C-9: IMMUNOLOGY UNIT-III Major Histocompatibility complexes class I & class II MHC , processing and presentation. NOTES Major Histocompatibility Complex MHC complex is a large genomic region or group of genes found in most vertebrates on a single chromosome that codes the MHC molecules which plays a vital role in immune system. Major histocompatibility antigens (also called transplantation antigens) mediate rejection of grafts between two genetically different individuals. HLA (human leukocyte antigens) were first detected on leukocytes and so they are called MHC antigens of humans. H-2 antigens are their equivalent MHC antigens of mouse. A set of MHC alleles present on each chromosome is called an MHC haplo-type.

Monozygotic human twins have the same histocompatibility molecules on their cells, and they can accept transplants of tissue from each other. Histocompatibility molecules of one individual act as antigens when introduced into a different individual. George Snell, Jean Dausset and Baruj Benacerraf received the Nobel Prize in 1980 for their contributions to the discovery and understanding of the MHC in mice and humans MHC gene products were identified as responsible for graft rejection.

MHC gene products that control immune responses are called the immune response genes. Immune response genes influence responses to infections. The essential role of the HLA antigens lies in the induction and regulation of the immune response and defence against microorganisms. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes.

There are two general classes of MHC molecules – Class I and Class II. Class I MHC molecules are found on all nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found on certain immune cells themselves, chiefly macrophages, B cells and dendritic cells, collectively known as professional antigen-presenting cells (APCs). These APCs specialize in the uptake of pathogens and subsequent processing into peptide fragments within phagosomes. The Class II MHC molecules on APCs present these fragments to helper T cells, which stimulate an immune reaction from other cells.

2. Structure of Major Histocompatibility Complex (MHC): The MHC complex resides in the short arm of and overall size of the MHC is approximately 3.5 million base pairs. The complete three-dimensional structure for both class I and class II MHC molecules has been determined by x-ray crystallography. The class I gene complex contains three loci A, B and C, each of which codes of α chain polypeptides.

The class II gene complex also contains at least three loci, DP, DQ and DR; each of these loci codes for one α and a variable number of β chain polypeptides. Class III region is not actually a part of the HLA complex, but is located within the HLA region, because its components are either related to the functions of HLA antigens or are under similar control mechanisms to the HLA genes. Class III antigens are associated with proteins in serum and other body fluids (e.g., C4, C2, factor B, TNF) and have no role in graft rejection. 5. Classification of MHC Molecules: 1. MHC Class I Molecule: MHC Class I is a membrane spanning molecule composed of two proteins. The membrane spanning protein is approximately 350 amino acids in length, with about 75 amino acids at the carboxylic end comprising the trans-membrane and cytoplasmic portions. The remaining 270 amino acids, as shown in the diagram, are divided into three globular domains Alpha-1, Alpha- 2 and Alpha-3 prime, with alpha-1 being closest to the amino terminus and alpha-3 closest to the membrane.

The second portion of the molecule is a small globular protein called Beta-2 Micro-globulin. It associates primarily with the alpha-3 prime domain and is necessary for MHC stability. The bound peptide sits within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity.

The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors.

2. MHC Class II Molecule: Although similar to Class I, the MHC Class II molecule is composed of two membrane spanning proteins. Each chain is approximately 30 kilodaltons in size, and made of two globular domains as shown in the diagram. The domains are named Alpha-1, Alpha-2, Beta-1 and Beta-2. The two regions farthest from the membrane are alpha-1 and beta-1. The two chains associate without covalent bonds.

The bound peptide is within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity. The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors. Class II molecules are dimers consisting of an alpha and beta polypeptide chain.

Each chain contains an immunoglobulin like region, next to the cell membrane. The antigen binding cleft, composed of two alpha-helices above a beta-pleated sheet, specifically binds short peptides, about 15 to 24 residues long. The amino acid sequence around the binding site, which specifies the antigen binding properties, is the most variable site in the MHC molecule.

Differences between Class I and Class II structures can explain the different length requirements for the bound peptide. The ends of the antigen binding cleft of Class I molecules taper and are blocked by bulky tyrosine that bind the N terminus of the peptide. These conserved residues are not found in Class II molecules where smaller residues (glycine or valine) replace the larger tyrosine.

3. MHC Class III Molecule: This class includes genes coding several secreted proteins with immune functions-components of the complement system (such as C2, C4 and B factor) and molecules related with inflammation (cytokines such as TNF-α, LTA, LTB) or heat shock proteins (hsp). Class-III

000002 molecules do not share the same function as class-I and II molecules, but they are located between them in the short arm of human chromosome 6. For this reason they are frequently described together.

Fig. MHC I are found on all nucleated body cells, and MHC II are found on macrophages, dendritic cells, and B cells (along with MHC I). The antigen-binding cleft of MHC I is formed by domains α1 and α2. The antigen-binding cleft of MHC II is formed by domains α1 and β1.

Antigen-Presenting Cells (APCs)

All nucleated cells in the body have mechanisms for processing and presenting antigens in association with MHC molecules. This signals the immune system, indicating whether the cell is normal and healthy or infected with an intracellular pathogen. However, only macrophages, dendritic cells, and B cells have the ability to present antigens specifically for the purpose of activating T cells; for this reason, these types of cells are sometimes referred to as antigen- presenting cells (APCs).

While all APCs play a similar role in adaptive immunity, there are some important differences to consider. Macrophages and dendritic cells are phagocytes that ingest and kill pathogens that penetrate the first-line barriers (i.e., skin and mucous membranes). B cells, on the other hand, do not function as phagocytes but play a primary role in the production and secretion of antibodies. In addition, whereas macrophages and dendritic cells recognize pathogens through nonspecific receptor interactions (e.g., PAMPs, toll-like receptors, and receptors for opsonizing complement or antibody), B cells interact with foreign pathogens or their free antigens using antigen-specific immunoglobulin as receptors (monomeric IgD and IgM). When the immunoglobulin receptors bind to an antigen, the B cell internalizes the antigen by endocytosis before processing and presentting the antigen to T cells.

Antigen Presentation with MHC II Molecules

MHC II molecules are only found on the surface of APCs. Macrophages and dendritic cells use similar mechanisms for processing and presentation of antigens and their epitopes in association with MHC II; B cells use somewhat different mechanisms that will be described

000003 further in B Lymphocytes and Humoral Immunity. For now, we will focus on the steps of the process as they pertain to dendritic cells.

After a dendritic cell recognizes and attaches to a pathogen cell, the pathogen is internalized by phagocytosis and is initially contained within a phagosome. Lysosomes containing antimicrobial enzymes and chemicals fuse with the phagosome to create a phagolysosome, where degradation of the pathogen for antigen processing begins. Proteases (protein- degrading) are especially important in antigen processing because only protein antigen epitopes are presented to T cells by MHC II.

APCs do not present all possible epitopes to T cells; only a selection of the most antigenic or immune dominant epitopes are presented. The mechanism by which epitopes are selected for processing and presentation by an APC is complicated and not well understood; however, once the most antigenic, immunodominant epitopes have been processed, they associate within the antigen-binding cleft of MHC II molecules and are translocated to the cell surface of the dendritic cell for presentation to T cells.

Figure .A dendritic cell phagocytoses a bacterial cell and brings it into a phagosome. Lysosomes fuse with the phagosome to create a phagolysosome, where antimicrobial chemicals and enzymes degrade the bacterial cell. Proteases process bacterial antigens, and the most antigenic epitopes are selected and presented on the cell’s surface in conjunction with MHC II molecules. T cells recognize the presented antigens and are thus activated.

Antigen Presentation with MHC I Molecules

MHC I molecules, found on all normal, healthy, nucleated cells, signal to the immune system that the cell is a normal “self” cell. In a healthy cell, proteins normally found in the cytoplasm are degraded by proteasomes (enzyme complexes responsible for degradation and processing of proteins) and processed into self-antigen epitopes; these self-antigen epitopes bind within

000004 the MHC I antigen-binding cleft and are then presented on the cell surface. Immune cells, such as NK cells, recognize these self-antigens and do not target the cell for destruction. However, if a cell becomes infected with an intracellular pathogen (e.g., a virus), protein antigens specific to the pathogen are processed in the proteasomes and bind with MHC I molecules for presentation on the cell surface. This presentation of pathogen-specific antigens with MHC I signals that the infected cell must be targeted for destruction along with the pathogen.

Before elimination of infected cells can begin, APCs must first activate the T cells involved in cellular immunity. If an intracellular pathogen directly infects the cytoplasm of an APC, then the processing and presentation of antigens can occur as described (in proteasomes and on the cell surface with MHC I). However, if the intracellular pathogen does not directly infect APCs, an alternative strategy called cross-presentation is utilized. In cross-presentation, antigens are brought into the APC by mechanisms normally leading to presentation with MHC II (i.e., through phagocytosis), but the antigen is presented on an MHC I molecule for CD8 T cells. The exact mechanisms by which cross-presentation occur are not yet well understood, but it appears that cross-presentation is primarily a function of dendritic cells and not macrophages or B cells.

Antigen processing Antigen processing, or the cytosolic pathway, is an immunological process that prepares antigens for presentation to special cells of the immune system called T lymphocytes. It is considered to be a stage of antigen presentation pathways. This process involves two distinct pathways for processing of antigens from an organism's own (self) proteins or intracellular pathogens (e.g. viruses), or from phagocytosed pathogens (e.g. bacteria); subsequent presentation of these antigens on class I or class II major histocompatibility complex (MHC) molecules is dependent on which pathway is used. Both MHC class I and II are required to bind antigen before they are stably expressed on a cell surface. MHC I antigen presentation typically (considering cross-presentation) involves the endogenous pathway of antigen processing, and MHC II antigen presentation involves the exogenous pathway of antigen processing. Cross-presentation involves parts of the exogenous and the endogenous pathways but ultimately involves the latter portion of the endogenous pathway (e.g. proteolysis of antigens for binding to MHC I molecules). While the joint distinction between the two pathways is useful, there are instances where extracellular-derived peptides are presented in the context of MHC class I and cytosolic peptides are presented in the context of MHC class II (this often happens in dendritic cells).

The endogenous pathway The endogenous pathway is used to present cellular peptide fragments on the cell surface on MHC class I molecules. If a virus had infected the cell, viral peptides would also be presented, allowing the immune system to recognize and kill the infected cell. Worn out proteins within the cell become ubiquitinated, marking them for proteasome degradation. Proteasomes break the protein up into peptides that include some around nine amino acids long (suitable for fitting within the peptide binding cleft of MHC class I molecules). Transporter associated with antigen processing (TAP), a protein that spans the membrane of the rough endoplasmic reticulum, transports the peptides into the lumen of the rough endoplasmic reticulum (ER). Also within the rough ER, a series of chaperone proteins, including calnexin, calreticulin, ERp57, and Binding immunoglobulin protein (BiP) facilitates the proper folding of class I MHC and its association with β2 microglobulin. The partially folded MHC class I molecule then interacts with TAP via tapasin (the complete complex also contains calreticulin and Erp57 and, in mice,

000005 calnexin). Once the peptide is transported into the ER lumen it binds to the cleft of the awaiting MHC class I molecule, stabilizing the MHC and allowing it to be transported to the cell surface by the golgi apparatus.

The exogenous pathway The exogenous pathway is utilized by specialized antigen-presenting cells to present peptides derived from proteins that the cell has endocytosed. The peptides are presented on MHC class II molecules. Proteins are endocytosed and degraded by acid-dependent proteases in endosomes; this process takes about an hour.[1] The nascent MHC class II protein in the rough ER has its peptide-binding cleft blocked by Ii (the invariant chain; a trimer) to prevent it from binding cellular peptides or peptides from the endogenous pathway. The invariant chain also facilitates MHC class II's export from the ER in a vesicle. This fuses with a late endosome containing the endocytosed, degraded proteins. The invariant chain is then broken down in stages, leaving only a small fragment called "Class II- associated invariant chain peptide" (CLIP) which still blocks the peptide binding cleft. An MHC class II-like structure, HLA-DM, removes CLIP and replaces it with a peptide from the endosome. The stable MHC class-II is then presented on the cell surface.

Cross-presentation processing In Cross-presentation, peptides derived from extracellular proteins are presented in the context of MHC class I. The cell starts off with the exogenous pathways but diverts the antigens (cytosolic diversion) to the endogenous pathway. This can allow the cell to skip the parts of the endogenous pathway that involve synthesis of antigens from the antigenic genes with cellular machinery upon infection, because the endogenous pathway can involve infection before being able to present antigens with MHC I, and cross-presentation saves them the effort needed for that and allows the professional antigen-presenting cells (dendritic cells) to process and present antigens without getting infected, which does not tend to happen to dendritic cells and is quite common scenario of antigen-processing using the endogenous pathway.[2] Not all antigen-presenting cells utilize cross-presentation.

Viral evasion of antigen processing Certain species in the Cytolomegavirus family can cause the infected-cell to produce proteins like US2, 3, 6, and/or 11. US11 and US2 mislead MHC I to the cytoplasm; US3 inhibits the transportation of MHC I in the ER (a part of the endogenous pathway and cross-presentation); US6 blocks peptide transportation by TAP to MHC I. Mycobacterium tuberculosis inhibits phagosome-endosome fusion, thus avoiding being destroyed by the harsh environment of the phagosome.[3] ICP47 from some herpesvirus block transport of the peptide by TAP. U21 from some human herpesvirus 7 binds and targets certain MHC I molecules for lysosomal degradation. E19 from some adenoviruses block the movement of MHC I to the proper locations for the endogenous pathway. Nef from some HIV strains enhance the movement of MHC molecules back into the cytoplasm, preventing them from presenting antigens.

000006 The role of Langerhans' dendritic cells in antigen processing Langerhans' cells are particular type of dendritic cells present in non lymphoid tissues together with interstitial cells. When these cells (in an immature state) come in contact with antigenic cells or disease causing viruses etc. these cells produce an inflammatory stimulus and start antigen processing and move toward lymph nodes where these APCs present antigen to mature T lymphocytes. T-dependent antigen – Antigens that require the assistance of T cells to induce the formation of specific antibodies. T-independent antigen – Antigens that stimulate B cells directly.

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