (LMP1 and (more...)

Relative to their chromosome position, the class I region is located nearest to the telomere, and the genes within this region encode for the class I molecule α-polypeptide chain. It is noteworthy that the gene encoding β2-microglobulin –the common light chain of the HLA class I molecules– is not located in the HLA complex but is on chromosome 15. Genes encoding HLA class I α-chain have a characteristic structure in which different domains of the protein are encoded by different exons. The leader peptide is encoded by exon 1, and the three extracellular domains (α1, α2, and α3) are encoded by exons 2, 3, and 4 respectively. Meanwhile the transmembrane anchor is encoded by exon 5, the cytoplasmic tail by exons 6 and 7, and the 3’ untranslated region by exon 8 (Figure 2).

Figure 2

Exon-intron organization of an HLA class I gene. Within the class I heavy chain each protein domain is encoded by different exons. Abbreviations: leader sequence (L); transmembrane region (TM); cytoplasmic tail (CYT).

Overall, there are three types of class I genes in the HLA region: HLA-A, -B, and -C, the so-called classic. Furthermore, there are other loci in this region called HLA-E, -L, -J, -K, -H, and -G, which encode the non-classical HLA molecules (4).

Likewise, the HLA class II molecules are heterodimers composed of α and β chains. The exon-intron organization of the class II genes is like that of the class I genes because the different domains of the protein are encoded by different exons. The α- and β-chain genes have a similar structure in which exon 1 encodes for the leader peptide, and exons 2 and 3 encode the two extracellular domains. In the β-chain genes, exon 4 encodes for the transmembrane domain, and exon 5 encodes for the cytoplasmic tail. In contrast, in both α-chain genes, the transmembrane and the cytoplasmic tail are encoded by exon 4 (Figure 3).

Figure 3

Exon-intron organization of the genes encoding α and β chains of HLA class II. Abbreviations: leader sequence (L); transmembrane region (TM); cytoplasmic tail (CYT).

As a whole, there are five isotypes of the class II HLA protein designated as HLA-DM, -DO, -DP, -DQ, -DR. To classify them into their respective loci on chromosome 6, there is a nomenclature of three letters: the first (D) indicates the class, the second (M, O, P, Q, or R) the family, and the third (A or B) the chain (α or β respectively) (4).

Meanwhile, the class III region (although less polymorphic) is also polygenic and more than 50 genes have been identified within it. Among the genes found, those encoding for factors C3, C4, and C5 of complement, heat shock proteins, and the TNF family including TNF-α are of interest (4).

Regulation of gene expression

Along with the CCAAT and TATA elements, which are involved in the binding and positioning of the basal transcription initiation complex, there are other relevant upstream DNA sequences in the HLA class I promoter region involved in the regulation of HLA class I gene expression. These conserved DNA sequences include the enhancer A element, the interferon-stimulated response element (ISRE), site α, and enhancer B element (Figure 4).

Figure 4

Schematic representation of the regulatory elements within the promoter of HLA class I genes. The Enhancer A (comprising the κB-binding sites), interferon-stimulated response element (ISRE), site α, Enhancer B, and CCAAT, and TATA elements (more...)

The enhancer A element, which encompasses two putative NF-kB binding sites (kB1 and kB2), plays an important role in the constitutive and cytokine-induced expression of HLA class I genes (5). For instance, the kB1 and kB2 sites of the HLA-A locus bind members of the NF-kB/rel family of transcription factors, e.g., p50, p65, and c-Rel. In addition, kB elements are also potential target sequences for zinc finger proteins such as MZF1, which bind with high affinity to the NF-kB binding sites of the HLA class I promoter.

Alternatively, HLA class I expression can be induced by members of the interferon (IFN) family (6). For instance, interferon regulatory factor 1 (IRF-1) acts as an activator of HLA class I transcription, whereas IRF-2 and IFN consensus sequence-binding protein (ICSBP) act as repressors of HLA class I transcription. In addition, IFN-γ induces the expression of the IRF-1 gene through the JAK/STAT pathway of signal transduction. IRF-1 subsequently binds to the conserved ISRE element in the HLA class I promoter, which is adjacent to the enhancer A element, thereby mediating the induction of HLA class I expression by IFN-γ.

Expression of HLA class II molecules is regulated at the transcriptional level by a complex process involving highly conserved sequences, which recruit specific binding factors and generate the HLA class II enhanceosome. At the DNA level, this regulatory unit consists of four sequences (W/S, X, X2, and Y boxes), the SXY module, which has been found in genes encoding the three human HLA class II isotypes (HLA-DP, HLA-DQ, and HLA-DR) (7) (Figure 5). The SXY module is also found in the promoters of the genes encoding invariant chain (Ii) and the non-classical HLA class II molecules, HLA-DM and HLA-DO. These are accessory proteins that are required for intracellular trafficking and peptide loading of HLA class II molecules. Moreover, sequences that resemble SXY module also contribute to the function of HLA class I promoters.

Figure 5

Schematic representation of the regulatory elements within the promoter of HLA class II genes. The W/S, X (comprising the X1 and X2 halves) and Y boxes together with the CCAAT, and TATA elements are indicated.

The Y box is ubiquitous in the genome and binds the heterotrimeric transcription factor NF-Y, which consists of NF-YA, NF-YB, and NF-YC subunits. NF-YB and NF-YC contain histone fold domains that are thought to bend the DNA at promoters, a process that may facilitate the efficient assembly of multiple transcription factors and provide easy access to RNA polymerase. The X2 box was found to bind to the AMP response element binding protein (CREB) which is important for the stability and assembly of the X1 box factor, and the regulatory factor X (RFX) at specific promoter SXY (8).

The main transacting factors that interact with the SXY module were identified in patients with Bare Lymphocyte Syndrome (BLS), a severe hereditary immunodeficiency disease in which there is defective synthesis of class II molecules. Mutations associated with this syndrome were found in genes encoding for RFX, RFX-associated ankyrin-containing proteins (RFXB) (9), and also for class II transactivator (CIITA) (10).

CIITA has been implicated in promoting transcription by various mechanisms: first, recruiting components such as transcription factor IID (TFIID) and TFIIB from the general transcription-initiation machinery; second, inducing phosphorylation of RNA polymerase II; third, interacting with the positive transcription elongation factor b (P-TEFb); and last, recruiting co-activators that alter chromatin accessibility by inducing histone acetylation. Briefly, CIITA does not bind DNA directly, but instead assembles at SXY promoters through direct interactions with the RFX-CREB-NF-Y complex (10,11). When bound, CIITA activates transcription through a potent acidic activation domain located in its N-terminal region. Through this domain, and other sequences, CIITA interacts with components of the basal transcription machinery. CIITA also interacts with three co-activators, including the CREB binding protein (CBP), p300 and p300/CBP associated factor (PCAF) (8). As a consequence, CIITA is known as the master control factor or master regulator of HLA class II genes and related genes (12) (Figure 6).

Figure 6

Regulation of the transcription of HLA class II genes. The SXY module that is mainly present in all classical HLA class II genes is bound cooperatively by four factors: the RFX factor, which is composed of RFX5, RFXAP, and RFXANK; the X2-box-binding factor (more...)

Origin and evolution of HLA

The acquired immunity and antigen presentation function of the HLA originates from ancestral vertebrates. Thus, the HLA is evolutionarily conserved, and this can be demonstrated not only at the functional level but also at the genetic level (13).

As was mentioned previously in the text, the HLA is an organized genetic system that encodes for a great variety of molecules that have key roles within biological pathways. Therefore, the expression and function of these molecules are strictly controlled. This control involves the action of chaperone molecules, which, despite their close relationship with the HLA function, are not located within the same gene complex. HLA generally contains genes which encode proteins whose function is closely related to each other. This clustering suggests that positive selection pressure occurred, and it would induce the binding of functionally related gene subsets. Moreover, this conservative selection pressure would prevent the separation of the loci within the system (13).

The HLA evolution has been explained by two successive duplications in the genome and their consecutive expansion. This hypothesis was proposed by Kasahara et al, after they discovered that the human genome contains at least three paralogous regions located on chromosomes 1, 9, and 19 of humans (14).

It is suggested that this evolutionary process took place by grouping the most basic functions and the most complex and specialized ones. Indeed, while the ancestral genes C3, C4, C5, and TNF (HLA III) acquired a role in both innate and acquired immunity, genes involved in antigen presentation such as HLA-A, -B, -C, -DR, -DQ, and-DP (HLA I and II) appear to have been generated more recently. For instance, molecules encoded in the HLA II became part of the acquired immunity system, which only appeared late in the evolution of the immune system with the advent of adaptive immunity. Hence, this late evolutionary progress shows the importance of higher immune system complexity and the requirement for an immune response that is not just more diverse but also more specific (15).

HLA characteristics

The HLA system is composed of three regions. The class I region corresponds to the genes coding for molecules HLA-A, -B, and -C. In addition, the class II region encodes HLA-DR, -DQ, and –DP. Finally, the class III region, in which genes are encoding for proteins of the complement system and TNF family genes. The function of HLA-encoded class I and class II molecules is to bind peptide antigens and display them for recognition by antigen-specific T lymphocytes. Peptide antigens associated with HLA class I molecules are recognized by CD8⁺ T Cells while HLA class II molecules are recognized by CD4⁺ T Cells (16).

Polymorphism

The HLA system contains the most polymorphic gene cluster in the entire human genome. To date, the IMGT/HLA Database has reported 9,154 HLA alleles (17) (Table 1 and Figure 7). Comparison of the sequences of alleles of the polymorphic class I and class II loci shows that nucleotide substitutions are concentrated in the exons that encode the peptide-binding groove and the site of interaction with the T Cell receptor (TCR). It is possible that these features on HLA sequences can be explained by selection pressures exerted by epidemics of infectious diseases because these pressures lead to the selection of the HLA alleles that have distinctive peptide-binding properties. However, this is an unproven hypothesis. Polymorphisms in HLA class I and HLA class II molecules affect which amino acids are in the peptide-binding groove and thus their binding specificity, but the more open structure of the HLA class II peptide-binding groove and the greater length of the peptides bound in it allow greater flexibility in peptide binding (18).

Table 1

Number of HLA Alleles.

Figure 7

Number of HLA class I and II alleles officially recognized between 1987 and 2013. Reprinted by permission from: Robinson J, et al. The IMGT/HLA database. Nucleic acids research. 41:D1222–7, copyright (2013)

The presence of multiple HLA alleles in the population will ensure that at least some individuals within a population will be able to recognize protein antigens produced by virtually any microbe, thus reducing the likelihood than a single pathogen can evade host defenses in all individuals in a given species.

Nomenclature of HLA alleles

The HLA system nomenclature is updated by an international committee (17). This nomenclature differs based on the detection method used. The HLA antigens, defined by serology, are designated by the denomination of the gene locus (e.g., HLA-A, HLA-DR) and followed by the numerical identification of the antigen (e.g., HLA-A1, HLA-DR1). The nomenclature of the C locus incorporates the letter “w” (e.g., HLA-Cw1, HLA-Cw2) to differentiate it from the complement system.

In contrast, the nomenclature of HLA alleles defined by molecular biology varies based on their class. For class I, the denomination HLA-A, HLA-B, and HLA-C is used to designate antigens defined by serology. An asterisk is added to define the method as being one used in molecular biology (e.g., HLA-A*), and two to eight digits are then added (e.g., HLA-A*02:01). The first two digits refer to antigen serological typing. The third and fourth are related to the denominations of specific alleles. The fifth and sixth describe allele variations, and the seventh and eighth represent variations at introns (5’ or 3’ gene regions).

For HLA class II, the procedure is not exactly the same. After the designation of the HLA and its gene locus, the letter “A” or “B” is added to represent the polymorphic α and β chains of the HLA-DR and HLA-DQ and only the letter “B” to represent the polymorphic β chain of the HLA-DP (e.g., HLA-DQA, HLA-DRB, HLA-DPB). Since some regions have several genes for the α and β chains, each locus receives a corresponding number (e.g., HLA-DQB1). Next, as defined for HLA class I, four to eight digits are added after an asterisk (e.g., HLA-DQB1*03:01).

In addition to the specific allele number, there are additional optional suffixes that may be added to an allele to indicate its expression status. Those alleles which have been shown to be alternatively expressed are defined in Table 2.

Table 2

Basis for HLA nomenclature alleles (17).

Structure of HLA class I and class II

Structure of HLA class I

HLA class I molecules consist of two non-covalently linked polypeptide chains, an HLA-encoded α chain or heavy chain (44 to 47 kD), and a non-HLA encoded subunit called β2microglobulin (12 kD) (19) (Figure 8). The α chain has three regions including a cytoplasmic region containing a peptide-binding groove made from the α1 and α2 domains, a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane, and a highly conserved α3 immunoglobulin-like domain to which CD8 binds (3).

Figure 8

Structure of HLA class I molecules. The schematic diagram illustrates the different regions of the HLA-A2 class I molecule. β2-microglobulin (β2m) is the light chain of the class I molecule. In addition, the α chain of the class (more...)

A peptide-binding groove is formed between the α1 and α2 helices with a β-pleated sheet as its floor. Usually, the groove will accommodate peptides of approximately 8–10 amino acids in length. There, they are ligated by a network of H-bonds between conserved α chain residues and the free amino acid carboxyl-terminus of the peptide. This allows peptides with different sequences at their termini to be bound the same way by different class I molecules (Figure 9). Nevertheless, peptides longer than 10 amino acids can occasionally bind class I molecules although the termini generally remain tucked into the cleft such that the central part of the peptide bulges upwards to accommodate the extra residues (19).

Figure 9

Peptide binding groove of HLA molecules. Crystal structures HLA molecules. A. Peptides bind to HLA class I molecules at each of their ends (upper panel). B. HLA class II molecules, the peptide extends beyond the peptide-binding groove and is held by interactions (more...)

The antigen-binding groove contains distinct pockets –A, B, C, D, E, and F– that vary in their chemical properties from one allele to another. There is a preferred sequence “motif” which generally contains two or three preferred primary anchor residues, and their amino acid side chains fit well into distinct pockets within the class I cleft where they provide significant energy binding (20). Dominant anchor sites usually occupy the B-pocket and F-pocket of class I molecules. The intervening residues between dominant anchor sites are much more varied in their side-chains. Moreover, the peptide often contains secondary anchor sites that contribute to HLA-binding but are usually more degenerate in the side chains they will accommodate at these sites and still support strong binding (21).

Furthermore, the antigen-binding groove is the region responsible for the high polymorphism of HLA class I molecules. Consequently, there will be changes in the electrostatic charge, hydrophobicity, and shape of the cleft which will thereby alter the peptide binding properties of allelic class I molecules (22).

Structure of HLA class II

Class II HLA molecules are composed of two non-covalently associated polypeptide chains, a 32 to 34 kD α chain, and a 29 to 32 kD β chain (23) (Figure 10). The genes encoding both chains of class II molecules are polymorphic and present in the HLA locus. Both chains have three regions including a cytoplasmic region containing a peptide-binding groove made up of the α1 and β1 domains, a transmembrane region containing hydrophobic amino acids by which the molecule is anchored in the cell membrane, and the highly conserved α2 and β2 domains to which TCR binds (3).

Figure 10

Structure of HLA class II molecules. The schematic diagram illustrates the different regions of the HLA-DR1 class II molecule. Each of the class II α and β chains has four domains: the peptide binding domain (α1 and β1), (more...)

A peptide binding groove is formed between the α1 and β1 domains with a β-pleated floor. Unlike class I molecules, the cleft of the class II molecules is open. As a result, class II molecules accommodate longer peptides than class I molecules do. Typically, the peptides bound to HLA class II molecules are 12 to 24 amino acids in length, but longer ones are not uncommon (20) (Figure 9). HLA class II molecules bind their peptides in an extended conformation with about a third of the peptide surface being accessible for interaction with the TCR. The termini of class II-bound peptides are not ligated by the same network of H-bonds that bind class I peptides so they may hang over the end of the cleft (23). The superantigens, in turn, bind class II as an interactive protein outside the conventional peptide antigen-binding site which explains their lack of restriction to any particular class II alleles (24).

Functions of HLA class I and class II

Class I and class II molecules are essential for T Cell-mediated adaptive immunity. The foreign antigens recognized by the TCR are peptides produced by intracellular protein degradation which are bound to class I or class II molecules on the surface of human cells. While degradation of foreign proteins to produce peptides is called antigen processing, the binding of peptides by HLA molecules to produce ligands for TCR is called antigen presentation (1).

When TCR recognizes HLA-associated peptides on an APC, several T Cell surface proteins and intracellular signaling molecules are rapidly mobilized to the site of T Cell-APC contact. This set of molecules includes TCR complex which consists of αβ TCR non-covalently linked to the CD3 and ζ proteins, CD4 or CD8 co-receptors, co-stimulatory molecules (e.g., B7 molecules that are involved in signal transduction), and accessory molecules (e.g., ICAM-1 and CD58, which are important in strengthening the adhesion between the T Cells and APCs).

The effector functions of CD8⁺ and CD4⁺ T Cells are different. CD8+ T Cells have a cytotoxic function that enables them to kill cells infected with viruses and cancer cells. CD4⁺ T Cells, in turn, have a wider range of effector functions, all of which involve the targeted delivery of cytokines. CD4⁺ T Cells are often called helper T Cells, and three distinct types of CD4⁺ T Cells have been distinguished: TH1 cells that secrete IFN-y, which mediate defense against intracellular microbes; TH2 cells that secrete IL-4 and IL-5, which favor IgE and eosinophil/mast T Cell-mediated immune reactions against helmints; and TH17 cells, which promote inflammation and mediated defense against extracellular fungi and bacteria.

In general, while endogenous antigens associated with HLA class I are recognized by CD8⁺ T Cells, exogenous peptides associate with HLA class II and are recognized by CD4⁺T Cells (Figure 11).

Figure 11

T cell and antigen presenting cell interaction. Schematic shows how the T cell co-receptors interact with HLA class I and HLA class II. T cell recognition of HLA-peptide complexes is aided by two surface glycoproteins called co-receptors: CD4 which binds (more...)

Antigen processing and presentation

HLA class I antigen presentation pathway

In general, foreign antigens presented by class I molecules are derived from intracellular infections caused by viruses, or from proteins synthesized in the cytosol, mature proteins which have fulfilled their cell cycle, or defective ribosomal products that are thought to be derived from poorly folded nascent proteins including a high proportion of all newly translated products in living cells, called DRIPs (25).

Assembly of class I molecules with antigenic peptides requires the coordination of multiple processes to first create peptides, then to transport and load them into the cleft of nascent class I molecules in the endoplasmic reticulum (ER) (26,27). Many of these polypeptides are ubiquitinated and are thus tagged for degradation by the proteasome (25). The peptides are transported into the ER by the adenosine triphosphate-dependent transporters associated with antigen processing (TAP) where they associate with heterodimers of HLA class I heavy chain and β2-microglobulin. The TAP-associated glycoprotein called tapasin functions to facilitate peptide loading of class I molecules in the ER. Tapasin bridges HLA class I molecules to TAP in association with the chaperone molecules calreticulin and the thioloxidoreductase ERp57. Together, these molecules (TAP, calreticulin, ERp57, and tapasin) make up the peptide loading complex. Likewise, Tapasin stabilizes the empty class I dimer by retaining it in the ER until peptide assembly by the peptide loading complex. When stably assembled, the HLA class I-peptide complex is transported to the cell surface via the ER and Golgi network to be recognized by the specific TCR on the CD8⁺ T Cell (27) (Figure 12 and Table 3).

Figure 12

Pathway by which intracellular antigens are processed and presented by HLA class I molecules. Proteins in the cytosol are degraded by proteosome to make small peptides which are transported by TAP protein into the lumen of ER. HLA class I molecules are (more...)

Table 3

Molecular chaperones involved in HLA class II antigen presentation.

HLA class II antigen presentation pathway

In general, the foreign antigens presented by class II molecules are derived from pathogens present in the extracellular spaces. APCs use specialized receptors to bind and internalize microbes in vesicles called phagosomes which may fuse with lysosomes to produce phagolysosomes or secondary lysosomes. Though this occurs less often, cytoplasmic and membrane proteins may be processed and displayed by HLA class II molecules. In this case, cytoplasmic proteins are trapped within membrane bound vesicles called autophagosomes. These vesicles fuse with lysosomes, and the cytoplasmic proteins are degraded by proteolysis. In both cases, degraded proteins are then able to bind to HLA class II molecules (25).

HLA class II α and β chains assemble in the ER with a non-polymorphic protein called invariant chain (Ii). The interaction with the Ii has the effect of stabilizing the structure of the HLA class II molecule while preventing the binding of peptides within the ER. Ii is anchored in the ER membrane, and the cytosolic portion of the molecule directs intracellular sorting of class II molecules through the Golgi to the HLA class II compartment (MIIC). Within MIIC, the Ii is degraded and can be replaced by a peptide derived from degradation in the endosomes or lysosomes of endocytosed material.

Afterwards, proteolytic enzymes such as cathepsins, which generate peptides from internalized proteins also act on the Ii to degrade it and leave only a 24 amino acid remnant called class II-associated invariant peptide (CLIP), which sits in the peptide binding groove (28). Then, CLIP is removed by the action of the HLA-DM. This HLA-DM action can be modulated by a second class II-related molecule called HLA-DO which synergises with HLA-DM under specific conditions. Complexes of HLA II and peptide are then taken to the plasma membrane where they can be recognized by CD4⁺ T Cells (29) (Figure 13 and Table 3).

Figure 13

Pathway by which extracellular antigens are processed and presented by HLA class II molecules. Extracellular proteins are taken into the cell by phagocytosis and are then degraded to peptides in endosomes and lysosomes. The peptides are then sorted into (more...)

Cross-presentation

In contrast to all other cells, APCs can present exogenous antigens to naïve CD8+ T lymphocytes. Two main intracellular pathways have been described for the cross-presentation of phagocytosed antigens (Figure 14):

Figure 14

Cross-presentation of antigens to CD8+ cells. After phagocytosis, exogenous antigens can be exported into the cytosol, where they are processed by the proteasome. The processed antigens can then be loaded on HLA class I molecules in the ER (the cytosolic (more...)

• Cytosolic pathway: Internalized proteins access the cytosol where they are degraded by the proteasome. Proteasome-generated peptides are transported to ER TAP-dependently. Then, peptides are cut by ER-associated aminopeptidase 1 (ERAP1) and endosomal insulin responsive aminopeptidase (IRAP) and, finally, loaded on HLA class I molecules (30).
• Vacuolar pathway: This is TAP-independent and insensitive to proteasome inhibitors. Internalized antigens are degraded into peptides by cathepsin S in the phagosome, and peptides are then loaded onto HLA class I molecules. Finally, the resulting complexes are transported to the cell surface through vesicular recycling. Note that there is evidence that the cytosolic pathway can also occur apart from TAP, possibly through another, as yet unidentified, peptide transporter (31).
• The origin of the HLA class I molecules involved in cross-presentation is also a matter of debate. Initially, it was suggested that cross-presenting HLA class I molecules originated from the plasma membrane and recycled to endosomes. A conserved tyrosine residue in their cytosolic tail which is required for internalization from the cell surface was shown to be crucial for cross-presentation. However, it was recently shown that CD74 promotes the trafficking of newly synthesized HLA class I molecules from the ER to endocytic compartments in DCs and that this routing is required for the cross-presentation of cell-associated antigens (32,33).

CD1 molecule and presentation of non-protein antigens

Regarding genetic structure, human CD1 is located on the long arm of chromosome 1 on band 1q23.1. The five CD1 genes that have been identified are designated CD1A, CD1B, CD1C, CD1D, and CD1E and correspond to five CD1 proteins: CD1a, CD1b, CD1c, CD1d, and CD1e (34). On the basis of sequence analysis, the CD1 isoforms can be classified into three groups: group 1 consists of CD1a, CD1b, and CD1c; group 2 consists of CD1d; and group 3 consists of CD1e.

All CD1 molecules produce heterodimers together with β2-microglobulin and are expressed on the cell surface (with the exception of CD1e which remains intracellular). Crystal analyses of CD1a, CD1b, and CD1d molecules have revealed a three dimensional structure resembling that of classical HLA class I molecules. CD1 proteins are organized into three extracellular domains (α1, α2, and α3), a transmembrane region, and a cytoplasmic tail. The membrane distal α1 and α2 domains adopt a conformation similar to that of other antigen-presenting molecules consisting of two antiparallel α-helices overlying a β-pleated sheet floor. These two distal domains are supported by the α3 domain that interacts with β2-microglobulin (35).

However, unlike HLA class I molecules, CD1 proteins bind alkyl chains in hydrophobic channels that reside beneath the surface of CD1 molecules. The hydrophilic head groups of the lipid antigens, in turn, protrude where the hydrophobic channels open to the membrane distal surface of the CD1 molecule. These head moieties are stabilized by hydrogen bonds, which also contribute to the correct positioning of the lipid antigens. Moreover, the different architecture of the antigen binding groove of different CD1 isoforms allows them to bind distinct lipid antigens (36) (Table 4).

Table 4

CD1-restricted lipid antigens (36).

CD1 trafficking in the endocytic pathway

Newly synthesized CD1 molecules have signal sequences for translocation into the lumen of the ER. Following synthesis, they rapidly become glycosylated to bind the ER chaperones calnexin and calreticulin (37). CD1d molecules, unlike CD1b, can move to the cell surface in the absence of β2-microglobulin. This difference is thought to be a result of the association of CD1d with the ERp57, which is important for the formation of disulfide bonds within the CD1 heavy chain prior to assembly with β2-microglobulin (38). Before leaving the ER, CD1 molecules are loaded with ER-resident lipids.

Following assembly in the ER, CD1b follows the secretory pathway through the Golgi directly to the plasma membrane (39). CD1d has also been observed to associate with HLA class II molecules and Ii chain, which can direct CD1 complexes with these proteins from the trans-Golgi network to endosomal compartments without first going to the plasma membrane (40).

After crossing the plasma membrane, CD1 molecules are internalized in endosomes. This clathrin-dependent pathway is common to proteins that contain well described tyrosine-based sorting motifs which bind adaptor protein complex 2 (AP2) and allow sorting of cargo proteins into clathrin-coated pits. In contrast to the other isoforms, CD1a does not contain any sorting motifs in its cytoplasmic tail, and yet, it is also internalized from the plasma membrane into endosomal compartments by an unknown mechanism (36).

After internalization by the early or sorting endosomes, the different CD1 isoforms follow different trafficking pathways. CD1a and CD1c head for the endocytic recycling compartment, whereas CD1b molecules move mainly through late endosomes and lysosomes. Thus, CD1b can bind both AP2, which mediates CD1 internalization from the plasma membrane, and AP3, which diverts these CD1 molecules from the early recycling pathway to late endosomes and lysosomes (41).

Lipid trafficking

Because of their hydrophobicity, lipids circulate in association with membranes or with lipid-transferproteins (LTP) (e.g., high density lipoproteins (HDL) and very low density lipoproteins (VLDL)). For instance, lipoproteins bound to Apolipoprotein E (ApoE) are internalized through specific receptors and reach the endosomal system. In acidic intracellular compartments, ApoE dissociates from VLDL and, then, associates with HDL and is secreted. During this exchange, intracellular lipids may become incorporated into membranes and remain in the cell or, instead, become associated with nascent HDL particles and thus be released into the extracellular space.

There are several ways exogenous lipids become internalized: through specific interaction of lipoproteins with lipoprotein-specific receptors, upon insertion into the plasma membrane, by binding of C-type lectins with mannose residues on glycolipids, or through internalization of apoptotic bodies (36).

The first mechanism involves lipoprotein-specific receptors which deliver lipids into clathrin coated pits allowing traffic through early recycling endosomes. Lipids such as sulfatide and sphingomyelin are internalized through clathrin- coated vesicles, then, reach late endosomes/lysosomes and are degraded. The second mechanism is related to internalization of extracellular lipids that associate with the plasma membrane. Glycosphingolipids such as gangliosides, globosides, and lactosylceramide are instead internalized clathrin-independently. The third mechanism involves the calcium-dependent binding of C-type lectins to carbohydrate moieties via their carbohydrate-recognition domains (CRDs).

Most C-type lectins bind mannosylated moieties which are present in several CD1 lipid antigens. Indeed, lipoarabinomannan has been shown to be internalized and delivered to late endosomal and lysosomal compartments by the macrophage mannose receptor. Finally, uptake of apoptotic cells is another important mechanism of lipid antigen internalization (42). During mycobacterial infection, phagocytic cells may die by apoptosis and apoptotic bodies are internalized by macrophages and dendritic cells which may simultaneously present both peptide and glycolipid antigens (43).

CD1a⁻, CD1b⁻, and CD1c-restricted T Cells were initially identified among the CD4⁻CD8− double negative T Cell subset but have now been recognized to be even more common in the CD4⁺ and CD8⁺ αβ TCR T Cell pool. Conversely, CD1d- restricted iNKT Cells comprise a large pool of T Cells that respond rapidly and display the features of innate immune cells. Overall, the antigen presentation pathway by CD1 molecules is highly important in infectious, tumoral, and autoimmune processes (41).

References

1.

Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. 7th ed. 2012. Cellular and Molecular Immunology; pp. 117–38.

2.

Dausset J. An interview with Jean Dausset. Am J Transplant. 2004;4:4–7. [PubMed: 14678028]

3.

Klein J, Sato A. The HLA system. First of two parts. N Engl J Med. 2000;343:702–9. [PubMed: 10974135]

4.

Goldsby RA, Kindt TJ, Osborne BA, Kuby J. Immunology. 5th ed. Mc Graw Hill; 2003. pp. 171–94.

5.

Gobin S, Keijsers V, Woltman A, Peijnenburg A, Wilson L, Van den Elsen P. Functional and Medical Implications. 1997;2:295–7.

6.

Zhou F. Molecular mechanisms of IFN-gamma to up-regulate MHC class I antigen processing and presentation. Int Rev Immunol. 2009;28:239–60. [PubMed: 19811323]

7.

Handunnetthi L, Ramagopalan SV, Ebers GC, Knight JC. Regulation of major histocompatibility complex class II gene expression, genetic variation and disease. Genes Immun. 2010;11:99–112. [PMC free article: PMC2987717] [PubMed: 19890353]

8.

Choi NM, Majumder P, Boss JM. Regulation of major histocompatibility complex class II genes. Curr Opin Immunol. 2011;23:81–7. [PMC free article: PMC3033992] [PubMed: 20970972]

9.

Masternak K, Barras E, Zufferey M, et al. A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients. Nat Genet. 1998;20:273–7. [PubMed: 9806546]

10.

Steimle V, Otten LA, Zufferey M, Mach B. Complementation cloning of an MHC class II transactivator mutated in hereditary MHC class II deficiency (or bare lymphocyte syndrome). Cell. 1993;75:135–46. [PubMed: 8402893]

11.

Lochamy J, Rogers EM, Boss JM. CREB and phospho-CREB interact with RFX5 and CIITA to regulate MHC class II genes. Mol Immunol. 2007;44:837–47. [PubMed: 16730065]

12.

Reith W, LeibundGut-Landmann S, Waldburger J-M. Regulation of MHC class II gene expression by the class II transactivator. Nat Rev Immunol. 2005;5:793–806. [PubMed: 16200082]

13.

Klein J, Figueroa F. Evolution of the major histocompatibility complex. Crit Rev Immunol. 1986;6:295–386. [PubMed: 3536303]

14.

Kasahara M. The chromosomal duplication model of the major histocompatibility complex. Immunol Rev. 1999;167:17–32. [PubMed: 10319248]

15.

Danchin E, Vitiello V, Vienne A, et al. The major histocompatibility complex origin. Immunol Rev. 2004;198:216–32. [PubMed: 15199965]

16.

Traherne JA. Human MHC architecture and evolution: implications for disease association studies. Int J Immunogenet. 2008;35:179–92. [PMC free article: PMC2408657] [PubMed: 18397301]

17.

Robinson J, Halliwell JA, McWilliam H, Lopez R, Parham P, Marsh SG. The IMGT/HLA database. Nucleic Acids Res. 2013;41:D1222–7. [PMC free article: PMC3531221] [PubMed: 23080122]

18.

Shiina T, Hosomichi K, Inoko H, Kulski JK. The HLA genomic loci map: expression, interaction, diversity and disease. J Hum Genet. 2009;54:15–39. [PubMed: 19158813]

19.

Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. Structure of the human class I histocompatibility antigen, HLA-A2. J Immunol. 2005;174:6–19. [PubMed: 15622565]

20.

Rammensee HG. Chemistry of peptides associated with MHC class I and class II molecules. Curr Opin Immunol. 1995;7:85–96. [PubMed: 7772286]

21.

Rammensee H-G. Peptides made to order. Immunity. 2006;25:693–5. [PubMed: 17098199]

22.

Deres K, Beck W, Faath S, Jung G, Rammensee HG. MHC/peptide binding studies indicate hierarchy of anchor residues. Cell Immunol. 1993;151:158–67. [PubMed: 8402926]

23.

Brown JH, Jardetzky TS, Gorga JC, et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature. 1993;364:33–9. [PubMed: 8316295]

24.

Jardetzky TS, Brown JH, Gorga JC, et al. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature. 1994;368:711–8. [PubMed: 8152483]

25.

Pamer E, Cresswell P. Mechanisms of MHC class I--restricted antigen processing. Annu Rev Immunol. 1998;16:323–58. [PubMed: 9597133]

26.

Heemels MT, Ploegh H. Generation, translocation, and presentation of MHC class I-restricted peptides. Annu Rev Biochem. 1995;64:463–91. [PubMed: 7574490]

27.

Williams A, Peh CA, Elliott T. The cell biology of MHC class I antigen presentation. Tissue antigens. 2002;59:3–17. [PubMed: 11972873]

28.

Ghosh P, Amaya M, Mellins E, Wiley DC. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature. 1995;378:457–62. [PubMed: 7477400]

29.

Morris P, Shaman J, Attaya M, et al. An essential role for HLA-DM in antigen presentation by class II major histocompatibility molecules. Nature. 1994;368:551–4. [PubMed: 8139689]

30.

Rock KL, Farfán-Arribas DJ, Shen L. Proteases in MHC class I presentation and cross-presentation. J Immunol. 2010;184:9–15. [PMC free article: PMC3094101] [PubMed: 20028659]

31.

Merzougui N, Kratzer R, Saveanu L, Van Endert P. A proteasome-dependent, TAP-independent pathway for cross-presentation of phagocytosed antigen. EMBO Rep. 2011;12:1257–64. [PMC free article: PMC3245693] [PubMed: 22037009]

32.

Lizée G, Basha G, Tiong J, et al. Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain. Nat Immunol. 2003;4:1065–73. [PubMed: 14566337]

33.

Basha G, Omilusik K, Chavez-Steenbock A, et al. A CD74-dependent MHC class I endolysosomal cross-presentation pathway. Nat Immunol. 2012;13:237–45. [PMC free article: PMC4933585] [PubMed: 22306692]

34.

Dascher CC. Evolutionary biology of CD1. Curr Top Microbiol Immunol. 2007;314:3–26. [PubMed: 17593655]

35.

Brigl M, Brenner MB. CD1: antigen presentation and T Cell function. Annu Rev Immunol. 2004;22:817–90. [PubMed: 15032598]

36.

Barral DC, Brenner MB. CD1 antigen presentation: how it works. Nat Rev Immunol. 2007;7:929–41. [PubMed: 18037897]

37.

Sugita M, Porcelli SA, Brenner MB. Assembly and retention of CD1b heavy chains in the endoplasmic reticulum. J Immunol. 1997;159:2358–65. [PubMed: 9278326]

38.

Kang SJ, Cresswell P. Calnexin, calreticulin, and ERp57 cooperate in disulfide bond formation in human CD1d heavy chain. J Biol Chem. 2002;277:44838–44. [PubMed: 12239218]

39.

Briken V, Jackman RM, Dasgupta S, Hoening S, Porcelli SA. Intracellular trafficking pathway of newly synthesized CD1b molecules. EMBO J. 2002;21:825–34. [PMC free article: PMC125873] [PubMed: 11847129]

40.

Kang S-J, Cresswell P. Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J. 2002;21:1650–60. [PMC free article: PMC125936] [PubMed: 11927549]

41.

Silk JD, Salio M, Brown J, Jones EY, Cerundolo V. Structural and functional aspects of lipid binding by CD1 molecules. Annu Rev Cell Dev Biol. 2008;24:369–95. [PubMed: 18593354]

42.

Schaible UE, Winau F, Sieling PA, et al. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med. 2003;9:1039–46. [PubMed: 12872166]

43.

Mori L, De Libero G. Presentation of lipid antigens to T Cells. Immunol Lett. 2008;117:1–8. [PubMed: 18243339]