The four stages of antigen presentation are outlined in Figure 8.1. In lymphoid organs, all four stages of the process can occur, resulting in T cell proliferation. However, antigen presentation can also occur to a more limited degree in tissues, resulting in cytokine production, but with little T cell division.

Fig. 8.1 Summary of the key intercellular signals in T cell activation

Association of APCs and T cells first involves non-specific, reversible binding through adhesion molecules, such as LFA-1 with ICAM-1 or ICAM-3. Antigen-specific recognition of the peptide antigen in the MHC molecule by the TCR, provides the specificity of the interaction, and results in prolonged cell–cell contact. A second signal (costimulation) is necessary for the T cell to respond efficiently, otherwise tolerance may result. Cytokine signals result in upregulation of cytokines and their receptors, including IL-2 and the IL-2 receptor (CD25) which drive T cell division. Expression of IL-2 receptor is enhanced by IL-1 from the APC.

Interactions with antigen-presenting cells direct T cell activation

The way in which a T cell first encounters antigen largely dictates how it will react subsequently. A wide spectrum of cells can present antigen, depending on how and where the antigen is first encountered by cells of the immune system. In a lymphoid organ, the three main types of APC are:

• DCs which are most effective at presentation to naive T cells;

• macrophages; and

• B cells (Fig. 8.2).

Fig. 8.2 Localization of antigen-presenting cells (APCs) in lymph nodes

A lymph node represented schematically showing afferent and efferent lymphatics, follicles, the outer cortical B cell area, and the paracortical T cell area. Different APCs predominate in these areas and selectively take up different types of antigen, which then persist on the surface of the cells for variable periods. Polysaccharides are preferentially taken up by marginal zone macrophages and may persist for months or years, whereas antigens on recirculating macrophages in the medulla may last for only a few days or weeks. The recirculating ‘veiled’ cells (Langerhans’ cells and dermal dendritic cells), which originally come from the skin, change their morphology to become interdigitating dendritic cells within the lymph node. Both these cells and the follicular dendritic cells have long processes, which are in intimate contact with lymphocytes.

Activation of naive T cells on first encounter with antigen on the surface of an APC is called priming, to distinguish it from the responses of effector T cells to antigen on the surface of their target cells and the responses of primed memory T cells.

Dendritic cells are crucial for priming T cells

Dendritic cells which are found in abundance in the T cell areas of lymph nodes and spleen, are the most effective cells for the initial activation of naive T cells. They pick up antigens in peripheral tissues, then migrate to lymph nodes, where they express high levels of adhesion and costimulatory molecules, as well as MHC class II molecules, which allow them to interact with CD4⁺ TH cells.

Once they have migrated, DCs stop synthesizing MHC class II molecules, but maintain high levels of MHC class II molecules containing peptides from antigens derived from the tissue where they originated. Interdigitating DCs are believed to be the major APCs involved in primary immune responses because they induce T cell proliferation more effectively than any other APC.

The majority of dendritic cells enter lymph nodes via afferent lymphatics. Originally it was thought that these cells were mostly derived from Langerhans’ cells in the skin, but it now appears that a substantial proportion of the early migrating DCs in afferent lymph are dermal dendritic cells (they do not express langerin (CD207) a marker of Langerhans’ cells). Moreover DCs derived from Langerhans’ cells tend to localize in the paracortex of the lymph node, whereas dermal DCs remain near lymphoid follicles. Dendritic cells arriving from the periphery of the body transport antigen to the lymph node and process it for presentation to T cells. A minor proportion of the DCs in lymph nodes, arrive from the blood across the HEV, using the same route as T cells and B cells (see Fig. 6.15), however these cells have not acquired antigen in the periphery and they can only acquire it from lymph or transfer from other cells. As they mature, dendritic cells express CCR7 which allows them to localize to the lymphoid tissues. There is also some evidence that DCs from skin and the gut have distinctive chemokine receptors, which allow them to selectively recirculate to their own lymphoid organs. As they mature, DCs also increase expression of key costimulatory molecules, including CD40, CD80 and CD86 (B7-1 and B7-2).

Macrophages and B cells present antigen to primed T cells

Macrophages and B cells are less effective than DCs at antigen presentation to naive T cells, partly because they only express appropriate costimulatory molecules upon infection or contact with microbial products. Although they migrate to lymph nodes, the numbers of macrophages in afferent lymph is relatively few by comparison with DCs and this too limits their effectiveness in activating naive T cells. Macrophages:

• ingest microbes and particulate antigens;

• digest them in phagolysosomes; and

• present fragments at the cell surface on MHC molecules.

Q. A number of bacterial components enhance the expression of MHC molecules and costimulatory molecules on macrophages. What effect would you expect this to have on the immune response? Would it be advantageous for the individual?

A. In the presence of infection, the action of the microbial components would enhance the ability of macrophages to present antigen to T cells. This would generally be advantageous because it would allow the immune system to respond more effectively to the infection. However, in some circumstances it might be disadvantageous because microbial components would also enhance unwanted immune responses such as autoimmune reactions.

B cells can:

• bind to a specific antigen through surface IgM or IgD;

• internalize it; and

• then degrade it into peptides, which associate with MHC class II molecules.

If antigen concentrations are very low, B cells with high-affinity antigen receptors (IgM or IgD) are the most effective APC because other APCs simply cannot capture enough antigen. Therefore, for secondary responses, when the number of antigen-specific B cells is high, B cells may be a major APC.

The properties and functions of some APCs are summarized in Figures 8.3 and 8.4.

Fig. 8.3 Antigen presentation

Mononuclear phagocytes (1), B cells (2), and DCs (3) can all present antigen to MHC class II restricted TH cells. Macrophages take up bacteria or particulate antigen via non-specific receptors or as immune complexes, process it, and return fragments to the cell surface in association with MHC class II molecules. Activated B cells can take up antigen via their surface immunoglobulin and present it to T cells associated with their MHC class II molecules. DCs constitutively express MHC class II molecules and take up antigen by pinocytosis.

Fig. 8.4 Antigen-presenting cells

Many APCs are unable to phagocytose antigen, but can take it up in other ways, such as by pinocytosis. Endothelial cells (not normally considered to be APCs) that have been induced to express MHC class II molecules by interferon-γ (IFNγ) are also capable of acting as APCs, as are some epithelial cells. Another example is the thyroid follicular cell, which acts as an APC in the pathogenesis of Graves’ autoimmune thyroiditis.

Antigen processing

Antigen processing involves degrading the antigen into peptide fragments. The vast majority of epitopes recognized by T cells are fragments from a peptide chain. Only a minority of peptide fragments from a protein antigen are able to bind to a particular MHC molecule. Furthermore, different MHC molecules bind different sets of peptides (see Chapter 5). For example, the great majority of the immune response in humans against the HIV matrix protein is directed against a single immunodominant region, i.e. one which is recognized by a large number of T cells. However, exactly which part of this region is recognized, depends on the MHC haplotypes of the individual (Fig. 8.5).

Fig. 8.5 Recognition of HIV protein gag

Overlapping polypeptide fragments from an immunodominant region of the matrix protein (p17) of the HIV protein Gag, are presented by different variant MHC molecules. The amino acid sequence is given in single letter code and the peptide presented by each MHC molecule is shaded in blue.

Antigens are partially degraded before binding to MHC molecules

The processing of antigens to generate peptides that can bind to MHC class II molecules occurs in intracellular organelles. Phagosomes containing endocytosed proteins fuse with lysosomes where a number of proteases are involved in breaking down the proteins to smaller fragments. The proteases include:

• cathepsins B and D;

• an acidic thiol reductase, γ-interferon-inducible lysosomal thiol reductase (GILT), which acts on disulfide-bonded proteins.

Alkaline agents such as chloroquine or ammonium chloride diminish the activity of proteases in the phagolysosomes and therefore interfere with antigen processing.

In laboratory studies, the requirement for internal degradation of antigen by APCs can be circumvented by the use of synthetic peptides. This ability to use synthesized peptides of known sequences has enabled researchers to readily identify epitopes recognized by T cells with different specificities.

The relative importance of different amino acids within a defined epitope can also be investigated by amino acid replacements at different sites to identify residues that bind to the MHC molecule and those that bind to the TCR.

MHC class I pathway

MHC class I-restricted T cells (CTLs) recognize endogenous antigens synthesized within the target cell, whereas class II-restricted T cells (TH) recognize exogenous antigen.

Manipulation of the location of a protein can determine whether it elicits an MHC class I- or class II-restricted response. For example:

• influenza virus hemagglutinin (HA), a glycoprotein associated with the membrane of an infected host cell, normally elicits only a weak CTL response, but influenza virus HA can be generated in the cytoplasm by deleting that part of its sequence that encodes the N terminal signal peptide (required for translation across the membrane of the endoplasmic reticulum [ER]) and, when this is done, there is a strong CTL response to HA;

• the introduction of ovalbumin into the cytoplasm of a target cell (using an osmotic shock technique) generates CTLs recognizing ovalbumin, whereas the addition of exogenous ovalbumin generates an exclusively TH cell response.

Proteasomes are cytoplasmic organelles that degrade cytoplasmic proteins

Although the assembly of MHC class I molecules occurs in the ER of the cell, peptides destined to be presented by MHC class I molecules are generated from cytosolic proteins. The initial step in this process involves an organelle called the proteasome – a multi-protein complex which forms a barrel-like structure (Fig. 8.6).

Fig. 8.6 Generation of immunoproteasomes by replacement of active subunits

The 20 S proteasome, shown in cartoon form, is composed of four stacked disks, two identical outer disks of α subunits, and two similar inner disks comprised of β subunits. Each disk has seven different subunits. Peptides enter into the body of the proteasome for cleavage into peptides. Only three of the β subunits are active. In normal proteasomes, these are called MB1, delta, and Z (1). Interferon-γ treatment of cells results in replacement of these three subunits by the two MHC-encoded proteins, PSMB8 and PSMB9, as well as a third inducible protein, PSMB10 (2). These subunits are shown adjacent to each other here, whereas they are actually in separate parts of the β ring and some would be hidden at the back of the structure shown.

Proteasomes provide the major proteolytic activity of the cytosol. They have a range of different endopeptidase activities and they degrade denatured or ubiquitinated proteins to peptides of about 5–15 amino acids (ubiquitin is a protein that tags other proteins for degradation).

Two genes, PSMB8 and PSMB9 located in the class II region of the MHC (Fig. 8.7), encode proteasome components that subtly modify the range of peptides produced by proteasomes. The expression of these genes is induced by interferon-γ (IFNγ). The proteins displace constitutive subunits of the proteasome and along with a third inducible proteasome component (PSMB10 encoded on a different chromosome) influence processing of peptides by creating a wider range of peptide fragments suitable for binding MHC class I molecules. Additional subunits associate with the ends of core (20 S) proteasomes and may influence antigen processing. These include interferon-inducible PA28 (proteasome-activator-28) molecules as well as a complex of proteins that result in a larger 26 S particle.