Basics of Immunology - A Course of Lectures by M. V. Skok - Kyiv 2002

Chapter II. Mechanisms of the Immune Response

Lecture 6. The Major Histocompatibility Complex. Antigen Processing and Presentation

The 1980 Nobel Prize in Physiology or Medicine was awarded to Baruj Benacerraf, Jean Dausset, and George D. Snell "for their discoveries concerning genetically determined structures on The Cell surface that regulate immunological reactions."

In the 1960s, B. Benacerraf observed that when immunized with the same antigen, animals even within a single population responded differently: some reacted by synthesizing Antibodies, while others did not. The GENETIC BASIS OF this phenomenon was discovered, and the genes determining whether an Immune Response would develop were designated as IR (immune response) genes. In 1965, Hugh McDevitt identified the MAJOR HISTOCOMPATIBILITY COMPLEX genes and their products, which had been first described by G. Snell as transplantation Antigens. Finally, Jean Dausset discovered a similar Gene complex in humans.

It was subsequently elucidated that the GENES OF THE major histocompatibility complex (MHC) are divided into two classes: MHC I comprises The genes of transplantation antigens, whereas MHC II comprises the genes of the immune response. They also came to be called Ia (immune associated) genes. It turned out that the products of these genes are necessary for antigen presentation during immune recognition and determine the so-called phenomenon of genetic restriction of the immune response. This phenomenon implies that the Cells that recognize the antigen (T lymphocytes) and the cells that present it (macrophages, dendritic cells, target cells in the case of a cytotoxic response) must be genetically identical, that is, originate from the same Organism or from animals of the same genetic line. In other words, T lymphocytes recognize an antigen only in a complex with syngeneic MHC products.

In the 1980s, X-Ray Structural Analysis of MHC I and II Proteins was performed, and the mechanisms of their Biosynthesis and functioning were elucidated.

Structure of MHC Genes and Their Products.

The MHC genes of mice and humans are the most extensively studied.

In the mouse, the MHC gene complex is designated as H-2 and includes the H-2K, H-2D, H-2L loci (MHC I) and the H-2A and H-2E loci (MHC II). In humans, this complex is designated as HLA and includes, respectively, the A, B, C loci (MHC I) and the DP, DQ, DR loci (MHC II). Among the MHC II genes, There are also the DM-A, DM-B, LMP, and TAP genes, which encode products important for ANTIGEN Processing AND presentation. Genes encoding Complement system C2, C4, and factor B are also located within the MHC complex and are designated as MHC III.

MHC genes are characterized by polygeny and polymorphism. Polygeny means that for each Class (I and II), there are multiple loci (A, B, C or DP, DQ, DR), which facilitates the efficient presentation of structurally diverse antigens in each individual. Polymorphism of MHC genes lies in the existence of numerous variants (alleles) whose products differ by up to 35% in their Amino Acid Composition, which increases the probability of survival upon encounter with a specific pathogen at the population level. Polymorphism is achieved through point Mutations, recombinations, homologous Crossing-over, and Gene Conversion. Unlike immunoglobulin genes, MHC genes are not subject to allelic exclusion, meaning that in every heterozygous organism, all possible MHC alleles are expressed.

Each of the MHC I gene loci encodes a single polypeptide chain—the α-chain. Each of the MHC II gene loci encodes two polypeptide chains: α and β. They are designated as DP_α, DP_β; DQ_α, DQ_β; DR_a, DR_β in humans and, correspondingly, as A_α, A_β; E_α, E_β in the mouse. In a heterozygote, each chain is represented by two alleles, meaning that four variants of MHC II proteins are possible for each locus (eight variants for DR, since There are two genes for the β-chain), and each cell thus bears 16 variants of MHC II proteins on its surface. E_α, DP_α, and DR_α are less polymorphic than the genes of other loci.

As we can see, MHC polygeny is somewhat similar to the polygeny of IMMUNOGLOBULINS, but the DIVERSITY OF IMMUNOGLOBULINS and MHC proteins is achieved through different pathways: in immunoglobulins, through combinations of genes in a multigene system, and in MHC, through the presence of various allelic variants at each locus. The Diversity of immunoglobulins is higher than the polymorphism of MHC proteins. Despite this, each individual possesses a unique set of MHC genes. MHC I gene products function as transplantation antigens and determine the impossibility of organ or tissue transplantation across species barriers. Susceptibility to certain diseases is associated with specific MHC II variants (type I Diabetes Mellitus, multiple sclerosis, myasthenia gravis).

MHC genes are expressed to varying degrees in the Cells of the organism. MHC I molecules are expressed on almost all Cells and Tissues (with the exception of some where the expression level is very low, such as the Cytology/practical/76.html">Cornea of the eye; which is why this tissue is quite easily transplanted). MHC II molecules are expressed only on certain cells—those capable of presenting an antigen in the immune response. These are primarily macrophages, dendritic cells, and B lymphocytes. MHC II expression is also found on activated human T Lymphocytes and on certain cells capable of presenting antigens in specialized tissues: Brain glial astrocytes, epithelial cells, and neutrophils.

The MHC I gene product is a heterodimer consisting of a heavy (43 kDa) α-chain and β₂-microglobulin (11 kDa), the gene for which is not part of the MHC gene complex. The heavy chain consists of three domains (α₁, α₂, α₃), a transmembrane region, and a cytoplasmic C-terminus (Fig. 7A). The α₃ domain and β₂-microglobulin are homologous to immunoglobulin domains, whereas the N-terminal α₁ and α₂ domains are polymorphic (i.e., it is here that the Amino Acid Substitutions distinguishing allelic forms of MHC I proteins are localized).

The MHC II gene product is also a heterodimer consisting of α and β chains (34 and 28 kDa, respectively). Each chain has two extracellular domains at the N-terminus, a transmembrane region, and a cytoplasmic region, and the overall structure resembles that of the MHC I protein (Fig. 7B). The α₁ and β₁ domains are polymorphic, whereas α₂ and β₂ are immunoglobulin-like.

X-ray structural Analysis of the MHC I protein was performed in 1987. The extracellular portion of HLA-A2 was cleaved with Papain and crystallized. The analysis revealed that the membrane-proximal domains, α₃ and β₂-microglobulin, closely resemble immunoglobulin domains, whereas the external ones—α₁ and α₂—resemble each other and form a cleft at the top of the molecule. By introducing amino acid substitutions (mutations) into various PARTS OF THE molecule, it was established that the residues interacting with the T-lymphocyte receptor are located On the surface of the MHC I molecule, while those participating in the binding of the antigenic peptide reside within the cleft. The cleft, shaped like a closed pocket, was found to be occupied by the antigenic peptide presented by the given MHC molecule (Fig. 8A). It was calculated that the optimal length of the peptide fitting into the pocket is 8-9 amino acid residues. In different allelic forms of MHC I, the shape of the pocket varies, and polymorphic residues accordingly bind Peptides of different structures.

X-ray structural analysis of the MHC II protein was accomplished only six years later, in 1993. The structure proved to be very similar to MHC I, but two important differences were found:

1) instead of a closed pocket where the antigenic peptide binds, a groove was discovered in which a peptide of 15-16 amino acid residues is accommodated in an extended conformation (Fig. 8B);

2) unlike MHC I, MHC II crystallized as a dimer (a dimer of heterodimers). It is believed that dimerization promotes greater avidity in The formation of the antigenic peptide – MHC II – T-cell receptor complex.

Antigen Processing and Presentation. The Dichotomy of Cellular and Humoral Responses.

As noted above, T lymphocytes recognize an antigen only in a complex with MHC proteins. In the course of a cellular immune response (for example, for the destruction of virus-infected host cells or transplanted allogeneic cells), the antigen is recognized in a complex with MHC I, whereas for a HUMORAL IMMUNE RESPONSE (antibodies against Bacteria and soluble toxins), the antigen must be presented with MHC II. To bind to MHC proteins, a protein antigen is degraded into short peptides (8-9 amino acid residues for MHC I and 12-15 residues for MHC II). This process is termed antigen processing.

How the immune system decides whether an antigen is to be presented with MHC I or MHC II and, accordingly, whether a humoral or cellular response develops, remained unclear for a long time. It was believed that the dose of the antigen, its form, and the route of administration into the organism mattered. A decisive experiment was conducted in 1986 by Morrison and Braciale. They derived lines of T lymphocytes specific to Influenza virus that recognized their antigen in a complex with MHC I (CTL I) or with MHC II (CTL II). It was found that CTL I destroyed only cells infected with live virus—that is, when viral antigens were synthesized inside the cell—whereas CTL II also destroyed cells when the virus was inactivated, meaning it was passively engulfed by the cell. It was concluded that endogenous (internal) antigens are presented with MHC I, while exogenous (external) ones are presented with MHC II.

These data were confirmed in numerous subsequent studies. The question arose as to where the binding of the antigen to MHC proteins takes place. It was logical to suggest that endogenous antigens bind to MHC I inside the cell, and exogenous antigens to MHC II on the outside. There was even a hypothesis that MHC II proteins are the primary receptors for antigens during an immune response. However, in the late 1980s, it became clear that free MHC proteins are extremely unstable and degrade rapidly, meaning they cannot exist on the cell surface in a free state. All events regarding the binding of the antigenic peptide, for both MHC I and MHC II, occur inside the cell along the pathway from The biosynthesis of MHC proteins to their expression on the membrane. There is a specialized system of chaperone proteins that retains MHC I and II chains inside the cell until they have bound an antigenic peptide, as well as a specialized system that ensures free MHC molecules do not appear on the surface. This system is Temperature-dependent: if the temperature of cell cultivation is lowered

to 26 - 28^oC (this is possible, for instance, when MHC molecules are artificially expressed in Drosophila cells), free MHC dimers appear on the surface.

Biosynthesis of MHC class I proteins (Fig. 9).

The MHC class I α-chain and β₂-microglobulin are synthesized separately and translocated into The Endoplasmic reticulum (ER). The α-chain is associated with the chaperone calnexin. Upon binding to β₂-microglobulin, the complex is transferred to another chaperone, calreticulin, and remains bound to it until an antigenic peptide is attached. Peptides enter the ER from the Cytosol, where they are generated through the processing of endogenous antigens. These may be degradation products of self-proteins, or viral and bacterial antigens synthesized intracellularly. Degradation is mediated by a specialized proteasome. The proteasome is a large multienzyme complex (20S, 700 kDa) composed of 15 subunits of two types, α and β, each 21–31 kDa in size. The subunits are arranged in a barrel-like structure. The peptide (protein) passes through the "barrel" and is cleaved by the proteasomal β subunits. The transport of antigenic peptides from the Cytoplasm to the ER is carried out by specialized transmembrane proteins known as TAPs (transporters of antigenic peptides). The proteasomal proteins are encoded by the LMP2 and LMP7 genes, which reside within the MHC gene complex alongside the TAP genes, with LMP2 and TAP sharing a common promoter. Peptides translocated by TAP associate with the α-chain/β₂-microglobulin complex with the assistance of the protein tapasin. Notably, additional proteolytic trimming of the peptide can occur even after binding to MHC class I. Subsequently, the MHC class I-peptide complex dissociates from chaperones and migrates in a vesicle toward The Plasma Membrane. En route, it passes through the Golgi apparatus, where it undergoes glycosylation. Initial hypotheses regarding antigenic peptide binding pointed to the Golgi apparatus as the primary site of interaction. However, experiments utilizing brefeldin A—a specific antibiotic that blocks protein export from the ER to the Golgi—demonstrated that the peptide binds to MHC class I prior to leaving the ER.

Biosynthesis of MHC class II proteins (Fig. 10).

The α and β chains, as well as a specialized invariant chain (Ii), are synthesized in the ER. The invariant chain is produced in two alternative isoforms, 31 and 41 kDa, resulting from alternative RNA splicing. The invariant chain Functions as a surrogate antigenic peptide. Its key functions are:

1) blocking the peptide-binding groove of MHC class II to prevent premature binding of endogenous peptides;

2) facilitating the release of MHC class II from ER chaperones;

3) directing the entire complex to the endosome.

Thus, the MHC class II complex—comprising the α, β, and invariant chains—leaves the ER, passes through the Golgi apparatus for glycosylation, and is targeted to the endosomal compartment, where it encounters the antigen.

Exogenous antigens enter the endosome via endocytosis. Various cell types that present exogenous antigens are capable of different forms of endocytosis. Macrophages perform phagocytosis, dendritic cells engage in macropinocytosis, and B lymphocytes utilize receptor-mediated endocytosis, which is the most efficient and specific mechanism. In all cases, the antigen is first engulfed by invaginations of the plasma membrane and subsequently internalized into vesicles coated externally with the protein clathrin. These vesicles move inward, fuse with existing intracellular compartments, and undergo a series of transformations: proton pumps are incorporated, lowering the pH; and Proteolytic Enzymes with an acidic pH optimum, known as cathepsins, become active. The final destination of this maturation pathway is the lysosome, where proteins are degraded down to Amino Acids. Along this pathway, a specialized compartment is formed where the antigenic peptide encounters the MHC class II molecule trafficking from the ER. This compartment is termed the MIIC (MHC class II compartment). In the MIIC, exogenous antigens already exist as a mixture of peptides. It was initially believed that the invariant chain simply dissociates from the α/β dimer under acidic pH conditions, allowing the antigenic peptide to take its place. It was later discovered that the invariant chain is first cleaved by proteases, leaving only a small peptide fragment within the MHC class II groove—known as CLIP (class II-associated invariant chain peptide, residues 81–105). In effect, CLIP acts as a surrogate antigenic peptide, and its 92–105 region inhibits the binding of other peptides within the MHC class II groove. The exchange of CLIP for an antigenic peptide is mediated by a specialized protein dimer, HLA-DM (composed of DM-α and DM-β chains, the genes for which are also located within the MHC locus). This protein stabilizes the intermediate MHC class II structures formed after CLIP dissociation, facilitating the binding of an exogenous antigenic peptide present in the same vesicle. If peptide binding fails, the α/β dimer aggregates and is degraded by endogenous proteases. It is worth noting that the alternative isoforms of the invariant chain (31 and 41 kDa) differ in their resistance to proteolysis: the heavier isoform contains a cathepsin L inhibitor domain. Consequently, different Regions of the invariant chain encode distinct functional signals: trimerization (residues 163–183), endosomal targeting (cytoplasmic tail), MHC class II groove binding (residues 92–105), and cathepsin L inhibition.

From the foregoing Discussion, it becomes clear that the dichotomy in presenting endogenous versus exogenous antigens—which underlies the fundamental division into humoral and cellular immune responses—is rooted in the distinct biosynthetic and Metabolic pathways of MHC class I and class II molecules.

However, there are exceptions to every rule. For instance, endogenous antigens can occasionally be presented via MHC class II. This occurs when such antigens are synthesized in Abundance (overexpression), triggering a salvage pathway that transports cytosolic proteins to the endosomes. Evidence suggests that the transport proteins in this scenario are heat Shock proteins (HSPs)—molecular chaperones whose intracellular concentrations rise markedly under cellular stress, such as heat shock or nutrient deprivation. The genes encoding heat shock proteins and MHC molecules are closely linked, and their protein products share structural similarities. For example, the Introduction/19.html">Primary Structure of the α1 and β1 domains of MHC molecules strongly resembles that of heat shock proteins. Both Protein Families are adapted for binding and transporting peptides: Hsp70 proteins facilitate the transport of defective intracellular proteins—which have failed quality control checkpoints and are targeted for degradation—to Lysosomes. Furthermore, Hsp70 proteins possess binding sites for clathrin, the coat protein that surrounds endosomes and lysosomes. Through this mechanism, self-proteins or viral proteins from the cytoplasm can gain access to endosomes and associate with MHC class II molecules.

Conversely, exogenous antigens can occasionally be presented via MHC class I molecules. This process, known as cross-presentation, occurs in dendritic cells with the assistance of tapasin and plays a crucial role in mounting immune responses against viral infections. This indicates that a retrograde transport pathway must also exist for moving antigenic peptides from endosomes back into the cytoplasm.

Lipid antigens and hydrophobic Proteins can be presented for recognition by T lymphocytes in the context of cell-surface CD1 molecules. It has been established that while this presentation pathway also utilizes endocytosis, its processing does not require HLA-DM, HLA-AP, or TAP. The antigen-binding groove of CD1 is notably rich in hydrophobic amino acid residues.

Beyond presenting antigenic peptides to T lymphocytes, MHC molecules perform several other vital functions. Regardless of the specific peptide presented, they serve as ligands for receptors on T lymphocytes and natural killer cells, acting as unique molecular signatures recognized, for instance, during graft rejection. They participate in shaping the T-cell repertoire during ontogeny (a topic to be covered in a separate lecture). MHC molecules also exhibit independent receptor functions; upon contacting a T lymphocyte, the antigen-presenting cell receives intracellular signals that can trigger its proliferation, differentiation, or apoptosis. Finally, MHC molecules determine the distinct individual odor of each member of a population, the recognition of which plays a key role in mate Selection and in preventing Inbreeding.

Summary

Immune recognition of antigens begins with their presentation to immune cells in complex with proteins encoded by the major histocompatibility complex (MHC). There are two main classes of Histocompatibility Antigens: MHC class I and class II. MHC class I proteins consist of a three-domain α-chain and β₂-microglobulin, whereas MHC class II proteins are composed of two two-domain chains (α and β), though their overall three-dimensional structures are remarkably similar. The peptide-binding groove forms a closed pocket in MHC class I molecules and an open-ended cleft in MHC class II molecules. MHC class I proteins typically present endogenous (intracellular) antigens, whereas MHC class II proteins present exogenous (extracellular) antigens. The generation of antigenic peptides from precursor proteins is termed antigen processing. Endogenous antigen processing occurs in the cytoplasm via the proteasome complex, whereas exogenous antigen processing takes place in endosomes with the help of endosomal enzymes, particularly cathepsins. The loading of MHC class I and class II molecules with antigenic peptides occurs along their biosynthetic pathways: within the ER for MHC class I and endogenous peptides, and within endosomes for MHC class II and exogenous peptides. MHC class I molecules are expressed on virtually all nucleated somatic cells, whereas MHC class II molecules are restricted to specialized professional antigen-presenting cells. Endogenous antigens presented via MHC class I predominantly stimulate cellular immune responses, whereas exogenous antigens presented via MHC class II drive humoral immune responses.